橡胶树PEPC基因的克

发布时间：2018-07-16 11:17

【摘要】：【目的】克隆橡胶树磷酸烯醇式丙酮酸羧化酶(PEPC)基因的全长cDNA,通过生物信息学和基因表达分析,为基因功能研究奠定基础。【方法】根据橡胶树PEPC基因片段序列设计引物,通过RACE获得cDNA全长,用DNAMAN软件预测编码多肽序列,用Prot Param工具分析多肽基本理化性质,用NCBI-Blast工具搜索同源的核酸及多肽序列,用Clustal X2.0软件比对不同植物来源的多肽序列,并用MEGA 6.06软件构建系统进化树,用Real-time RT-PCR分析基因表达模式。【结果】从橡胶树胶乳中克隆到一个新的PEPC基因的全长cDNA,其长度为3206 bp,包含一个2898 bp的开放读码框(ORF),112 bp的5’UTR和196 bp的3’UTR。预测该基因编码965个氨基酸的多肽(HbPPC2),其分子量为110.35 kDa,等电点为5.76;该基因的编码区核苷酸序列和其编码多肽氨基酸序列分别与早期克隆的橡胶树HbPPC1基因(KJ721228)的编码区和编码多肽序列有81.3%和92.1%的同源性。预测HbPPC2定位于细胞质,为不稳定蛋白,其活性受多种因素调控。预测HbPPC2酶活性中心是其第172位残基His,磷酸化位点是其第11位残基Ser,单泛素化位点是其第628位残基Lys。其第178、179、226和366位残基Arg在三维结构中靠近,构成葡萄糖-6-磷酸(G6P)的结合位点;其第450、641、753、767位残基Arg在三维结构中靠近,构成PEP的结合位点。HbPPC2基因在叶片、树皮、胶乳、雄花、雌花均有表达,在胶乳表达量最高;乙烯利处理显著下调该基因在胶乳的表达。【结论】本研究可为橡胶树PEPC功能研究提供理论基础。
[Abstract]:[objective] to clone the full-length cDNAof Hevea phosphoenolpyruvate carboxylase (PEPC) gene and lay a foundation for the study of gene function by bioinformatics and gene expression analysis. [methods] Primer was designed according to the sequence of PEPC gene fragment in Hevea. The full-length cDNA was obtained by race, the coding peptide sequence was predicted by DNAMAN software, the basic physicochemical properties of the peptide were analyzed by Prot Param tool, the homologous nucleic acid and peptide sequences were searched by NCBI-Blast tool, and the peptide sequences from different plant sources were compared with Clustal X2.0 software. The phylogenetic tree is constructed with MEGA6.06 software. Real-time RT-PCR was used to analyze the gene expression pattern. [results] A new full-length cDNAof PEPC gene was cloned from rubber latex with a length of 3206 BP, containing a 2898 BP open reading frame (ORF) of 112bp 5UUTR and 196bp 3UUTR. The peptide encoding 965 amino acids (HbPPC2) was predicted, with a molecular weight of 110.35 kDaand a isoelectric point of 5.76.The coding region of HbPPC1 gene (KJ721228) and the amino acid sequence of HbPPC1 gene (KJ721228) cloned in the early stage of HbPPC1 were compared with those of HbPPC1 gene (KJ721228). There were 81.3% and 92.1% homology with the coding polypeptide sequence. HbPPC2 was predicted to be an unstable protein located in the cytoplasm, and its activity was regulated by many factors. It is predicted that HbPPC2 enzyme activity center is its 172nd residue His, phosphorylation site is its 11th residue Seran, and monoubiquitin site is its 628th residue Lys. The binding site of glucose-6-phosphoric acid (G6P) was formed by the Arg at positions 178179226 and 366.The Arg at position 450641753767 was close to the three-dimensional structure, and the binding site of HbPPC2 gene was found in leaves, bark, latex and male flowers. Ethephon treatment significantly down-regulated the expression of the gene in latex. [conclusion] this study may provide a theoretical basis for the study of PEPC function of rubber tree.
【作者单位】：中国热带农业科学院橡胶研究所"农业部橡胶树生物学与遗传资源利用重点实验室;
【基金】：中国热带农业科学院基本科研业务费专项资金(1630022012011,1630022017023)
【分类号】：S794.1

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