《植物生理学报》 2011, 47(1): 49-56

大豆 GmcpSecA 基因的克隆及表达特异性

党利洁, 李鹏丽, 刘栋, 诸锡莉, 龚清秋, 王宁宁*

党利洁, 李鹏丽, 刘栋, 诸锡莉, 龚清秋, 王宁宁*

通信作者：王宁宁；E-mail: wangnn@nankai.edu.cn；Tel: 022-23504096

摘　要：

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 1.5 kb 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。

关键词：大豆; GmcpSecA基因; 表达特异性; 回补

收稿：2010-08-02   修定：2010-08-18

资助：国家高技术研究发展计划(“863” 计划) (2007AA10Z105)、国家自然科学基金(30670193)和天津市自然科学基金(08JCYBJC03800)。

Cloning and Expressional Characterization of Soybean GmcpSecA Gene

DANG Li-Jie, LI Peng-Li, LIU Dong, ZHU Xi-Li, GONG Qing-Qiu, WANG Ning-Ning*

College of Life Sciences, Nankai University, Tianjin 300071, China

Corresponding author: WANG Ning-Ning; E-mail: wangnn@nankai.edu.cn; Tel: 022-23504096

Abstract:

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 reported 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 agy1 mutant with p35S::GmcpSecA, and the results showed that GmcpSecA can partialy restore the phenotype of agy1. Our data indicates that the GmcpSecA gene has a function similar to AtcpSecA, and that GmcpSecA plays an important role in chloroplast biogenesis.

Key words: soybean; GmcpSecA gene; expression pattern; complementation