森林草莓DDM1基因RNAi干扰载体的构建及功能分析

Construction and functional analysis of FveDDM1 gene RNAi interference vector in forest strawberries

DNA甲基化对植物的生长发育、器官分化等生物过程有着很重要的影响。研究通过构建森林草莓DDM1基因(Decrease in DNA methylation1)RNA干扰(RNA interference,RNAi)载体,对该基因的功能进行初步探究。以森林草莓的cDNA为模板克隆出基因正向片段DDM1-1和反向片段DDM1-2,然后将其分别连接到内含子的干涉表达载体上,构建RNAi表达载体;再通过冻融法将其转入到根癌农杆菌EHA105感受态细胞中,利用叶盘法进行森林草莓遗传转化;最后通过Gus染色对阳性苗进行鉴定,然后对转基因草莓植株进行了表型观察和FveDDM1基因表达量分析。结果显示,与对照植株相比,转基因植株FveDDM1基因的表达量降低,叶片出现了明显的畸形,叶边缘变窄,缺刻变深。研究结果可为进一步探讨DDM1基因的功能及甲基化对植物叶片生长发育的影响提供一定的基础。

DNA methylation plays an important role in plant growth,organ differentiation and other biological processes.In this study,the function of DDM1 gene for forest strawberry was preliminarily investigated by constructing RNA interference(RNAi)expression vector.cDNA of forest strawberry was used as template,the forward fragment DDM1-1 and the reverse fragment DDM1-2 were cloned,and then they were respectively connected to interference expression vectors containing introns to construct RNAi expression vectors;it was transferred into Agrobacterium tumefaciens EHA105 receptor cells by freeze-thaw method,leaf disc method was used for genetic transformation of forest strawberry,and Gus staining was used to screen the positive seedlings;the phenotypic observation and expression analysis of transgenic strawberry plants were carried out.The results showed that the expression level of FveDDM1 gene in RNAi transgenic plants was lower than that in control plants,and the leaves showed obvious deformities,with narrowed leaf margins and deeper nicks.The results of this study can provide a basis for further exploration of the function and methylation of DDM1 gene on plant leaf growth and development.