小麦穗发芽抗性相关Vp1基因启动子的分离及功能验证

doi:10.3724/SP.J.1006.2011.01743

摘要/Abstract

摘要： 成熟期穗发芽严重影响小麦产量和品质。Vp1是调节胚发育, 促进胚成熟和休眠的重要转录因子, 对小麦种子休眠和穗发芽抗性具有重要作用。本研究分离了普通小麦B基因组Vp1基因的启动子, 生物信息学预测结果表明, 其含有9个脱落酸响应元件ABRE、2个DREB和6个MYB干旱响应元件、3个赤霉素响应元件GARE、1个水杨酸响应元件TCA-E、2个茉莉酸甲酯响应元件TGACG-motif、4个SKn-1和1个RYREPE胚乳特异表达元件。采用5′端缺失的方法, 构建了系列含Vp1启动子不同区段融合GUS报告基因的瞬时表达载体和植物表达载体。通过基因枪转化小麦愈伤组织, 瞬时表达结果显示, Vp1启动子在无诱导的情况下不能启动GUS基因表达, 在低温、ABA、GA、PEG和NaCl诱导后可以启动GUS基因表达, 表现诱导表达特性, 且其诱导表达强度随启动子缺失片段长度变短而减弱。利用Gateway方法成功构建了6个启动子各缺失片段类型的植物表达载体, 并通过农杆菌介导转化四倍体小麦Stewart, 获得转基因植株。该启动子可有效启动GUS基因在转基因植株的花药、糊粉层、穗轴及根中表达, 其他组织中没有表达。当启动子片段大于660 bp时, 外源ABA可诱导启动子启动GUS基因在转基因植株茎节中的表达。

关键词: 穗发芽, Vp1启动子, 功能分析, 转基因小麦

Abstract: Viviparous1 (Vp1) plays an important role in regulating embryo development, seed dormancy, and germination in higher plants. Although we previously demonstrated a close correlation between Vp1B allelic variation and the different levels of tolerance to pre-harvest sprouting (PHS) in common wheat, little is known about the potential cis-elements in Vp1B promoter region, which may be involved in the control of PHS by Vp1B. In this study, a 2 232 bp Vp1B upstream sequence was isolated. In silico analysis revealed the presence of nine ABRE, two DREB, six MYB, three GARE, one TCA-E, two TGACG-motif, four SKn-1, and one RYREPE cis-elements in the isolated sequence.Based on this, we constructed various plasmid vectors with 5′-truncated Vp1B promoters fused with the GUS (encoding β-glucuronidase) reporter gene. Transient expression assay in wheat callus indicated that Vp1B promoter activity was inducible by low-temperature, ABA, or GA treatment. However, the level of induced GUS activity declined as promoter length decreased. Subsequently, six truncated promoter reporter fusions were introduced into the genome of durum wheat by Agrobacterium-mediated transformation and stable transgenic lines were obtained. Analysis of the transgenic lines indicated that Vp1B promoter could drive GUS expression in the anther, axis,aleurone and root, but not in the leaves, stems and nodes. However, in the plants with the transgenic Vp1B promoter fragment longer than 660 bp, GUS expression could be induced in the nodes by ABA treatment.

Key words: Pre-harvest Sprouting, Vp1 promoter, Functional analysis, Transgenic wheat

孙永伟, 聂丽娜, 马有志, 徐兆师, 夏兰琴. 小麦穗发芽抗性相关Vp1基因启动子的分离及功能验证[J]. 作物学报, 2011, 37(10): 1743-1751.

SUN Yong-Wei, NIE Li-Na, MA Wei-Zhi, XU Zhao-Shi, JIA Lan-Qin. Cloning and Functional Analysis of Viviparous-1 Promoter in Wheat[J]. Acta Agron Sin, 2011, 37(10): 1743-1751.

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://zwxb.chinacrops.org/CN/10.3724/SP.J.1006.2011.01743

https://zwxb.chinacrops.org/CN/Y2011/V37/I10/1743

参考文献

[1]Xiao S-H(肖世和), Yan C-S(闫长生), Zhang H-P(张海萍), Sun G-Z(孙果忠). Studies for Preharvest Sprouting of Wheat (小麦穗发芽研究). Beijing: China Agricultural Science and Technology Press, 2002. pp 266–292 (in Chinese)
[2]He Z-T(何震天), Chen X-L(陈秀兰), Han Y-P(韩月澎). General situation of study on ear sprouting resistance of white-seed-coat wheat. Seed(种子), 2000, (2): 36–38 (in Chinese with English abstract)
[3]Bailey P C, McKibbin R S, Lenton J R, Holdsworth M J, Flintham J E, Gale M D. Genetic map location for orthologous VP1 genes in wheat and rice. Theor Appl Genet, 1999, 98: 281–284
[4]Nakamura S, Toyama T. Isolation of a VP1 homologue from wheat and analysis of its expression in embryos of dormant and non-dormant cultivars. J Exp Bot, 2001, 52: 875–876
[5]Wilkinson M D, Mckibbin R S. Use of comparative molecular genetics to study pre-harvest sprouting in wheat. Euphytica, 2002, 126: 27–33
[6]Jones H D, Peters N C B. Genotype and environment interact to control dormancy and differential expression of the VIVIPAROUS 1 homologue in embryos of Avena fatua. Plant J, 1997, 12: 911–920
[7]Yang Y, Ma Y Z, Xu Z S, Chen X M, He Z H, Yu Z, Wilkinson M, Jones D H, Shewry R P, Xia L Q. Isolation and characterization of Vp-1 genes in wheat varieties with distinct pre-harvest sprouting tolerance and ABA sensitivity. J Exp Bot, 2007, 58: 2863–2871
[8]Yang Y, Zhao X L, Xia L Q, Chen X M, Xia X C, Yu Z, He Z H. Development and validation of a Vp-1 STS marker for pre-harvest sprouting in Chinese wheats. Theor Appl Genet, 2007, 115: 971–980
[9]Xia LQ, Ganal M W, Shewry P R, He Z H, Yang Y, Roder M. Evaluation of the Viviparous-1 gene alleles in the European wheat varieties. Euphytica, 2008, 159: 411–417
[10]Yang Y, Chen X M, He Z H, Roder M & Xia L Q. Distribution of Vp-1 alleles in Chinese white-grained landraces, historical and current wheat cultivars. Cereal Res Commun, 2009, 37: 169–177
[11]Utsugi S, Nakamura S, Noda K, Maekawa M. Structural and functional prop-erties of Viviparous1 genes in dormant wheat. Genes Genet Syst, 2008, 83: 153–166
[12]Ng D W, Chandrasekharan M B, Hall T C. The 5′-UTR negatively regulates quantitative and spatial expression from the ABI3 promoter. Plant Mol Biol, 2004, 54: 25–38
[13]Cao X, Costa L M, Biderre-Petit C, Kbhaya B, Dey N, Perez P, McCarty D R, Gutierrez-Marcos J F, Becraft P W. Abscisic acid and stress signals induce Viviparous-1 (Vp1) expression in seed and vegetative tissues of maize. Plant Physiol, 2007, 143: 720–731
[14]He Y, Jones H D, Chen S, Chen X M, Wang D W, Li K X, Wang D S, Xia L Q. Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L. var. durum cv. Stewart) with improved efficiency. J Exp Bot, 2010, 61: 1567–1581
[15]McCarty D R, Hattori T, Carson C B Vasil V, Lazar M, Vasil I K. The Viviparous-1 developmental gene of maize encodes a novel transcriptional activator. Cell, 1991, 66: 895−905
[16]Giraudat J, Hauge B M, Valon C, Smalle J, Parcy F, Goodman H M. Isolation of the Arabidopsis ABI3 gene by positional cloning. Plant Cell, 1992, 4: 1251−1261
[17]Bobb A J, Eiben H G, and Bustos M M. PvAlf, an embryospecific acidic transcriptional activator enhances gene expression from phaseolin and phytohemagglutinin promoters. Plant J, 1995, 8:331–343
[18]Li C D, Ni P X. Genes controlling seed dormancy and pre-harvest sprouting in rice-wheat- barley comparison. Funct Integr Genomics, 2004, 4: 84-93
[19]Derera N F, Sci D A, Dip P B. Pre-harvest Field Sprouting in Cereals. Florda: CRC Press, 1989. pp 27–61
[20]Maarten J, Chrispeels, Varner J E. Gibberellic acid-enhanced synthesis and release of amylase and ribonuclease by isolated barley and aleurone layers. Plant Physiol, 1967, 42: 398–406
[21]Suzuki M, Ketterling M G. Viviparous1 alters global gene expression patterns through regulation of abscisic acid signaling. Plant Physiol, 2003, 132: 1664–1677
[22]Nakashima K, Fujita Y, Katsura K, Maruyama K, Narusaka Y, Seki M, Shinozaki K, Yamaguchi-Shinozaki K .Transcriptional regulation of ABI3- and ABA-responsive genes including RD29B and RD29A in seeds, germinating embryos, and seedlings in Arabidopsis. Plant Mol Biol, 2006, 60: 51–68
[23]Rouster J, Leah R, Mundy J, Cameron-Mills V. Identification of a methyl jasmonate-responsive region in the promoter of a lipoxygenase 1 gene expressed in barley grain. Plant J, 1997, 11: 513–523
[24]Shen Q, Ho T H. Functional dissection of an abscisic acid (ABA)-inducible gene reveals two independent ABA-responsive complexes each containing a G-box and a novel cis-acting element. Plant Cell, 1995, 7: 295–307
[25]Shen Q, Zhang P, Ho T H. Modular nature of abscisic acid (ABA) response complexes: composite promoter units that are necessary and sufficient for ABA induction of gene expression in barley. Plant Cell, 1996, 8: 1107–1119

相关文章 15

[1]	宋裕祯, Bheel Chander Kumar, 王跃, 张颖星, 郭娟, Khound Rituraj, Santra Dipak Kumar, 曹晓宁, 王瑞云. 糜子AP2亚家族全基因组鉴定及PmAP2-1和PmAP2-9耐盐功能分析[J]. 作物学报, 2026, 52(4): 1127-1139.
[2]	汪玲, 胡好, 宋家凤, 程洁蓝, 陈颖, 郑婷婷, 吕钊彦, 朱晓彪, 侯华兰. 马铃薯UDP-糖基转移酶基因StUGT52的克隆及功能验证[J]. 作物学报, 2026, 52(3): 665-676.
[3]	张晴, 杨昱, 郭茜, 岳霈尧, 殷丛丛, 牛景萍, 赵晋忠, 杜维俊, 岳爱琴. 大豆GmARA6a的克隆及响应盐胁迫的功能分析[J]. 作物学报, 2026, 52(2): 480-493.
[4]	马婷婷, 郭晓江, 李豪, 邓梅, 蒲至恩, 李伟, 张亚洲, 王凤涛, 崔凤娟, 魏育明, 王际睿, 蒋云峰, 陈国跃. 利用小麦农家种孝感麦协同改良蜀麦753产量与抗病耐逆性的育种实践[J]. 作物学报, 2026, 52(1): 56-71.
[5]	张顺杰, 吴维泰, 冉禧玥, 赵梓含, 韩永辉, 吴正丹, 张凯. 甘薯β淀粉酶基因IbBAM₄₈₈₂₉的功能解析[J]. 作物学报, 2025, 51(11): 3096-3104.
[6]	李旭娟, 李纯佳, 田春艳, 孔春艳, 徐超华, 刘新龙. 甘蔗硝酸盐转运蛋白1/肽转运蛋白家族6.4基因(ScNPF6.4)克隆及其调控分蘖功能分析[J]. 作物学报, 2024, 50(8): 2131-2142.
[7]	李闻娟, 王利民, 齐燕妮, 赵玮, 谢亚萍, 党照, 赵丽蓉, 李雯, 徐晨梦, 王琰, 张建平. 亚麻LuWRI1a在旱盐胁迫响应中的功能分析[J]. 作物学报, 2024, 50(7): 1750-1761.
[8]	李海芬, 鲁清, 刘浩, 温世杰, 王润风, 黄璐, 陈小平, 洪彦彬, 梁炫强. 花生赤霉素3-β-双加氧酶(AhGA3ox)基因家族的全基因组鉴定及表达分析[J]. 作物学报, 2024, 50(4): 932-943.
[9]	张宝华, 刘佳静, 田晓, 田旭钊, 董阔, 武郁洁, 肖凯, 李小娟. 小麦TaSPX1基因的克隆、表达及耐低氮逆境的功能研究[J]. 作物学报, 2024, 50(3): 576-589.
[10]	李艳, 方宇辉, 王永霞, 彭超军, 华夏, 齐学礼, 胡琳, 许为钢. 不同磷胁迫处理转OsPHR2小麦的转录组学分析[J]. 作物学报, 2024, 50(2): 340-353.
[11]	张馨月, 秦阳, 李瑞, 黄全生, 王逸茹, 郑军. 玉米穗发芽突变体vp2的基因克隆及功能研究[J]. 作物学报, 2024, 50(11): 2712-2719.
[12]	杨晨曦, 周文期, 周香艳, 刘忠祥, 周玉乾, 刘芥杉, 杨彦忠, 何海军, 王晓娟, 连晓荣, 李永生. 控制玉米株高基因PHR1的基因克隆[J]. 作物学报, 2024, 50(1): 55-66.
[13]	苏亚春, 李聪娜, 苏炜华, 尤垂淮, 岑光莉, 张畅, 任永娟, 阙友雄. 甘蔗割手密种类甜蛋白家族鉴定及栽培种同源基因功能分析[J]. 作物学报, 2021, 47(7): 1275-1296.
[14]	唐锐敏, 贾小云, 朱文娇, 印敬明, 杨清. 马铃薯热激转录因子HsfA3基因的克隆及其耐热性功能分析[J]. 作物学报, 2021, 47(4): 672-683.
[15]	杨阳, 李淮琳, 胡利民, 范楚川, 周永明. 白菜型油菜srb多室性状的遗传分析与分子鉴定[J]. 作物学报, 2021, 47(3): 385-393.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed