农杆菌VirD2蛋白的定位特异性改造

摘要

Agrobaeterium tumefaciens VirD2 protein performs functions in binding a single-stranded T-DNA (ssT-DNA) covalently, transferring it into plant nuclear, and integrating it efficiently into the nuclear genome. If it can be modified to target plastid, the VirD2 can pilot foreign genes there, and a new method of plastid transformation can be established. In this study, the localization signals of VirD2 were modified, which included the coding sequence of the nuclear localization signal (NLS) at its N terminal was point-mutated and its C terminal containing a bipartite-type NLS was truncated using an overlapping PCR method. This mutated VirD2 (mVirD2) was then fused with an eGFP (enhanced green fluorescent protein) reporter gene at the 3' end, and with or without the coding sequence of plastid-targeting peptide from Arabidopsis thaliana cold-regulated gene (AtCORlSA) at the 5' end, to construct chimeric genes pt-mVirD2-eGFP and mVirD2-eGFP, respectively. The chimeric genes were controlled by CaMV 35S promoter and were integrated into tobacco (Nicotina tabacum) nuclear genome viaAgrobacterium-mediated transformation. The fluorescence was only concentrated in chloroplasts for the transformants expressing pt-mVirD2-eGFP while dispersed among the cytoplasm for those with mVirD2-eGFP, and was not observed in the nuclear for both of them. The results showed that the expressing fused protein pt-mVirD2-eGFP targeted plastid specifically without nuclear localization, which bring forth an idea to explore efficient plastid transformation mediated by protein pt-mVirD2 as a guider of foreign DNA.%农杆菌(Agrobacterium tumefaciens)VirD2蛋白具有与单链T-DNA(ssT-DNA)共价结合并将其转运至植物细胞核,将外源基因高效整合进核基因组的功能；如果将该蛋白改定位于质体,则有可能利用其将外源基因携带进质体,建立质体转化新方法.本研究对VirD2蛋白的定位信号进行了改造,采用重叠延伸PCR突变技术对Vir2的N端核定位信号(NLS)编码序列进行点突变,对C端编码序列实施缺失突变,并在合成的突变VirD2(mVirD2)基因3’端连接上增强绿色荧光蛋白(eGFP)报告基因,在其5’端加上或不加源自拟南芥(A rabidopsis thaliana)冷调节基因(AtCOR15A)的质体定位信号肽编码序列,分别构成pt-mVirD2-eGFP和mVirD2-eGFP嵌合基因,由CaMV 35 S启动子控制,经农杆菌介导整合进烟草(ic otiana tab ac um)核基因组.荧光检验显示,mVirD2带质体定位信号时绿色荧光集中于叶绿体,mVirD2不带质体定位信号时绿色荧光弥散在细胞质中,且两者的细胞核均无荧光,表明pt-mVirD2-eGFP嵌合基因表达的蛋白具有质体定位特性,并失去了核定位能力.该结果为今后探讨利用pt-mVirD2融合蛋白将外源DNA主动定向牵引进质体,实现质体高效转化研究提供基础资料.