research and application of plant immunity
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Research and Application of Plant Immunity
Institute of Plant ProtectionChinese Academy of Agricultural Sciences
Dewen Qiu
主要内容
Application of plant immunity Application of plant immunity
Mechanism of plant immunityMechanism of plant immunity
Concept of plant immunityConcept of plant immunity
Mammals immunity Plant disease resistance and immunity: L. Yarwood (), “Mechanism of acquired immunity to a plant rust” Proc Natl
Acad Sci U S A. 1954, 40(6): pp. 374–377 KUC J, ULLSTRUP AJ, QUACKENBUSH FW, Production of fungistatic
substances by plant tissue after inoculation, Science. 1955,Dec16;122(3181):1186-7
Ross, AF . Systemic acquired resistance induced by localized virus infections in plants. Virology 1961, 14, 340-358.
Plant immunity conceptPlant immunity concept
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Cell
Plant immunology- Hot Areas
New discovery
Jen Sheen, Researchers discover mechanism of plant resistance to pathogens, Plants have effective mechanisms aimed at protecting themselves against bacteria and fungi. Science Daily Feb. 28, 2002.
Jonathan D. G. Jones1 & Jeffery L. Dangl,The plant immune system,Nature 444, 323-329 (16 November 2006)
Shen QH, et al.Nuclear Activity of MLA Immune Receptors Links Isolate-Specific and Basal Disease-Resistance Responses, Science, 23 February 2007: Vol. 315 no. 5815 pp. 1098-1103
Sang-Wook Park, Evans Kaimoyo, Dhirendra Kumar*, Stephen Mosher† and Daniel F. Klessig. Methyl Salicylate is a Critical Mobile Signal for Plant Systemic Acquired Resistance. Science, 5 October 2007: Vol. 318 no. 5847 pp. 113-116
New research results
Liqun Du1, Gul S. Ali3, Kayla A. Simons1, Jingguo Hou2,4, Tianbao Yang1, A. S. N. Reddy3 & B. W. Poovaiah1, Ca2+/calmodulin regulates salicylic-acid-mediated plant immunity. Nature 457, 1154-1158 (26 February 2009)
Thomas Boller1,* and Sheng Yang He, Innate Immunity in Plants: An Arms Race Between Pattern Recognition Receptors in Plants and Effectors in Microbial Pathogens. Science 8 May 2009: Vol. 324 no. 5928 pp. 742-744
Boudsocq M, Willmann MR, McCormack M, Lee H, Shan L, He P, Bush J, Cheng SH, Sheen J, Differential innate immune signalling via Ca(2+) sensor protein kinases. Nature. 2010 Mar 18;464(7287):418-22
Presidential Awards by EPA in 2001
Messenger --Cornell University and EDEN Bioscience,
主要内容
Application of plant immunity Application of plant immunity
Mechanism of plant immunityMechanism of plant immunity
Concept of plant immunityConcept of plant immunity
Panstruga R, Parker JE, Schulze-Lefert P. SnapShot: Plant immune response pathways.Cell. 2009 Mar 6;136(5):978.e1-3.
Plant Growth Promoting Rhizobacteria
Trichoderma and Rhizobacteria -induced plant immunity
SA→PR →induced plant immune (SAR)? JA/ET→LOX →PAL → induced plant immune(ISR)?JA →MAPK?
Plant immunity mechanismPlant immunity mechanism
Multiple and complex effects (Plant stress and disease resistance
Plant growth promoting)
Multiple and complex effects (Plant stress and disease resistance
Plant growth promoting)
Plant immunity signaling ( Protein elicitor and
Chitosan Oligosaccharide )
Plant immunity signaling ( Protein elicitor and
Chitosan Oligosaccharide )
Salicylic acid Jasmonic acid Ethylene NO
多年来我们一直试图寻找专化性高效免疫诱抗剂。
经研究表明蛋白质、寡糖、枯草芽孢杆菌及木霉菌等免疫诱抗剂所诱导的免疫反应基本比较一致,主要集中在水杨酸、茉莉酸和乙烯的途径。
本人分析认为植物的免疫作用可能与哺乳动物或昆虫等动物的免疫反应不同, 不形成IGG或YGG等免疫结构,植物的免疫是一个原始初级的免疫反应,植物的免疫反应通过大致相同的路径来抵御病虫害的侵入,也就是植物诱抗剂所诱导的免疫反应具有广谱性或多功能。
植物免疫的诱导或激活是一个初级的免疫反应
主要内容
Application of plant immunity Application of plant immunity
Mechanism of plant immunityMechanism of plant immunity
Concept of plant immunityConcept of plant immunity
Products of Elicitor
Eden Bioscienes, “Messenger® ” Redox Chemicals, “Oxycom” Morse, “ KeyPlex” Syngenta , “Actigard” Nippon Kayaku Co Ltd, “NCI” “Chitosan”
Application of Elicitor
Messenger, Promotes root growth, vigor, disease and stress resistence, increased flowering and fruit set.
“Oxycom”, “KeyPlex”, for control of leaf spot disease in Orange and Banana.
“Chitosan”, used in seed treatment and plant growth enhancer, against fungal infections, control of diseases vegetables.
“Actigard”, for downy mildew in leafy vegetables, bacterial leaf spots in tomatoes and blue mold in tobacco.
HR –inducing protein elicitorPevD1;Hrip1--- Extracellular
Non HR protein elicitorPeaT1;PebC1;PemG1- Intracellular
Protein elicitor from fungi
Disease resistance and growth were enhanced by protein elicitor
75.5%
11.09%
Treatment Control
11.9%
PeaT1
CK
SAR against TMV induced by protein elicitor PeaT1
Purification of protein
Gene clone
1
2
3
protein application
Protein expression and function
Application
Mechanism4
5
Alternaria tenuissima
Botrytis cinerea
Magnaporthe grisea
Penicillium spp
Aspergillus Flarres
Rhizoctonia solani
Trichoderma spp
Fusarium spp
Fungi species
PeaT1
一、Protein purification
PebC1
PemG1PeaT1
PebC1
Purification of PevD1
HR
HP Q HiTrap™ Ion exchange chromatography
hydrophobic chromatography
SDS-PAGE
2
1, 2, 3, 4, 5 and 6 represents pH 2, 4, 6, 8, 11 and 13
HR –inducing protein elicitorPevD1;Hrip1--- Extracellular
Non HR protein elicitorPeaT1;PebC1;PemG1- Intracellular
Fungi elicitor protein
PeaT1
Purification of Protein elicitor
PebC1
PemG1PeaT1
PebC1
Purification of Protein elicitor
HR2
HP Q HiTrap™ Ion exchange chromatography
hydrophobic chromatography
SDS-PAGE
1, 2, 3, 4, 5 and 6 represents pH 2, 4, 6, 8, 11 and 13
离子交换的色谱 分子筛的色谱和电泳图
目的蛋白
PeaT1蛋白纯化与检测
peptides fingerprint
Trypsin T Chromtrypsin CTGlu-C V8
Gene cloning of protein elicitor
The mass-spectrometry/proteomic experiment
1. NILFVINKPDVYKSPSSNTWIIFGEAK2. DIELVMQQASVSR3. ALKENDNDIVNSIMALSI4. RPKNILFVINQPDVYK
PeaT1 protein sequence
1. NILFVINKPDVYKSPSSNTWIIFGEAK2. DIELVMQQASVSR3. ALKENDNDIVNSIMALSI4. RPKNILFVINQPDVYK
3‘RACE获得该基因的3’全长
交链孢菌激活蛋白的全基因序列207 amono acids MANPRIEELPDEPEKKNVQIEEDESSDESEGEEGEVSVPAGSSVAVHSRNEKKARKAIAKLGLKHIDGITRVTLRRPKNILFVINQPDVYKSPSSNTWIIFGEAKIEDLNSQAQASAAQQLAQAEAASHDHAGHDHGDEASKGKGKAVEDKKDEEEEDDDEEIDDSGLEAKDIELVMQQASVSRKKAVKALKENDNDIVNSIMALSI
NAC Domain:49-108aa
Alternaria tenuissima NAC protein sequence
1 MSNPRIEELP DNEEPTKQQV TAEDEGSDSS DSEAEGEEVA GIPAGSQVAF SRNEKKARKS
61 IAKLGLTRVP GITRVTLRRP KNILFVINQP EVYKSPTSNT YIVFGEAKIE DLNSQAQASA
121 AAQLAAQESH DHAGHDHSGH DHSHDHGKGK AVDTEEKKEE EEDDTEEVDA TGLEDKDIEL
181 VMTQASVSRN KAVKALKEND NDIVNSIMAL SI
Botrytis cinerea protein sequence
Fusarium spp αNAC和βNACαNAC
βNAC
MSNPRVEELPDEEPKKTTVQEHEDDSSDDSEVEEVGEGQLPAGSTVIHNRNEKKARKALEKLHLTRIPGITRVTLRRPKNILFVINTPEVYKSPNSNTYIVFGEAKIEDVNAAAQQAAAAQLASQNAEDHSGHNHGEPSKAVEADEKKEDKEDDEDEEEEEEEEVDASGLEDKDIELVMTQANVSRNKAVKALKENDNDIVNSIMALSI(210aa)
MSDVQERLKKLGLGARTGESNKLIYNTGGKGTPRRKVKRAPARSGADDKKLQLALKKLNTQPIQAIEEVNMFKQDGNVIHFAAPKVHAAVPSNTFAIYGNGEDKELTELVPGILNQLGPDSLASLRKLAESYQNLQKEKGEDDDEIPDLVEGENFEGEPKVE (162aa)
Gene:624bp
ATGGCCAACCCCCGCATTGAAGAGCTCCCCGACGAGCCCGAGAAGAAGAACGTCCAGATCGAGGAGGATGAGTCCAGCGACGAGTCTGAGGGCGAGGAGGGCGAGGTCAGCGTACCCGCGGGCTCCTCCGTCGCTGTCCACTCCCGCAACGAGAAGAAGGCTCGCAAGGCCATCGCCAAGCTCGGCCTGAAGCACATCGACGGCATCACACGCGTCACCCTCCGCCGACCCAAGAACATCCTCTTTGTCATCAACCAGCCCGACGTCTACAAGTCCCCTTCAAGCAACACCTGGATCATCTTCGGTGAGGCCAAGATCGAGGACCTCAACTCCCAGGCTCAGGCTTCCGCCGCCCAGCAGCTTGCTCAGGCCGAGGCCGCATCCCACGACCACGCCGGCCACGACCACGGCGACGAGGCCAGCAAGGGCAAGGGCAAGGCTGTCGAGGACAAGAAGGACGAGGAGGAGGAGGATGACGATGAGGAGATTGACGACTCTGGCCTTGAGGCCAAGGACATCGAGCTCGTCATGCAGCAGGCCAGCGTTTCGCGGAAGAAGGCCGTCAAGGCCCTCAAGGAGAACGATAACGATATAGTCAACTCCATCATGGCGCTGAGCATATAG
Protein:207aa
MANPRIEELPDEPEKKNVQIEEDESSDESEGEEGEVSVPAGSSVAVHSRNEKKARKAIAKLGLKHIDGITRVTLRRPKNILFVINQPDVYKSPSSNTWIIFGEAKIEDLNSQAQASAAQQLAQAEAASHDHAGHDHGDEASKGKGKAVEDKKDEEEEDDDEEIDDSGLEAKDIELVMQQASVSRKKAVKALKENDNDIVNSIMALSI
PeaT1 (Alternaria tenuissima )
Doman analysis of PeaT1
Structure of protein PeaT1
207Amino acidMANPRIEELPDEPEKKNVQIEEDESSDESEGEEGEVSVPAGSSVAVHSRNEKKARKAIAKLGLKHIDGITRVTLRRPKNILFVINQPDVYKSPSSNTWIIFGEAKIEDLNSQAQASAAQQLAQAEAASHDHAGHDHGDEASKGKGKAVEDKKDEEEEDDDEEIDDSGLEAKDIELVMQQASVSRKKAVKALKENDNDIVNSIMALSI
NAC Domain:49-108aa
Genes Fungi GenBank
Accession No Patens
pemG1 Magnaporthe spp EF062504 ZL200510011580.5 peaT1 Alternaria tenuissima EF030819 ZL200610152700.8 pebC1 Botrytis cinerea FJ748868 -- PevD1 Verticillium dahliae HQ540585 201010593488.5 Hrip1 Alternaria tenuissima HQ713431 201110144389.3
Genes and patents
1. PET-28a/PeaT1
2. PET-28a
3. Marker
SDS-PAGE
Expression of protein elicitor
Pichia pastoris
E.coli
PeaT1
PeaT1
PeaT1的X-射线晶体学
Crystallization X-ray diffraction
Conditions:20% PEG 3350,0.2M MgCl2,0.1M Mes pH5.5 20℃
分辨率:2.4Å
Crystal and X-ray diffraction
a-Helix b-Sheet 0.161 0.307
Structure of protein elicitor (Peat1)
NAC domain was constructed. NAC domain formed a homodimerfrom reverse direction. This structure outline was similar with a barrel, with a hole at the center and 6 intercrossed β strands surrounded
Structure of NAC domain in PeaT1
TCTP1 TCTP2
Intermediate file
据目前研究表明:TCTP家族蛋白功能:在人体内为胞外作用,引起过敏反应,与免疫有关
TCTP1:暴露在蛋白的表面,亲水性强,结构无固定规则----为可能的活性位点
TCTP2:无规则卷曲,固定在在两个折叠之间
1. PET-28a/PeaT1
2. PET-28a
3. Marker
SDS-PAGE
Expression of protein elicitor
Pichia pastoris
E.coli
PeaT1
PeaT1
PeaT1
CK
SAR against TMV induced by protein elicitor PeaT1
Protein elicitor products
Certificate for Bio-fertilizer
Certificate for pesticide
Treatment
Before
treated
Treated 3 times Treated 4 times
7d 15d 7d 15d
Disease
index
Disease
index
Effect
(%)
Disease
index
Effect
(%)
Disease
index
Effect
(%)
Disease
index
Effect
(%)
Bingdu A 9.48 34.80 37.01 35.64 40.75 36.51 42.09 36.89 44.87 99zhibao 11.73 25.09 54.58 25.17 58.16 22.78 63.87 21.95 67.15 Protein 11.02 21.55 60.99 20.10 66.59 18.75 70.26 17.02 74.53 Protein
+Bingdu A 9.82 20.95 62.08 19.21 68.07 17.71 71.91 15.84 76.29
Protein+
99 zhibao 10.63 55.24 65.11 17.75 70.49 13.24 79.00 11.60 82.64
ck 10.79 \ 60.16 \ 63.04 \ 66.81 \
Control of pepper virus disease
Increase pepper yield
Treatment
Treated 3 times Treated 4 times
7d 15d 7d 15d
Disease
index
Effect
(%)
Disease
index
Effeact
(%)
Disease
index
Effect
(%)
Disease
index
Effect
(%)
Bingdu A 47.68 40.15 47.31 46.29 44.84 50.32 39.35 58.16
99 Zhibao 40.24 49.79 38.69 56.08 27.59 69.43 25.99 72.37
Protein 29.08 63.72 28.07 68.14 18.73 79.25 14.44 84.65
Protein+
Bingdu A 31.39 60.83 23.61 73.20 16.82 81.36 11.36 87.92
Protein+
99 Zhibao 17.20 78.54 12.41 85.91 6.16 93.18 3.56 96.21
CK 80.15 \ 88.09 \ 90.26 \ 94.05 \
Hengyang, Hunan
Control of virus disease in tomato
Treatment
Before
treated
Treated 3 times Treated 4 times
7d 15d 7d 15d
Disease
index
Disease
index
Effect
(%)
Disease
index
Effect
(%)
Disease
index
Effect
(%)
Disease
index
Effect
(%)
Bingdu A 9.48 34.80 37.01 35.64 40.75 36.51 42.09 36.89 44.87 99zhibao 11.73 25.09 54.58 25.17 58.16 22.78 63.87 21.95 67.15 Protein 11.02 21.55 60.99 20.10 66.59 18.75 70.26 17.02 74.53 Protein
+Bingdu A 9.82 20.95 62.08 19.21 68.07 17.71 71.91 15.84 76.29
Protein+
99 zhibao 10.63 55.24 65.11 17.75 70.49 13.24 79.00 11.60 82.64
ck 10.79 \ 60.16 \ 63.04 \ 66.81 \
Control of pepper virus disease
Treatment
Treated 3 times Treated 4 times
7d 15d 7d 15d
Disease
index
Effect
(%)
Disease
index
Effeact
(%)
Disease
index
Effect
(%)
Disease
index
Effect
(%)
Bingdu A 47.68 40.15 47.31 46.29 44.84 50.32 39.35 58.16
99 Zhibao 40.24 49.79 38.69 56.08 27.59 69.43 25.99 72.37
Protein 29.08 63.72 28.07 68.14 18.73 79.25 14.44 84.65
Protein+
Bingdu A 31.39 60.83 23.61 73.20 16.82 81.36 11.36 87.92
Protein+
99 Zhibao 17.20 78.54 12.41 85.91 6.16 93.18 3.56 96.21
CK 80.15 \ 88.09 \ 90.26 \ 94.05 \
Hengyang, Hunan
Control of virus disease in tomato
Treatment 20 days after treatment 45 days after treatment
Morbidity(%)
Disease index
Effect(%)
Morbidity(%)
Disease index
Effect (%)
Activator WP
8.07 3.95 72.87 9.1 4.32 73.93
bingdubike 8.35 401 71.84 9.56 4.95 70.13
shengwujunsu
11.65 5.72 60.71 14.75 7.23 56.37
Water 27.11 14.56 — 32.25 16.57 —
Control of tobacco virus disease
CK 处理
60%
40%65%
Treatment Control
Treatment Control Treatment Control
Increase disease resistance and improve yields of strawberry and grape
Chinese cabbage, Cucumber, Pepper, Celery, Rice, Peach
TreatmentControl
Protein gene expressed in trangenical rice and cotton
pemG1转基因水稻:对稻瘟病防治效果为 42.53%
利用农杆菌介导的方法,成功地将激发子基因pemG1、peaT1和pebC1转入不同水稻品种(粳稻、恢复系)
水稻
A:转化 B:棉铃 C:卡那筛选 D:抗性棉苗
2号
对照 0
100
200
300
400
500
对照 2号
取食
面积
/平方
毫米
取食量
棉铃虫取食量差异 卷叶螟取食量差异
棉花
花粉管通道法:获得转PeaT1抗性棉株
茎粗增加,棉铃数增加,表现出一定抗虫性
农杆菌介导法转化棉花下胚轴: Coker312和CCRI24获得再生棉苗
无菌苗
继代
侵染
共培养
胚状体
成苗
胚性愈伤
转昆虫蜕皮基因烟草饲喂棉铃虫1天后的生测结果
转昆虫蜕皮基因烟草饲喂棉铃虫1天后的生测结果
转昆虫蜕皮基因烟草饲喂棉铃虫5天后的生测结果
昆虫取食后5天脱皮体式显微镜图
Steinernema glaserisurface coat protein suppresses the
immune response of Popilliajaponica larvae
Cuticle protein on gel
昆虫病原线虫:
多种害虫寄生天敌
表皮蛋白
抑制昆虫免疫
一种什么蛋白,编码基因,到目前为止还没有报道
昆虫免疫抑制蛋白
Small-scale laboratory entomopathogenic nematode culture
The insect great wax moth is used as a culture medium
(a) Healthy (cream);(b) Steinernema feltiae - infected (brown);
(c) Steinernema carpocapsae - infected (tan) and (d) Heterorhabditis bacteriophora -
infected (red) larvae of Galleria mellonella.
3-5days
10 days
抗昆虫免疫蛋白抗昆虫免疫蛋白
The nematode culture room
Vegetal medium: soybean powders,flour, egg powder, lard, termoamyl
Semi-vegetal medium:soybean powder, flou r, egg powder, lard, insect pupa homogenate,
Animal source medium:Pig liver extract, eggs, lard
Wash the nematodes out from sponge
Wash residue culture medium using the “The Big funnel”
Infective stage
0.5% sodium hypochlorite for 15minStore in distilled water with bubbled air for 2 days
Extraction in ice-cold ethanol
for 30 mins and lyophilized
Dissolve the freeze-dry powder in millQ water and incubate on ice for 30 mins with gently vortex
Centrifuge at 13000 rpm, 4 .c
Lyophilize the extraction
Lipid layer
The “sandwich structure”
Supernatant:
Protein solution
Pellet:
Surface coat debris
12% SDS-PAGE
Dissolve in 1% oNG
8% native PAGE
Marker
97kd,66kd,44kd,22kd,14kd
How do they correspond?
pH4 pH7
2-D results of 35% EtOH extraction proteins
IEF with ampholine
SDS-PAGEpH4 pH6
新昆虫免疫抑制蛋白:分离纯化、基因克隆及表达
72kDa
15% sds page 2-DE
72kDa55kDa
XeGroEL (from Xenorhabdus ehlersii) 共生菌 1647bp Xbecotin (from Xenorhabdus bovienii) 共生菌 483bp Haemocoel Insecticidal toxin
(from Xenorhabdus budapestensis D43)共生菌
ATGAAAAAGTATCTACTTCCTTTAGCCGCTATGATGGTTTCGGTTTCTACTTTTGCTCAGGCTGATAAGAAACTTGAAGATGTCGCTCCTTATCCTAAAGCTTCAGAGGGAATGGTACGCAATGTCATTGACCTTGCACCAGAAGAGGATGAAGGCGATTACATGGTTGAACTGATGATTGGCAAGGATATAAAAGTAGACTGTAATCATCACTGGTTTGGTGGGCAACTTGAAACAAAAACCTTGGAGGGGTGGGGATACGATTATTATGTATTGAATAATGTCACCGGCCCAGCGTCAACCCACATGGGTTGTTCAGGACAAAAAGAAACCGTGCGCTTTGTTCAAGTGCAGTTAGGCAAAGATGCGCTTATACGCTATAACAGCAAATTACCTATCGTGGTATATACGCCAAAATCAATGACAGTCAAATATCGTATTTGGCAGGCATTGGATGATGTCGATAATGCAGTAATCAAATAA
Xbecotin:483bp
1.0 *107 coupled Microspherses(3ul)
In vivo phagocytosis test
Microinjection
Cherry red microspheres in larva body
12 hours
Bleeding
Grace insect medium
DAPI treatment
(cell nucleolus stain)
Fluorescent microscope imaging
Engulfment of microspheresKeratin and Enolase protein coupled microspheres
BSA coupled microspheres
Transparent blue: Cell nucleolus
Bright red: Microspheres
Lipid layerSupernatant:
Protein solutionPellet:
Surface coat debris
Bioassay抑制昆虫血细胞免
疫BSA SCP2
Phagocytosis
2D electrophoresis
蛋白斑点
MAIDI-TOF-MS/MSLC-MS/MS
YGLDATAVGDEGGFAPNIQDNKAAVPSGASTGIHEALELRACNCLLLK
秀丽隐杆线虫、单一异尖线虫、旋毛线虫、刺尾蝎、拟南芥、番茄、油菜
GenBank:enolase
eno1: 1311bpEno1: 436aa
GenBank: EU333864
1 MPITRIHARQIYDSRGNPTVEVDLHTEKGVFRAAVPSGAS
41 TGIHEALELRDQDKAVHHGKGVEKAVANVIEKIAPALIAK
81 NFDVTDQVAIDKFMIELDGTENKSSLGANAILGVSLAVAK
121 AGAVHKGVPLYKHLADLAGVSKVVLPVPAFNVINGGSHAG
161 NKLAMQEFMILPVGAKSFKEAMRMGSEIYHHLKAEIKKRY
201 GLDATAVGDEGGFAPNIQDNKEGLDLLNTAIKLAGYTGLV
241 SIGMDVAASEFYKENEKKYDLDFKNPNSDPSKWINGDELA
281 ALYQSFIKDYPVVSIEDAFDQDDWANWSKLMGNTSIQLVG
321 DDLTVTNPKRIQMAVDQKACNCLLLKVNQIGSITESIEAA
361 KLSRANGWGVMVSHRSGETEDCFIADLVVGLATGQIKTGA
401 PCRSERLAKYNQLLRIEEELGADAVYAGENFRNPQI
专利申请200910119907.9
表达蛋白:具有抑制昆虫免疫的活性
格式线虫enolase基因序列: 5’UTR GACTCACGCGGCAACCCAACTGTTGAAGTCGACCTCCACACCGAGAAGGGTGAGTCTTTGC
TTGTCTTTGCAGTAGAATAACTGAATCGATTTAGGTGTGTTCCGCGCTGCTGTCCCCAGCGGAGCCTCCACTGGCATCCACGAGGCTCTGGAACTCCGCGACCAGGACAAGGCTGTCCACCACGGAAAGGGAGTCGAGAAGGCCGTCGCCAACGTCATCGAGAAGATCGCGCCGGCCCTCATCGCCAAGAACTTCGATGTGACCGACCAGGTCGCCATCGACAAGTTC
ORF:1032bp: ATGATCGAGCTTGACGGAACCGAGAACAAGTCGAGCCTCGGAGCCAACGCCATCCTCGGC
GTGTCGCTCGCCGTCGCCAAGGCCGGTGCCGTGCACAAGGGAGTGCCCCTCTACAAGCACTTGGCCGATCTCGCCGGAGTGAGCAAGGTTGTCCTCCCCGTTCCTGCCTTCAACGTCATCAACGGAGGCTCGCACGCCGGAAACAAGCTCGCCATGCAGGAGTTCATGATCCTCCCCGTCGGAGCCAAGAGCTTCAAGGAGGCCATGCGCATGGGATCCGAGATCTACCACCACCTCAAGGCCGAGATCAAGAAGCGCTACGGACTCGACGCCACCGCCGTCGGAGATGAGGGAGGATTCGCCCCCAACATCCAGGACAACAAGGAGGGTCTTGACCTCCTCAACACCGCTATCAAGCTTGCCGGATATACCGGACTGGTGTCCATCGGCATGGACGTCGCCGCCTCCGAGTTCTACAAGGAGAACGAGAAGAAGTACGACTTGGACTTCAAGAACCCCAACTCCGACCCCAGCAAGTGGATCAACGGAGACGAGCTCGCCGCGCTCTACCAGTCGTTCATCAAGGACTATCCCGTCGTCTCCATCGAGGACGCCTTCGACCAGGACGACTGGGCGAACTGGAGCAAGCTGATGGGCAACACCTCCATCCAGCTCGTCGGAGATGATCTCACCGTCACCAACCCCAAGCGCATCCAGATGGCCGTCGACCAGAAGGCGTGCAACTGCCTTCTCCTCAAGGTCAACCAGATCGGATCCATCACCGAGTCCATCGAGGCCGCCAAGCTGTCCCGCGCCAACGGATGGGGCGTCATGGTTTCCCACCGTTCTGGAGAAACCGAAGACTGCTTCATCGCTGATCTTGTTGTTGGCCTCGCTACTGGACAGATCAAGACCGGAGCCCCTTGCCGATCTGAGCGTCTCGCCAAGTACAACCAGCTTCTTCGCATCGAGGAGGAGCTCGGAGCCGATGCCGTGTACGCCGGAGAGAACTTCCGCAACCCCCAGATCTAA
3’UTR-98bp: ATGACCACCGTACTTTAATCTATCGTGTAGTTTGTAGTTTGACATTGTGATGAGTGGTTAAT
TGTATAATACGTAATTGTTGGTCAAAAAAAAAAAAA
蛋白序列:MIELDGTENKSSLGANAILGVSLAVAKAGAVHK
GVPLYKHLADLAGVSKVVLPVPAFNVINGGSHAGNKLAMQEFMILPVGAKSFKEAMRMGSEIYHHLKAEIKKRYGLDATAVGDEGGFAPNIQDNKEGLDLLNTAIKLAGYTGLVSIGMDVAASEFYKENEKKYDLDFKNPNSDPSKWINGDELAALYQSFIKDYPVVSIEDAFDQDDWANWSKLMGNTSIQLVGDDLTVTNPKRIQMAVDQKACNCLLLKVNQIGSITESIEAAKLSRANGWGVMVSHRSGETEDCFIADLVVGLATGQIKTGAPCRSERLAKYNQLLRIEEELGADAVYAGENFRNPQI
昆虫拒食蛋白 CB6蛋白对大蜡螟的生物活性测定: 1)大蜡螟的注射活性
取总蛋白5微升进行注射大蜡螟(5龄老虫),总蛋白浓度:1.890mg/mL,大约不到5分钟,大蜡螟体色很快变成黑灰色,表现为中毒现象。
取GeAB蛋白回收条带III,也同样进行大蜡螟注射活性验证得到和总
蛋白相同的现象。怀疑致使大蜡螟血腔毒素蛋白即可能就是这一单个蛋白(约为62kD左右)。
毒素III的胃毒活性还在进行中,待定。
处理 CK
双向电泳与结果分析: 1)采用GE公司的IPGphor-3等电聚焦仪进行第一相,具体的操作
由刘华指导完成。
60kD
60kD
Acknowledgement
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