烟草抗PVY^N突变株eIF4E基因的克隆及表达差异分析

Cloning and Expression Analysis of e IF4E Gene in Tobacco Mutants Resistant to PVY^N

为了明确抗马铃薯Y病毒病的相关基因、进一步揭示其抗病机制,以烟草高抗马铃薯Y病毒脉坏死株系(Potato virus Y vein necrosis strain,PVYN)突变株SN01和SN02为试验材料,研究了突变株的抗病相关基因翻译起始因子4E(e IF4E)在寄主中的抗病作用。结果表明:从抗病突变株SN01和SN02中克隆到目的全长基因,分别命名为e IF4E01与e IF4E02,其开放阅读框ORF长度均为669 bp,编码222个氨基酸;利用MAGA4.1和CLUSTAL软件进行序列同源性比对分析表明,e IF4E01和e IF4E02与已报道的烟草e IF4E(Gen Bank:AY702653.1)基因序列相似度均达到99%,其开放阅读框序列完全一致。并与甜椒、番茄、拟南芥等物种的e IF4E家族基因也具有高度同源性;Real-time PCR结果表明,在PVYN侵染初期(接种后0~9 d),突变株SN01和SN02中的e IF4E基因表达量明显低于其感病亲本NC89和K326。说明这两个抗病突变株中的e IF4E基因的抑制表达与烟草对PVYN的抗性密切相关。

In order to explore PVY resistance-related gene and further reveal its resistance mechanism, the tobacco mutants SN01 and SN02 highly resistant to potato virus Y vein necrosis strain （PVY^N） were experimented to study the role of translation initiation factor eIF4E of PVY resistance-related gene in the mutants. The results showed that full length target genes were successfully cloned from the mutants SN01 and SN02. The genes were referred to as eIF4E01 and eIF4E02, respectively; their open reading frame （ORF） was 669 bp in length and encoded a protein of 222 amino acids. The homology analysis by MEGA 4.1 and CLUSTAL showed that these genes possessed 99% sequence similarities to and exactly the same ORF sequence as reported eIF4E in tobacco （GenBank： AY702653.1）, they were highly homologous to the eIF4E family genes in Capsicum annuum, Lycopersicon esculentum and Arabidopsis thaliana. Real-time PCR results indicated that the expression level of eIF4E in the mutants was obviously lower than that in their susceptible parents NC89 and K326 at the early stage of PVYN infection （0-9 days after inoculation）, it indicated that the suppression of eIF4E expression in the two mutants was closely related to the resistance of tobacco to PVY^N.