高山离子芥铁转运蛋白基因编码序列的克隆及其抗逆性表达的实时定量分析

Molecular Cloning and Analysis of IRT Gene Expression via Real-time PCR in Chorispora bungeana fisch & C.A.Mey

铁转运蛋白(iron transport protein,IRT)是具有跨膜运输离子功能的特殊蛋白质,属于金属离子转运家族的成员.本研究运用RACE方法从高山离子芥(Chorispora bungeana)中克隆得到完整的铁转运蛋白cDNA,命名为CbIRT(基因登录号为EU330924).该基因全长1290 bp,包含1个1035 bp的开放阅读框(ORF),编码344个氨基酸的蛋白.系统进化树分析显示,该基因与拟南芥AtIRT1和遏蓝菜TcIRT-G的亲缘关系最近,同源性分别达到了87.4%和86.5%,而在氨基酸序列水平与拟南芥AtIRT1的同源性达到了89%,表明克隆得到的CbIRT属于金属离子转运体家族成员.实时荧光定量方法对高山离子芥CbIRT基因在不同温度和铁营养水平条件下的表达情况进行分析表明,CbIRT对零下低温和零上低温的表达水平呈截然不同的反应;正常铁营养状态下,CbIRT是微量表达的,而缺铁及低温处理都可以大幅度地促进该基因的表达,富铁可以抑制该基因的表达.显示了该蛋白在转运Fe离子方面的重要作用.

Iron transport protein（IRT） is a unique protein that transports ion across membrane and belongs to the member of iron transport family-ZIP.The full-length IRT gene in Chorispora bungeana and its expression under different temperatures and different Fe levels were studied by using 3′ and 5′ RACE-PCR cloning and real-time quantitative PCR.Sequence alignment and phylogenetic tree analyses showed that the IRT gene in Chorispora bungeana（named CbIRT）,comprising 1 290 bp with a complete open reading frame of 1 035 bp,encoding a peptide of 344 amino acid residues,was highly homologous to AtIRT1（87.4%） and TcIRT-G（86.5%）,respectively,and the sequence of deduced amino acids has the 89% homology to that of AtIRT1.Expression analysis by real-time quantitative PCR revealed that CbIRT gene expression can be induced either by shorten Fe treatment or low temperature stress,while depressed by additional Fe treatment.This indicated that CbIRT gene might play key roles not only in Fe metabolism but also in low temperature stress.