水稻线粒体基因atp6超表达载体的构建及遗传转化

摘要

[目的]克隆水稻线粒体基因atp6,构建转基因表达载体,获得35S∷Rf1b5′∷atp6转基因水稻植株。[方法]设计目的基因的特异引物,采用TRIzol法提取水稻叶片总RNA,借助Toyobo反转录试剂盒反转录为cDNA,通过PCR扩增得到atp6全序列;将atp6连接到含线粒体信号肽（Rf1b5′）的pCAMBIA1302双元表达载体上,采用农杆菌介导的愈伤组织侵染法进行水稻的遗传转化[结果]利用目的基因atp6成功构建了35S∷Rf1b5′∷atp6植物表达载体,并获得了转atp6基因的阳性植株。[结论]为探讨水稻中超表达atp6对水稻生长的影响奠定基础。%[Objective] This study aimed to clone the gene atp6 from rice mitochondria, construct the binary expression vector 35S :: Rflb5' :: atp6 and obtain transgenic plants with atp6. [Method] The special primers were designed according to the sequence of target gene. With the TRIzol method the total RNA was extracted from rice seedlings and reverse transcripted to cDNA. The ORF of atp6 was amplified by PCR, and ligated to binary expression vector pCAMBIA1302 which contains se- quence of signal peptide from mitochondria (Rflb5'). The recombinant plasmid was then transformed into rice callus mediated by Agrobacterium. [Result] The binary expression vector 35S :: Rflb5' :: atp6 was constructed, and positive transgenic plants were obtained. [Conclusion] This study lays foundation for understanding influence of atp6 gene over-expression on rice growth.