茶树丙氨酸氨基转移酶基因的克隆与表达分析

doi:10.13305/j.cnki.jts.2016.04.009

摘要/Abstract

摘要： 丙氨酸氨基转移酶（Alanine Aminotransferase,AlaAT）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CsAlaAT1的cDNA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 kD,理论等电点（pI）为5.82。同源比对结果表明,CsAlaAT1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AtAlaAT1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CsAlaAT1在茶树各组织中均有表达,在根中的表达量最高;CsAlaAT1基因表达对氮素的响应研究表明,成熟叶中CsAlaAT1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CsAlaAT1上调表达,低氮诱导CsAlaAT1下调表达。

关键词: 茶树, 丙氨酸氨基转移酶, 克隆, 表达, 氮素诱导

Abstract: 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.

Key words: tea plant (Camellia sinensis), Alanine aminotransferase, cloning, expression, nitrogen induction

中图分类号:

S571.1
Q52

白培贤, 王丽鸳, 韦康, 阮丽, 成浩, 张芬, 张成才. 茶树丙氨酸氨基转移酶基因的克隆与表达分析[J]. 茶叶科学, 2016, 36(4): 405-413. doi: 10.13305/j.cnki.jts.2016.04.009.

BAI Peixian, WANG Liyuan, WEI Kang, RUAN Li, CHENG Hao, ZHANG Fen, ZHANG Chengcai. Cloning and Expression Analysis of Alanine Aminotransferase Gene in Camellia sinensis[J]. Journal of Tea Science, 2016, 36(4): 405-413. doi: 10.13305/j.cnki.jts.2016.04.009.

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