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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。
According to the sequencing results of the SSH-cDNA library of upland cotton, the EST ESTs were expressed in ESTs of cotton ESTs, and one EST sequence specifically expressed in secondary wall thickening stage of cotton fiber was obtained. The sequence encoded pectin methylesterase (PME) ). Using RACE technology to obtain its full-length cDNA sequence, named GhPME6. Gene structure analysis showed that GhPME6 contains a 1560 bp open reading frame, two exons and one intron, encoding a protein containing 519 amino acids. Its amino acid sequence has two conserved domains of PMEI and Pectinesterase. By comparing and analyzing the PME6 genes in different cotton genotypes, PME6 was found to be highly conserved during evolution. qRT-PCR analysis showed that GhPME6 was overexpressed in the thickened secondary wall of the fiber, suggesting that this gene may have an important effect on the specific strength of cotton fiber. The construction of the E. coli expression system of GhPME6 gene and obtaining the target protein with the same expected size laid the foundation for further study on the effect of GhPME6 on the specific strength of cotton fiber.