茶树丙氨酸氨基转移酶基因的克隆与表达分析  

Cloning and Expression Analysis of Alanine Aminotransferase Gene in Camellia sinensis

作　　者：白培贤 王丽鸳[1] 韦康[1] 阮丽[1] 成浩[1] 张芬[1,2] 张成才[1,2] BAI Peixian;WANG Liyuan;WEI Kang;RUAN Li;CHENG Hao;ZHANG Fen;ZHANG Chengcai(Tea Research Institute, Chinese Academy of Agricultural Sciences, National Center for Tea Improvement, Key Laboratory of Tea Biology and Resource Utilization, Ministry of Agriculture, Hangzhou 310008, China;Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China)

机构地区：[1]中国农业科学院茶叶研究所/国家茶树改良中心/农业部茶树生物学与资源利用重点实验室,浙江杭州310008 [2]中国农业科学院研究生院,北京100081

出　　处：《茶叶科学》2016年第4期405-413,共9页Journal of Tea Science

基　　金：国家自然科学基金(31570695);国家现代农业产业技术体系(nycytx-23);浙江省农业新品种选育重点专项(2012C2905-4)

摘　　要：丙氨酸氨基转移酶(Alanine Aminotransferase,Ala AT)是与碳氮代谢相关的一种重要酶类。采用反转录PCR的方法克隆了茶树Cs Ala AT1的c DNA序列,该序列全长1 747 bp,包含一个完整的ORF(1 626 bp),编码541个氨基酸,推导的蛋白质分子量为59.4 k D,理论等电点(p I)为5.82。同源比对结果表明,Cs Ala AT1含有丙氨酸氨基转移酶亚家族保守的辅酶磷酸吡哆醛结合位点,其氨基酸序列与拟南芥At Ala AT1蛋白的相似性为84%。二级结构预测显示该蛋白由α-螺旋(40.67%)、无规则卷曲(29.57%)、β-折叠(13.68%)和延伸链(16.08%)组成,定位于线粒体,不含信号肽与跨膜结构。实时荧光定量PCR(RT-PCR)检测发现Cs Ala AT1在茶树各组织中均有表达,在根中的表达量最高;Cs Ala AT1基因表达对氮素的响应研究表明,成熟叶中Cs Ala AT1受氮素诱导上调表达,高浓度(1 mmol·L-1 NH4NO3)氮素的诱导效应比低浓度(0.1 mmol·L-1 NH4NO3)氮素诱导效应更强烈;在根中,处理24 h后,高氮诱导Cs Ala AT1上调表达,低氮诱导Cs Ala AT1下调表达。Alanine Aminotransferase (AlaAT) is a critical enzyme involved in carbohydrate and nitrogen metabolisms.In this study, a cDNA (1 747 bp) with a complete ORF (1 626 bp) of AlaAT1 was isolated from tea plant (Camellia sinensis). The cDNA encodes a protein with 541 amino acids, which has a molecular mass of 59.4 kD and a theoretical isoelectric point (pI) of 5.82. The deduced sequence of protein CsAlaAT1 shared 84% similarity with AlaAT1 in Arabidopsis thaliana, which contains a highly-conserved pyridoxal 5′-phosphate binding site. Secondary structure prediction showed that the CsAlaAT1 was comprised of alpha helix (40.67%), random coil (29.57%), beta turn (13.68%) and extended strand (16.08%), localized in mitochondrion and had no signal peptide or transmembrane structure. The expression levels of CsAlaAT1 in various tissues and its responses to different N concentration were investigated by real-time fluorescent quantitative RT-PCR. The results of RT-PCR showed that CsAlaAT1 expressed in all tissues of tea plant and the highest transcript level was observed in roots. The transcript abundance of CsAlaAT1 was up-regulated by N in both shoots and mature leaves, especially under high N condition.Interestingly, the expression of CsAlaAT1 in roots was highly induced high N condition, but showed an opposite trend under low N treatment for 24 h.

关 键 词：茶树 丙氨酸氨基转移酶 克隆 表达 氮素诱导 

分 类 号：S571.1[农业科学—茶叶生产加工] Q52[农业科学—作物学]