黑籽南瓜NBS类抗病基因的鉴别及关键基因分析

The identification and key genes analysis of NBS type disease-resistance gene from Cucurbita ficifotia

以云南栽培的枯萎病抗病品种绿皮黑籽南瓜(Cucurbita ficifolia)为材料,采用二代转录组和全长转录组测序相结合的方法,对黑籽南瓜NBS类抗病基因进行鉴别和筛选。结果显示:以NB-ARC作为参考氨基酸序列,共鉴定了43条CfNBS(NBS-type gene from C.ficifolia)类基因全长序列,分属于TNL、CNL、TN、RPW8-N和N类5个亚基因家族,典型的TNL和CNL类分别有2个和13个。系统进化树分析表明,CfNBS类基因可以分为4大类群,黑籽南瓜的NBS类抗病基因数量较少但其基因进化类型比其他瓜类物种丰富。与二代转录组进行比对,筛选获得11个差异表达的黑籽南瓜NBS类关键基因,包括4个抗病蛋白、2个蛋白激酶、2个TMV抗性蛋白和3个未知功能蛋白,qRT-PCR分析表明有10个Cf NBS类关键基因在枯萎病菌接种后48和96 h出现上调或下调表达,可能参与了黑籽南瓜对枯萎病菌侵染不同时间点的抗病应答过程。11个CfNBS类关键基因的GO(Gene Ontology)功能富集分析表明,其涉及的生物学过程显著富集在防卫反应、谷胱甘肽转运、改良氨基酸转运等10条GO条目中,涉及分子功能富集在ADP结合、受体丝氨酸/苏氨酸激酶结合、细胞壁结构成分3个GO条目中。而富集通路最多的是与TMV抗性蛋白同源的2个基因(CL19588contig1和CL21402contig1),说明这2个基因在抗病应答过程中起到了重要作用。11个CfNBS类关键基因qRT-PCR组织表达特异性分析表明,在根、茎、叶和果实中表达量最高的基因分别是CL34065Contig1、CL52011Contig1、CL6035Contig1和CL19588Contig1。以上结果可为开展黑籽南瓜NBS类抗病基因的克隆和功能验证,以及解析黑籽南瓜响应枯萎病菌胁迫的抗性应答机制提供参考。

The black-seeded pumpkin(Cucurbita ficifolia)is one of characteristic germplasm crops in Yunnan that has strong resistant abilities to Fusarium wilt caused by Fusarium oxysporum.In this study,the NBS type genes of the variety’Green Peel’which is highly resistant to F.oxysporum were identified and screened out by the combination analysis of second-generation and third-generation transcriptome.Using NB-ARC as the conserved domain,the results of third-generation transcriptome analysis showed that 43 CfNBS(NBS-type gene from C.ficifolia)genes were identified,which belonged to five subgene families:TNL,CNL,TN,RPW8-N and N.Among them,2 and 13 of CfNBS genes were classified into the typical TNL and CNL respectively.The phylogenetic tree analysis results indicated that CfNBS genes could be classified into four groups,and CfNBS genes’evolution types were more abundant than other Cucurbita species despite of a smaller quantity of NBS type resistance genes.After contrasing 43 CfNBS genes in second-generation transcriptome,we have obtained 11 differentially expressed CfNBS resistant genes,including 4 disease resistance protein genes,2 protein kinases genes,2 TMV resitance protein genes,and 3 unkown protein genes.Among 11 CfNBS genes,10 genes were up-regulated or down-regulated after inoculation with F.oxysporum,suggesting these genes were potentially involved in the process of the response to F.oxysporum stress at different infection stages.Subsequent GO(Gene Ontology)enrichment analysis results showed that 11 differentially expressed genes were significantly enriched in 10 biological process GO terms,in which the top three were denfense response,glutathione transport and modified amino acid transport.And the enriched molecular function GO terms were ADP binding,receptor serine/threonine kinase binding and structural constituent of cell wall.Two genes(CL19588 contig1 and CL21402 contig1),which were orthologous of TMV resistance proteins,involved in the most enrichment pathways,implying the two genes played important roles in the process of diseases-resistance.The results of tissue specific expression analysis showed that the genes of CL6035 Contig1,CL19588 Contig1,CL52011 Contig1 and CL34065 Contig1 highly expressed in leaf,root,stem and fruit respectively.Our acquried results might provide concrete foundation for further research of the CfNBS genes,including cloning gene,identifying gene function and revealing the mechanism of C.ficifolia defence response to F.oxysporum infection.