烟草(云烟87)NBS-LRR类同源抗病基因克隆及分析

Cloning and analysis of NBS-LRR resistance gene analogs in tobacco(Yunyan 87)

【目的】克隆并分析烟草NBS-LRR类抗病基因的同源序列(RGAs),为采用同源克隆技术挖掘烟草抗病基因提供理论参考。【方法】从基于NBS-LRR类抗病基因保守序列设计的简并引物中筛选扩增效果较好的引物用于扩增烟草RGAs,采用DNAMAN 6.0翻译成氨基酸序列,并与已知的其他植物抗病基因进行聚类分析。【结果】克隆获得6条与参比抗病基因具有高度同源性的烟草RGAs,大小在500 bp左右,但仅有4条RGAs具有连续的开放阅读框(ORF)及编码NBS功能结构域的核苷酸序列,命名为TRGA-87-1~TRGA-87-4。这4个烟草RGAs编码的氨基酸序列均含有NBS类抗病蛋白的多个典型保守基序,与已知的多种植物抗病基因编码的氨基酸序列均具有较高的相似性,其中,与烟草相关抗病基因编码的氨基酸序列相似性最高,为97.00%~100.00%,与其他植物的抗病基因编码氨基酸序列相似性为29.00%~53.00%,这些序列可分为3个亚类,其中TRGA-87-2与烟草N和亚麻L6聚为一类(TRGAⅠ),属于TIR-NBS-LRR类;TRGA-87-3和TRGA-87-4与水稻Xa-1和番茄Mi聚为一类(TRGAⅡ),属于non-TIR-NBS-LRR类,TRGA-87-1与拟南芥RPM1聚成一类(TRGAⅢ),属于non-TIR-NBS-LRR类。【结论】同源扩增技术可用于烟草中抗病基因的克隆、功能分析及定位等研究。

【Objective】To clone and analyze the resistance gene analogs(RGAs)of NBS-LRR resistance genes in tobacco,provide theoretical reference for using homologous cloning technology to mine tobacco resistance genes.【Method】The degenerate primers designed based on the conserved sequences of NBS-LRR resistance genes were selected to amplify RGAs of tobacco.The vector sequences in the sequenced fragments were removed by DNAMAN 6.0,and then translated into amino acid sequences.Homology analysis was performed in NCBI database,and cluster analysis was performed with other known disease resistance genes.【Result】Six tobacco RGAs with high homology with the reference disease resistance genes were cloned and their sizes were about 500 bp.Only four RGAs had continuous open reading frames(ORFs)and nucleotide sequences encoding NBS functional domain,named from TRGA-87-1 to TRGA-87-4.The amino acid sequences encoded by these four RGAs all contained several typical conserved motifs of NBS like resistance proteins,and shared high similarity with known amino acid sequences encoded by many plant disease resistance genes.Among them,the amino acid sequences of tobacco related disease resistance genes had the highest similarity(97.00%-100.00%)and 29.00%-53.00%with other plant disease resistance genes.These sequences could be divided into three subgroups.TRGA-87-2,tobacco N and flax L6 were clustered into one group(TRGA I),belonging to TIR-NBS-LRR resistance genes.TRGA-87-3,TRGA-87-4,rice Xa-1 and tomato Mi clustered into a subgroup(TRGAⅡ),belonging to non-TIRNBS-LRR resistance genes.TRGA-87-1 and arabidopsis RPM1 clustered into a subgroup(TRGA III),belonging to nonTIR-NBS-LRR resistance genes.【Conclusion】Homologous amplification can be used for cloning,functional analysis and localization of disease resistance genes in tobacco.