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。
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.
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