茶树丙氨酸氨基转移酶基因的克隆与表达分析

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1747 bp，包含一个完整的 ORF（1626 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。%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.