烟草抗马铃薯Y病毒病相关基因NtPsaN的克隆及表达分析

Cloning and Expression Analysis of the PVY Resistance-Related Gene NtPsaN in Tobacoo (Nicotiana tabacum)

【目的】从高抗PVY烟草品种VAM中克隆抗病相关基因,分析该基因的序列特征、进化关系和表达特性,研究其在烟草抗PVY防御反应中的作用,为培育抗PVY烟草新品种奠定基础。【方法】利用SSH结合cDNA芯片筛选出来的病害诱导特异表达基因片段,通过RACE技术克隆烟草抗病相关基因;生物信息学分析该基因保守结构域以及序列特征;采用MEGA4.0软件构建系统进化树;利用实时荧光定量RT-PCR技术研究该基因的表达特性。【结果】克隆了1个烟草抗病相关基因,命名为NtPsaN,该基因具有PsaN基因家族保守结构域,OFR长度507bp,编码168个氨基酸;构建了PsaN亚基系统进化树;实时荧光定量RT-PCR结果显示接毒之后该基因表达与不接毒对照相比有明显上调趋势。【结论】克隆得到一个烟草PVY抗病相关基因NtPsaN,其表达受PVY侵染诱导,该基因可能在烟草抵御PVY病害侵染过程中起重要作用。

【Objective】 The common tobacco （Nicotiana tabacum） variety VAM was highly resistant to PVY. A PVY resistance-related gene NtPsaN was cloned from VAM. The sequence features,evolutionary relationships and expression characteristics of NtPsaN were analyzed and the molecular mechanism of tobacco resistance response was studied,and thus provide a theoretical foundation for PVY-resistant tobacco breeding. 【Method】 A virus resistance-related gene fragment was screened through Suppression Subtractive Hybridization （SSH） and cDNA chip technologies,the full-length cDNA sequence was cloned through RACE,the conserved domains and sequence features of the gene were analyzed by bioinformatic methods,a neighbor-joining phylogenetic tree was constructed using the MEGA4.0 program and its relative expression characters were explored by real-time PCR. 【Result】 The tobacco resistance-related gene,named NtPsaN,was cloned. The gene possesses conserved domains of PsaN superfamily. The open reading frame （ORF） of the full length cDNA was 507 bp in length,encoding 168 amino acids. The PsaN subunit phylogenetic tree was constructed. The results of real-time PCR showed that the expression of NtPsaN was obviously up-regulated in leaves infected with PVY. 【Conclusion】 A PVY resistance-related gene NtPsaN was cloned from tobacco,and it was induced by PVY infection. The NtPsaN gene may play a critical role in the PVY-resistance of tobacco.