一步法RT—PCR克隆水稻线粒体磷转运蛋白基因及其序列特征分析

Cloning and Sequence Analysis of Rice Mitochondrial Phosphate Transporter Gene by One-step RT-PCR

摘要:

以水稻广亲和品种Cpslo17幼穗为材料，用一步法RT—PCR（逆转录聚合酶链式反应）克隆了一个长度为1118bp的编码线粒体磷转运蛋白的OsMPT基因。序列分析表明其包含了基因完整的编码序列，编码由368个氨基酸组成的线粒体磷转运蛋白，它与玉米、大豆、Lotus japonicus、Betula pendula、拟南芥的线粒体磷转运蛋白氨基酸序列相似率分别为93.5％，85.6％，83.8％，83.7％，81.1％。氨基酸疏水谱分析显示它有线粒体磷转运蛋白家族高度保守的6个跨膜结构域。水稻线粒体磷转运蛋白N端富含精氨酸（Arginine）、丙氨酸（Alanine）和丝氨酸（Serine）。iPSORT预测其蛋白N端具有定位于线粒体的信号肽序列，进一步分析表明此编码区段有6个外显子和5个内含子。RT—PCR结果表明，OsMPT基因在水稻两个亚种粳稻和籼稻的叶片中均有表达，在Cpslo17营养器官和生殖器官中都有高水平表达。水稻线粒体磷转运蛋白的克隆和表达分析将为研究其结构和生物学功能奠定基础。

Abstract:

Gene-specific primers were designed according to the known sequence of mitochondrial phosphate transporter gene （MPT）, and the rice MPT （OsMPT） cDNA fragment of 1118 bp was cloned by One-step reverse transcription PCR （RT-PCR） from Cpslo17, a wide-compatibility variety of rice （Oryza saliva）. DNA sequencing analysis indicated that it carried an entire open reading frame and encoded a protein of 368 amino acid residues. The deduced amino acid sequence of OsMPT showed similarities of 93.5%, 85.6%, 83.8%, 83.7% and 81. 1% with those from maize, soybean, Lotus japonicus, Betula pendul and A rabidopsis thaliana, respectively. The hydropathy profiles of the deduced amino acid sequence predicted the existence of six membrane-spanning domains, which are highly conserved in the mitochondrial transporter family. The N-terminal amino acids were enriched with arginine, alanine and serine, and a signal peptide targeted in mitochondria was predicted by iPSORT. Further analysis of ecoding segment of OsMPT revealed that it contained 6 exons and 5 introns. The average length of introns and exons were 467 bp and 186.3 bp respectively; and the average GC content of introns and exons are 35.5% and 54.0% respectively. RT-PCR results showed relative high-level expression of OsMPT in leaves of both jopnica and indica rices, so was in both vegetative and reproductive tissues of Cpslo 17. Cloning and expression analysis of OsMPT will provide a base for the researches of its structure and biological function.