利用甘薯质体同源片段构建多顺反子表达载体

Construction of Multicistron Expression Vector by Using Plastid Homologous Sequences of Sweet Potato

本研究利用质体基因组的进化保守性,根据烟草质体基因组全序列设计引物,从甘薯(Ipomoea batatas L.)质体基因组中克隆了两个相邻的功能基因rbcL(GenBank登录号为AY942199)和accD(GenBank登录号为AY942200),以此作为定点整合外源基因的同源重组片段。选用水稻质体基因组强启动子prrn及psbA3'非翻译区调控选择标记基因aadA和报告基因gfp,构建多顺反子表达盒prrn-aadA-gfp-psbA-3',之后将表达盒克隆进甘薯质体同源片段中,获得甘薯质体多顺反子定点整合表达载体pSAG,酶切鉴定结果表明所构建的载体符合预期设计。这为后期甘薯质体转化体系的建立和通过质体基因工程手段将更多目的基因导入甘薯进行遗传改良奠定了基础。

Plastid transformation in higher plants offers several advantages over nuclear transformation, including maternal inheritance of transgene, lack of position effects and gene silencing, and high levels of transgene expression, because the high ploidy level of the plastome in cells and the genes of interest are integrated into the plastome via homologous recombination. Based on the highly conservative features of rbcL and accD genes during the plastid genome evolution of higher plants, we constructed a distinct construction protocol of species-specific transforma tion vector of sweet potato. We designed the primers according to the plastome sequence of tobacco and PCR- amplified two adjacent functional genes rbcL （GenBank accession No. AY942199） and accD （GenBank accession No. AY942200） from plastome of sweet potato, we constructed the sweet potato-specific plastid expression vector named pSAG that carries the expression cassette of prrn-aadA-gfp-psbA-3＇. Then, we verified it by digestion with restriction enzymes. It indicated that this sweet potato-specific plastid expression vector pSAG is effective and desirable to be applied in traits improvement of sweet potato via plastid genetic transformation.