苎麻α-amylase基因的克隆与表达  

Molecular Cloning and Characterization of Bn-α-amylase Gene from Ramie(Boehmeria nivea)

作　　者：余伟林[1] 钟英丽[1,2] 揭雨成[1,3,4] 周清明[4] 周精华[1] 朱守晶[1] 

机构地区：[1]湖南农业大学苎麻研究所,长沙410128 [2]湖南农业大学生物科学与技术学院,长沙410128 [3]湖南省种质创新与资源利用重点实验室,长沙410128 [4]湖南农业大学农学院,长沙410128

出　　处：《农业生物技术学报》2014年第1期27-36,共10页Journal of Agricultural Biotechnology

基　　金：湖南省科技计划重点项目(No.2012FJ4063);国家科技支撑计划(No.2012BAD20B05-04);湖南省教育厅重点项目(No.09A042);湖南省教育厅优秀人才项目(No.10B050);省部共建国家重点实验室培育基地科学基金开放项目(No.10KFXW09)

摘　　要：α-amylase基因不仅参与植物糖代谢与生物能量的调动,而且与植物抗逆功能相关。为了克隆Bn-α-amylase基因,分析其序列及表达特征,本研究以湘苎3号苎麻(Boehmeria nivea)转录组文库中Unigene4746序列为基础,采用RT-PCR技术克隆该基因的全长编码序列,并进行生物信息学分析;利用Real-time PCR分析该基因在不同组织和不同胁迫条件下的表达特征。研究结果表明,苎麻Bn-α-amylase的编码区序列长2 295 bp,编码765个氨基酸(GenBank登录号:KF860891),推导该蛋白质的等电点和分子量分别为5.685和86.11 kD,该蛋白在羧基端含有两个保守的结构域,亚细胞定位预测显示该蛋白位于细胞质中,不存在信号肽和跨膜结构域。与苹果(Malus×domestic)、拟南芥(Arabidopsis thaliana)、猕猴桃(Actinidia chinensis)、蓖麻(Ricinus communis)、大豆(Glycine max)的α-amylase基因的核苷酸序列相似性分别为80%、76%、76%、73%和67%,与之编码的氨基酸序列相似性分别为68%、65%、65%、69%和51%。进化树分析表明Bn-α-amylase与大豆、葡萄、猕猴桃、蓖麻的α-amylase基因亲缘关系较近,与拟南芥、水稻、高粱的α-amylase基因亲缘关系次之,与卷柏的α-amylase基因亲缘关系较远。实时荧光定量PCR分析表明,Bn-α-amylase在苎麻根、茎皮、茎木质部、茎尖、叶片中均有表达,其中,在叶中表达量最高,在根中表达量最低,且受干旱、高盐胁迫表达增强,受ABA胁迫表达降低。本研究获得了Bn-α-amylase基因的编码区序列,其编码的蛋白具有植物α-amylase典型的结构域,且该基因响应ABA、干旱和高盐逆境胁迫,提示该基因可能与苎麻抗逆境机制密切相关。α-amylase is not only involved in plant sugar metabolism and biological energy transfer, but also associates with the stress resistance of plants. The aim of this study was to clone the Bn-α-amylase gene,analyze the gene sequence and the gene expression pattern. The full-length coding sequence of Bn-α-amylase gene was cloned by RT-PCR methods, based on the sequence of Unigene4746 in the transcriptome library of ramie（Boehmeria nivea）. Then the sequence was analyzed using bioinformatics methods. And the expression patterns of Bn-α-amylase in various ramie tissues or under different stress conditions were analyzed by Real- time PCR. The full-length coding sequence of Bn-α-amylase was 2 295 bp（GenBank accession no. KF860891）. The encoded protein contained 765 amino acids, whose predicted pI was 5.685 and molecular weight was 86.11 kD. Bioinformatics analysis demonstrated that two conserved domain located in the carboxyl terminal, Subcellular localization prediction showed that the protein was located in the cytoplasm, there was no signal peptide and Transmembrane domain, the protein sequence was highly identical to α-amylase of other species. Bioinformatics analysis demonstrated that the sequence was highly identical to α-amylase of other species. It shared a 80% nucleotide identity to Malus x domestic, 76% to Arabidopsis thaliana and to Actinidia chinensis, 73% to Ricinus communis, and 67% to Glycine max. And it also shared the high protein similarity to these species with the valure of 68%, 65%, 65%, 69% and 51%, respectively. Phylogenetic tree analysis showed that the α-amylase of Glycine max, Vitis vinifera, Actinidia chinensis and Ricinus communis was closer to Bn-ct-amylase than the α-amylase of Arabidopsis thaliana, Oryza sativa and Sorghum vulgare, then the α-amylase of Selaginella tamariscina was less closer to to Bn-α-amylase than the α-amylase of Arabidopsis thaliana, Oryza sativa, Sorghum vulgare. The results of Real-time PCR analysis suggested that Bn-α-amylase expressed in root, ba

关 键 词：苎麻α-amylase 基因表达 抗逆 

分 类 号：S852.65[农业科学—基础兽医学]