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【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 objective of this study was to clone the disease-resistance-related gene from VAM of high resistance PVY tobacco variety, analyze the sequence, evolutionary relationship and expression characteristics of this gene, study its role in defense against PVY in tobacco and lay a foundation for cultivating new tobacco varieties resistant to PVY basis. 【Method】 The disease-specific gene fragments selected by SSH and cDNA microarray were cloned and the genes related to disease resistance were cloned by RACE. The bioinformatics analysis of the conserved domains and sequence features of the genes was carried out. The phylogenetic tree was constructed by MEGA 4.0 software ; Using real-time fluorescent quantitative RT-PCR technology to study the expression characteristics of the gene. 【Result】 A tobacco resistance-related gene was cloned and named as NtPsaN. The gene has a conserved PsaN gene family with an ORF of 507 bp encoding 168 amino acids. A phylogenetic tree of PsaN subunits was constructed. Real-time fluorescence quantitative RT- PCR results showed that the expression of the gene was significantly up-regulated compared with the untreated control after receiving the virus. 【Conclusion】 A tobacco PVY resistance-related gene, NtPsaN, was cloned and its expression was induced by PVY infection. This gene may play an important role in tobacco defense against PVY disease.