紫花苜蓿F-box蛋白基因MsFTL的克隆及功能分析

Cloning and Functional Analysis of F-box Protein Gene MsFTL in Alfalfa(Medicago sativa L.)

FTL(F-box Triple LRR protein)是F-box蛋白家族的成员,具有F-box保守结构域,在植物抵御逆境胁迫过程中起重要作用。本研究参考低温胁迫下紫花苜蓿转录组数据设计引物,通过RT-PCR克隆获得紫花苜蓿MsFTL基因,该基因的全长1422 bp,编码473个氨基酸。该蛋白含有1个F-box结构域及3个LRR重复。系统进化分析表明,MsFTL与蒺藜苜蓿XP_003626345.1 F-box/FBD/LRR-repeat protein亲缘关系最近。两者蛋白序列比对发现共有11个差异位点。在低温、盐、干旱以及外源ABA处理下,MsFTL基因受到诱导,表达量上调。构建植物过表达载体pCBM-MsFTL,通过农杆菌介导法转化烟草。对经过抗性筛选、PCR和Real-time PCR验证的转基因植株进行低温抗性鉴定。在-4℃低温胁迫下,野生型烟草叶片出现了明显的萎蔫失水现象,而转基因烟草萎蔫程度相对较轻。生理检测结果表明,4℃处理24 h之后,转基因烟草的可溶性蛋白含量、可溶性糖含量、SOD活性,CAT活性高于野生型,MDA含量低于野生型。本研究表明,MsFTL基因在提高植物对低温胁迫的抗性方面具有重要的作用。

FTL(F-box Triple LRR)protein belongs to the family of F-box proteins that play an important role in tolerance to low temperature. In this study,an MsFTL gene was isolated by candidate gene approach from alfalfa,and this gene was differently expressed in leaf under cold stress. The full-length cDNA of MsFTL gene was 1422 bp,which putatively encoded for 473 amino acids. By bioinformatic analysis,the MsFTL protein was found to carry an F-box domain and three LRR repeats in C-terminus. MsFTL showed homology closely to XP003626345.1,a member of F-box/FBD/LRR-repeat protein in Medicago truncatula,with 11 differential sites between the two proteins. The expression of MsFTL was induced by low temperature,salt,drought stress and ABA treatments. We generated the transformation construct and transformed the MsFTL into tobacco. Under-4 ℃,wild-type tobacco leaves showed remarkable wilting,while the transgenic plants presented slightly stressed phenotype. Transgenic plants accumulated higher content of soluble protein and soluble sugar than that of wildtype. The activity of SOD and CAT in transgenic plants was higher than those of wild type under low temperature(4 ℃)for 24 h. Moreover,the MDA content in transgenic plants was lower than that of wild type. Thus,these results suggested that overexpressing MsFTL might significantly improve tobacco plant tolerance to low temperature stress.