红麻线粒体基因atp6过表达载体的构建与转化

Construction and Transformation of Overexpression Vector of atp6 in Kenaf

【目的】构建红麻线粒体基因atp6过表达载体遗传转化体系,为红麻线粒体基因的遗传转化及功能研究打下基础。【方法】以红麻UG93A花药cDNA为模板,通过同源克隆技术克隆atp6基因编码区(CDS)的全长序列;利用In-Fusion基因融合技术构建植物过表达载体,通过农杆菌介导法转化野生型烟草,并对转基因阳性烟草植株进行抗性筛选和PCR验证。【结果】红麻UG93A的atp6基因CDS全长为1182 bp,成功构建红麻atp6基因全长过表达载体pBI121-atp6-EGFP,并获得4株转基因烟草植株(B1、B2、B3和B4),使用过表达载体上3对不同位点的引物组合对转基因植株进行PCR验证,发现有2株转基因烟草植株在3对不同引物中稳定表达,即获得2株含pBI121-atp6-EGFP的T0代转基因阳性植株(B1和B2)。【结论】构建的红麻线粒体基因atp6过表达载体遗传转化体系,可用于指导后续atp6基因调控红麻细胞质雄性不育(CMS)分子机理研究。

【Objective】In order to establish the genetic transformation system of kenaf mitochondrial gene atp6 overexpression vector and lay a foundation for the genetic transformation and functional research of kenaf mitochondrial gene.【Method】The full-length CDS sequence of atp6 was obtained with anther cDNA of kenaf UG93A by homologous cloning and the plant overexpression vector was constructed by In-Fusion gene fusion technology.The wild-type tobacco was transformed by Agrobacterium tumefaciens-mediated method and transgenic plants were identified through resistance screening and target gene PCR verification.【Result】The total length of atp6 gene obtained in kenaf UG93A was 1182 bp.Overexpression vector pBI121-atp6-EGFP was successfully constructed and 4 transgenic tobacco plants were obtained(B1,B2,B3 and B4).The transgenic plants were amplified by PCR using three pairs of primer combinations at different sites on the overexpression vector.Two transgenic tobacco plants were stably expressed in three pairs of different primers,so two T0 generation transgenic positive plants with pBI121-atp6-EGFP were obtained in this study(B1 and B2).【Conclusion】The genetic transformation system of the overexpression vector of kenaf mitochondrial gene atp6 was constructed,which will lay a foundation for further study on the molecular mechanism of atp6 regula-ting plant cytoplasmic male sterility(CMS).