大豆GmPM31基因的功能分析及拟南芥转化

Functional Analysis of Soybean GmPM31 Gene and Transformation into Arabidopsis thaliana

大豆GmPM31基因编码蛋白属于植物小分子热激蛋白sHSP-CI(Class I)亚家族的成员,为研究其在大豆抗逆过程中的生物学功能,对GmPM31蛋白进行亚细胞定位分析;采用酵母双杂交和双分子荧光互补方法验证Gm2-MMP与GmPM31蛋白在体内的互作关系;构建GmPM31启动子5’端不同缺失片段的植物表达载体,通过烟草叶片瞬时表达系统分析其驱动GUS基因的表达水平;利用农杆菌侵染法将GmPM31基因转化拟南芥。结果表明:大豆基质金属蛋白酶Gm2-MMP与小分子热激蛋白GmPM31存在体内互作关系。经40℃高温高湿胁迫处理,与对照组相比,P2062与P1800启动子驱动表达的GUS活性显著增加,说明-1800~-1535 bp区段对GmPM31基因响应高温高湿胁迫信号起关键作用。繁种过表达GmPM31转基因拟南芥植株,筛选得到5株T_(2)代拟南芥阳性植株。

Soybean GmPM31 gene encoding protein is a member of sHSP-CI(Class I)subfamily of plant small molecule heat shock protein.In order to study the biological function of GmPM31 protein in soybean stress tolerance,subcellular localization analysis of GmPM31 protein was performed.Yeast two-hybrid and Bimolecular Fluorescent Complimentary methods were used to verify the interaction between Gm2-MMP and GmPM31 protein in vivo.Plant expression vectors with different missing fragments at the 5′end of GmPM31 promoter were constructed,and the expression level of drived GUS gene was analyzed by transient expression system in tobacco leaves.GmPM31 gene was transformed into Arabidopsis thaliana by Agrobacterium tumefaciens infection.The results showed that soybean matrix metalloproteinase Gm2-MMP interacts with small molecule heat shock protein GmPM31 in vivo.Compared with the control group,the activity of GUS driven by the P2062 and P1800 promoters was significantly increased under high temperature and high humidity stress at 40℃,indicating that-1800~-1535 bp region plays a key role in the response of the GmPM31 gene to high temperature and high humidity stress signals.Transgenic Arabidopsis plants overexpressing GmPM31 were propagated,and five T_(2)-generation Arabidopsis positive plants were screened.