低温胁迫下龙眼碳酸酐酶基因（CA）的克隆与表达分析

 应用蛋白质组学研究低温胁迫下龙眼叶片蛋白质组变化时发现碳酸酐酶（CA）蛋白下调表达。利用RT-PCR方法克隆CA基因的全长cDNA，GenBank登录号JN033201，长度为1 119 bp，包括1个966 bp的开放阅读框，编码321 bp的氨基酸序列，同源性分析表明，12个不同植物同源性为81% ~ 88%。龙眼CA基因具有典型的CA结构域，并且非常保守。实时荧光定量分析结果表明，CA在龙眼根、茎、叶中都有表达，为组成型表达，在叶中的表达量最高，茎和根中的表达量最少。CA基因在低温胁迫下随着低温胁迫时间的延长而发生变化。将CA在大肠杆菌中表达，获得1个约40.5 kD的外源蛋白。推测CA表达与低温胁迫有关。

Cloning and Expression of Longan Carbonic Anhydrase Gene Under Low Temperature Stress

The protein expressions of longan(Dimocarpus longan Lour.)under low temperature stress were studied using proteomics method.The results showed that the expression of carbonic anhydras(eCA) protein was down-regulated.The CA(GenBank accession number:JN033201)cDNA obtained by RT-PCR was 1 119 bp of full length with a 966 bp open read frame which encodes a putative CA gene with 321 amino acids.Comparison of the amino acids sequences homology in CA from 12 different species indicated that CA had a range of 81%to 88%identity in amino acids sequence with homologues of other plants.The deduced amino acids sequence not only contained a typical CA domain,but also was very conservative.Quantitative real-time PCR results showed that the CA expressed in root,stem and leaf,while the amount of expressions were different in different organs.The mRNA of CA was the most abundant in leaf,the least in root and stem.Furthermore,the mRNA of CA was changed with time extension under low temperature stress.A 40.5 kD heterologous protein was obtained when CA gene was expressed in E.coli.These results suggested that CA might be involved in function of chilling stress.