大豆GmcpSecA基因的克隆及表达特异性

Cloning and Expressional Characterization of Soybean GmcpSecA Gene

Sec途径是将核编码的叶绿体蛋白输入到类囊体腔的蛋白分选途径之一,对叶绿体正确行使其功能有重要作用。前期研究获得了拟南芥AtcpSecA功能缺失的突变体agy1,其叶片呈黄白色,叶绿体发育缺陷,内部缺少类囊体片层结构。我们从大豆中克隆了拟南芥AtcpSecA的同源基因GmcpSecA基因的全长cDNA序列和5'端ATG上游1.5kb的启动子序列,通过RT-PCR的方法对GmcpSecA基因表达的器官特异性进行了初步分析;并构建了GmcpSecA::GUS和35S::GmcpSecA融合基因,以农杆菌介导的转化方法获得转基因拟南芥。GUS组织化学染色结果表明:在转基因拟南芥的子叶、叶片、花萼等绿色组织中都有较强的GUS表达,而在非绿色组织中没有GUS表达。通过将过表达载体p35S::GmcpSecA转化agy1,结果表明GmcpSecA能够部分回补拟南芥agy1突变体的表型。推测GmcpSecA基因具有与AtcpSecA基因相似的功能,在叶绿体发育过程中发挥重要作用。

The Sec-dependent protein sorting pathway is essential for protein import into the thylakoid lumen and is important for the proper function of the chloroplast. We have previously reposed the characterization of a loss-of-function mutant of cpSecA, the ATPase subunit of the Sec protein complex. The homozygous mutant is albino and seedling lethal, and lacks the thylakoid structure. We cloned the full-length cDNA of GmcpSecA and its 1.5 kb promoter sequence upstream of the coding region from soybean, and examined the expression patterns of GmcpSecA by RT-PCR in soybean. In this study, we constructed GmcpSecA：：GUS and 35S：：GmcpSecA fusion genes and introducted into Arabidopsis by Agrobacterium-mediated floral diping. Histochemical staining of GUS revealed that GmcpSecA is strongly expressed in all green tissues including cotyledons, rossette leaves, and sepals whereas no expression could be detected in non-green tissues such as roots, inflorescencs, and siliques. Then we transformed the agyl mutant with p35S：：GrncpSecA, and the results showed that GmcpSecA can partialy restore the phenotype of agyl. Our data indicates that the GrncpSecA gene has a function similar to AtcpSecA, and that GmcpSecA plays an important role in chloroplast biogenesis.