cloning and characterization of a multifunctional promoter from maize (zea mays l.)

Cloning and Characterization of a MultifunctionalPromoter from Maize (Zea mays L.)

Qing Dong & Haiyang Jiang & QianQian Xu &

Xiaoming Li & Xiaojian Peng & Haibing Yu & Yan Xiang &

Beijiu Cheng

Received: 23 June 2014 /Accepted: 23 September 2014# Springer Science+Business Media New York 2014

Abstract The use of tissue-specific promoters to drive the expression of target genes duringcertain developmental stages or in specific organs can prevent unnecessary gene expressioncaused by constitutive promoters. Utilizing heterologous promoters to regulate the expressionof genes in transgenic receptors can help prevent gene silencing. Here, we engineeredheterologous maize promoters that regulate gene-specific expression in rice plant receptors.We performed a histochemical and quantitative β-glucuronidase (GUS) analysis of the Zeamays legumin1 (ZM-LEGF) gene promoter and detailed detection of stably transformed riceexpressing the GUS gene under the control of the promoter of ZM-LEGF (pZM-LEGF) and itstruncated promoters throughout development. When the promoter sequence was truncated, thelocation and intensity of GUS expression changed. The results suggest that the sequence from−140 to +41 is a critical region that confers the expression of the entire promoter. Truncation ofpZM-LEG (3′-deleted region of pZM-LEGF) markedly increased the GUS activity, with thecore cis-elements located in the −273 to −140 regions, namely pZM-LEG6. Detailed analysisof pZM-LEG6::GUS T2 transformant rice seeds and plant tissues at different developmentalstages indicated that this promoter is an ideal vegetative tissue-specific promoter that can serveas a valuable tool for transgenic rice breeding and genetic engineering studies.

Keywords Vegetative tissue .Promoter.Oryza sativa .Maize (ZeamaysL.) .Heterologousgene

Introduction

Constitutive promoters can drive target gene expression throughout a plant’s lifecycle, but atsome point, this gene expression may not be necessary, sometimes leading to the accumulation

Appl Biochem BiotechnolDOI 10.1007/s12010-014-1277-4

Qing Dong and Haiyang Jiang are the co-first authors.

Q. Dong :H. Jiang : Q. Xu :X. Li : X. Peng :Y. Xiang (*) : B. Cheng (*)Key Lab of Biomass Improvement and Conversion, Anhui Agricultural University, Hefei 230036, Chinae-mail: [email protected]: [email protected]

H. YuSchool of Plant Sciences, Anhui Science and Technology University, Chuzhou 239000, China

of intracellular metabolites, increasing the burden on the cell and wasting energy [8, 36, 58].The use of tissue-specific promoters to drive target gene expression during certain develop-mental stages or in specific organs [57] can prevent unnecessary gene expression induced byconstitutive promoters. In addition, the safety of genetically modified foods (GMFs) hasbecome an increasing concern of both the public and governments. There is currently nointernational standard for GMFs. Restrictions are placed on most transgenic crops, especiallythose carrying non-plant or non-crop genes, for instance, the Bacillus thuringiensis (Bt) genefrom Bacillus [53], Bar from Streptomyces hygroscopicus [4], dehydration-responsive ele-ment-binding gene 1A (DREB1A) from Arabidopsis [41], and so on. The use of tissue-specificpromoters, especially those not expressed in seeds, can prevent the expression of exogenousgenes in crop seeds, which may help relieve the widespread concern about the safety of GMFs.

Previous studies have led to the isolation of different types of tissue-specific promoters fromvarious plants, including those expressed in pollen, vascular bundles, roots, leaves, stems, seedsor fruit, parenchyma, meristems, and so on. For instance, Liu et al. [34] detected a sequence at−1677 to −1380 bp of Arabidopsis profiling 2 promoters that can drive gene-specific expressionin vascular bundles. Cai et al. [7] obtained an interesting and useful rice green tissue-specificpromoter,PD540, that when integratedwith cry1Ac produces good insect resistance in transgenicrice, while the cry1Ac protein is undetectable in seeds. Azria and Bhalla [3] isolated a pollen-specific Orys1 promoter from rice. Tolley et al. [51] identified a light-regulated, cell-specificmethylation promoter in maize in the upstream region of the phosphoenolpyruvate carboxylase(PEPC) promoter. Wheat TaPR61 gene is an important hydrophobic signal peptide lipidtransfer protein gene whose promoter is specifically expressed in wheat seeds [29]. Ye et al.[63] identified two novel positive rice green tissue-specific cis-acting elements (GSE1 andGSE2) that are specifically expressed in green tissues in rice.

Rice is one of the most important cereal crops, and it also serves as a model organism inplant molecular biology. Great progress has been made in the genetic transformation of rice,and studies of tissue-specific promoters are crucial for rice breeding and modern molecularbiology. Plant genetic transformation is an important way to improve crops through the use ofexogenous genes. With the continued progress of transgenic research, the expression ofexogenous genes in receptor plants is becoming increasingly precise. The use of heterologouspromoters to regulate gene expression in transgenic receptor plants can help prevent genesilencing caused by the homologous promoter sequence [32].

Currently, there are few reports of promoters that are solely expressed in the vegetative organsof rice (roots, stems, leaves, glumes) and not in the reproductive organs (flowers, embryos,endosperms). Here, we are giving emphasis on the reporting of a novel promoter, pZMLEG6,which is efficiently expressed in vegetative tissues, especially leaves, but not in reproductivetissues. This promoter is likely to be quite useful in genetic engineering and plant breeding.

Materials and Methods

Plant Materials and Transformation Methods

Zhonghua 11 (Oryza sativa L. subsp. japonica) was used as the transgenic receptor throughoutthis study. The rice plants were cultivated in controlled chambers. Constructs carrying thedesired sequences were transformed into Agrobacterium tumefaciens EHA105 and were thentransferred into rice (Zhonghua 11, O. sativa L. subsp. japonica) [23]. Mature seeds weredehulled and sterilized with 70 % alcohol for 1 min, followed by 0.15 % HgCl2 for 15 min.The seeds were then washed with sterile water 4–5 times, placed onto callus induction medium

Appl Biochem Biotechnol

and incubated in the dark at 26±1 °C for approximately 4 weeks. The resulting calli wasimmersed in an Agrobacterium suspension for 30 min (OD600 0.8–1.0) and cultured in the darkat 19–20 °C for 3 days. Then, 250 μL hygromycin (Hyg; 50 mg/L) and 200 ppm cephalo-sporin (Cep; 500 mg/L) were added to the selection medium, and the immersed calli werecultured for 2–3 cycles. Plantlets were grown at 30 °C under a 16/8-h light/dark cycle and werethen planted in a rice paddy field.

Promoter Analysis, Cloning, and Vector Construction

The cis-elements of the Zea mays legumin1 gene (pZM-LEGF, GRMZM2G174883) promoter,namely pZM-LEGF, were analyzed using Plant Cis-Acting Regulatory Elements (PlantCARE)(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [33] and PLACE (http://www.dna.affrc.go.jp/PLACE/) [20], and the transcription star site (TSS) was predicted at the website(http:// linux1.softberry.com). A 2.1-kb promoter of the translation initiation site (ATG) wasisolated from genomic DNA of maize plants at the three-leaf stage [12, 64].

To produce the pZM-LEGF::GUS constructs, the 3′- and 5′ flanking regions of pZM-LEG,pZM-LEG1, pZM-LEG2, pZM-LEG3, pZM-LEG4, pZM-LEG5, and pZM-LEG6 were ampli-fied by PCR using forward and reverse primers containing overhanging HindIII and BglIIrecognition sites, respectively. All the primer pairs were used in this study are listed in Table 1,and fused with the β-glucuronidase (GUS)-coding sequence, and the resulting constructs wereused to transform rice. Reactions were performed with Taq polymerase (TransGen Biotech-nology, Beijing) under the following PCR conditions: initial denaturation for 5–10 min at94 °C, followed by 34 cycles for 1 min at 94 °C, 1 min at 68 °C, and 3 min at 72 °C, and thefinal extension of 10 min at 72 °C. All the fragments were digested and inserted into thepCAMBIA1301 vector and were confirmed by sequencing.

Table 1 PCR primers for promoters used in this study

Name Forward Reverse Size(bp)

ZM-LEGF 5′-CCCAAGCTTTACCGCAGTACTGCTACATTTCACA-3′

5′-GGAAGATCTCATTGCTGCGCTGCCTCTGCTCGCT-3′

2091

ZM-LEG 5′-CCCAAGCTTAACTGCTGGGAGATGGAACTCCA-3′

5′-GGAAGATCTCTTTGCTTTGCCGTGATAGGAGCTG-3′

1910

ZM-LEG1 5′-CCCAAGCTTAGACCCTTTATGAAATGCGCCACAA-3′


1608

ZM-LEG2 5′-CCCAAGCTTGCTTCTTTGCATCGCTGTATCGTGG-3′


1314

ZM-LEG3 5′-CCCAAGCTTCTGTCTTCGTTCGTGTTAGCCTGGT-3′


1002

ZM-LEG4 5′-CCCAAGCTTGTAACAATACTCACACTTTCACATA-3′


705

ZM-LEG5 5′-CCCAAGCTTAGCATTTGTCAAGGCTGCCTCATTA-3′


362

ZM-LEG6 5′-CCCAAGCTTTGTCAGCACAAGCAACAAGTCCAAA-3′


133

GUS 5′-GTGAATCCGCACCTCTGGCAAC-3′ 5′-ATCGCCGCTTTGGACATACCAT-3′ 720

The underlined sites are the sites for the digestion of restriction enzymes HindIII. The underlined italicized sitesare the sites for the digestion of restriction enzymes BglII


http://bioinformatics.psb.ugent.be/webtools/plantcare/html/

http://www.dna.affrc.go.jp/PLACE/

http://www.dna.affrc.go.jp/PLACE/

Southern Blot Analysis

Genomic DNA was isolated using the improved cetyl trimethylammonium bromide (CTAB)method [CTAB extraction buffer: 2 % CTAB, 0.1 M Tris-Cl (pH 8.0), 20 mM ethylenediaminetetraacetic acid (EDTA, pH 8.0), 1.4 M NaCl, 2 % β-mercaptoethanol]. Then, HindIII-digestedDNA (100–150 μg) was immobilized on a positively charged nylon membrane (10 mL/100 cm2) and probed with a digoxigenin (DIG)-labeled GUS probe primers (Table 1) (2 μLDIG probe/1 mL hybridization solution) according to the methods described in the DIG-HighPrime DNA Labeling and Detection Starter Kit I manual (Roche, Basel, Switzerland). Primersused to amplify promoters and GUS gene of the transgenic rice were included before theanalysis of Southern blot. The plasmid of fusion promoter vector was used as positive control,and empty vector transgenic and untransformed rice was used as negative control.

Abiotic Stress and Hormone Treatments

To explore the expression patterns of the pZM-LEGF, seedlings of T1 rice transformantharboring pZM-LEGF were separated and exposed to various stress conditions includingtreatment with jasmonic acid methyl ester (MeJA; 100 μM), polyethylene glycol 6000 (PEG6000; 20 %), abscisic acid (ABA; 100 μM), and low temperature (4 °C) after 0, 3, 6, and9 days of germination. Fifty transformic seeds or seedlings were soaked in a solution of100 μMMeJA, 20 % PEG 6000, 100 μM ABA, and distilled water (4 °C) at 2, 6, 12, 24, and48 h. Positive (transformed 35S rice) and negative (untransformed rice) control seedlings werealso treated at the same times and conditions.

Histochemical GUS Assay

GUS activity in various tissues of transformed plants at different developmental stages wasdetected via histochemical staining using 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc). Specifically, the tissues were vacuum-infiltrated with 0.5 mM K3Fe(CN)6, 0.5 mMK4Fe(CN)6, 100 mM sodium phosphate (pH 7.0), 10 mM EDTANa2 (pH 8.0), 0.1 % TritonX-100, and 1 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide [24], incubated at 37 °C for 3–12 h, and rinsed with 75 % ethanol to remove the chlorophyll. Quantitative analysis of GUSfluorescence was used to detect the average GUS activity levels in the transformants comparedwith a standard. Bovine serum albumin (BSA) in dilution buffer was used to construct astandard curve for protein concentration using the Bradford method, and a dilution series of 4-methylumbelliferone (4-MU) in dilution buffer was used to construct a GUS standard curve.GUS activity was expressed as nanomole 4-MU per microgram protein per minute [27]. Thefluorescence values were determined at excitation and emission wavelengths of 365 and455 nm, respectively, using a fluorescence spectrophotometer (Hitech U2001).

Results

Promoter Isolation and Analysis

To obtain useful promoters for crop improvement, we selected 20 tissue-specific genes usingdata from the genome-wide gene expression atlas of maize inbred line B73 (GSE27004, [45]).We selected GRMZM2G174883_T01 as the gene of interest, as this Zea mays legumin1 gene(ZM-LGEF) is transcribed in starchy endosperm and aleurone cells [56]. A 2091-bp promoter


sequence of genomic-DNA upstream of the translation initiation site (ATG) was amplifiedusing the primer pair (Table 1).

The results of the analysis showed that the sequence contained not only core cis-elements(TATA box and CAAT box) but also several known cis-elements including six types of light-r e spons ive e lemen t s (Box4 , GAG-mot i f , GT1-mot i f , CATT-mot i f , andREALPHALGLHCB21), four types of tissue expression elements (ROOTMOTIFTAPOX1and RAV1AAT, a known cis-acting element related to root-specific expression; AACA motifand AAAG-motif, involved the endosperm-specific expression), and seven types of inducibleelements (AuxRR-core, cis-acting regulatory element involved in auxin responsiveness; P-box, gibberellin-responsive element; ABRE, cis-acting element involved in the abscisic acidresponsiveness; CGTCA-motif and TGACG-motif, cis-acting element involved in the MeJAresponsiveness; MBS, MYB binding site involved in drought inducibility; and LTR, cis-actingregulatory element involved in low-temperature responsiveness), and the specific results wereshown in Table 2.

GUS Gene Expression of pZM-LGEF

T0 transgenic lines were detected via RT-PCR, the primes of pZM-LGEF and GUS gene(Table 1), and the results verified the presence of the pZM-LGEF and GUS gene in eachtransgenic line evaluated (figure not shown). The GUS histochemical staining and GUSfluorescent quantitative analysis of these lines revealed that pZM-LGEF::GUS was notexpressed in any tissues. Through cis-element prediction, we determined that pZM-LGEFcontained seven types of inducible elements. The results of different treatments by 100 μMMeJA, 20 % PEG 6000, 100 μM ABA, and distilled water (4 °C) at 2, 6, 12, 24, and 48 hshowed that pZM-LGE::GUS was still not expressed in any tissue examined (date not shown).

To further understand the developmental regulation of this promoter and to elucidate itspossible function, we constructed 3′- and 5′-deleted promoters of pZM-LEGF (−2050 to +41,2091 bp) and fused them to the β-glucuronidase (GUS)-coding sequence. The resultingconstructs were used to transform rice; the 3′-deleted promoter of pZM-LEGF was designatedpZM-LEG (−2050 to −140, 1910 bp). Stable gene integration and copy numbers in pZM-LEGtransgenic T0 rice were estimated by Southern blot analysis (Fig. 1). Of the six independenttransgenic rice lines examined, lines 1 and 4 had single copies of the transgene, lines 2 and 7had three copies, and line 6 had weak bands. T1 transgenic rice lines were detected via GUShistochemical staining and GUS fluorescence quantitative analysis. The results showed that noGUS activity was detected in leaves, stems, buds, scabbard, roots, glumes, flowers, endo-sperm, or embryos, but it was detected in callus (Fig. 2a, b).

GUS Gene Expression of pZM-LEG and Its Truncated Promoters

Six 5′-deleted sequences of pZM-LEG, namely pZM-LEG1 (−1748 to −140, 1608 bp), pZM-LEG2 (−1454 to −140, 1314 bp), pZM-LEG3 (−1142 to −140, 1002 bp), pZM-LEG4 (−845 to−140, 705 bp), pZM-LEG5 (−502 to −140, 362 bp), and pZM-LEG6 (−273 to −140, 133 bp)were produced (Fig. 3), and Southern blot data of transgenic T0 rice were also estimated (datenot shown). More than ten independent transgenic rice plants were obtained harboring thefollowing promoter–GUS fusion constructs: pZM-LEG1::GUS, pZM-LEG2::GUS, pZM-LEG3::GUS, pZM-LEG4::GUS, pZM-LEG5::GUS, pZM-LEG6::GUS, and CaMV35S (posi-tive control); wild-type rice was used as the negative control.

The results of histochemical analysis and quantitative GUS fluorescence analysis are shownin Fig. 4. GUS expression was weak in leaves, stems, roots, and lemma but strong in callus. In


particular, in addition to expression in callus, the GUS expression patterns were as follows:pZM-LEG1::GUS was only expressed in lemma (lemma hairs); pZM-LEG2::GUS in leaves(mature leaves); pZM-LEG3::GUS in leaves (young leaves); pZM-LEG4::GUS in leaves, roots,and lemma (lemma hairs); pZM-LEG5::GUS in leaves (young leaves), roots (lateral root base),and lemma, as well as endosperm; and pZM-LEG6::GUS in leaves, stems (vascular bundles),roots, and lemma. None of the truncated promoters were expressed in flowers, endosperm, orembryos, except for pZM-LEG5::GUS.

Functional analysis indicated that the tissue-specific expression of pZM-LEGF is regulatedby multiple cis-elements. The region −140 to +41 contains negative regulatory elements thatmay suppress GUS expression in callus. Comparative analysis of the function of pZM-LEGand its 5′-deleted promoters revealed that these promoters may contain positive and negativeregulatory elements that regulate GUS gene expression in leaves, stems, roots, glumes, and

Table 2 Known cis-acting regulatory elements in the ZM-LGEF promoter using the PlantCARE and PLACEdatabases

Types Elements No. Sequence Position

CE TATA box [16] 24 TATA −894,−904,−906,−914,−988,−1266,−1366,−1589,−1619,−1630,−1671,−1679,−1703,−1705,−1796,−1925,−1943,−1967,−1969

ATTATACA −1945TTTTA −77,−1080,−1245

CAAT box [46] 16 CAAT −685,−815,−842,−888, −917,−1167,−1255,−1810

CAAAT −746,−796.−804,−1778CCAAT −889,−1168,−1256,−1811

LRE Box4 [50] 2 ATTAAT −482,−786GAG-motif [5] 1 GGAGATG −2043GT1-motif [18] 1 GGTTAA −1293Sp1 [22] 2 GGGCGG −1263,−1475CATT-motif [31] 1 TTACTTAA −49REALPHALGLHCB21 [11] 3 AACCAA −432,−891,−965

TEE ROOTMOTIFTAPOX1 (root) [14] 4 ATATT −987,−1165,−1384,−1588RAV1AAT (root) [26] 5 CAACA −97,−262,−335,−775,−1896AACA-motif (endosperm) [49] 20 AACA −11,−92,−96,−261,−334,−443,−687,

−710,−774,−798,−809,−844,−860,−919,−1174,−1481,−1715,−1766,−1895,−1979

AAAG-motif (endosperm) [60] 7 AAAG −145,−150,−342,−561,−724,−851,−1490

IE AuxRR-core [6] 1 GTCCAT −553P-box [40] 1 CCTTTTG −285ABRE [47] 2 ACGT −173,−1196CGTCA-motif [44] 1 CGTCA −1299TGACG-motif [43] 1 TGACG −468MBS [2] 1 CAACTG −1671LTR [13] 1 CCGAAA −525

CE core elements, LRE light responsive elements, TEE tissue expression elements, IE inducible elements


endosperm. The region −2050 to −1748 suppresses GUS gene expression in lemma hairs,while the region −1712 to −1454 both activatesGUS expression in lemma hairs and suppressesits expression in leaves. The region −1142 to −845 may suppress GUS expression in roots andlemma hairs, while the region −845 to −502 may suppress GUS expression in endosperm.Finally, the region −502 to −273 both activates GUS expression in stems and suppresses itsexpression in endosperm.

pZM-LEG6 Drove High Vegetative Tissue-Specific Expression of GUS Gene

The levels of GUS expression were also compared in different tissues of plants harboring thesesix deleted promoters. As the size of the deleted sequence site decreased, the GUS activityincreased. Compared with the other five truncated promoters, the activity of pZM-LEG6::GUSwas obviously high in various vegetative tissues, especially leaf and root tissue. In leaves, theactivity of pZM-LEG6::GUS was approximately 9.77-, 23.13-, 10.14-, and 14.69-fold higherthan those of plants transformed with pZM-LEG2::GUS, pZM-LEG3, pZM-LEG4::GUS, andpZM-LEG5::GUS, respectively. In roots, the activity of pZM-LEG6::GUS was approximately5.88- and 6-fold higher than those of pZM-LEG4::GUS and pZM-LEG5::GUS, respectively. Inglumes, the activity of pZM-LEG6::GUS was approximately 1.07-, 2.05-, and 1.62-fold higherthan those of pZM-LEG1::GUS, pZM-LEG4::GUS, and pZM-LEG5::GUS, respectively.

Fig. 1 Southern blot analysis of pZM-LEG T0 transgenic lines. M: Lambda DNA/EcoRI + HindIII markers; 3:positive control (positive plasmid); 1, 2, 4, 5, 6, 7: transgenic rice lines

Fig. 2 The expression patterns of pZM-LEG T1 transgenic rice lines via GUS histochemical staining and GUSfluorescent quantitative analysis. a Histochemical analysis of GUS expression in different tissues of transgenicrice lines. A, callus; B, leaf; C, stem; D, ligule; E, root; F, glume; G, anther; H, endosperm and embryo; A, B, C,D, E, F, H, scale bars=1 mm; G, scale bars=0.5 mm. b GUS fluorescent quantitative analysis of callus atdifferent time intervals. CK−, negative control (untransformed rice); CK+, 35S transgenic rice lines. Valuesrepresent the mean±SD of triplicates


Further detailed analysis was performed on T2 transformant rice harboring pZM-LEG6::GUS as the expression, location, and intensity of GUS facilitated the analysis in seedsat various stages of germination and in plants at different developmental stages. GUS was notexpressed in seeds until germination. As shown in Fig. 5, GUS expression was not detected atthe milky-ripe, waxy-ripe, or full-ripening stages, while GUS expression was observed ingerminated seeds at 2, 5, and 7 days after sowing, indicating that pZM-LEG6 is a vegetativetissue-specific promoter.

Discussion

In this study, we utilized maize promoters (heterologous genes) to regulate GUS geneexpression in transgenic rice receptors. To obtain the best heterologous maize promoter, wefirst examined 20 tissue-specific genes in maize. In a previous study, the promoters of ZM-LEG1 (Zea mays legumin1, −1039 to +41) and Zm-LEG1B (Zea mays legumin1, −768 to +41)were used to regulate gene expression in transgenic maize (Hi-II) endosperm from 10 to40 days after pollination (DAP); these genes were not expressed in leaf tissue [1]. By thecurrent study, we determined that the promoter of ZM-LEGF::GUS (Zea mays legumin1,−2050 to +41) was not expressed by the GUS gene in any rice tissue before pre- and post-treatment; pZM-LEG (−2050 to −140) was merely by expressed the GUS gene in rice callus(Fig. 2). When the promoter sequence of pZM-LEG was truncated, the expression pattern andintensity changed. None of the truncated promoters were expressed in rice reproductive organsexcept pZM-LEG5. Consequently, our findings conflict with previous results. Results similarto ours were obtained for other species. For example, the promoter of Commelina yellow

Fig. 3 Structures of truncated promoters of pZM-LEG fused with GUS. Putative cis-elements were predictedusing the PlantCARE and PLACE databases; the transcription start site designated as +1 which is red letter; +41is the translation initial site which are blue letters (ATG); the colored bars represent different cis-elements. LRElight responsive elements, TEE tissue expression elements, IE inducible elements; AACA, AACA-motif


mottle virus (CoYMV) drives gene expression in phloem, axial parenchyma, and anthers intobacco [37], and in oat, and this promoter is expressed in phloem and associated cells,developing ovaries, and seed scutellum, but not in anthers [52]. The Agrobacterium rhizogenesrolC promoter is expressed in vascular cells of potato [17], in the phloem of rice [35], and inembryonic tissues of carrot cells [15].

The observation that pZM-LEG::GUS was specifically expressed in callus, not in othertissues (Fig. 2), suggests that this promoter may be used as a callus-specific promoter tomediate the expression of selectable marker genes. Since this promoter may be used to expressselectable marker proteins in callus, not in plant tissues, perhaps it can be used to improve theefficiency of positive plant transformant line selection [28, 38]. Wakasa and Takaiwa [55]utilized a rice callus-specific promoter (CSP) that fused a mutated rice acetolactate synthase(mALS) gene, which can be used as a selectable marker, to select transgenic rice seedsaccumulating proteins of interest. Conventional selective methods utilize phosphinothricinacetyltransferase (BAR), hygromycin phosphotransferase (HPT), and neomycin phosphotrans-ferase genes derived from bacteria as selectable markers during Agrobacterium-mediatedtransformation. Genes from non-plant or non-crop sources are considered by some to reducethe safety of a genetically modified organism, suggesting that the use of native selectablemarkers causes less environmental damage.

Skadsen et al. [48] reported that sequences within the 5′-untranslated region (5′-UTR)might not be necessary for a promoter activity and identified the upstream region located at−80 to −3 as inducing strong expression in the lemma. In the current study, the region of -1748

Fig. 4 GUS histochemical staining and GUS fluorescent quantitative analysis of T1 transgenic rice linesexpressing six truncated promoters of pZM-LEG::GUS in seven tissues. a GUS histochemical staining; L, St,R, G, S, scale bars=1 mm; F, scale bars=0.5 mm; (L leaf, St stem, R root, G glume, F flower, S seed). b GUSfluorescent quantitative analysis. CK−, negative control (untransformed rice);CK+, 35S transgenic rice lines. P1–P6 represent pZM-LEG1::GUS, pZM-LEG2::GUS, pZM-LEG3::GUS, pZM-LEG4::GUS, pZM-LEG5::GUS, andpZM-LEG6::GUS, respectively. Values represent the mean±SD of triplicates. The asterisks indicate that thecorrelation coefficients were highly significantly different (*P<0.05, **P<0.01)


to -140 (pZM-LEG1) region can drive GUS expression specifically in lemma hairs, while thefull-length pZM-LEGF (−2050 to +41) sequence containing the 5′-UTR did not drive lemmahair-specific GUS gene expression. When the 5′ sequences were further deleted, the GUSactivity gradually increased, but the lemma hair-specific expression pattern was lost (Fig. 4).

The pZM-LEG2::GUS, pZM-LEG4::GUS, and pZM-LEG6::GUS gene fusions wereexpressed in two or more tissues. In particular, pZM-LEG6::GUS was expressed in allvegetative tissues, and it had the shortest sequence and the highest expression level(Fig. 4b). The cis-elements of pZM-LEG6 are not typical basal cis-elements (Fig. 3), incontrast to those of the rosette leaf- and root-specific cis-element RAV1AAT (CAACA)[26], the AACA-motif (involved the endosperm-specific expression) [49], and two AAAG-motifs that are essential elements in the complex Dof–DNA interaction [59, 60], which may be

Fig. 5 GUS histochemical staining analysis of pZM-LEG6::GUS expression in T2 transformant rice lines atdifferent developmental stages. a callus, b young leaf, c young stem, d young root, emature leaf, fmature stem, gmature root, h glume, i peduncle, j anther, k pollen, lmilky-ripe stage 1,mmilky-ripe stage 2, nmilky-ripe stage3, o waxy-ripe stage, p full-ripening stage, q germinated seed at 2 days after sowing, r germinated seed at 4 daysafter sowing, s germinated seed at 6 days after sowing, t germinated seed at 8 days after sowing. The scale bar ofJ is 0.5 mm, the others are 1 mm


involved in light regulation [9, 61], the regulation of endosperm-specific gene expression [54],and guard cell-specific GUS or GFP reporter gene expression using (T/A)AAAG cis-elements[19, 42]. In the current study, the pZM-LEG6 promoter regulated gene expression in vegetativeorgans, primarily in roots, stems (vascular bundles), leaves, and lemma, with expression levelsin leaves comparable to those obtained with the 35S promoter. Expression analysis of pZM-LEG::GUS 6 in T2 transformant rice seeds indicated that this gene was not expressed in seedsuntil germination (Fig. 5), indicating that pZM-LEG6 is a vegetative tissue promoter.

Previous reports have identified many green tissue-specific promoters that drive theexpression of resistance genes in many crops, not only to increase disease-resistant abilitybut also to prevent exogenous gene expression in seeds, which reduces food safety concerns.Green tissue-specific promoters drive Bt gene expression only in green tissues, not in seeds [7,10, 62]; Molla et al. [39] demonstrated that a green tissue-specific promoter driving the riceoxalate oxidase gene can increase tolerance to sheath blight pathogen (Rhizoctonia solani). Inthe current study, pZM-LEG6::GUS was expressed in vegetative tissues, not only in greentissues but also in roots. Roots are vital plant vegetative organs involved in absorbing andtransporting water and nutrients, and they are also an important component of plant survivalunder adverse conditions and of resistance to plant diseases and insect pests [21, 25, 30].Therefore, pZM-LEG6 represents an ideal promoter for driving the expression of pest- anddisease-resistant genes in rice to enhance rice production. In our study, we selected fivespecific rice promoters by GUS histochemical staining and GUS fluorescent quantitativeanalysis and analyzed the possible core and regulatory region. The ZM-LEGF gene-truncated promoters show multifunctional roles in rice. So specific promoters containingdifferent cis-elements or motifs should be validated in future research.

Acknowledgments The financial support was provided by the National Major Special Project of China on NewVarieties Cultivation for Transgenic Organisms (2011ZX08-010-002-003), the Genetically Modified OrganismsBreeding Major Projects (2013ZX003-002), and the Scientific Research Projects of Anhui (KJ2012A118,1408085MC47).

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cloning and characterization of a multifunctional promoter from maize (zea mays l.)

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