蒜芥茄SsVe1和SsVe2基因的克隆及表达分析

Cloning and Expression Analyses of SsVe1 and SsVe2 Genes from Solanum sisymbriifolium L.

黄萎病是危害茄子生产的主要病害之一,对黄萎病抗性基因研究是解决该病危害严重的有效途径。本研究以黄萎病抗性材料野生蒜芥茄(Solanum sisymbriifolium)为研究对象,在前期转录组测序的基础上,通过RT-qPCR技术克隆得到2个Ve同源基因的c DNA全长,分别命名为SsVe1和SsVe2。其中,SsVe1 cDNA序列有3615 bp,含3168 bp开放阅读框,编码1061个氨基酸;SsVe2 cDNA序列有3756 bp,含3456 bp开放阅读框,编码1157个氨基酸。2个Ve基因所编码的氨基酸具有极高的相似性,均富含抗病基因共有的结构域—亮氨酸重复序列(LRR),可编码R蛋白;亚细胞定位预测基因产物均在细胞壁上;此外,2个基因编码蛋白的二级、三级结构预测结果也高度相似,以上结果预示着SsVe1和SsVe2功能的相似性。与已报道的其它植物Ve基因对比分析结果表明:SsVe1和SsVe2基因与野生茄S.torvum中Ve基因的关系较近。实时荧光定量PCR检测结果表明:蒜芥茄接种大丽轮枝菌(Verticillium dahliae)后,SsVe1和SsVe2均表现出先下调(0~12 h)后上调(12~48 h)的趋势;同时,Ve基因在蒜芥茄不同组织中的表达有一定的差异,SsVe2基因的表达量高于SsVe1,且两者在根、茎部的表达量高于叶片。本研究结果为探究Ve基因在茄子黄萎病抗性分子机制中的作用和功能奠定了基础。

Verticillium wilt is one of the most serious diseases in eggplant production.The study of resistance genes to Verticillium wilt is an effective way to control this disease.On the basis of transcriptome sequencing,two Ve homologous genes,named SsVe1 and SsVe2,were isolated from Solanum sisymbriifolium(a wild eggplant species)by RT-qPCR.The full-length of c DNA of SsVe1 was 3615 bp with an open reading frame of 3168 bp,and encoded a putative protein of 1061 amino acids.The full-length of cDNA of SsVe2 was 3756 bp with an open reading frame of 3456 bp,and encoded a putative protein of 1157 amino acids.The amino acids encoded by SsVe1 and SsVe2 have extremely high similarity.SsVe1 and SsVe2 were rich in leucine repeat sequence(LRR),a domain common to disease resistance genes,and encoded R protein.Subcellular localization prediction showed that the gene products of SsVe1 and SsVe2 were on the cell wall.In addition,the secondary and tertiary structure prediction results of the two Ve proteins were highly similar.The above all indicated the similarity of their functions.Phylogenetic tree analysis showed that the proteins encoded by SsVe1 and SsVe2 genes were closely related to Ve genes in S.torvum.Real-time quantitative PCR revealed that after V.dahliae infection,the expression of SsVe1 and SsVe2 showed a down-regulated trend(0~12 h)and then up-regulated trend(12~48 h).The expression level of Ve genes in different tissues of S.sisymbriifolium was different.The expression level of SsVe2 gene was higher than SsVe1,while the expression level of root and stem was higher than that of leaf.The above results laid a foundation for exploring the role and function of Ve gene in the molecular mechanism of eggplant resistance to Verticillium wilt.