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.
[1] Raun WR, Solie JB, Johnson GV, et al.Improving nitrogen use efficiency in cereal grain production with optical sensing and variable rate application[J]. Agron J, 2002, 94(4): 815-820.
[2] de Sousa CAF, Sodek L. Alanine metabolism and alanine aminotransferase activity in soybean (Glycine max) during hypoxia of the root system and subsequent return to normoxia[J]. Environ Exp Bot, 2003, 50(1): 1-8.
[3] 吴洵, 茹国敏. 茶树对氮肥的吸收和利用[J]. 茶叶科学, 1986, 6(2): 15-24.
[4] 周月琴, 庞磊, 李叶云, 等. 茶树硝酸还原酶基因克隆及表达分析[J]. 西北植物学报, 2013, 33(7): 1292-1297.
[5] 冯素花. 茶树硝酸根转运蛋白NRT1.2、NRT1.5、NRT2.5基因的克隆与表达[D]. 杭州: 中国农业科学院茶叶研究所, 2014.
[6] 汪进, 添先凤, 江昌俊. 茶树硝酸盐转运蛋白基因的克隆与表达分析[J]. 植物生理学报, 2014, 50(7): 983-988.
[7] 徐乾. 茶树中与氨基酸合成相关的三个基因的克隆与分析[D]. 合肥: 安徽农业大学, 2012.
[8] 林郑和, 钟秋生, 陈常颂. 茶树叶片GDH、GS、GOGAT基因的克隆及荧光定量PCR分析[J]. 茶叶科学, 2012, 32(6): 523-529.
[9] 胡娟, 王丽鸳, 韦康. 茶树CsCDF1基因克隆及表达分析[J]. 茶叶科学, 2015, 35(5): 501-511.
[10] Good AG, Johnson SJ, De Pauw M.Engineering nitrogen use efficiency with alanine aminotransferase[J]. Can J Bot, 2007, 85(3): 252-262.
[11] Miyashita Y, Dolferus R, Ismond KP, et al.Alanine aminotransferase catalyses the breakdown of alanine after hypoxia in Arabidopsis thaliana[J]. Plant J, 2007, 49(6): 1108-1121.
[12] Rocha M, Sodek L, Licausi F.Alalysis of alanine aminotransferase in various organs of soybean (Glycine max) and in dependence of different nitrogen fertilisers during hypoxic stress[J]. Amino Acids, 2010, 39(4): 1043-1053.
[13] Igarashi D, Miwa T, Seki M, et al.Identification of photorespiratory glutamate:glyoxylate aminotransferase (GGAT) gene in Arabidopsis[J]. Plant J, 2003, 33(6): 975-987.
[14] Liepman AH, Olsen LJ.Alanine aminotransferase homologs catalyze the glutamate: glyoxylate aminotransferase reaction in peroxisomes of Arabidopsis[J]. Plant Physiol, 2003, 131(1): 215-227.
[15] Rech J, Crouzet J.Partial purification and initial studies of the tomato L-alanine: 2-oxoglutarate aminotransferase[J]. Biochim Biophys Acta, 1974, 350(2): 392-399.
[16] Kikuchi H, Hirose S, Toki S, et al.Molecular characterization of a gene for alanine aminotransferase from rice (Oryza sativa)[J]. Plant Mol Biol, 1999, 39(1): 149-159.
[17] Son D, Kobe A, Sugiyama T.Nitrogen-dependent regulation of the gene for alanine aminotransferase which is involved in the C4 pathway of panicum miliaceum[J]. Plant Cell Physiol, 1992, 33(8): 507-509.
[18] Son D, Sugiyama T.Molecular cloning of an alanine aminotransferase from NAD-malic enzyme type C4 plant Panicum miliaceum[J]. Plant Mol Biol, 1992, 20(4): 705-713.
[19] Shrawat AK, Carroll RT, DePauw M. Genetic engineering of improved nitroge use efficiency in rice by the tissue-specific expression of alanine aminotransferase[J]. Plant Biotechnology Jorunal, 2008, 6(7): 722-732.
[20] Ruan J, Gerendas J, Hardter R, et al.Effect of nitrogen form and root-zone pH on growth and nitrogen uptake of tea (Camellia sinensis) plants[J]. Ann Bot, 2007, 99(2): 301-310.
[21] Ricoult C, Echeverria LO, Cliquet JB, et al.Characterization of alanine aminotransferase (AlaAT) multigene family and hypoxic response in young seedlings of the model legume Medicago truncatula[J]. J Exp Bot, 2006, 57(2): 3079-3089.
[22] Kendziorek M, Paszkowski A, Zagdanska B.Differential regulation of alanine aminotransferase homologues by abiotic stresses in wheat (Triticum aestivum L.) seedlings[J]. Plant Cell Rep, 2012, 31(6): 1105-1117.
[23] Gupta KJ, Zabalza A, van Dongen JT. Regulation of respiration when the oxygen availability changes[J]. Physiol Plant, 2009, 137(4): 383-391.
[24] 阚君杰, 丁仕波. 山东日照市茶树无性系良种引种试验初报[J]. 茶业通报, 2015, 37(2): 66-70.
[25] Muench DG, Good AG.Hypoxically inducible barley alanine aminotransferase: cDNA cloning and expression analysis[J]. Plant Mol Biol, 1994, 24(3): 417-427.
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