大豆抗SMV相关基因Gm39300的克隆与功能分析

The gene Gm39300 was preliminarily confirmed to be the candidate gene responsible for the soybean mosaic virus (SMV) resistance and stress tolerance-related gene by fine mapping and real-time fluorogenic quantitative polymerase chain reaction (PCR) analysis. Based on the genome database and bioinformatics method, the sequences of Gm39300 were cloned from the resistant and susceptible cultivars. Semi-quantitative and quantitative PCR were utilized to investigate the tissue specific expression pattern of Gm39300 and sequence difference between resistant and susceptible cultivars. The quantitative expression levels of Gm39300 induced by SMV and soybean cyst nematode (SCN) infection; abiotic stress treatment including salt, drought, low temperature; and salicylic acid (SA) treatment were detected and analyzed in order to examine whether the Gm39300 gene is a related gene for the biotic and abiotic stress reaction in soybean. Further the functional position of the protein expression of Gm39300 in the cell was determined by the subcellular locating method. The transgenic and virus-induced gene silencing (VIGS) technique were used to examine and confirm the function of the Gm39300 in the SMV resistance process. This project will provide an opportunity to further understand the molecular mechanism of soybean SMV resistance, and is critical importance in improving soybean resistance by biotechnology method.

通过精细定位及荧光定量PCR表达分析，在初步确定Gm39300为大豆抗SMV以及耐逆相关基因基础上，利用大豆基因组数据库，通过生物信息学手段，鉴定并克隆Gm39300在大豆抗病和感病品种中的全长，半定量和荧光定量PCR进行组织表达分析并检测该基因在SMV和SCN侵染，盐碱、伤害、干旱、低温和SA等胁迫处理后的荧光定量PCR表达水平，以明确Gm39300在抗感品种间的序列异同及其在大豆不同器官中的表达特征，阐明该基因是否是大豆调控生物、非生物因子胁迫的相关基因。通过亚细胞定位确认Gm39300编码的蛋白在细胞中发挥功能的具体部位。利用转基因和VIGS技术将抗SMV相关基因Gm39300构建到过表达载体和VIGS载体中以研究和验证其在大豆抗SMV过程中的功能。本项目的成果将有助于明确大豆抗SMV的分子机制，同时对利用生物技术改良大豆的抗性也具有重要应用价值。

E3泛素连接酶在参与植物的细胞周期、抗性反应、信号转导和转录调控等途径中起着重要的调控作用。本研究发现大豆E3泛素连接酶基因Gm39300全长有1885 bp，开放阅读框为1320 bp，编码439个氨基酸，具有U-box保守结构域。组织表达分析表明其在大豆的根、茎、叶、花、荚、子粒中均有不同程度的表达，但是表达水平不同，其中在茎中的表达量最高，种子中的表达量最低。荧光定量PCR表达分析表明Gm39300基因受SMV、SCN、盐害、冷害、ABA和SA等因子的诱导调控。亚细胞定位结果显示，Gm39300-GFP融合蛋白定位于细胞核和细胞膜上。转基因拟南芥研究表明Gm39300受冷害、干旱和外源激素GA、SA等诱导上调表达，能显著提高拟南芥的抗旱性。利用VIGS技术研究该基因的功能还发现沉默后的拟南芥比对照提早抽薹和开花。发表SCI论文2篇，国内核心期刊4篇，出席全国性的会议并进行分会场报告1次。联合博士后1名并出站，联合培养硕士研究生1名，已获得硕士学位；另有1名博士研究生在读。