栽培番茄与野生番茄NBS-LRR类抗病基因家族的全基因组鉴定及表达分析

Genome-wide identification and evaluation of NBS-LRR disease-resistant gene family in cultivated tomatoes and wild tomatoes

【目的】对栽培番茄和野生番茄NBS-LRR类抗病基因家族进行全基因组鉴定及表达分析,为NBS-LRR类抗病基因功能研究及其在植物抗病育种中的应用提供理论参考。【方法】应用生物信息学和比较基因组学方法,从栽培番茄和野生番茄[潘那利番茄(Solanum pennellii)和醋栗番茄(Solanum pimpinellifolium)]基因组中鉴定出NBS-LRR类抗病基因家族成员,明确其类型,并进行序列特征、染色体定位、系统进化及生物胁迫下表达模式分析。【结果】从栽培番茄、潘那利番茄和醋栗番茄基因组中分别鉴定获得238、202和217个NBS-LRR类抗病基因家族成员,根据这些抗病基因编码的结构域差异,将其分为TNL(TIR-NBS-LRR)、CNL(CC-NBS-LRR)、RNL(RPW8-NBS-LRR)、TN(TIRNBS)、CN(CC-NBS)、RN(RPW8-NBS)、NL(NBS-LRR)和N(NBS)8种类型,其中TNL、CNL和RNL型基因分别为58、87和13个。番茄NBS-LRR类抗病蛋白整体呈弱酸性,具有不稳定性和亲水性,以丝氨酸(Ser)磷酸化为主,二级结构以α-螺旋和无规则卷曲为主,定位于细胞核、细胞质和叶绿体。栽培番茄和野生潘那利番茄的NBS-LRR类抗病基因不均匀地散布在13条染色体(0号虚拟染色体~12号染色体)上,分别有58.47%和52.48%的基因形成多个基因簇,有35.59%和22.77%的基因形成串联重复。栽培番茄和2个野生番茄共477个NBS-LRR类抗病蛋白可聚为十大类群,其中N端为TIR的蛋白基本聚在一起,但N端为CC和RPW8结构域的蛋白未能很好分开,聚在多个类群中;NL和N型基因散布于各类群中。在477个番茄NBS-LRR类抗病基因中共发现115对直系同源基因和8对旁系同源基因,主要分布在4号和5号染色体上,其中51.57%的NBS-LRR类抗病基因以同源基因形式存在,大多数Ka/Ks均小于1,说明其进化中主要受到纯化选择。基于转录组测序数据(RNA-Seq)分析结果表明,多数NBS-LRR类抗病基因能响应不同细菌胁迫。【结论】番茄NBS-LRR类抗病基因具有成簇存在的特点,在进化过程中经重复扩张,已形成大量同源基因,且能响应多种细菌胁迫。

【Objective】Genome-wide identification and expression analysis of NBS-LRR disease-resistantgenes in cultivated tomatoand its wild relatives were performed,to provide a theoretical basis for the research on the function of NBSLRR disease-resistant genes and the application in plant disease resistance breeding.【Method】NBS-LRR disease-resistant gene family members were identified and analysed on classification,sequence characteristics,chromosome location,phylogeny,and expression patterns under biotic stresses from cultivated(Solanum lycopersicum)and two wild(S.pennellii and S.pimpinellifolium)tomato species genomes using bioinformatics and comparative genomics methods.【Result】In total,238,202 and 217 NBS-LRR disease-resistant genes were identified in cultivated tomato and S.pennellii and S.pimpinellifolium genomes,respectively.Based on the differences in the domains encoded by these disease-resistant genes,they were divided into eight types,which were TNL(TIR-NBS-LRR),CNL(CC-NBS-LRR),RNL(RPW8-NBS-LRR),TN(TIR-NBS),CN(CC-NBS),RN(RPW8-NBS),NL(NBS-LRR)and N(NBS),with 58 TNL,87 CNL and 13 RNL genes,respectively.NBS-LRR disease-resistant genes were weakly acidic in general,and were unstable and hydrophilic,with serine(Ser)phosphorylation and secondary structure of alpha-helix and random coil,which were mainly located in the nucleus,cytoplasm and chloroplast.Chromosome localization revealed that the NBS-LRR disease-resistant genes in cultivated tomato and S.pennellii genomes were unevenly distributed on 13 different chromosomes(virtual chromosome 0-chromosome12),with 58.47%and 52.48%genes forming multiple gene clusters,35.59%and 22.77%genes forming tandem duplicates,respectively.Systematic clustering divided 477 NBS-LRR disease-resistant genes in the three genomes into ten groups,the N-terminal TIR-type genes were basically clustered together,but CC-and RPW8-type genes were not well separated and clustered in multiple groups;NL-and N-type genes were scattered in various groups.Gene homology analysis revealed 115 pairs of orthologous genes and 8 pairs of paralogous genes in 477 NBS-LRR disease-resistantgenes,equivalent to 51.57%of NBS-LRR genes had a homologous gene,and that they were mainly distributed on chromosomes 4 and 5,with most Ka/Ks values being less than 1,indicating that purification selection had occurred in evolution.It revealed that the most NBS-LRR disease-resistant genes could respond to different bacterial stresses by transcriptome sequencing(RNA-seq)data analysis.【Conclusion】The tomato NBS-LRR disease-resistant genes exist in cluster.During the evolution,a large number of homologous genes have appeared through gene expansion.Also,they can respond to a variety of bacterial stresses.