普通烟草抗渗透胁迫基因NtP5CS的克隆与表达分析

从普通烟草中克隆脯氨酸合成的关键酶基因P5CS,分析其序列特征、进化关系、表达模式,以期为烟草的抗逆研究奠定基础。设计兼并引物获得P5CS基因中间片段,利用RACE技术扩增其全长cDNA;采用生物信息学技术分析该基因的序列结构及其编码蛋白的保守域及基本特性;利用RT-PCR研究其表达模式。从受干旱胁迫诱导的普通烟草品种中烟14中克隆到全长为2584bp的烟草P5CS基因,命名为NtP5CS,GenBank登录号为HM854026,开放读码框为2160 bp,编码719个氨基酸。经Blast同源性比对分析,该序列与番茄tomPRO2基因在核苷酸和氨基酸水平上高度同源。系统进化树分析显示,NtP5CS与番茄tomPRO2基因可能是直系同源基因。RT-PCR分析表明,干旱、低温、ABA、高盐等胁迫条件均可诱导NtP5CS基因的上调表达,且在旺长期和胁迫条件下根和叶中表达量最高。首次从普通烟草中克隆得到P5CS基因,该基因对水分胁迫响应,可能参与普通烟草抗渗透胁迫反应。

Cloning and expression analysis of osmotic stress tolerance gene NtP5CS in Nicotiana tabacum

Sequence signature,evolutionary relationship,and expression profile of key enzyme of P5CS gene cloned from tobacco(Nicotiana tabacum)for Pro synthesis were analyzed to establish foundation for research in tobacco stress tolerance.Degenerate primers were designed to obtain segment of P5CS,and 5’ and 3’ ends of the cDNA were amplified by RACE.Bioinformatics was used to analyze sequence structure,coded protein’s conserved domains,and basic characteristics.Expression pattern was studied by RT-PCR.The P5CS gene cloned from tobacco induced by drought stress,which was 2,584 bp in full length,were termed NtP5CS(GenBank accession HM854026).The ORF was 2160 bp in length,which encoded proteins of 719 amino acid residues.GenBank Blast analysis showed that the sequence is in high homologous with tomPRO2 gene from tomato in level of nuleotide and amino acid.According to phylogenetic tree,NtP5CS and tomPRO2 may be Ortholog gene.RT-PCR results showed that NtP5CS could be induced by drought,high salt,low temperature,ABA,and it had the highest expression in root and leaf of tobacco’s prosperous long-term and water stress condition.It was the first time to obtain P5CS from tobacco.From the response to water stress,it may be inferred that it played a role in resistance to osmoticstress.