Cloning and characterization of a pectin methylesterase gene brpme1 in chinese cabbage | 大白菜果胶甲酯酶基因BrPME1的克隆及特征分析

Chinese. 【目的】克隆大白菜果胶甲酯酶基因,为进一步探讨果胶代谢在大白菜育性调控中的分子机制提供帮助。【方法】利用cDNA-AFLP技术分析大白菜核雄性不育两用系‘AB02’可育株(msms)和不育株(Msms)花蕾的基因表达谱,在可育株混合花蕾cDNA中扩增出1条特异条带TDF-24,通过RACE技术扩增该基因的cDNA全长序列,采用生物信息学软件分析所克隆基因的编码蛋白特性,利用荧光定量PCR技术分析基因时空表达模式。【结果】该基因编码大白菜果胶甲酯酶(EC 3.1.1.11),被命名为BrPME1(GenBank登录号：HM185497)。BrPME1全长cDNA序列为1 290 bp,编码1个包含363个氨基酸的前体蛋白。BrPME1蛋白N末端1―23位为信号肽,成熟蛋白含有保守的果胶甲酯酶酶活结构域,而不含酶活抑制位点。预测BrPME1蛋白含有10个磷酸化位点、1个酰胺化位点和6个N端豆蔻酰基化位点。BrPME1在两用系不育株花蕾中表达量很低,在可育株的大花蕾以及成熟花药中高水平表达。【结论】BrPME1是一个受大白菜细胞核雄性不育基因抑制的果胶甲酯酶基因家族成员。

English. 【Objective】 This paper aims at cloning a pectin methylesterase gene in order to provide assistance to study the roles of the pectin metabolism in the fertility regulation of Chinese cabbage.【Method】Analysis of gene differential expression was performed by cDNA-AFLP in the genic male sterile line ‘AB02’of Chinese cabbage, and a differentially expressed cDNA fragment, TDF-24, was found only in fertile plants. The full-length cDNA of the gene related to TDF-24 was amplified by RACE and RT-PCR, and the characteristics of the deduced protein were analyzed using bioinformatics softwares. Gene expression characteristics were proved by Real-time PCR 【Result】 The novel pectin methylesterase (PME,EC 3.1.1.11) cDNA(BrPME1, GenBank accession number HM185497), was isolated from Chinese cabbage. The cDNA was 1 290 bp long, encoding a putative preprotein of 363 amino acids with a signal peptide of 23 amino acids, which contains ten phosphorylation sites, six N-myristoylation site, one amidation site and one conserved PME domain, but no PMEI (pectin methylesterase inhibitor) site. BrPME1 was highly expressed in big flower buds and mature anthers of fertile plants, with an extremely low expression level in sterile buds.【Conclusion】 The results indicated that BrPME1 is a member of the PME gene family inhibited by genic male sterile gene in Chinese cabbage.