miR172超表达载体的构建及其在micro-Tom番茄中的遗传转化

Construction of mi R172 Over-expression Vector and Its Transformation into micro-Tom Tomato

为了进一步阐明mi RNA在番茄不同生长发育阶段尤其是在果实成熟阶段的调控途径,利用聚合酶链式反应(PCR)从番茄(Solanum lycopersicum)的c DNA克隆出mi R172基因核心片段,将该基因片段,以及p CAMBIA1300-221载体通过特异性的限制性内切酶双酶切,然后将mi R172片段正向连接到载体上,转化大肠杆菌Trans5α,鉴定重组质粒正确后,利用冻融法将重组载体转入农杆菌EHA105中。再次回转大肠杆菌验证获得的重组载体,通过PCR以及双酶切鉴定是否符合预期结果,将测序结果与NCBI上的基因序列相比较,同源性高达100%。结果成功构建了适用于番茄农杆菌遗传转化的植物表达载体,继续进行转基因植株的培育和鉴定。为通过mi R172基因进一步研究果实成熟衰老机理奠定了物质基础。

To reveal the pathway of mi RNA regulating tomato at different growth and development stages,especially in fruit ripening, we obtained tomato（Solanum lycopersicum） mi R172 gene by the polymerse chainreaction technique（PCR）, then used the restriction enzymes to digest especially gene segments andp CAMBIA1300-221 vector, and connected the gene to the plasmid p CAMBIA1300-221, then transformed therecombinant plasmid into Agrobacterium by Freeze- thaw method when it was proved accurate. Therecombinant plasmid was transformed again into Escherichia coli in order to obtain the exact plasmid by PCRand double enzyme digestion in the research, and the sequence result was homology to NCBI database up to100%. The results indicated that tomato expression vector p CAMBIA1300- 221- mi R172 was constructedsuccessfully and then the transgenic plants were cultivated and identified. The research would provide base forgenetic research in fruit ripening and aging through mi R172 transgenic tomatoes.