sequence characterization of goat tlr genes -...

RESULTS

48

RESULTS

Sequence characterization of goat TLR genes

4.1. Isolation and quantification of RNA

Total RNA was extracted from different tissues of goat and used for cDNA

synthesis to amplify goat TLR4 and TLR8 genes for cloning and sequencing. The RNA

extracted by TRIzol method was further purified using RNeasy MinElute Clean-up kit

(Qiagen). The concentration and purity of RNA was measured by Nanodrop (ND1000).

The concentration and optical density (OD) ratio at 260 and 280nm of RNA isolated from

different tissues was estimated, the values for which have been given in Table 4.1. The

RNA concentration in different tissues was found to be between 564.4 ng/µl (heart) to

4984 ng/µl (intestine). For all the samples except for intestine the 260/280 ratio was

around 1.9 to 2.0, indicating good quality RNA without any RNA contamination and

suitable for further use.

Table. 4.1. Concentration and Purity of RNA isolated from goat tissues.

Tissue Conc.(ng/µl) 260/280nm

Liver 4983 1.9

Lungs 4284 1.9

Kidney 3844 2.0

Skin 1082 2.0

Ovary 3513 2.0

Placenta 4876 1.9

Spleen 4975 1.87

Lymph node 4446 1.9

Heart 564.4 2.0

Mammary gland 1292 2.0

Intestine 4984 1.8

Skeletal muscle 2881 2.0

4.2. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

cDNA was synthesized using RevertAid™ First Strand cDNA Synthesis Kit (MBI,

Fermentas). This kit is designed for preparation of full-length first strand cDNA from

RNA templates. The kit relies on a genetically engineered version of the Moloney Murine

Leukemia Virus Reverse Transcriptase (RevertAid™ M-MuLV RT) with low RNaseH

activity. This allows the synthesis of full-length cDNA from long templates (up to 13 kb).

cDNA was synthesized from various RNA samples using Oligo(dT)18 primers,

helping in synthesis of larger cDNA fragments for multiple genes from the same reaction.

4

RESULTS

49

The cDNA synthesized from the same reaction was further utilized for full gene

amplification as well as real-time PCR analysis of both TLR4 and TLR8 genes of goat.

4.3. PCR amplification

Complete coding regions corresponding to goat TLR4 and TLR8 were amplified

from the cDNA synthesized from RNA of spleen tissue by using single primer pair

covering the complete ORF. Amplified products were checked by using 1.5% agarose gel

electrophoresis. Specific PCR products of expected sizes of goat TLR4 and TLR8 gene

were obtained in the amplification reactions. The full length amplified PCR products were

purified by using QIAquick PCR purification kit (Qiagen). After purification, the obtained

products were found to be free from primer dimers or any nonspecific amplification (Fig.

4.1).

(a) (b)

Fig. 4.1. Ethidium bromide stained agarose gel showing the purified PCR products

of (a) TLR4 (2.5Kb) and (b) TLR8 (3.2 Kb) genes of goat.

4.4. Cloning of goat TLR4 and TLR8 genes

4.4.1. Ligation of PCR products

The kit purified PCR products of goat TLR4 and TLR8 genes were ligated in

pGEM®

-T Easy Vector (Promega) by using manufacturer‟s instructions. The ligated

recombinant plasmid DNA having goat TLR4 and TLR8 inserts were used to transform

CaCl2 treated competent E. coli DH5-α cells. Recombinant clones were identified by

blue/white i.e. „lac Z‟ selection system. Blue colonies were indicating the non-recombinant

clones, where as white colonies were recombinant ones (Fig. 4.2). Most of the colonies

obtained from the transformation experiment were white, which indicated good ligation

and transformation efficiency.

TLR4 ORF

1Kb ladder

TLR8 ORF

1Kb ladder

RESULTS

50

Fig.4.2. LB-agar plate showing recombinant (white) and non-recombinant (blue)

colonies.

4.4.2. Confirmation of recombinant clones

Plasmid DNA isolated from four white colonies each of TLR4 and TLR8 genes

were screened to confirm the recombinant plasmid having insert of desired gene by

different methods described below.

Plasmid isolation from transformed cells

Plasmid DNA was isolated from the recombinant clones by alkali lysis method, as

well as kit (Qiagen Midi Prep) using manufacturer‟s instructions and was checked by 1%

agarose gel electrophoresis. Good quality plasmid was obtained without any contamination

of genomic DNA or RNA.

PCR from culture and plasmid

PCR was carried out directly from the culture of white colonies as well as plasmid

DNA isolated from white colony cultures using the gene specific primers. PCR products

were runned on 1.5% agarose gel which showed the presence of TLR4 and TLR8 inserts in

all the four clones screened with the insert of 2582 bp size for TLR4 and 3238 bp for

TLR8 genes, when run along with 1 kb molecular marker (Fig. 4.3 a,b).

(a) (b)

Fig. 4.3. Ethidium bromide stained agarose gel confirming the inserts of (a) TLR4

(2.5 kb) and (b) TLR8 (3.2 kb) by PCR of recombinant clones.

TLR4

ORF

1Kb ladder

TLR8 ORF

1Kb ladder

Non-recombinant blue

colonies

Recombinant

white colonies

RESULTS

51

Restriction enzyme digestion

The presence of correct size insert in the recombinant plasmid was further

confirmed by restriction endonuclease digestion method. After single digestion with EcoRI

restriction enzyme the products were checked on 2% agarose gel, which confirmed the

presence of recombinant clones by release of insert of specific size. Digestion with EcoRI

resulted in release of TLR4 fragment from the recombinant plasmid vector and yielded two

fragments of approximately 2.5 kb insert and 2.8 kb vector (Fig. 4.4a). Similarly, digestion

of TLR8 with EcoRI resulted in the release of 2 fragments (3.2 kb insert and 2.8 kb

Vector) (Fig. 4.4 b).

(a) (b)

Fig. 4.4. Agarose gel showing EcoRI digested products of cloned Goat TLR4 (a) Lane

1: Restriction digested product, Lane 2: Uncut recombinant plasmid DNA,

Lane3: 1 Kb DNA ladder and TLR8 (b) Lane 1: Uncut recombinant plasmid

DNA, Lane 2: Restriction digested product, Lane3: 1 Kb DNA ladder.

Confirmation by Sequencing

Recombinant plasmids having TLR4 and TLR8 gene inserts were further

confirmed by sequencing from both ends by using M13 (forward and reverse) universal

primers. The sequencing of recombinant plasmids also confirmed the presence of TLR4

and TLR8 gene inserts on the basis of homology shown with reported TLR4 and TLR8

genes after BLASTN analysis. After confirmation, recombinant plasmids were subjected

to full length sequencing which was carried out by primer walking/internal gene specific

primers.

4.5. Sequence analysis of goat TLR4

Sequence Characterization

Different overlapping fragments of goat TLR4 gene sequences generated by

internal primers were assembled into 2664 bp long contig, which included 2526

nucleotides long coding sequence (CDS) along with 108 bp long partial 5‟UTR of and 29

TLR 4kb

1Kb ladder

Uncut

plasmid Vector

TLR4 ORF

1Kb ladder

Uncut

plasmid

TLR8 ORF

Vector

1 kb

ladder

RESULTS

52

Fig. 4.6. Tertiary Structure visualized by PyMole

(Red- signal peptide, Green-TM, Yellow –TIR).

bp long 3‟UTR. The sequence was submitted to NCBI (GenBank accession no. JF825527).

The predicted open reading frame (ORF) of goat TLR4 gene was translated into protein

and was found to be 841 amino acids long. Secondary structure of goat TLR4 protein was

analysed by using CLC Main Workbench5 showing presence of 46 alpha-helices and 16

beta-sheets (Table 4.2).

Domain structure analysis

Amino acid sequence of goat TLR4 protein was further analyzed by using online

program Simple Modular Architecture Research Tool (SMART), which revealed amino

acids 1-629 to be lying in ectodomain, out of which amino acids 1-25 were part of

signaling peptide, amino acid 630-819 were found in cytoplasmic domain containing TIR

domain (674-819) as shown in Fig. 4.5. The ectodomain of goat TLR4 displayed different

regions, which included 13 leucine-rich repeats (LRRs), one leucine rich repeat of C-

terminal domain (LRRCT), one transmembrane domain and a conserved toll/interleukin-1

receptor domain (TIR) with their respective amino acid positions.

Fig. 4.5. Predicted domain structure of goat TLR4 by SMART analysis.

Tertiary structure of goat TLR4

Insilico translation of predicted coding

DNA sequence of goat TLR4 when subjected

to the online tool Modeller for the prediction of

tertiary structure, distinctive horseshoe shaped

structure was formed, this structure was formed

by the repeating amino acid sequence, termed as

“leucine-rich repeats”. The different functional

domains have been distinguished by different

colors, shown in (Fig. 4.6.). The structure was

found to be similar as that of the structure

reported for the human and mouse TLR4.

Cytoplasmic TIR domain

Trans membrane domain Signal peptide

Ectodomain (LRR)

RESULTS

53

Comparison of goat TLR4 with other livestock species

The nucleotide sequence of caprine TLR4 gene was compared on the basis of

alignment, percent similarity, phylogeny and secondary structures with other reported

livestock species. Goat TLR4 was found to have 2526 nucleotides long ORF, like other

livestock species except in horse, which was 2532 nucleotides long (Table 4.2).

Phylogenetic analysis on the basis of nucleotides and amino acid, showed goat being

placed closest to sheep (Fig. 4.7). The protein structure domains predicted by SMART

analysis was compared with other livestock species, which showed goat and sheep having

the similar domain structures (Fig. 4.8). The secondary structure of different livestock

species was predicted and the number of α-helices and β-sheets are shown in (Table 4.2),

which showed goat, cattle, buffalo and sheep having almost similar secondary structures

whereas, non-ruminant species pig showed higher number of beta strands as compared to

other species.

The SMART structure of goat TLR4 when compared with other livestock species

showed almost similar structure among the ruminant species but in non-ruminant species

the position of some LRRs varied. Prediction of different functional domains based on the

predicted amino acid sequences was attempted in goat in comparison to other livestock

species by aligning them altogether. The signal peptide was found to be conserved among

the ruminant species with some variation in non-ruminant species. The LRRs (1-4) were

also found to be conserved among the ruminants with some variations in non-ruminants.

LRR5 was more similar in goat and sheep as compared to cattle and buffalo, where they

shared the amino acid changes with horse and pig. Again, LRRs (6-10) were found to be

conserved among the ruminants, but LRR11 was found to be conserved between goat and

sheep in comparison to buffalo, cattle, horse and pig. LRR12, 13 and C-terminal LRR were

again found to be conserved among the ruminants as compared to non-ruminants, followed

by TIR domain (Fig. 4.9). Overall the results indicate that the ligand specificity in

ruminants may be conserved as compared to non-ruminants.

Fig. 4.7. Phylogenetic analysis of goat TLR4 nucleotides after alignment with other

livestock species.

RESULTS

54

Fig. 4.8. SMART analysis of goat TLR4, comparison of its domain structures with other

livestock species (variations are shown in boxes).

Table 4.2. Comparison of goat TLR4 gene structural attributes with other livestock

species.

Species ORF Amino

acid

CDS identity

(%) with goat

Amino acid identity

(%) with goat

Alpha-

Helices

Beta-

Sheets Accession no.

Goat 2526 841 - - 46 16 JF825527

Buffalo 2526 841 95.9 93.8 45 15 HQ343416

Cattle 2526 841 96.1 94.5 42 15 NM_174198.6

Sheep 2526 841 99.1 98.5 44 16 NM_001135930

Pig 2526 841 85.6 79.4 47 26 GQ503242

Horse 2532 843 83.3 77.6 49 15 NM_001099769

Buffalo

Cattle

Goat

Pig

Sheep

Horse

RESULTS

55

Goat MMARARRAAALIPAMAILSCLRTESWDPCVQVVPNISYQCMELNLYKIPDNIPVSTEMLD

Sheep -MARARRAAALIPAMAILSCLRTESWDPCVQVVPNISYQCMELNLYKIPDNIPMSTEMLD

Buffalo MMARARLAAALIPATAILSCLRTESWDPCVQVVPNISYQCMELNLYKIPDNIPISTKMLD

Cattle MMARARLAAALIPATAILSCLRTESWDPCVQVVPNISYQCMELNLYKIPDNIPISTKMLD

Pig MIPRIRLAVATIPAMAFLSCLRSESWDPCVQVVPNISYQCMELNFYKIPDNIPTSVKILD

Horse MMPPTRLAGTLIPAMAFLSCLRPESWDPCVQVVPNTTYQCMDLNLYKIPENIPTSTKELD

:. * * : *** *:*****.************ :****:**:****:*** *.: **

Goat LSFNYLRHLGSHNFSRFPELQVLDLSRCEIKIIEDNTFQGLNHLSTLILTGNPIQSLAWG

Sheep LSFNYLRHLGSHNFSRFPELQVLDLSRCEIKIIEDNTFQGLNHLSTLILTGNPIQSLAWG

Buffalo LSFNYLRHLGSHNFSSFPELQVLDLSRCEIKIIEDDTFQGLNHLSTLILTGNPIQSLAWG

Cattle LSFNYLRHLGSHNFSSFPELQVLDLSRCEIKIIEDDTFQGLNHLSTLILTGNPIQSLAWG

Pig LSFNYLSHLDSNSFSSFPELQVLDLSRCEIQTIDDDAYQGLNYLSTLILTGNPIQSLALG

Horse LSFNPLKELGSHSFSNFPELQVLDLSRCEIEMIEDDAYQGLNHLSTLILTGNPIRSLALG

**** * .*.*:.** **************: *:*:::****:***********:*** *

Goat AFSGLSSLQKLVAVETNLVSLDDFPIGHLKTLKELNVAHNFIHSFKLPEYFSNLPNLEHL

Sheep AFSGLSSLQKLVAVETNLVSLDDFPIGHLKTLKELNVAHNFIHSFKLPEYFSNLPNLEHL

Buffalo AFSGLSSLRKLVAVETNLVSLHDFPIGHLRALKELNVAHNFIHSFKLPEYFSNLPNLEHL

Cattle AFSGLSSLQKLVAVETNLVSLNDFPIGHLKNLKELNVAHNFIHSFKLPEYFSNLPNLEHL

Pig AFSGLPSLQKLVAVETNLASLEDFPIGHLKTLKELNVAHNHIHSFKLPEYFSNLPNLEHL

Horse AFSGLSSLQTLVAVETKLSSLEKFPIGHLKTLKELNVAHNLIHSFKLPEYFSKMPNLEHL

*****.**:.******:* **..******: ********* ***********::******

Goat DLSNNKIQNIYYEDVKVLHQMPLLNLSLDLSLNPLEFIEPGTFKEIKLNGLTLRSNFNSS

Sheep DLSNNKIQNIYYEDVKVLHQMPLLNLSLDLSLNPLEFIEPGTFKEIKLNGLTLRSNFNSS

Buffalo DLSNNKIQNIYYEDVKVLHQMPLLNLSLALSLNPLDFIEPGTFKEIKLNGLTLRSNFNSS

Cattle DLSNNKIQNIYYEDVKVLHQMPLLNLSLDLSLNPLDFIEPGTFKEIKLNGLTLRSNFNSS

Pig DLSKNKIENIYHEHLQVLHQVPLLNLSLDLSLNPLNFIEPGAFNKIRLNGLTLRSNFNSS

Horse DLSNNKIQNISHEDLRVLHQMPLLNLSLDLSLNPLEFIQPDAFKEIKLHKLTLRSNFDSI

***:***:** :*.::****:******* ******:**:*.:*::*:*: *******:*

Goat YVMKTCIQGLAGLKINRLVLGEFKNERKLQKFDRSCLEGLCNLTIEQFRIAYLNKFSR-N

Sheep DVMKTCIQGLAGLKINRLVLGEFKNERKLQKFDRSCLEGLCNLTIEQFRIAYLSKFSW-N

Buffalo HVMKTCIQGLAGLKTNRLVLGEFKNERKLQRFDRSFLEGLCNLTIEQFRIAYLDKFSG-D

Cattle HVMKTCIQGLAGLKTNRLVLGEFKNERKLQRFDRSFLEGLCNLTIEQFRIAYLDKFSG-D

Pig DVMKTCIQGLAGSKINQLVLGEFKNERNLESFDKSVLEELCNLTLEQFRIAHFGEFPD-D

Horse DVMKSCIQGLAGLKVNRLVLGEFKNERKLERFDTSALRGLHNLTIEEFRLAYIDNYSSKD

***:******* * *:**********:*: ** * *. * ***:*:**:*::.::. :

Goat DTDLFNCLANVSMISLLSIPLGSLQALLKDFRWQHLEMINCDFDKFPALELRSLKKFVFT

Sheep DTDLFNCLANVSVISLLSISLGSLQALLKDFRWQHLEMINCDFDKFPALKLRSLKKFVFT

Buffalo DTDLFNCLANVSVISLLSISLGSLQALLKDFRWQHLEIINCDFDKFPALKLSSLKKFVFT

Cattle DTDLFNCLANVSVISLLSISLGSLQALLKDFRWQHLEIINCDFDKFPALKLSSLKKFVFT

Pig VSDLFNCLANASVISLLSLNLRGLEALPNDFRWQHLEVVNCKLKQFPALKFNSLKKFVFK

Horse SIDLLNCLADISKISLVSLDLGNLKDFPKGFGWQDFELVNCRIEGFPTLELTSLKRLVFT

**:****: * ***:*: * .*: : :.* **.:*::** :. **:*:: ***::**.

Goat DNKDVSSFTKTELPSLQYLDLKRNHLSFKTCCSHTDFGTTNLKHLDLSFNDVITLSSNFM

Sheep DNKDVSTFTKTELPSLQYLDLKRNHLSFKSCCSHTDFGTTNLKHLDLSFNDVITLGSNFM

Buffalo DNKDISTFTEFQLPSLQYLDLKRNHLSFKGCCSHTDFGTTNLKHLDLSFNDVITLGSNFM

Cattle DNKDISTFTEFQLPSLQYLDLKRNHLSFKGCCSHTDFGTTNLKHLDLSFNDVITLGSNFM

Pig DNKHMHTFTEINLPNLQFLDLSGNHLSFKGCCSHNEFGTTKLKHLDLSFNEIITMKSNFM

Horse SNKDMKSFNEVKLPSLEFLDLSRNRLSFKSCCSEADLKTTRLKHLDLSFNDVISMSSNFM

.**.: :*.: :**.*::***. *:**** ***. :: **.*********::*:: ****

Goat GLEQLEHLDFQHSTLKQINAFSTFLSLRNLRYLDISYTNIRIVFHGIFTGLVSLQTLKMA

Sheep GLEQLEHLDFQHSTLKQINAFSTFLSLRNLRYLDISYTNIRIVFHGIFTGLVSLQTLKMA

Buffalo GLEQLEHLDLQHSTLKQINAFSAFLSLRNLRYLDISYTNIRIVFHGIFTGLVSLQTLKMA

Cattle GLEQLEHLDFQHSTLKQINAFSAFLSLRNLRYLDISYTNIRIVFHGIFTGLVSLQTLKMA

Pig GLEQLEYLDFQHSSLKQANDFSIFLSLRNLHYLDISYTNIHVVFRGIFAGLVSLQTLKMA

Horse GLEQLEHLDFQHSTLKQASDFPVFLSLKNLRYLDISYTNTRVVFHGIFDGLVSLQVLKMA

******:**:***:*** . *. ****:**:******** ::**:*** ******.****

Goat GNSFQNNLLPDIFTELTNLTILDLSKCQLEQVSWTAFHSLPSLQVLNMSHNKLLSLDTFL

Sheep GNSFQNNLLPDIFTELTNLTILDLSKCQLEQVSWAAFHSLPSLQVLNMSHNKLLSLDTFL

Buffalo GNSFQNNLLPDIFTELTNLTVLDLSKCQLEQVAQTAFHSLSSLQVLNMSHNKLLSLDTFL

Cattle GNSFQNNLLPDIFTELTNLTVLDLSKCQLEQVAQTAFHSLSSLQVLNMSHNKLLSLDTFL

Pig GNSFQNNLLPDVFTDLTNLILLDLSKCQLEQVSQRAFHSLPRLQVLNMSHNRLLFLDTLP

Horse GNSFKDNFLPNIFREMTNLTTLDLSKCNLEQVSQEAFCLLPRLRVLNMSHNNLLFLDMLP

****::*:**::* ::*** ******:****: ** *. *:*******.** ** :

Signaling Sequence (1-25)

LRR 1 (77-100)

LRR 3 (149-173)

LRR 4 (174-200) LRR 5 (201-225)

LRR 6 (372-393) LRR 7 (398-420)

LRR 8 (421-442) LRR 9 (446-469)

LRR 10 (470-494) LRR 11 (495-518) LRR 12 (519-542)

LRR 2 (100-124)

RESULTS

56

Goat YEPLHSLRILDCSFNRITASKEQELRSLPRNLTWLNLTQNEFACVCEHQSFLQWVKDQRQ

Sheep YEPLHSLRILDCSFNRITASKEQELRNLPRNLTWLNLTQNEFACVCEHQSFLQWVKDQRQ

Buffalo YEPLHSLRILDCSFNRIMASKEQELQNLPRSLTWLNLTQNAFACVCEHQSFLQWVKDQRQ

Cattle YEPLHSLRILDCSFNRIMASKEQELQNLPRSLTWLNLTQNAFACVCEHQSFLQWVKDQRQ

Pig YKPLHSLRILDCSYNLIVASKEQELQHLPRSLAFLNLTKNDFSCACEHQTFLQWVKDQKQ

Horse YKPLHSLQILDCSFNRIVAFKWQELQHFPSSLASLNLTQNDFACVCEYQSFLQWVKDQRQ

*:*****:*****:* * * * ***: :* .*: ****:* *:*.**:*:********:*

Goat LLVGAEQMMCAEPLDVKDMPVLGFRNATCQMSKTIISVSVVTVLLVSVVGVLVYKFYFHL

Sheep LLVGAEQMMCAEPLDVKDMPVLGFRNATCQMSKTIISVSVVTVLLVSVVGVLVYKFYFHL

Buffalo LLVGAEQMMCAEPLDMEDMPVLSFRNATCQLSKTIISVSVVTVLLVSVVGVLVYKFYFHL

Cattle LLVGAEQMMCAEPLDMEDMPVLSFRNATCQLSKTIISVSVVTVLLVSVVGVLVYKFYFHL

Pig LLVGAEQMVCTQPLEMQDLPVLSFRNATCQISEAVISASVLTFLLVSVAGILVYKFYFHL

Horse LLVEVEHLVCAIPLQMRGMPVLGFNNATCQISKTIVGGSVFSILMVSVIAVLVYKFYFHL

*** .*:::*: **::..:***.*.*****:*::::. **.:.*:*** .:*********

Goat MLLAGCKKYGRGESTYDAFVIYSSQDEAWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAI

Sheep MLLAGCKKYGRGESTYDAFVIYSSQDEAWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAI

Buffalo MLLAGCKKYGRGESTYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAI

Cattle MLLAGCKKYGRGESXYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAI

Pig LLFVGCKKYGRGESTYDAFVIYSSQDEDWVRNELVKNLEEGVPPFHLCLHYRDFIPGVAI

Horse MLLAGCKKYGRGESIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAI

:*:.********** ************ *****************:**************

Goat AANIIQEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKLEKSL

Sheep AANIIQEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKLEKSL

Buffalo AANIIQEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIVFIVLQKLEKSL

Cattle AANIIQEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKLEKSL

Pig AANIIQEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLRSHAGIIFIVLQKLEKSL

Horse AANIIQEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLHKLEKSL

****************************************** *:***:****:******

Goat LRQQVELYRLLNRNTYLEWEDSVLGRHVFWRRLRKALLAGKPRSPEGTADAETNPQEATTST-

Sheep LRQQVELYRLLNRNTYLEWEDSVLGRHVFWRRLRKALLAGKPRSPEGTADAETNPQEATTST-

Buffalo LRQQVELYRLLSRNTYLEWEDSVLGRHVFWRRLRKALLAGKPTSPEGTADAETNAQEVTTST-

Cattle LRQQVELYRLLSRNTYLEWEDSVLGRHVFWRRLRKALLAGKPQSPEGTADAETNPQEATTST-

Pig LRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLKKALLDGKPWSPEGTEDSESNQHDTTAFT-

Horse LRQQVELYRLLNRNTYLEWEDSVLGRHIFWRRLRKALLDGKPWSPAGTADAAESRQHDAETST-

***********.***************:*****:**** *** ** ** *: . :. : :

Fig. 4.9. Comparison of the domain architecture of goat, buffalo, cattle, sheep, pig

and horse TLR4 protein sequence. The domains were predicted using the

SMART program (Schultz et al., 1998). LRR- leucine-rich repeat; NT—N-

terminal repeat; CT—C-terminal repeat; TIR—toll/interleukin-1 receptor

domain.

4.7 Sequence analysis of goat TLR7 gene

Sequence characterization

A total of 3.4 kb long sequence data of caprine TLR7 gene was generated by

sequencing of seven overlapping PCR amplicons ranging from 424 to 1186 bp. The

sequence obtained was submitted to GenBank and Accession No. GU289401 was assigned

to the sequence. ORF of Caprine TLR7 was 3150 nucleotides long coding for 1049 amino

acids long protein, similar to sheep. Comparison of goat TLR7 genomic sequence with

cattle TLR7 genomic sequence reported in Ensemble (ENSBTAT00000029895) showed

goat TLR7 ORF limiting to exon3 only, different from cattle. The predicted amino acids

LRR 13 (543-566) LRR CT (579-629)

TIR (674-819)

TM (635-657)

RESULTS

57

Fig. 4.11. Tertiary Structure visualized by PyMole

(Red- signal peptide, Green-Extracellular domain,

Magenta –TIR)

were subjected to CLC Main workbench5 for the determination of secondary structure

which showed the presence of 39 alpha-helices and 40 beta-sheets (Table 4.3).

Domain Structure analysis

Amino acid sequence of goat TLR7 protein was further analyzed by using online

program Simple Modular Architecture Research Tool (SMART), which revealed amino

acids 1-853 to be lying in ectodomain, amino acids (837-853) were the part of low

complexity region, amino acids 854-1046 were found in cytoplasmic domain containing

TIR domain (887-1033) (Fig 4.10). The ectodomain of goat TLR7 displayed different

regions, which included 20 leucine-rich repeats (LRRs), one leucine rich repeat each of C-

terminal domain (LRRCT) and N-terminal domain (LRRNT) respectively, one low

complexity region and a conserved toll/interleukin-1 receptor domain (TIR) with their

respective amino acid positions represented below (Fig. 4.10).

Fig. 4.10. Predicted domain structure of goat TLR7 by SMART analysis.

Tertiary structure of goat TLR7

The translation of predicted coding

DNA sequence of TLR7 of goat when

subjected to the online tool Modeller for the

prediction of tertiary structure, distinctive

horseshoe shaped structure was formed, this

structure was formed by the overlapping of

“leucine-rich repeats”. The different

functional domains have been distinguished

by different colors, shown in (Fig. 4.11).

Ectodomain LRRs

Low complexity region


58


The extensive homology of goat TLR7 nucleotide sequence with that of sheep and

cattle also corresponded with extensive homologies of the deduced amino acids among

these species (Table 4.3). When phylogenetic analysis was carried out, as expected goat

shared the closest phylogenetic relationship with sheep followed by cattle and buffalo (Fig.

4.12). All the ruminant species branched off separately from other non-ruminants, whereas

among other livestock species, pig was closer to ruminant species than horse. The

secondary structure analysis showed little variation in the number of alpha and beta- sheets

in ruminants as compared to non-ruminants.

The domain structures of TLR7 gene for goat, sheep, cattle, buffalo, pig, horse

were predicted (Fig.4.13) and goat TLR7 structure was found similar to that of pig, cattle

and sheep, which consisted same numbers of extracellular domains with leucine-rich

repeats (LRRs), a transmembrane domain and cytoplasmic TIR, Toll-interleukin-1 receptor

domain. Other domains with high inter-species conservation included the N and C-

terminal cysteine-flanked LRRs and the transmembrane domains.

A number of amino acid changes were observed in different functional domains of

TLR7 of goat compared to the sequences of other species. Predicted amino acid sequence

of caprine TLR7 showed a total of four and 17 amino acid changes compared to that of

ovine and cattle sequences, respectively. The TIR domain was found to be conserved

among ruminant species goat, sheep, buffalo and cattle. One amino acid change, His34Cys

in the TIR domain was found to be ruminant specific as compared to other species

compared. Phylogenetic analysis based on nucleotide sequence of TIR domain showed

results similar to whole TLR7 gene sequence analysis, four ruminant species grouping

together. However, among four ruminant species, differentiation was more in goat and

sheep clustering little apart from each other as well as cattle and buffalo.The numbers of

LRR motifs in goat TLR7 were found to be similar to sheep but higher than cattle among

ruminants. Transmembrane (TM) domain was also absent in all the livestock species

except for pig (Fig. 4.13).

59

Fig.4.12.Phylogenetic analysis of goat TLR7 based on nucleotide homology with

reported TLR7 gene sequence of other species.

Table. 4.3. Comparison of goat TLR7 gene structural attributes with other livestock

species.

Fig. 4.13. SMART analysis of goat TLR7 comparing with other reported livestock

species.

Species ORF Amino

acid

CDS identity

(%) with goat

Amino acid

identity(%)

with goat

Alpha-

Helices

Beta-

Sheet

s

Accession no.

Goat 3150 1049 - - 39 40 GU289401

Buffalo 3153 1050 98.0 98.4 37 31 GU903503

Cattle 3153 1050 97.3 97.1 37 34 DQ333225

Sheep 3141 1046 99.5 99.6 39 30 NM001135059

Pig 3153 1050 90.5 90.8 42 27 NM001097434

Horse 3153 1050 89.6 88.5 43 27 NM001081771

60

Goat -VFPMWTLKRQFPILFNMILISELLGARWFPKTLPCDVTLDAPNAHVIVDCTDKHLTEIP

Sheep ----MWTLKRQFPILFNMILISELLGARWFPKTLPCDVTLDAPNAHVIVDCTDKHLTEIP

Buffalo MVFPMWTLKRQFPILFNMILISELLGARWFPKTLPCDVTLDAPNAHVIVDCTDKHLTEIP

Cattle MVFPMWTLKRQFPILFNMILISGLLGARWFPKTLPCDVTLDAPNTHVIVDCTDKHLTEIP

Pig MVFPMWTLKRQFLILFNIVLISELLGARWFPKTLPCDVSLDAPNAHVIVDCTDKHLTAIP

Horse MMFPMWTLKREFLILFNMILISKLLGARWFPKTLPCDVFLDAPKAQVIVDCTDKHLTDIP

******:* ****::*** *************** ****:::*********** **

Goat GGIPANATNLTLTINHIAGISPASFHRLDHLVEIDFRCNCIPVRLGPKDNVCTKRLQIKP

Sheep GGIPANATNLTLTINHIAGISPASFHRLDHLVEIDFRCNCIPVRLGPKDNVCTKRLQIEP

Buffalo GGIPANATNLTLTINHIAGISPASFHRLDHLVEIDFRCNCIPVRLGPKDNVCTKRLQIKP

Cattle GGIPANATNLTLTINHIAGISPASFHRLDHLVEIDFRCNCVPVRLGPKDNVCTKRLQIKP

Pig GGIPTNATNLTLTINHIASITPASFQQLDHLVEIDFRCNCIPVRLGPKDNLCTRRQQIKP

Horse GGIPTNATNLTLTINHIPGISPASFHQLENLLEIDFRCNCVPVRLGPKDNVCKNRLQIKP

****:************..*:****::*::*:********:*********:*..* **:*

Goat NSFSKLTYLKSLYLDGNQLLEIPQDLPPSLQLLSLEANNIFLIMKENLTELANLEILYLG

Sheep SSFSKLTYLKSLYLDGNQLLEIPQDLPPSLQLLSLEANNIFLIMKENLTELANLEILYLG

Buffalo NSFSKLTYLKSLYLDGNQLLEIPQDLPPSLQLLSLEANNIFLIMKENLTELANLEILYLG

Cattle NSFSKLTYLKSLYLDGNQLLEIPQDLPPSLQLLSLEANNIFLIMKENLTELANLEILYLG

Pig SSFSKLTYLKALYLDGNQLLEIPRDLPPSLQLLSLEANNIFWIMKENLTELANLEMLYLG

Horse RSFSRLTNLKSLYLDGNQLLEIPQDLPPSLQLLSLEANNIFLIMKKNLTELANIEMLYLG

***:** **:************:***************** ***:*******:*:****

Goat QNCYYRNPCNVSFTIEKDAFLNMRNLKLLSLKDNNISAVPTVLPSSLTELYLYNNIITKI

Sheep QNCYYRNPCNVSFTIEKDAFLNMRNLKLLSLKDNNISAVPTVLPSSLTELYLYNNIITKI

Buffalo QNCYYRNPCNVSFTIEKDAFLNMRNLKLLSLKDNNISAVPTVLPSSLTELYLYNNIITKI

Cattle QNCYYRNPCNVSFTIEKDAFLNMRNLKLLSLKDNNISAVPTVLPSSLTELYLYNNIITKI

Pig QNCYYRNPCNVSFSIEKDAFLSLRNLKLLSLKDNNISAVPTVLPSTLTELFLYNNIIAKI

Horse QNCYYRNPCNVSFFIEKDAFLNLKNLKLLSLKDNNITAVPTILPSSLTELYLYNNIIAKI

************* *******.::************:****:***:****:******:**

Goat QEDDFNNLSQLQVLDLSGNCPRCYNVPFPCTPCENNSPLQIDLNAFDALTELQVLRLHSN

Sheep QEDDFNNLSQLQVLDLSGNCPRCYNVPFPCTPCENNSPLQIDLNAFDALTELQVLRLHSN

Buffalo QEDDFNNLSQLQVLDLSGNCPRCYNVPFPCTPCENNSPLQIDPNAFDALTELQVLRLHSN

Cattle QEDDFNNLSQLQVLDLSGNCPRCYNVPFPCTPCENNSPLQIDPNAFDALTELQVLRLHSN

Pig QEDDFNNLSQPQVLDLSGNCPRCYNVPFPCTPCENNAPLQIHLHAFDALTELQVLRLHSN

Horse QEDDFKMLNQLQILDLSGNCPRCYNVPYPCTPCENNSPLQIHANAFDALTELQVLRLHSN

*****: *.* *:**************:********:****. :****************

Goat SLQHVPQRWFKNINKLKELDLSQNFLAKEIGDAKFLHLLHNLVNLDLSFNYDLQVYHAVI

Sheep SLQHVPQRWFKNINKLKELDLSQNFLAKEIGDAKFLHLLHNLVNLDLSFNYDLQVYHAVI

Buffalo SLQHVPQRWFKNINKLKELDLSQNFLAKEIGDAKFLHLLHNLVNLDLSFNYDLQVYHAVI

Cattle SLQHVPQRWFKNINKLKELDLSQNFLAKEIGDAKFLHLLHNLVNLDLSFNYDLQVYHAVI

Pig SLQYVPQRWFQNLNKLKELDLSQNFLAKEIGDAKFLHLLPNLVKLDLSFNYELQVYHTFM

Horse SLQYVPQRWFKNINKLKELDLSQNFLAKEIGDAKFLHLLHNLVQLDLSFNYELQVYHASM

***:******:*:************************** ***:*******:*****: :

Goat NLSDAFSSLKKLKVLRIKGYVFKELNSLNLFPLCNLPSLEVLDLGTNFIKIADLSIFKQF

Sheep NLSDAFSSLKKLKVLRIKGYVFKELNSLNLFPLRNLPSLEVLDLGTNFIKIADLSIFKQF

Buffalo NLSDAFSSLKKLKVLRIKGYVFKELNSLNLFPLHNLPNLEVLDLGTNFIKIANLSIFNQF

Cattle NLSDAFSSLKKLKVLRIKGYVFKELNSLNLFPLHNLPNLEVLDLGTNFIKIANLSIFNQF

Pig NLSDSFSSLKNLKVLRIKGYVFKELKSLNLSPLRNLPNLEVLDLGTNFIKIANLSIFKQF

Horse NLSEAFSSWKNLRVLRIKGYVFKELKDLNLSPLRNLSNLEVLDLGTNFIKIADLSIFKQF

***::*** *:*:************:.*** ** **..**************:****:**

Goat KTLKFIDLSVNKISPSGDSTEGGFCSNMRTSVEGHGPQVLEALHYFRYDEYARSCRSKSK

Sheep KTLKFIDLSVNKISPSGDSTEGGFCSNMRTSVEGHGPQVLEALHYFRYDEYARSCRSKSK

Buffalo KTLKFIDLSVNKISPSGDSPEGGFCSNMRTSVAGHGPQVLETLHYFRYDEYARSCRSKSK

Cattle KTLKFIDLSVNKISPSGDSPEGGFCSNRRTSVEGHGPQVLETLHYFRYDEYARSCRSKSK

Pig KTLKFIDLSVNKISPSGDSSESGFCSGMRTSAESHGPQVLESLHYFRYDEYARSCRFKNK

Horse KTLKVIDLSMNKISPSGESSEVGFCSNTRTSVAGHGPQVLETLHYFRYDEYARSCWFKNR

****.****:*******:*.* ****. ***. .*******:************* *.:

Goat EPPSFLPLNEDCYMYGQTLDLSRNNIFFIKSSDFQHLSFLKCLNLSGNSISQTLNGSEFQ

Sheep EPPSFLPLNEDCYMYGQTLDLSRNNIFFIKSSDFQHLSFLKCLNLSGNSISQTLNGSEFQ

Buffalo EPPSFLPLNEDCYMYGQTLDLSRNNIFFIKPSDFQHLSFLKCLNLSGNSISQTLNGSEFQ

Cattle EPPSFLPLNEDCYMYGQTLDLSRNNIFFIKPSDFQHLSFLKCLNLSGNSISQTLNGSEFQ

Pig EPSSSLPLNEDCSMYGQTLDLSRNNIFFIRSSEFQHLTFLKCLNLSGNSISQALNGSEFQ

Horse ETSSFLPFNEGCYMYGQTLDLSKNNIFFIKSSDFRHLSFLKCLNLSGNSISQTLNGSEFQ

*..* **:**.* *********:******:.*:*:**:**************:*******

Signal peptide (1-22) LRR1 (29-65)

LRR2 (122-141) LRR3 (167-189)

LRR4 (199-222) LRR5 (220-239)

LRR6 (246-278) LRRTYP (285-308)

LRR7 (309-330) LRR8 (335-360)

LRR9 (392-412)

LRR10 (416-436)

LRR11 (493-512) LRR12 (513-537)

61

Goat PLVELKYLDFSNNRLDLLYSTAFEELHNLEVLDISSNSHYFQSEGITHMLNFTKNLKVLK

Sheep PLVELKYLDFSNNRLDLLYSTAFEELHNLEVLDISSNSHYFQSEGITHMLNFTKNLKVLK

Buffalo PLVELKYLDFSNNRLDLLYSTAFEELHNLEVLDISSNSHYFQSEGITHMLNFTKNLKVLK

Cattle PLVELKYLDFSNNRLDLLYSTAFEELHNLEVLDISSNSHYFQSEGITHMLNFTKNLKVLR

Pig PLVELKYLDFSNNRLDLLHSTAFEELRNLEVLDISSNSHYFQSEGITHMLDFTKNLKVLK

Horse PLVELKYLDFSNNRLDLLYSTAFEELRNLEVLDISSNSHYFQSEGITHMLNFTKNPKVLR

******************:*******:***********************:**** ***:

Goat KLMMNYNDIATSTSRTMESESLQILEFRGNHLDILWRDGDNRYLKFFKNLLNLEELDISE

Sheep KLMMNYNDIATSTSRTMESESLQILEFRGNHLDILWRDGDNRYLKFFKNLLNLEELDISE

Buffalo KLMMNYNDIATSTSRTMESESLQILEFRGNHLDILWRDGDNRYLKFFKNLLNLEELDISE

Cattle KLMMNYNDIATSTSRTMESESLQILEFRGNHLDILWRDGDNRYLKFFKNLLNLEELDISE

Pig KLMMNNNDIATSTSTTMESESLRILEFRGNHLDILWRDGDNRYLKFFKNLHKLEELDISE

Horse KLMMNNNDISTSTSRTMESESLTILEFRGNHLDVLWKDGDNRYLKFFKNLLNLKELDISE

***** ***:**** ******* **********:**:************* :*:******

Goat NSLSFLPLGVFDSMPPNLKTLSLAKNGLKSFSWKGLQSLKNLETLDLSFNQLKTVPERLS

Sheep NSLSFLPLGVFDSMPPNLKTLSLAKNGLKSFSWKGLQSLKNLETLDLSFNQLKTVPERLS

Buffalo NSLSFLPLGVFDSMPPNLKTLSLAKNGLKSFSWEKLQSLKNLETLDLSFNQLKTVPERLS

Cattle NSLSFLPLGVFDSMPPNLKTLSLAKNGLKSFSWERLQSLKNLETLDLSFNQLKTVPERLS

Pig NSLSFLPSGVFDGMPPNLKTLSLAKNGLKSFNWGKLQYLQNLETLDLSYNQLKTVPERLS

Horse NSLSFLPPGVFESMPPNLKTLYLVNNKFKSFNWGKLQLLKNLETLDLSYNQLRTVPERLS

******* ***:.******** *.:* :***.* ** *:********:***:*******

Goat NCSRSLKKLILKNNQIRCLTKYFLQGAFQLRHLDLSSNKIQVIQKTSFPENVLNNLNILF

Sheep NCSRSLKKLILKNNQIRCLTKYFLQGAFQLRHLDLSSNKIQVIQKTSFPENVLNNLNILF

Buffalo NCSRSLKKLILKNNQIRCLTKYFLQGAFQLRHLDLSSNKIQVIQKTSFPENVLNNLNILF

Cattle NCSRSLKKLILKNNQIRCLTKYFLQGAFQLRHLDLSSNKIQVIQKTSFPENVLNNLNILF

Pig NCSRSLKKLILKNNEIRNLTKYFLQDAFQLRHLDLSSNKIQVTQKTSFPENVLNNLQILF

Horse NCSRSLKKLILKNNQIRRLTKYFLQDAFQLRYLDLSSNKIQIIQKSSFPENVLNNLDMLL

**************:** *******.*****:*********: **:**********::*:

Goat LHHNRFLCNCDAVWFVWWVNHTEVTIPYLATDVTCMGPGAHKGQSVVSLDLYTCELDLTN

Sheep LHHNRFLCNCDAVWFVWWVNHTEVTIPYLATDVTCMGPGAHKGQSVVSLDLYTCELDLTN

Buffalo LHHNRFLCNCDAVWFVWWVNHTEVTIPYLATDVTCMGPGAHKGQSVVSLDLYTCELDLTN

Cattle LHHNRFLCNCDAVWFVWWVNHTEVTIPYLATDVTCMGPGAHKGQSVVSLDLYTCELDLTN

Pig LHHNRFLCNCDAVWLVWWVNHTEVTIPFLATDVTCMGPGAHKGQSVVSLDLYTCELDLTN

Horse LHRNRFLCTCDAVWFVWWVNHTEVTIPYLATDVTCTGPGAHKGQSVVSLDLYTCELDLTN

**:*****.*****:************:******* ************************

Goat FILFSLSISAILSLMMITTANHLYFWDVWYSYHFCKAKIKGYRRLISPNSCYDAFIVYDT

Sheep FILFSLSISAVLSLMMITTANHLYFWDVWYSYHFCKAKIKGYRRLISPNSCYDAFIVYDT

Buffalo FILLSLSISAVLSLMMITTANHLYFWDVWYSYHFCKAKIKGYRRLISPNSCYDAFIVYDT

Cattle FILFSLSISAVLSLMMITIANHLYFWDVWYSYHFCKAKIKGYRRLISPNSCYDAFIVYDT

Pig FVLFSLSLSAVLFLIVITIANHLYFWDVWYSYHFCKAKIKGYQRLISPNSCYDAFIVYDT

Horse LILFSLSMSMALFLMVVTTANHLYFWDVWYSYHFCKAKIKGYQRLTLTDSCYDAFIVYDT

::*:***:* * *:::* ***********************:** .:***********

Goat KDPAVTEWVLDELVAKLEDPREKCFNLCLEERDWLPGQPVLENLSQSIQLSKKTVFVMTD

Sheep KDPAVTEWVLDELVAKLEDPREKCFNLCLEERDWLPGQPVLENLSQSIQLSKKTVFVMTD

Buffalo KDPAVTEWVLDELVAKLEDPREKCFNLCLEERDWLPGQPVLENLSQSIQLSKKTVFVMTD

Cattle KDPAVTEWVLDELVAKLEDPREKCFNLCLEERDWLPGQPVLENLSQSIQLSKKTVFVMTD

Pig KDPAVTEWVLDELVAKLEDPREKHFNLCLEERDWLPGQPVLENLSQSIQLSKKTVFVMTD

Horse KDPAVTEWVLDELVAKLEDPREKHFNLCLEERDWLPGQPVLENLSQSIQLSKKTVFVMTD

*********************** ************************************

Goat KYAKTENFKIAFYLSHQRLMDEKVDVIILIFLEKPLQKSKFLQLRKRLCGSSVLEWPTNP

Sheep KYAKTENFKIAFYLSHQRLMDEKVDVIILIFLEKPLQKSKFLQLRKRLCGSSVLEWPTNP

Buffalo KYAKTENFKIAFYLSHQRLMDEKVDVIILIFLEKPLQKSKFLQLRKRLCGSSVLEWPTNP

Cattle KYAKTENFKIAFYLSHQRLMDEKVDVIILIFLEKPLQKSKFLQLRKRLCGSSVLEWPTNP

Pig KYAKTEKFKIAFYLSHQRLMDEKVDVIILIFLEKPLQKSKFFQLRKRLCGSSVLEWPTNP

Horse KYAKTENFKIAFYLSHQRLIDEKVDVIILIFLEKPLQKSKFLQLRKRLCGSSVLEWPRNP

******:**********************************:*************** **

Goat QAHPYFWQCLKNALATDNHVTYSQVFKETA

Sheep QAHPYFWQCLKNALATDNHVTYSQVFKETA

Buffalo QAHPYFWQCLKNALATDNHVTYSQVFKETA

Cattle QAHPYFWQCLKNALATDNHVTYSQVFKETA

Pig QAHPYFWQCLKNALATDNHVTYSQVFKETA

Horse QAHPYFWQCLKNALATDNHVTYSQVFKETV

*****************************.




terminal repeat; CT—C-terminal repeat; LC- Low complexity; TIR—

toll/interleukin-1 receptor domain.

TIR domain (887-1033)

LRR13 (538-564) LRR14 (592-615)

LRRTYP (646-669)

LRR15 (671-690) LRRTYP (695-718)

LRR16 (719-738) LRR17 (743-764)

LRRCT (780-831) LC (837-853)

62

Prediction of different functional domains based on the predicted amino acid

sequences was attempted in goat in comparison to other livestock species by aligning them

altogether. The signal peptide was found to be conserved among the ruminant species with

some variation in non-ruminant species. The LRR (1-6) were also found to be conserved

among the ruminants with some variations in non-ruminants. LRRTYP was more similar

in goat and sheep as compared to cattle and buffalo, where they shared the amino acid

changes with horse and pig. Again, LRRs (7,8) were found to be conserved among the

ruminants, but LRR9 was found to be conserved between goat and sheep in comparison to

buffalo, cattle, horse and pig. LRR10 was also conserved within ruminants as compared to

non-ruminant species, LRR11 was also found to be more similar in sheep and goat as

compared to other species. LRR12-17 and C-terminal LRR and 2 LRR_TYP were again

found to be conserved among the ruminants as compared to non-ruminants, followed by

low complexity region and TIR domain (Fig. 4.14). Overall the results indicate that the

ligand specificity in ruminants may be conserved as compared to non-ruminants.

4.8. Sequence analysis of goat TLR8 gene

Sequence characterisation

Recombinant plasmid of goat TLR8 was sequenced by primer walking and

sequence was assembled into 3238 nucleotides long contig by using SeqMan program of

Lasergene software. In the assembled mRNA sequence, TLR8 gene ORF was identified

with the help of EditSeq program. It showed goat TLR8 ORF to be 3102 nucleotides long,

coding for 1033 amino acids, with partial 5‟UTR of 90 bp and 3‟UTR of 45 bp, which was

submitted to NCBI (GenBank accession no. HM769860).

Domain Structure analysis

Further, SMART analysis revealed amino acids, 1-817 lying in ectodomain, out of

which amino acids 1-18 were part of signal peptide, amino acids 818-840 being part of the

transmembrane region of TLR8 and amino acids 841-1033 were found in the cytoplasmic

domain containing TIR (871-1017), as shown in Fig. 4.15. The ectodomain of goat TLR8

had different regions, which included 15 leucine-rich repeats (LRR), 3 leucine rich repeats

of typical subfamily (LRR-TYP), one leucine rich repeat of C-terminal domain (LRRCT),

one transmembrane domain and a conserved toll-interleukin1-receptor (TIR) domain.

Secondary structure prediction of the protein sequence carried out using CLC Main

Workbench, which revealed goat TLR8 amino acid sequence having 41 alpha-helices and

33 beta-sheets (Table 4.4).

63

Fig. 4.16. Tertiary structure visualized by PyMole

(Red- signal peptide, Green-TM, Yellow-TIR)

Fig. 4.15. Predicted domain structure of goat TLR8 by SMART analysis, showing

different functional domains.

Tertiary structure prediction of TLR8 gene of goat

Insilico translation of predicted coding

DNA sequence of TLR8 of goat, when subjected

to the online tool Modeller for the prediction of

tertiary structure, the distinctive horseshoe shaped

structure was observed. This structure was formed

by the overlapping of repeating amino acid

sequence, termed a “leucine-rich repeats”. The

different functional domains could be

distinguished by different colors (Fig. 4.16).


When nucleotide sequence of goat TLR8 was compared, with other livestock

species, goat TLR8 was found to have same ORF of 3102 nucleotides long, except pig

(3087 nucleotides) and horse (3117 nucleotides) as given in Table 4.4. Phylogenetic

analysis both at nucleotide and amino acid level showed goat being placed closer to

buffalo, then to cattle, sheep, pig and horse. Horse was again placed most distant from the

other livestock species Fig. 4.17.

The prediction of different functional domains in the goat TLR8 gene was carried

out by online software SMART, results of which were further compared to other reported

livestock species. All the ruminant species shared almost the similar domain structure but

the non-ruminant species pig and horse varied in the number of LRRs and their positions

in the structure (Fig. 4.18).

Ectodomain LRRs


Trans membrane domain Signal peptide

64

Fig. 4.17. Phylogenetic analysis of goat TLR8 based on comparison with other

livestock species.

Fig. 4.18. SMART analysis of goat TLR8 and its comparison with predicted

structures of other reported livestock species (Domain variations are

shown in boxes).

Table 4.4. Comparison of goat TLR8 characteristics with other livestock species.

Species

ORF

lengt

h

No. of

amino

acids

Nucleotide (%)

identity with

goat

Amino acid (%)

identity with goat

Alpha-

Helices

Beta-

Sheets Accession no.

Goat 3102 1033 - - 41 33 HM769860

Buffalo 3102 1033 99.1 98.5 43 35 GQ499855

Cattle 3102 1033 98.1 97.0 2 38 EF583902

Sheep 3102 1033 97.8 96.8 4 33 NM_001135929

Pig 3087 1028 84.0 79.3 5 34 AB258452

Horse 3117 1038 81.8 77.2 43 28 EF090622

65

Goat MTLRFLLLTSLFLLISDSCEFFTEASYPRSYPCDVKNENGSFIAECNSRRLQEVPQTVDK

Buffalo MTLRFLLLTSLFLLISDSCEFFTEASYPRSYPCDVKNENGSFIAECNSRRLQEVPQTVDK

Cattle MTLHFLLLTSLFLLISDSCEFFTEASYPRSYPCDVKNENGSFIAECNGRRLQEVPQTVDK

Sheep MTLHFLLLTSLFLLISDSCEFFTEASYPRSYPCDVKNKNGSFIAECNSRRLQEVPQTVDK

Pig MTLHFLLLTCLFLLIPDSCEFFTGANYSRSYPCDERKENGSVIAECNNRQLQEVPRRVGN

Horse MILQPLLLTCLFLIISDSCEFFTETNYSRSYPCDEKRENVSVIAECNDRRLEEVPQTVGK

* *: ****.***:*.******* :.*.****** :.:* *.*****.*:*:***: *.:

Goat DVTEVDLSDNFITRITNESFQGLQNLTKINLNHNAKSQ---SGNPAVKKAMTITDGAFLN

Buffalo DVTEVDLSDNFITQITNESFQGLQNLTKINLNHNAKSQ---SGNPAVKKAMTITDGAFLN

Cattle DVTEVDLSDNFITRVTNESFQGLQNLTKINLNHNAKSQ---SGNPAVKKAMTITDGAFLN

Sheep AVTEVDLSDNFITRITNESFQGLQNLTKINLNHNAKSQ---SGNPAVKKAMTITYGAFLN

Pig YVTELDLSDNFIRRITNESFQGLQNLTKINLNHNAKLW---PQS---ENGMTITDGAFLN

Horse YVTELDLSDNFIIHITNESFQGLQNLTKINLNHNGKARSQNENPDVNKNGMNITDGAFLN

***:******* ::*******************.* ::.*.** *****

Goat LKHLRELLLEDNQLQQIPAGLPESLKKLSLIQNNIITLTKKNTSGLGNLESLYLGWNCYF

Buffalo LKHLRELLLEDNQLQQIPAGLPESLKKLSLIQNNIITLTKKNTSGLGNLESLYLGWNCYF

Cattle LKHLRELLLEDNQLQQIPAGLPESLKKLSLIQNNIITLTKKNTSGLGNLESLYLGWNCYF

Sheep LKHLRELLLEDNQLQEIPAGLPESLKELSLIQNNIITLTKKNTSGLGNLERLYLGWNCYF

Pig LHHLRELLLEDNQLREIPTGLPESLRELSLIQNKIILLNTKNMFGLRKLESLYLGWNCYF

Horse LQNLRELLLEDNQLDKIPTGLPGSLRELSLIQNRITSVTKKNTSGLMNLEYLYLGWNCYF

*::*********** :**:*** **::******.* :..** ** :** *********

Goat A--CDKKFSIENGAFQNLTKLKVLSLSFNPLHSVPPSLPSSLTELYLSNTHIGNVSEEDF

Buffalo A--CDKKFTIENGAFQNLTKLKVLSLSFNPLHSVPPSLPSSLTELRLSNTHIGNVSEEDF

Cattle A--CDKKFTIENGAFQNLTKLKVLSLSFNPLHSVPPSLPSSLTELYLSNTHIGNVSEEDF

Sheep A--CNKKFSIENGAFQNLTKLKVLSLSFNPLHSVPPSLPSSLTELYLSNTHIGNVSEEDF

Pig T--CNETFIIDEGAFENLTNLKVLSLSFNTLYRVPPKLPSSLTKLYLSNTKIRNINQEDF

Horse GNICNKTFDIEDGTFERLTNLKVLSLSFNNLSHVPPKLPNSLRELYLSNTKIKNITQEDF

*::.* *::*:*:.**:********* * ***.**.** :* ****:* *:.:***

Goat KELSNLRVLDLSGNCPRCFNAPFPCVPCQGGASIQIHPLAFQTLTQLRYLNLSSTSLRKV

Buffalo KELSNLRVLDLSGNCPRCFNAPFPCVPCQGGASIQIHPLAFQTLTQLHYLNLSSTSLRKV

Cattle KELSNLRVLDLSGNCPRCFNAPFPCVPCQGDASIQIHPLAFQTLTQLRYLNLSSTSLRKV

Sheep KELSNLRVLDLSGNCPRCFNAPFPCVPCQGGASIQIHPLAFQTLTQLRYLNLSSTSLRKV

Pig KGLENLRVLDLSGNCPRCFNAPFPCNPCPGDASIQIHPLAFRYLTELRYLNLSSTSLRRI

Horse KELRNLTVLDLSGNCPRCFNAPFPCTPCERDSSIQIHPLAFQDLTELRYLNLSSTSLRKV

* * ** ****************** ** .:*********: **:*:**********::

Goat PASWFDNMHNLKVLDLEFNYLMDEIASGEFLTKLPSLEILDLSYNYELKKYPQYTNISKN

Buffalo PASWFDNMHNLKVLDLGFNYLMDEIASGEFLTKLPSLEILDLSYNYELKKYPQYINISKN

Cattle PASWFDNMHNLKVLDLEFNYLMDEIASGEFLTKLPSLEILDLSYNYELKKYPQHINISKN

Sheep PASWFDNMHNLKVLDLEFNYLMDEIASGEFLTKLPSLEILDLSYNYELKKYPQYINISKN

Pig PATWFENLHHLKVLHLEFNYLMDEIASGEFLAKLPSLEILDLSYNYEQKKYPQYINISHY

Horse PAIWFDNMHHLKVLHLEFNYLVQEIASGEFLTKLPSLEILDLSFNYIVTKYPKYIEISPN

** **:*:*:****.* ****::********:***********:** .***:: :**

Goat FSKLISLQMLHLRGYVFQELRKKDFNPLRHLSNLTTINLGVNFIKQIDFSIFPWFPNLKI

Buffalo FSKLISLQMLHLRGYVFQELRKKDFNPLRHLSNLTTINLGVNFIKQIDFSIFPWFPNLKI

Cattle FSKLISLQMLHLRGYVFQELRMEDFEPLRHLSNLTTINLGVNFIKQIDFSIFQWFPNLKI

Sheep FSKLISLQMLHLRGYVFQELRRKDFKPLQHLSNLTTINLGVNFIKQIDFSIFHWFPNLKI

Pig FANLTSLQILHLRAYVFQELRKEDFQPLRNLLHLKFINLGINFIKQIDFTIFSEFSNLSI

Horse FSNLTSLQILHLRGYVFQEVRKEHFRPLMSLSNLKTINLGVNFIKQIDFTFFQHFPNLTV

*::* ***:****.*****:* :.*.** * :*. ****:********::* *.**.:

Goat IYLSENRISPLVSDTEQHDANGTSFQGHILKRRSADIQFDPHSNFYHDTRPLIKTECSRL

Buffalo IYLSENRISPLVSDTEQHDANGTSFQSHILKRRSADIQFDPHSNFYHNTRPLIKTECSRL

Cattle IYLSENRISPLVSDTEQHDANGTSFQSHILKRRSADIQFDPHSNFYHNTRPLIKTECSRL

Sheep VYLSENRISPLVSDTEQHDANGTFFQSHILKRRSADIQFDPHSNFYHNTHPLIKTECSRL

Pig IYLSENRISPLVNNTGQKNGDRPSFQSHVLKPRSATPKFDPHSNFYHNTKPLIKPQCSRY

Horse IYLSENRISPLVNDSLQNYTNGSAFQSHILKRRSADFEFNPHSNFYHNTNPLIKPQCTAY

:***********.:: *: : . **.*:** *** :*:*******:*.****.:*:

Goat GNALDLSLNSISFIGVNQFQDFGNISCLNLSSSGNGQVLNGTEFSRLSGIKYLDLTNNRL

Buffalo GNALDLSLNSIFFIGVNQFQDFGNISCLNLSSNGNGQVLNGTEFSRLSGIKYLDLTNNRL

Cattle GNALDLSLNSIFFIGVNQFKDFGNISCLNLSSNGNGQVLNGTEFSCLSGIKYLDLTNNRL

Sheep GNALDLSLNSIFFIGVNQFKDFGNISCLNLSSNGNGQVLNGTEFSRLSGIKYLDLTNNRL

Pig GKALDLSLNSIFFIGPNQFEAFKDIACLNLSSNGNGQVLHGSEFSHLPGIKYLDLTNNRL

Horse GKALDLSLNSIFFIGQKQFKAFHDIACLNLSSNGIGQPLHGTEFSAVPHIKYLDLTNNRI

*:********* *** :**: * :*:******.* ** *:*:*** :. **********:

Signal Sequence (1-18)

LRR 1 (59-80)

LRR 3 (163-192)

LRR 5 (238-256)

LRR_TYP 4 (193-

213)

LRR 6 (279-302)

LRR 7 (303-322) LRR 8 (329-353)

LRR 9 (386-409)

LRR 10 (410-432)

LRR 11 (522-542)

LRR_TYP 2 (118-141)

66

Goat DFDDDAAFSELPLLEVLDLSYNAHYFRIAGVTHRLGFIEHLTNLRVLNLSNNDIFTLTET

Buffalo DFDDDAAFSELPLLEVLDLSYNAHYFRIAGVTHRLGFIEHLTNLRVLNLSNNDIFTLTET

Cattle DFDDDAAFSELPLLEVLDLSYNAHYFRIAGVTHRLGFIEHLTNLRVLNLSNNDIFTLTET

Sheep DFDDDAAFSELPLLEVLDLSYNAHYFRIAGVTHRLGFIEHLTNLRVLNLSNNDIFTLTET

Pig DFDDDAAFSELPLLEVLDLSYNSHYFRIAGVTHRLGFIQNLPQLRVLNLSHNSIFTLTET

Horse DFDDDNALRELPELEVLDFSYNAHYFRIAGVTHRLGFIQNLTQLRVLNLSHNSIYTLTEY

***** *: *** *****:***:***************::*.:*******:*.*:****

Goat QLKSASLGELVFSGNRLDLLWNAQDVRYWQIFQNLTNLTRLDLARNNLRHISSQAFLNLP

Buffalo QLKSASLGELVFSGNRLDLLWNAQDVRYWQIFQNLTNLTRLDLARNNLRHISSQAFLNLP

Cattle QLKSASLGELVFSGNRLDLLWNAQDVRYWQIFQNLTNLTRLDLARNNLRHISSQAFLNLP

Sheep QLKSASLGELVFSGNRLDLLWNAQDVRYWQIFQNLTNLTRLDLARNNLQHISSQAFLNLP

Pig YLKSTSLKELVFSGNRLDLLWNAQDDRYWQIFKNLSTLTHLDLSSNNLQHIPSEAFLNLP

Horse NMNSMSLEELVFSGNRLDLLWNAEDRRYRKIFKCLRNLTRLDLSFNNLQHIPDEAFLNLP

::* ** ***************:* ** :**: * .**:***: ***:**..:******

Goat RTLTDLYINDNMLNFFNWSLLEYFPYLRLLDLSGNQLFSLTNSLSTFASSLETLLLSGNR

Buffalo RTLTDLYINDNMLNFFNWSLLEYFPYLRLLDLSGNQLFFLTNSLSTFASSLETLLLSGNR

Cattle RTLTDLYINDNMLNFFNWSLLEYFPHLRLLDLSGNQLFFLTNSLSTFASSLETLLLSRNR

Sheep RTLTDLYINDNMLNFFNWSLLEYFPHLRLLDLSGNQLFFLTNSLSTFASSLETLLLSRNR

Pig QTLTELYISDNRLNFFNWSLLQQFPNLTLLDLSGNELSFLTDSLSKFTTSLQTLILRQNR

Horse QNLTELYINDNRLHFFNWTLLQHFPHLHLLDLSRNKLSSLTNNLSKFSPSLRTLLLSQNK

:.**:***.** *:****:**: ** * ***** *:* **:.**.*:.**.**:* *:

Goat ISHLPSDFLSGASSLIHLDLSSNQLKMLNRSTFETKTATRLTVLELGSNPFDRTCDFGDF

Buffalo ISHLPSDFLSGASSLIHLDLSSNQLKMLNRSTFETKTATKLTVLELGSNPFDCTCDFGDF

Cattle ISHLPSDFLSGASSLIHLDLNSNQLKMLNRSTFETKTATKLTVLELGGNPFDCTCDLGDF

Sheep ISHLPSDFLSGASSLIHLDLSSNQLKMLNRSTFETKTATKLTILELGSNPFDCTCDFGDF

Pig ISYLPSGLLSEASSLTHLDLSSNQLKMVNISKLHAKTTTNLAILKLDRNPFDCTCDIRDF

Horse ISHLPSGFFSEASGLIHLDLRFNRLKMINKTTLQTKTTINLAVLELGGNPFDCTCDIGDF

**:***.::* **.* **** *:***:* :.:.:**: .*::*:*. **** ***: **

Goat REWMDGNLNVRIPGLTDVICASPGDQEGKSIVSLDLTTCVSDTIAAIFCFLTFSVTISVM

Buffalo REWMDGNLNVRIPRLTDVICASPGDQGGKSIVSLDLTTCVSDTIAAIFCFLTFSVTISVM

Cattle LEWMDRNLNVRVPRLTDVICASPGDQEGKSIVSLDLSTCVSDTIAAIFCFLTFSVTISVM

Sheep REWMDRNLNVRVPRLTDVICASPGDQEGKSIVSLDLTTCVSDTIAAIFCFLTFSVTISVM

Pig RKWMDENLKVTIPRLTDVICASPGDQRGRSIVSLELTTCVSDTIAAIICFFTFFVTSTVM

Horse RRWMDENLNIAIPRLADVICASPGDQRGKSIVSLELTTCVSDAIAAILCFFTFFITVTVM

.*** **:: :* *:********** *:*****:*:*****:****:**:** :* :**

Goat LAALAHHWFYWDAWFIYHVCLAKVKGYRSLSTSQTFYDAYISYDTKDASVTDWVINELRF

Buffalo LAALAHHWFYWDAWFIYHVCLAKVKGYRSLSTSQTFYDAYISYDTKDASVTDWVINELRF

Cattle LAALAHHWFYWDAWFIYHVCLAKVKGYRSLSTSQTFYDAYISYDTKDASVTDWVINELRF

Sheep LAALAHHWFYWDAWFIYHVCLAKVKGYRSLSTSQTFYDAYVSYDTKDASVTDWVINELRF

Pig LVALAHHWFYWDAWFIYHVCLAKVKGYRSLPTSQTFYDAYVSYDTKDASVTDWVMNELRF

Horse LAALAHHWFYWDVWFIYHMCLAKIKGYRSLSTSQTFYDAYVSYDTKDASVTDWVINELRF

*.**********.*****:****:******.*********:*************:*****

Goat HLEESEDKNVLLCLEERDWDPGLAVIDNLMQSINQSKKTIFVLTKKYAKNWNFKTAFYLA

Buffalo HLEESEDKNVLLCLEERDWDPGLAVIDNLMQSINQSKKTIFVLTKKYAKNWNFKTAFYLA

Cattle HLEESEDKNVLLCLEERDWDPGLAIIDNLMQSINQSKKTIFVLTKKYAKNWNFKTAFYLA

Sheep HLEESEDKNVLLCLEERDWDPGLAIIDNLMQSINQSKKTIFVLTKKYAKNWNFKTAFYLA

Pig HLEESEGKNVLLCLEERDWDPGLAIIDNLMQSINQSKKTIFVLTKKYAKNWNFKTAFYLA

Horse HLEESEEKNVLLCLEERDWDPGLAIIDNLMQSINQSKKTIFVLTKKYAKNWNFKTAFYLA

****** *****************:***********************************

Goat LQRLMEENMDVIVFILLEPVLQHSQYLRLRQRICKSSILQWPDNPKAEGLFWQSLKNVVL

Buffalo LQRLMEENMDVIVFILLEPVLQHSQYLRLRQRICKSSILQWPDNPKAEGLFWQSLKNVVL

Cattle LQRLMEENMDVIVFILLEPVLQHSQYLRLRQRICKSSILQWPDNPKAEGLFWQSLKNVVL

Sheep LQRLMEENMDVIVFILLEPVLQHSQYLRLRQRICKSSILQWPDNPKAEGLFWQSLKNVVL

Pig LQRLMDENMDVIVFILLEPVLQHSQYLRLRQRICKSSILQWPDNPKAEGLFWQSLKNVVL

Horse LQRLMDENMDVIVFILLEPVLQHSQYLRLRQRICKSSILQWPDNPKAEGLFWQSLKNVVL

*****:******************************************************

Goat TANDSRYNNLYVNSIKQY

Buffalo TANDSRYNNLYVNSIRQY

Cattle TANDSRYNNLYVNSIKQY

Sheep TANDSRYNNLYVNSIKQY

Pig TENDSRYNSLYVNSIK--

Horse TENNSRYNNLYVDSIKQY

* *:****.***:**:




terminal repeat; CT—C-terminal repeat; TIR—toll/interleukin-1 receptor

domain.

LRR 12 (576-599)

LRR_TYP -13 (630-653)

LRR 14 (679-702)

LRR 15 (704-726) LRR 16 (728-750)

LRR CT 17 (764-815) TM (818-840)

TIR (871-1017)

67

The amino acid sequence of goat TLR8 was subjected to the SMART online

programme for the prediction of different functional domains in goat, by aligning them

with other livestock species. The signal peptide was found to be conserved among the

ruminant species with some variation in non-ruminant species. The LRRs (1-4) were also

found to be conserved among the goat, buffalo and cattle but with some variation found in

sheep and non-ruminants. Again, LRRs (5-7) were found to be conserved among the

ruminants but LRR8 was different in goat as compared to other ruminant and non-

ruminant species. LRR9 was found to be conserved among the ruminants but LRR10

showed variation in sheep as compared to goat, cattle and buffalo. LRRs (11-13) were

found to be conserved among goat, sheep and cattle and LRR14 was found to be different

in goat as compared to other species. LRRs (15-17) showed little variation, but

transmembrane region and TIR domain were found to be almost conserved in all the

ruminant species (Fig. 4.19).

4.9. Characterization of TLR8 5’-upstream region

Nucleotide sequence of PCR products, representing 5‟-upstream region of goat

TLR8 gene in overlapping fragments were assembled into 1.01 kb contig and submitted to

NCBI GenBank (Accession Number JQ911705). When compared with other livestock

species, the nucleotide sequence of goat TLR8 showed a maximum homology of 88% with

cattle and relatively low level of homology was observed with rest of the species. When

the sequence based phylogenetic analysis was carried out, goat shared the closest place

with cattle, as compared to other reported species (Fig. 4.20).

Fig. 4.20. Nucleotide sequence based phylogenetic analysis of 5’-upstream of TLR8

gene comparing goat with other species.

When the goat TLR8, 5‟-upstream region was compared with that of cattle it showed

the maximum variability in the region start codon to 808 nucleotides upstream.

Transcription factor binding sites analysis in goat TLR8 revealed presence of total 70

potential sites, located at different recognition sequences. Whereas, in cattle a total of 76

putative binding sites were found (Fig. 4.21). Important factors like PU-1, which is an

68

essential binding factor that regulates both cytokine-dependent proliferation and

differentiation of granulocyte/macrophage progenitors was found to be present in 5‟UTR

sequence of goat TLR8 as compared to cattle where it was found to be absent. Another

important factor C/EBP, which has been classified in six different classes and is said to be

involved in different cellular responses, such as control of cellular proliferation, growth

and differentiation, metabolism, and in immunity was found to present in both goat and

cattle in different forms in TLR8 sequence. Another major factor GATA-1, which is

important in erythroid development, was found to be present in goat and cattle both.

Binding factor ETS, implicated in the development of different tissues as well as cancer

progression was found to be present in goat but not in cattle. Activator Protein-1 (AP-1)

was found to be present in both goat and cattle TLR8 sequence.

Further comparison of TLR8 5‟-upstream sequence of goat with cattle, showed the

insertion of 5 nucleotide sequence at two different positions. The insertion at position 149

upstream to open reading frame was found to have an extra important binding factor Oct-1

site which was found to be missing in cattle. Another insertion which was found at

nucleotide 657 upstream to coding region in goat but it did not show any extra binding

factor site.

Goat --GTACAAAAACACTAAATGCCATGTCTTGCCCGCAAAAGTTTACAAAAG------CAAA

Cattle TAATTAACATTTTAAGATAACAGAAGGTTGAATCCAAAGACTGTCCTTAGGCAGAATACA

.*:.*.*:: :.:.*::.*..:. ***.. ****.. * :*.::** *.*

Goat AGAATGGCTATTAATACCAGTGTCTGTATGATAAGGGATATATAAGTGGTAGCCTCCCTA

Cattle TGATTGTTATATAATCCTAAGGAATGTGGAGAAAGAAAAAAAATTTGTCCTTTCTTCCTC

:**:** :::****.* *. *:.***. ..:***..*:*:*::: : ** ***.

Goat CAAAAAACTTTCTGAATGTTGTCTACATTTGAAGATGTAGTCTAACATAAAGATTTCAAA

Cattle CTTGAGAATTCCAGACCCCTCTCTCCTTGGGGACCCCTAGACTCCCTATCAACCTGCCTA

*::.*.*.** *:**. * ***.*:* *.* . ***:**..*:::.*.. * *.:*

Goat ATGAGATTGTGTAGCATGATGGTTAGTGGTTAAAAGCACCATGTTGGAATCTGATGGACT

Cattle GG--AAATGGCTCTCTCAATGGTTAGTGGTTAAAAGCACCATGTTGGAATCTGATGGACT

. .*:** *. *: .******************************************

Goat CGTCTCACTTCTGCAGTGTATCATCCTTCTGAAAAGTAGTTAAGTCTCTGAGTTTGAGTC

Cattle CGTCTCACCTCTGCAGTGTATTATCCTTCTGAAAATTAGTTAAGTCTCTGAGTTGGAGTC

******** ************ ************* ****************** *****

Goat TCACTTACAACCTATGGATAATAATGGAACCTATTGCATTGTATTGTTGGGAGCATAAAC

Cattle TCACTTACAACCTATGGATAATAATGGAACCTATTGCATTG-----TAGGGAGCATAAAC

***************************************** *:************

Goat ATGGGTGAGAAAATGTTCGTGTTTAGCATAACACGTGACACTTTGCTATTATTATAAACG

Cattle ATGGGTGGGAAAATGTTCATGTTTAGCATAACACGTGACACTTTGCTATTATTATAAACC

*******.**********.****************************************

Goat GAAAGTGTCCACCTTCCTGTCATGCTGGGACATGAGAGAATTTGATACATACCATCATGG

Cattle AAAAGTGTCCACTTTCCTGTCATGCTGGGACATGAGAGAATTTGATACATACCATCATAG

.*********** *********************************************.*

Goat AGGTATAGACAGATTGTGGGAAATATCAATAAAATATATGGTTGTATAGATAAGGTAGAG

Cattle AGGTATAGACAGATTGTGGGAAATATCAATAAAATATATGGTTGTGTAGATAAGGTAGAG

*********************************************.**************

c-Ets-1

GATA-1

c/EBPalp

Oct-1

PTF1-β USF

Sp1

69

Goat AGACTCTAGCTTGGAAATCTTGAAGGATGGCCTAGTAGAAGCTGGATCTGGACTCTCCTT

Cattle AGACTCTGGCTTGGAAATCTTGAAGGATGGCCTAGTAGAAGCTGGATCTGGACTCTCCTT

*******.****************************************************

Goat GGGGCTGGCCAGAGTTTCCTTGAGTTATGCTAATAGATTGAAGCAAAAGACATATACCTT

Cattle GGGGCTGGCCAGCGTTTCCTTGAGTTATGCTAATAGACTGAAGCAACAGACATATACCTT

************.************************ ********.*************

Goat GCTCTGCTATAGGTCTGGCATGAACCATAGCCAGGATCCAATATCAGGCCCCCTGTGATG

Cattle GCTCTGCTATAGGTCTGGCATGAACCATAGCCAGGATCCAACATCAGGCCCCCTGTGATG

***************************************** ******************

Goat GTGGGGCATCTTGATGTGTGCTTCTGCCAAAGCTTTTCAGGTTTTCTTGTCATCACATCT

Cattle GTGGGGCATCTTGCTGTGTGCTTCTGCCAAAGCTTTCCATGTTTTCTTGTCATCACATCT

*************.********************** ** ********************

Goat CAGATAGCTTCATCTTTTGCTTCTTTGAACATCAGGAAGCTGAACAGGGT-GAAAACTAA

Cattle CAGATAACTTCATCTTTTGCTTCTTTGAACATCAAGAAACTGAACAGGGTGAAAAACTAA

******.***************************.***.*********** .********

Goat AACCATTCTGAACTTAACTTACTGCACTTTCTTTAACTGTTCAGAGAACTCCTACTTCAG

Cattle AACCATTCTG-----AACTTACTGCAATTTCTTTAACCGTTCAGAGAACTCCTACTTCAG

********** ***********.********** **********************

Goat AACTCATTCAAAGTATGATCAAAGCACTGTGAATTTCATCCTGAAATGCCTAGAAAATAT

Cattle AACTCATTCAAAGTATGGTCAAAGCACTGCGAATTTCATCCTGAAATGCCTAGAAAATAC

*****************.*********** *****************************

Goat TGCCAGTCTAACATTGTGACTCCACTTTGATTTTCCTTAGGAAAACATGACCCTTCACTT

Cattle TGCCAGTCTAACATTGTGACTCTACTTTGATTTTCCTTAGGAAAACATGACCCTTCACTT

********************** *************************************

Fig. 4.21. Alignment of 5’-upstream sequence of goat TLR8 showing variable putative

transcription factor binding sites as compared to cattle.

4.10. Comparative structure analysis of goat TLR genes

Total length of three TLR genes varied from each other with bacterial LPS binding

TLR4 being 841 amino acids, whereas viral ligand recognizing TLR7 was 205 amino acids

longer than TLR4 and 13 amino acid longer than TLR8 respectively. Another viral ligand

recognizing gene TLR8 was 192 amino acids longer than TLR4 and was found to be of

1033 amino acids. The variation in length was mainly in the ligand binding LRR

ectodomain region. The length of transmembrane, intracellular and TIR domains was

almost similar in these genes (Table 4.5).

Table 4. 5. Comparison of domain structure lengths of goat TLR4 and TLR8 genes.

Gene Amino

acids length

Extracellular

domain

Trans-

membrane

Intracellular

domain

TIR

domain

TLR4 841 1-634 635-657 658-841 674-819

TLR7 1046 1-853 - 854-1046 887-1033

TLR8 1033 1-817 818-840 841-1033 871-1017

PU.1

AP-1

Oct-1

TBP

NF-1

YY1

ISGF-3

Sp1

Sp-1

GCN

c/EBP beta

ORF

70

To compare goat TLR4, TLR7 and TLR8 gene sequences generated in this study,

they were analyzed for phylogeny by comparing with other seven reported goat TLR gene

sequences, following NJ algorithm with 1000 bootstrap re-sampling. Viral TLRs 7, 8 and 9

were clustered together under a single clade while the other major gene sub-family

including TLRs 1, 2, 6 and 10 clustered together (Fig. 4.22). TLRs 3, 4 and 5 of goat were

found to be clustering as singletons. Within the TLR1 gene family, TLRs 1 and 6 were

found to be closer with 100% bootstrap value. Interestingly, TLRs 1, 2, 6 and 10 of goat

clustered separately within the same clade indicating, the higher sequence similarity

between them rather than their respective TLRs of goat. Similarly, within TLR7 gene sub-

family (TLRs 7, 8 and 9), members were found to be closely related.

In order to know the unambiguous relationship of goat among different ruminant

species and to avoid the confounding effect of distantly related species, multi-dimensional

scaling (MDS) analysis was performed. MDS analysis reduces the dimension of data and

allows visualization of relationship among subjects in a two or three dimensional

geometric space. In the present study, pair-wise amino acid differences were calculated

among the four ruminant species viz. goat, sheep, cattle, buffalo which were utilized to

draw the 2D MDS plot. Buffalo and goat were found to be closely related only with

respect to TLR4, while TLR8 was found in separate dimension. TLR8 of buffalo and

sheep whereas were in the same dimensions (4.23).In TLR7 also the buffalo and goat were

in different dimensions as same as for the other viral TLR i.e. TLR8 while cattle and

buffalo shared the same dimension.

71

Fig. 4.22. Phylogenetic analysis of different goat TLRs following NJ algorithm, to

delineate the grouping of TLR4, TLR7 and TLR8 compared to other

reported TLR genes.

Fig. 4.23. Multidimensional scaling plot of pair-wise differences among TLR4, TLR7

and TLR8 of different ruminant species compared to goat.

Goat TLR6

Goat TLR1

Goat TLR10

Goat TLR2

Goat TLR5

Goat TLR4

Goat TLR3

Goat TLR9

Goat TLR 8

Goat TLR7

0.1

72

Polymorphism analysis of goat TLR genes

4.11. DNA isolation

Genomic DNA was isolated from the blood samples collected from the different

breeds of goat. The quality of genomic DNA was checked after running the samples in

0.6% submerged agarose gel. Good quality of DNA was ascertained with concentration of

DNA, sufficient for further experiments (Fig. 4.24 a). Quantity and quality of DNA was

also estimated spectrophotometrically using Nanodrop (ND-1000), which again showed

good quality DNA free from proteins and RNA with ratio of OD260/OD280 around 1.8 for

all the samples. The working concentration of genomic DNA was prepared by diluting

stock to approximately 80 ng/µl, which was further checked on 0.6% agarose gel (Fig.

4.24 b).

Fig. 4.24a. Gel picture showing goat grand stock genomic DNA runned in 0.6%

agarose gel.

Fig. 4.24b. Gel picture showing working concentration of goat genomic DNA in 0.6%

agarose gel.

For the screening of polymorphism in caprine TLR4, TLR7 and TLR8 genes, a

panel of 24 DNA samples representing 12 different goat breeds was prepared. The primers

were used to amplify the complete coding and parts of noncoding regions as well as 5‟-

upstream sequences. Presence of synonymous/non-synonymous SNPs in different domains

of the TLRs was revealed by SMART analysis. After developing the genotyping protocols

for selected SNPs, they were screened in large goat populations and allelic and genotypic

frequencies were determined.

4.12. Identification of SNPs in goat TLR4 gene

For the screening of polymorphism in goat TLR4 gene, a total of nine primer sets

were designed covering entire coding region from exon1 to exon3. Fragments were

amplified from genomic DNA (Fig. 4.25) and sequenced. Assembled sequence of goat

TLR4 was 3755 nucleotides in length and submitted to NCBI (GenBank accession number

JN786600). For gene structure analysis genomic sequence was aligned with previously

73

published goat TLR4 mRNA sequence (Accession no. JF825527) and found to be having a

total of three exons as reported in other livestock species. Goat TLR4 exon1 consisted of

both partial 5‟UTR and CDS of 93 nucleotides, exon2 with full coding region consisted of

167 nucleotides and maximum coding region was present in exon3, which spread across

2266 nucleotides with CDS and partial 3‟UTR. Genomic organization of goat TLR4 with

their exon and intron boundaries is shown in Fig 4.26. Comparative sequence analysis of

goat TLR4 in 12 breeds revealed 27 polymorphic sites, including two in 5‟UTR, two in

exon1, one in intron1, one in exon2, maximum 20 in exon3 and one in 3‟UTR (Table 4.6).

Upon further analysis it was observed that out of these 27 SNPs, 25 were found to be

transitional changes and the rests two were transversions. The allele frequency of each

SNP in total 24 animals was also calculated and shown in Table 4.6.

Fig. 4.25. PCR amplification of goat TLR4 gene in overlapping fragments. Products

are shown with 100 bp marker in agarose gel.

Fig. 4.26. TLR4 gene structure in goat, designed from genomic DNA sequencing.

74

Table 4.6. Polymorphic nucleotide sites detected in goat TLR4 gene by comparative

sequence analysis of 12 goat breeds with their allele frequencies.

* Position of SNP w.r.t. Goat submitted sequence Acc. No. JN786600

Nucleotide Amino acid Transversion/ transition Region Domain Allele frequencies

31 G >A Transition 5‟UTR G 0.16

A 0.84

134 C>T Transition C 0.75

T 0.25

364 T>C Transition EXON-1

T 0.75

C 0.25

370 C>A Transversion

C 0.84

A 0.16

449T>C Transition INTRON-1

T 0.84

C 0.16

772G>A 54Val>Ile Transition EXON-2

G 0.87

A 0.13

2049C>T 368Thr>Ile Transition EXON-3 LRR C 0.79

T 0.21

2112C>G 389Thr>Ser Transversion LRR

C 0.84

G 0.16

2189A>G 415Ser>Gly Transition LRR

A 0.77

G 0.23

2227 G>A Transition

G 0.70

A 0.30


T 0.12

2602 T>C Transition

T 0.23

C 0.77

2616C>T 557Thr>Met Transition

LRR

C 0.75

T 0.25

2640A>G 565Arg>Gln Transition LRR

A 0.86

G 0.14

2655G>A 570Asn>Ser Transition LRR G 0.77

A 0.33

2668A>G Transition A 0.86

G 0.14

2834 A>C 630Met>Leu Transition LRR A 0.75

C 0.25

2862 T>C Transition T 0.65

C 0.35

2876C>T Transition C 0.18

T 0.82

2900 A>G Transition G 0.29

A 0.80

2913 G>A Transition G 0.73

A 0.27


G 0.34


T 0.09

3090A>C Transition A 0.60

C 0.40


G 0.05

3411G>A 822Arg>Gln Transition TIR G 0.80

A 0.20

3493 G>A Transition 3‟UTR G 0.66

A 0.34

75

Effect of amino acid changes in TLR4 on structure and function-PolyPhen analysis

In goat TLR4 a total of 27

polymorphic sites were observed, out

of which 9 changes were found to be

non-synonymous. Thinking that these 9

non-synonymous changes might have

an impact on the functionality and

structure of the coded protein, all the 9

non-synonymous amino acid changes

were subjected to online tool

PolyPhen2. It revealed all the changes

to be benign without any possible

damaging effect, with the sensitivity

score near 1 and specificity between 0.5

to 1(Fig. 4.27).

Effect of amino acid changes- predicted structure

Nine non-synonymous SNPs found in goat TLR4 were also analysed for their

effect on structure predicted by Modeller software. Out of these nine amino acid changes,

two were non-polar to non-polar, two polar to polar, three polar to non-polar and two

positively charge to polar. Tertiary structure of goat TLR4 was predicted using homology

model (crystal structure of the human TLR4-human MD-2-E.coli LPS Ra complex, PDB-

3FXI) to predict the effect of these amino acid changes on the structure. There were two

polar to non-polar variations Thr368IIe and Ser415Gly, showing structural changes in

ectodomain region (LRR) of goat TLR4. Other two variations, which were positively

charged amino acid to polar Arg565Gln in LRR domain and Arg822Gln in TIR domain

also showed effect on protein‟s secondary structure. Structure was also predicted including

all the nine amino acid changes possible, to see the effects on structure compared to

overall native structure of the goat TLR4 (Fig. 4.28).

Fig. 4.27. Non-synonymous SNPs with their sensitivity

and specificity score (Polyphen2) of goat

TLR4 gene.

76

Fig. 4.28. Effect of various non-synonymous amino acid changes found in goat TLR4

on its structure as predicted by Modeller and PyMol software.

At position Thr 368 Ile

( (

T368I

TIR

TM

At position Val 54 Ile

( (

V54I

TIR

TM

At position Thr 389 Ser

( (

T389S

TIR

TM

At position Ser 415 Gly

( (

TIR

TM

S 415 G

T 557 M

TIR

TM

At position Thr 557 Met

( (

At position Arg 565 Gln

( (

R565Q

TIR

TM

At position Asn 570 Ser

( (

N 570 S TIR

TM

At position Met 630 Leu

M 630 L TIR

TM

At position Arg 830 Gln

( (

R 830 Q TIR

TM

Native structure of Goat TLR4

( (

TIR

TM

Effect of all non-synonymous

TIR

TM

77

Genotyping of goat TLR4 SNPs by PCR-RFLP

ApeKI restriction enzyme based PCR-RFLP technique was developed for

genotyping of the polymorphic nucleotide site 2112 C>G, within the coding region of the

caprine TLR4 lied in the LRR domain. PCR-RFLP helped in the determination of

frequency distribution for two alleles (C, G) and three genotypes (C/C, G/G, C/G) in 165

goat samples representing 5 breeds. The DNA restriction fragments corresponding to C/C,

G/G, C/G genotypes were: 463 bp; 396 bp, 67 bp and 463 bp, 396 bp and 67 bp

respectively (Fig. 4.29). The genotypic and allelic frequencies of ApeKI variants are listed

in Table 4.7. Different goat breeds analysed, demonstrated the variation in proportion of

genotype frequencies. Out of these 5 breeds screened, Barbari and Jamunapari were found

to have the zero genotype frequency for GG genotype, whereas Kanniadu, Black Bengal

and Sirohi were found to have mixed frequencies of all the three genotypes.

(a) (b)

(c)

Fig.4.29. (a) Chromatogram showing variation in nucleotide at position 2112 C>G,

(b) Designing of PCR-RFLP using NEB cutter programme, (c) EtBr stained

2% Agarose gel showing all the three genotypes C/C, G/G, C/G, in goat

TLR4 gene across different breeds, using ApeKI restriction enzyme.

C/G C/C G/G C/C C/G

78

Table 4.7. Genotype and allelic frequencies of goat TLR4 SNP 2112 C>G, across

different breeds.

4.13. Identification of SNPs in goat TLR7 gene

For the screening of polymorphism in goat TLR7 gene, a total of seven primer sets

were designed covering the entire coding region. Fragments were amplified from genomic

DNA (Fig.4.30.) and sequenced. By sequence analysis of 24 animals, total 22 SNPs were

identified in the transcribed region of the goat TLR7 gene, which resulted into nine

haplotypes which has been named as Hap1-Hap9 with the highest frequency of Hap-1 of

0.542 and the lowest one being 0.021 which was shared by four different haplotypes Hap

6-Hap9 (Fig. 4.31). Since numbers of animals taken per breed were just two, breed-wise

haplotype frequencies could not be calculated. Two transversional and 20 transitional

changes were observed and seven out of 19 SNPs in the coding region, were found to be

non-synonymous type, from which, some of the SNPs were present in the functional

domains of goat TLR7 gene (Table 4.8).

Fig. 4.30. Goat TLR7 amplified into 7 different PCR framents for SNP detection.

Breed Total no of

Samples

No. of genotype Genotype frequency Allele frequency

GG G/C CC GG G/C CC G C

Kanniadu 40 16 14 10 0.40 0.35 0.25 0.58 0.43

B. Bengal 40 4 16 20 0.10 0.40 0.50 0.30 0.70

Sirohi 29 2 16 11 0.07 0.55 0.38 0.34 0.66

Barbari 28 0 11 17 0.00 0.39 0.61 0.20 0.80

Jamunapari 28 0 16 12 0.00 0.57 0.43 0.29 0.71

Total 165 22 73 70 0.13 0.44 0.42 0.35 0.65

79

Table 4.8. Polymorphism in goat TLR7 gene with their positions and domains.

*Positions as per Accession No. GU289401

Nucleotide Amino acid

Transition/Transversion Domain Allele frequencies


T 0.07

123 T>C Transition LRRNT T 0.97

C 0.03

130 G>A 44 Ala>Thr Transition LRRNT G 0.97

A 0.03


T 0.03

377 C>T 127 Thr>Met Transition LRR C 0.89

T 0.11

813 C>G Transversion Pfam LRR_1 C 0.97

G 0.03

845T>C 282 Leu>Pro Transition T 0.97

C 0.03

885 C>T Transition LRR_TYP C 0.97

T 0.03


T 0.03

1177 T>C 393 Cys>Arg Transition LRR T 0.97

C 0.03

1340 T>G 448 Met>Arg Transversion T 0.97

G 0.03

1722 T>C Transition T 0.97

C 0.03

1938 C>T Transition LRR_Typ C 0.97

T 0.03

2082 A>G Transition A 0.77

G 0.30


T 0.03


G 0.03

2548 A>G 850 Ile>Val Transition Low Complexity

Region

A 0.97

G 0.03

2731 C>T Transition TIR Domain C 0.97

T 0.03

2802 T>C 935Leu>Ser Transition TIR Domain T 0.97

C 0.03


T 0.07


G 0.03

3371 G>A Transition G 0.97

A 0.03

80

Fig.4.32. Non-synonymous SNPs with their

sensitivity and specificity score in

goat TLR7 (Polyphen2)

Fig. 4.31. Haplotypes observed and their frequencies in goat TLR7 gene.

Effect of amino acid changes on structure and function-PolyPhen analysis

In goat TLR7 a total of 22 polymorphic sites were

observed, out of which 7 changes were found to be

non-synonymous. To see the effect of these 7 non-

synonymous changes on the structure and function of

the coded protein, all the 7 non-synonymous amino

acid changes were subjected to online tool

PolyPhen2. It revealed two of the changes at

positions 127 Thr/Met and 935Leu/Ser to be possible

damaging ones with the sensitivity score near to 0.9

and specificity was also 0.9, and rest five changes

being benign without any possible damaging effect,

with the sensitivity score near 1 and specificity

between 0.5 to 1 (Fig. 4.32). These damaging amino

acids changes also showed their effects on the structure

of the protein as predicted by Modeller and PyMol software (Fig. 4.33). One of the

damaging amino acid changes 935Leu/Ser was found to be in the TIR domain and another

127Thr/Met was in one of the LRR domains. Another amino acid change which did not

showed any damaging effect on protein structure by PolyPhen but predicted model showed

breakage of structure was 393Cys/Arg.

Hap1

Hap2

Hap3

Hap4

Hap5

Hap6

Hap7

Hap8

Hap9

Haplotype

frequency

81

Fig. 4.33. Effects of non-synonymous amino acid changes due to polymorphism in

goat TLR7, on its structure as predicted by Modeller and PyMol software.

44 Ala>Thr

44 A>T

TM

127 Thr>Met

127 T>M

TIR

282 Leu>Pro

282 L>P

TIR

850 IIe>Val

850 I>V TIR

935 Leu>Ser

935 L>S

TM

393 Cys>Arg

393 C>R

TIR

448 Met>Arg

448 M>R

TIR

82

4.14. Polymorphism in goat TLR8 gene

For the detection of SNPs in the goat TLR8 gene, DNA sample of twelve animals

representing twelve breeds were amplified in eight overlapping fragments, covering 3488

nucleotides long region encompassing single exon, adjoining upstream and downstream

sequences (Fig. 4.34 a and b). Assembled sequence was submitted to GenBank (Accession

number JF825528). Goat TLR8 gene sequence comparison revealed presence of only four

SNPs and all were found to be synonymous (Table 4.9). They were found to be present in

different functional domains of the gene. Two out of these four SNPs were found to be

within LRR domains and one each was in transmembrane and TIR domain. Further

analysis showed all the changes to be transitional in nature.

Fig. 4.34.a. Goat TLR8 amplified into 8 different PCR framents for SNP detection.

Fig. 4.34.b. Genomic organization of goat TLR8.

Table 4.9. Polymorphism in goat TLR8 gene with their position and domain.

*Position as per accession JF825528.

SNP Amino acid Transition/

Transversion

Domain Genotyping

protocol

Allele

frequencies

2388A>G Thr747Thr Transition LRR HpyCH4V A 0.80

G 0.20

2463T>C Phe772 Phe Transition LRRCT T 0.84

C 0.16

2607A>G Ala820 Ala Transition Transmembrane HpyCH4IV A 0.71

G 0.79

3038T>C Met964 Met Transition TIR T 0.59

C 0.41

G/G

A/G

83

Genotyping of goat TLR8 SNPs by PCR-RFLP

For one of the SNPs 2388A>G, within LRR domain, HpyCH4V restriction enzyme based

PCR-RFLP genotyping protocol was developed to screen different breeds of goat, yielding

three genotypes and the fragments corresponding to three genotypes A/A, G/G, A/G (Fig.

4.35 a,b) : 487 bp, 280 bp; 351 bp, 280 bp and 136 bp; 487 bp, 351 bp, 280 bp and 136 bp

respectively. For another SNP, 2607A>G within transmembrane region, HpyCH4IV

restriction enzyme based PCR-RFLP genotyping protocol was developed and for this SNP

also three genotypes A/A, G/G, A/G (Fig. 4.35 c,d) resulted in the corresponding

fragments of 524 bp, 243 bp; 412 bp, 243 bp and 112 bp; 524 bp, 412 bp, 243 bp and 112

bp. The changes in nucleotides and chromatograms are shown in Fig. 4.35. Breed wise and

total allele and genotype frequencies of both of these SNPs have been shown in (Table

4.10 and 4.11). At locus 2388A>G, all the breeds screened viz. Barbari, Black Bengal,

Jamunapari, Boer and Sirohi showed higher frequency of „G‟ allele compared to allele „A‟

(Table 4.10). At locus 2607A>G, there was no significant variation in the allele

frequencies and a mixed allele frequencies were recorded (Table 4.11), except for Boer in

which the frequency of „G‟ allele recorded was zero as compared to other breeds.

(a) (b)

(c) (d)

Fig. 4.35 (a) PCR-RFLP genotyping of two SNPs 2388 A >G (a,b) and 2607A>G (c,d)

identified in goat TLR8 gene.

A/A

100bp ladder

Undigested

product

A/A G/G A/G

100bp ladder

G/G A/A

A/G

100bp ladder

84

Table 4.10. Genotype and allelic frequencies of goat TLR8 gene SNP 2388 A>G,

across different breeds.

Table 4.11. Genotype and allelic frequencies of goat TLR8 gene SNP 2607A>G,

across different breeds.

Haplotype analysis of goat TLR4 and TLR8

When the haplotype count among the goat TLR4 and TLR8 gene was carried out on the

basis of genotyping data recorded for all the breeds screened, a total of 42 animals were

found to be common in all the three SNPs genotyped and sharing a total of 9 haplotypes

with in them (Table 4.12). A major haplotype found was “AGC” with 20 animals sharing

this haplotype among them with a frequency of 0.476. Another major haplotype found was

“AGS” with 11 animals sharing this haplotype with a frequency of 0.262. Four minor

haplotypes “GAS, GGC, RRC, AGG” were found with a frequency of 0.238 shared by

four different animals. The haplotype “GAC” was shared by 2 animals with a frequency of

0.0476 and other haplotype which was shared by 3 animals was “RGC”. Using principal

Co-ordinate analysis based on haplotype frequencies, three different goat breeds Barbari,

Jamunapari and Black Bengal could be easily classified into distinct breed clusters (Fig.

4.36).

Breed Total no. of

Samples

No. of Genotype Genotype Frequency Allele Frequency

AA AG GG AA AG GG A G

Barbari 31 22 4 5 0.71 0.13 0.16 0.77 0.23

Black Bengal 30 28 2 0 0.93 0.07 0.00 0.97 0.03

JamunaPari 11 8 3 0 0.73 0.27 0.00 0.86 0.14

Boer 8 8 0 0 1.00 0.00 0.00 1.00 0.00

Total 80 66 9 5 0.83 0.11 0.06 0.88 0.12

Breed Total no. of

Samples

No. of Genotype Genotype Frequency Allele Frequency

GG AG AA GG AG AA G A

Barbari 28 23 1 4 0.82 0.04 0.14 0.84 0.16

Black Bengal 30 30 0 1.00 0.00 0.00 1.00 0.00

Jamunapari 15 15 0 0 1.00 0.00 0.00 1.00 0.00

Boer 7 6 1 0 0.86 0.14 0.00 0.93 0.07

Sirohi 15 13 1 1 0.87 0.07 0.07 0.90 0.10

Total 95 87 3 5 0.92 0.03 0.05 0.93 0.07

85

Table 4.12. Haplotypes and frequency distribution of goat TLR4 and TLR8 SNPs

among different breeds.

Fig. 4.36. Haplotype distribution based classification of three breeds of goats. JAM-

Jamunapari, BB- Black Bengal, BAR- Barbari by principal co-ordinate

analysis.

4.15. Tissue distribution of goat TLR genes

Quantitative real-time PCR was used to examine the TLRs expression in various

goat tissues. Relative expression was analyzed by calculating normalized Ct (threshold

cycle) values of all the three TLR genes against the housekeeping gene RPS15. Expression

of all the three TLRs was observed in all the tissues included in the present study viz.:

liver, lungs, kidney, skin, ovary, spleen, lymph node, heart, mammary gland, intestine,

skeletal muscle. Higher the ∆Ct value, lower will be the expression as it represents the

difference between mean Ct values of housekeeping and the reference gene. To ensure the

specific product without any non-specific amplification, dissociation curve analysis was

carried out for each gene which showed a single peak (Fig. 4.37) and efficiency of real-

time-PCR was also confirmed by running housekeeping gene with ten folds diluted cDNA,

Haplotype No. of haplotype Frequency of haplotype

AGS 11 0.262

AGC 20 0.476

GAS 1 0.0238

GGC 1 0.0238

GAC 2 0.0476

RGC 3 0.0714

RRC 1 0.0238

AGG 1 0.0238

RGS 2 0.0476

Total 42 1.000

B

B

86

resulting in single line curve having a slope of 3.5 and efficiency of 1.9 (Fig. 4.38). The

house keeping gene RPS15 showed stable expression across all the tissues with very small

variation (Table 4.14).

The highest expression of both TLR4 and TLR8 genes of goat was found in kidney

followed by spleen, lungs, lymph node, mammary gland and the lowest expression was

seen in intestine, heart and skin (Table 4.14). Intestine showed lowest expression of both

the genes. The highest expression of TLR7 gene was found in liver followed by kidney,

spleen, lungs and ovary and the lowest expression was seen in skeletal muscle, Intestine,

skin, heart.

Table 4.14. Real-time PCR based expression profiling, showing ∆Ct values of TLR4,

TLR7 and TLR8 genes.

Tissues RPS15

(Ct)

TLR4

(Ct)

TLR7

(Ct)

TLR8

(Ct)

∆Ct

TLR4

∆Ct

TLR7

∆Ct

TLR8

Liver 27.13 25.76

21.66

26.48 -1.37

-5.47

-0.64

Lungs 27.08 22.37

25.49

24.52 -4.71

-1.59

-2.56

Kidney 27.22 21.30

24.065

23.64 -5.91

-3.155

-3.57

Skin 27.01 29.61

30.17

27.87 2.59

3.155

0.85

Ovary 27.58 25.77

26.165

26.86 -1.81

-1.42

-0.72

Spleen 27.32 22.23

24.685

24.19 -5.09

-2.635

-3.12

Lymph node 26.98 24.12

25.73

24.35 -2.85

-1.25

-2.63

Heart 27.285 31.54

35.17

27.87 4.25

7.885

0.58

Mammary

gland 26.98 24.65

26.02

26.98 -2.33

-0.965

-0.005

Intestine 27.27 34.26

28.85

31.84 6.99

1.58

4.57

Skeletal

muscle 27.33 25.31

28.59

28.52 -2.02

1.255

1.18

87

(a) (b)

(c) (d)

(e) (f)

Fig. 4.37. Dissociation curve and amplification plots of (a) and (b) goat TLR4, (c)

and (d) goat TLR7 gene (e) and (f) goat TLR8 gene.

(a) (b)

Fig. 4.38.a.b. Standard curve and amplification plots of serially diluted cDNA of

house-keeping gene RPS15 to analyze the amplification efficiency of

reactions.

88

Cloning and expression in prokaryotic expression vector

4.16. Amplification of TIR domain of goat TLR4 and TLR8 and preparation of

vector

The TIR domain sequence of both the genes was used for designing of primers and

further amplification was done by employing the primers having the restriction sites for

HindIII and BamHI restriction enzymes at both the ends to facilitate directional cloning.

The amplified TIR domain of both TLR4 and TLR8 goat genes were then purified and

quantified by running in 1.5% agarose gel. The prokaryotic vector (pQE30) was also

digested by using the same enzymes, whose sequences have been incorporated in primers.

After digestion vector was purified and was quantified by running in gel (Fig. 4.39). After

the quantification the restriction enzyme digested PCR products and the purified vector

were ligated and transformed in SG13009 strain of E. coli. This expression vectors doesn‟t

allow the color selection of clones, so the precaution was taken at the time of digestion.

(a) (b)

Fig. 4.39. Ethidium bromide stained 1.5% Agarose gel showing Digested (a) pQE30,

TLR4 (498bp) and (b) pQE30, TLR8 (485bp) DNA.

4.17. Confirmation of recombinant clones

Plasmid DNA isolated from four white colonies for each of TLR4 and TLR8 TIR

domain cloned and transformed E.coli cells, and were screened to confirm recombinant

plasmid having insert of both the genes by different methods described below.

10

0b

p la

dd

er

Undigested

pQE30

10

0b

p l

ad

der

Undigested

pQE30

Digested

pQE30

Digested

TIR domain

of TLR8

Digested

pQE30

Digested

TIR domain

of TLR4

10

0b

p l

ad

der

Undigested

pQE30

89

Plasmid isolation from transformed cells

Plasmid DNA was isolated from the recombinant clones by alkali lysis as well as

kit purification (Qiagen Midi prep), using manufacturer‟s instructions and was checked by

1% agarose gel electrophoresis. Good quality plasmid was obtained without any

contamination of genomic DNA or RNA.

PCR of recombinant plasmid

PCR was carried out directly from the isolated recombinant plasmid DNA using

TLR4 and TLR8 gene specific primers. PCR products were runned on agarose gel

electrophoresis showing the presence of TIR domains of TLR4 and TLR8, with the insert

of approximately 500 bp size for each gene as expected, when runned along with 100 bp

molecular size marker (Fig. 4.40).

(a) (b)

Fig. 4.40. Confirmation of positive clones having TIR domains of (a) TLR4 (498 bp)

(b) TLR8 (485 bp) by plasmid PCR.

Confirmation by restriction enzyme digestion

The presence of correct size insert in the recombinant plasmids was also confirmed

by restriction endonuclease digestion. After double digestion with HindIII and BamHI

restriction enzymes the products were checked on 2% agarose gel, which confirmed the

presence of recombinant clones (Fig. 4.41). Digestion with HindIII and BamHI restriction

enzymes resulted in the release of TLR4 fragment from the plasmid vector and yielded two

fragments of approximately 498 bp insert and 3.4 kb vector (Fig 4.41 a). Similarly,

digestion of TLR8 with the same enzymes resulted in the release of 2 fragments of 485 bp,

and 3.4 kb (Fig. 4.41 b).

100bp

ladder 100bp

ladder

485bp

Insert

498bp

insert

M

M

90

(a) (b)

Fig.4.41. Confirmation of positive clones by restriction enzyme digestion using

HindIII and BamHI enzyme: (a) TLR4, (b) TLR8.

4.18. Characterization of expressed proteins on SDS-PAGE

The positive clones thus confirmed were induced for expression of cloned TIR

domains by using different concentrations of IPTG and the culture was pelleted at different

time intervals, the pelleted culture was mixed with Laemmli‟s sample buffer and incubated

in boiling water for 15 min, before it was loaded on 16% SDS-PAGE gel. The expressed

protein bands of approximately 17.5 KDa size for TLR4 (Fig. 4.42) and 17 KDa for TLR8

were characterized and confirmed by running a molecular size marker along with it (Fig.

4.43).

Undigested

Plasmid

Digested Plasmid

1K

b l

ad

der

498bp

3.4Kb

Undigested

Plasmid

Digested plasmid

1K

b l

ad

der

3.4Kb

485bp

91

Fig. 4.42. SDS-PAGE analysis of expressed TIR domain of goat TLR4 after IPTG

induction of recombinant plasmid vector pQE and harvested at different

interval.

Fig. 4.43. SDS-PAGE analysis of expressed TIR domain of goat TLR8 after IPTG

induction of recombinant plasmid vector pQE harvested at different

intervals.

INDUCED UNINDUCED

INDUCED UNINDUCED

O/N 5hr 0hr M 0hr 5hr O/N pQE

17.5KDa

30KDa

sequence characterization of goat tlr genes -...

Documents