陆地棉果胶甲酯酶GhPME6的克隆及功能分析

Cloning and Functional Analysis of the Pectin Methylesterase Gene GhPME6 in Upland Cotton

结合陆地棉SSH-cDNA文库的测序结果,与棉花EST数据库进行序列比对和分析,获得1条在棉纤维次生壁加厚期特异表达的EST序列,该序列编码果胶甲酯酶(PME)。利用RACE技术获得其全长cDNA序列,命名为GhPME6。基因结构分析表明,GhPME6包含1个长度为1560 bp的开放阅读框、2个外显子和1个内含子,编码含有519个氨基酸的蛋白。其氨基酸序列具有PMEI和Pectinesterase两个保守结构域。通过对不同棉属基因组中的PME6基因进行比对分析,发现PME6在进化过程中具有高度保守性。qRT-PCR分析显示,GhPME6在纤维发育次生壁加厚期大量表达,推测该基因可能对棉纤维比强度有重要影响。构建GhPME6基因的大肠杆菌表达体系,获得与预期大小一致的目的蛋白,为深入研究GhPME6对棉纤维比强度的影响奠定了基础。

With the suppressive subtractive hybridization result of a cotton fiber cDNA library, an expressed sequence tag （EST） sequence, which is specifically expressed during the cotton fiber secondary wall thickening stage and encodes pectin methylesterase, was identified from the cotton EST database. The complete cDNA sequence was obtained by rapid amplification of cDNA ends（RACE） technology, and named GhPME6. Gene structural analysis showed that GhPME6 contained a complete open reading frame of 1,560 bp, including two exons and one intron, and the gene encodes 519 amino acids. Its amino acid sequence consisted of two conservative structural domains： a pectin methylesterase inhibitor region and a pectinesterase region. The PME6 sequence was found to have been highly conserved during the evolution of different Gossypium genomes. Quantitative real-time PCR analysis revealed that GhPME6 was highly expressed during the secondary cell wall thickening stage of fiber development, so is predicted to influence the cotton fiber strength. We built an Escherichia coil expression system of GhPME6, which resulted in production of a target protein of expected size, thus laying the foundations for the further study of GhPME6 function in cotton fiber strength.