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.

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