茶树谷丙转氨酶基因的克隆及其表达分析  

Cloning and Expression Analysis of the Gene Encoding Alanine Aminotransferase in Camellia sinensis

作　　者：崔新[1] 刘志薇 吴致君[1] 李辉[1] 王文丽[1] 庄静[1] 

机构地区：[1]南京农业大学园艺学院茶叶科学研究所,南京210095

出　　处：《西北植物学报》2016年第12期2361-2369,共9页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(31570691)

摘　　要：该研究基于茶树的转录组数据,采用RT-PCR方法从茶树‘黄金芽’cDNA中克隆获得茶树谷丙转氨酶基因(CsAlaAT),利用荧光定量PCR方法,对CsAlaAT在茶树材料‘迎霜’和‘黄金芽’不同组织、温度胁迫与激素处理的表达进行分析。结果显示:CsAlaAT基因开放阅读框长度为1 662bp,编码553个氨基酸,含有天冬氨酸转氨酶家族(Aspartate aminotransferase family)典型的AAT-like保守结构域。多序列比对显示,该序列与多个相关物种的序列一致性达78.83%,与磷酸吡哆醛(PLP)结合的10个氨基酸残基以及第358位赖氨酸催化位点在物种间高度保守。茶树CsAlaAT蛋白属亲水性蛋白,相对分子质量为60 877.5D,等电点为6.11,碱性、酸性、脂肪族和芳香族氨基酸比例分别为12%、11%、22%和8%,无序化特征不明显,与大麦HvAlaAT具有相似的三维结构。实时定量PCR分析表明,CsAlaAT在茶树‘迎霜’和‘黄金芽’中的表达均具有组织特异性,且均在根部的表达量最高;CsAlaAT响应高温(38℃)和低温(4℃)胁迫的表达上调;外源施用脱落酸(ABA)和赤霉素(GA)能够抑制茶树中CsAlaAT基因的表达。Alanine aminotransferase（AlaAT）is a pyridoxal-5′-phosphate-dependent（PLP）enzyme which plays a critical role in linking carbon and nitrogen metabolism.AlaAT is also involved in plant responses to abiotic stress.In this study,based on the transcriptome data of tea plant,we cloned CsAlaATgene encoding alanine aminotransferase from cDNA of‘Huangjinya＇by RT-PCR method.In addition,the expression profiles of the CsAlaATgene in different tissues and under extreme temperatures and hormone treatments in two tea plant varieties（‘Huangjinya＇and‘Yingshuang＇）were analyzed by quantitative real time PCR.Results showed that the length of open reading frame（ORF）of CsAlaATgene was 1 662 bp,encoding 553 amino acids.CsAlaAT contains typical conserved AAT-like domain which belongs to Aspartate aminotransferase family.Multiple alignments of CsAlaAT with related plant species showed that the identity of them was 78.83%.The PLP binding sites and the catalytic residue of 358 Lys were highly conserved.The CsAlaAT is hydrophilic protein.Its theoretical relative molecular weight is 60 877.5D.Theoretical isoelectric point is 6.11.Percentages of basic,acidic,aliphatic and aromatic amino acids were12%,11%,22% and 8%,respectively.A three-dimension crystal structure of CsAlaAT was established on the basis of the HvAlaAT which share identity of 78.38% with CsAlaAT.Quantitative real-time PCR analysis of the expression profiles showed that the CsAlaATgene was tissue-specific expressed in two tea plant varieties‘Huangjinya＇and‘Yingshuang＇.The highest expression level was found in the root.The CsAlaATgene could respond to high temperature（38℃）and low temperature（4℃）treatments.Exogenous application of abscisic acid（ABA）and gibberellins（GA）could down-regulated the CsAlaAT.

关 键 词：茶树 谷丙转氨酶 多序列比对 温度胁迫 激素 表达分析 

分 类 号：Q785[生物学—分子生物学] Q786