普通小麦TaCAT 9基因的克隆与表达分析

Cloning and Expression Analysis of Cationic Amino Acid TaCAT 9 Gene from Wheat

氨基酸转运蛋白是植物体内一类负责氨基酸运输的蛋白,是植物氮代谢的重要媒介。CAT9(阳离子氨基酸转运蛋白9)是氨基酸转运蛋白家族的一员,为深入了解小麦中该基因的序列特征及表达特性,采用同源克隆的方法从普通小麦品种豫麦49-198中获得TaCAT9两个部分同源基因的cDNA序列。因两基因分别位于小麦6A和6B染色体长臂上,故分别命名为TaCAT9-A和TaCAT9-B。生物信息学分析结果表明,两个TaCAT9基因的CDS长度均为1 818bp,编码605个氨基酸;它们的编码蛋白等电点分别为8.23和8.27,分子量分别为64.04kDa和64.08kDa,属于疏水稳定蛋白。并且两蛋白均含有阳离子氨基酸转运蛋白的C末端和13个跨膜区。进化分析结果表明,小麦CAT9蛋白与乌拉尔图小麦和山羊草的CAT9蛋白亲缘关系密切。实时荧光定量反转录PCR结果表明,TaCAT9基因在根、茎、叶和籽粒中都有表达,但在叶中的表达量最高;在氮饥饿条件下,该基因的表达上调,推测该基因参与小麦低氮胁迫应答。

Amino acid transporter is a type of protein responsible for amino acid transport in plants.It is an important mediator in plant nitrogen metabolism.CAT9（Cationic Amino Acid Transporter 9）is a member of the amino acid transporter family.In order to understand the sequence characteristics and expression patterns of this gene in wheat,reverse transcription PCR（RT-PCR）was used to clone the cDNA sequences of two homologous genes of TaCAT9 from common wheat（Triticum aestivum L.cv.Yumai 49-198）,named TaCAT9-Aand TaCAT9-B because of their locations on the long arms of chromosome 6 Aand 6 B,respectively.Bioinformatics analysis showed that the CDS length of both TaCAT9 genes were 1 818 bp and encoded a polypeptide of 605 amino acids residues.The estimated molecular weight of the two putative proteins were 64.04 kDa and 64.08 kDa,and the estimated isoelectric point were 8.23 and 8.27,respectively.They all belonged to the hydrophobic stable proteins.Conserved domain analysis showed that both proteins contained the C terminal of the cationic aminoacid transporter family.Transmembrane structure analysis showed that each of the two proteins had13 transmembrane domains.The Phylogenetic tree indicated that TaCAT9 shared high similarity to CAT9 protein from Triticum urartu and Aegilops tauschii.Real-time fluorescence quantitative PCR showed that the expression level of TaCAT9 genes had significant differences in roots,stems,leaves and seeds,and the expression level in leaves was the highest.Under nitrogen starvation,the gene expression level in leaves was up-regulated compared to the control.The fact implied that it might be involved in low nitrogen stress signaling pathway in wheat.These findings provide the basis for further study on the function of TaCAT9.