甜荞柠檬酸转运蛋白基因FeFRD3的克隆及表达分析

Cloning and Expression Analysis of FeFRD3 Citrate Efflux Transporter Gene in Fagopyrum esculentum

该研究采用同源克隆策略,从甜荞中克隆到1个柠檬酸转运蛋白基因FeFRD3(GenBank登录号为MG462907)。FeFRD3基因含一个1 554bp开放阅读框,编码517个氨基酸,预测蛋白分子量为55.83kD,等电点为8.48。生物信息学分析显示,FeFRD3蛋白含有8个跨膜区,定位于质膜和液泡膜上。蛋白序列分析结果表明,FeFRD3与拟南芥、大豆和水稻的FRD3同源蛋白有较高的序列一致性。系统进化树分析表明,FeFRD3属于具有将铁由根向地上部位长距离转运功能的柠檬酸转运蛋白,且与拟南芥AtFRD3亲缘关系最近。qRT-PCR分析结果表明,FeFRD3基因在甜荞根、茎、叶和种子中均有表达,但在根中的表达量最高,在种子中的表达量最低;缺铁胁迫没有影响FeFRD3基因在根中的表达,但高铁胁迫明显诱导了该基因在根中的表达。研究结果为进一步深入研究FeFRD3基因在甜荞铁长距离转运中的功能奠定了基础。

Citrate efflux transporters play an important role in iron long-distance transport from root to shoot.A citrate efflux transporter gene FeFRD3(GenBank ID:MG462907)from Fagopyrum esculentum was cloned by using homology cloning strategy.FeFRD3 gene had an open reading frame(ORF)of 1 554 bp which encoded 517 amino acid residues with a predicated molecular weight of about 55.83 kD and an isoelectric point of 8.48.Bioinformatics analysis showed that FeFRD3 protein had eight transmembrane domains and located in plasma membrane or tonoplast.Protein sequence analysis indicated that FeFRD3 had a high degree of identity with FRD3 homology proteins from Arabidopsis,soybean and rice.Phylogenetic tree analysis showed that FeFRD3 belongs to the class of citrate efflux transporter with function in iron long-distance transport from root to shoot,and was most closely related to AtFRD3 of Arabidopsis.qRT-PCR analysis indicated that FeFRD3 gene was expressed in root,stem,leaf and seed.Moreover,the expression level in root and seed was highest and minimal,respectively.Deficiency of iron had no effect on the expression level of FeFRD3 in root.In contrast,higher iron treatment had obviously induced for the expression of FeFRD3 in root.Research result laid a foundation for further study the function of FeFRD3 in iron long-distance transport from root to shoot in F.esculentum.