黄瓜CC-NBS-LRR家族基因鉴定及在霜霉病和白粉病胁迫下的表达分析

Genome-Wide Identification and Analysis of CC-NBS-LRR Family in Response to Downy Mildew and Powdery Mildew in Cucumis sativus

【目的】对黄瓜全基因组的CC-NBS-LRR(CNL)基因家族进行生物信息学和表达模式分析,为深入研究CNL基因家族在黄瓜生长、发育和病害胁迫响应中的功能提供参考。【方法】以拟南芥CNL为参考序列,利用本地Perl语言和Pfam等软件检索黄瓜‘9930’基因组并确定黄瓜CNL基因家族成员。通过ExPASy、GSDS2.0、MEGA、MEME、Tbtools、Mev等工具对黄瓜CNL家族基因进行生物信息学分析。根据转录组数据库、霜霉病菌(Pseudoperonospora cubensis)、白粉病菌(Podosphaera xanthii)接种处理和实时荧光定量PCR技术分析该类基因的表达模式。【结果】从黄瓜全基因组中鉴定得到17个CsCNL,这些基因分布在除1号染色体外的6条染色体上,其编码蛋白与其他植物CNL结构相似,均含有CC、NBS和LRR保守结构域,蛋白质大小介于197—1148 aa,分子量在22.6—131.3 kD,等电点在5.71—8.38。共线性分析表明其中不存在片段重复和串联重复基因,多物种系统进化关系表明,CsCNL基因家族成员在葫芦科植物之间具有较高的结构和功能相似性。CsCNL启动子中存在多种与抗病相关的顺式作用元件。CsCNL表达具有组织特异性。在接种霜霉病菌2 d和5 d后,Csa3G815400、Csa3G822360和Csa7G420890在抗性品种中均显著上调表达,Csa4G015850在抗性品种中下调表达,在敏感品种中显著性表现不一;在接种白粉病菌2 d和5 d后,Csa2G008000和Csa3G684170在敏感品种中显著上调表达,在抗性品种中显著下调表达;Csa4G016360、Csa7G420890和Csa7G425940在抗性品种中显著上调表达,在敏感品种中表达量无变化或显著下调。【结论】CsCNL家族成员具有组织表达特异性并且多数基因能够响应霜霉病菌和白粉病菌的胁迫。推测Csa3G815400、Csa3G822360和Csa7G420890表达量增加,Csa4G015850表达量减少可诱发抗病黄瓜品种的霜霉病抗病反应;Csa4G016360、Csa7G420890和Csa7G425940表达量增加,Csa2G008000和Csa3G684170表达量减少可诱发抗病黄瓜品种的白粉病抗病反应;而Csa7G420890表达量的增加能同时诱发抗病黄瓜品种的霜霉病和白粉病的抗病反应。

【Objective】The bioinformatics and expression pattern analysis of the CC-NBS-LRR(CNL)gene family of the whole cucumber(Cucumis sativus)genome was carried out to provide a reference for further study of the functions of the CsCNL gene family in growth,development and disease stress response.【Method】Taking the CNL gene sequence of Arabidopsis thaliana as the reference,the genome of cucumber‘9930’was searched by local Perl language and Pfam software,and the members of CsCNL gene family were identified.CsCNL gene family was analyzed by some bioinformatics tools,such as ExPASY,GSDS2.0,MEGA,MEME,Tbtools and Mev.Transcriptome data,inoculating with downy mildew and powdery mildew pathogens(Pseudoperonospora cubensis and Podosphaera xanthii)and real-time quantitative PCR(qRT-PCR)were used to evaluate the expression of the genes.【Result】A total of 17 Cs CNL genes were identified from cucumber genome.Cs CNL genes are distributed on 6 chromosomes except chromosome 1.The encoded proteins are similar in structure to other plant CNLs.All CsCNL proteins contain conserved domains of CC,NBS and LRR.The amino acid sequence size ranged from 197 to 1148 aa,with a molecular weight(MW)of 22.6 to 131.3 kD,and a protein isoelectric point(PI)ranged from 5.71 to 8.38.Collinear analysis showed that there were no segmental duplication and tandem duplication genes,and the multi-species phylogenetic relationship showed that CNL gene family members had high structural and functional similarities among Cucurbitaceae plants.There were many cis-acting elements related to disease resistance in the promoter of Cs CNL genes.The expression of Cs CNL genes was tissue-specific.At 2 d and 5 d after inoculation with P.cubensis,Csa3G815400,Csa3G822360 and Csa7G420890 were all significantly up-regulated in resistant variety,Csa4G015850 was down-regulated in resistant variety,and significantly different in susceptible variety.At 2 d and 5 d after P.xanthii inoculation,Csa2G008000 and Csa3G684170 were significantly up-regulated in susceptible variety and significantly down-regulated in resistant variety.Csa4G016360,Csa7G420890 and Csa7G425940 were significantly up-regulated in resistant variety,while unchanged or significantly down-regulated in susceptible variety.【Conclusion】Cs CNL gene family has specific expression pattern in different tissues,and most of the Cs CNL genes were induced by P.cubensis and P.xanthii.It is speculated that the increased expression of Csa3G815400,Csa3G822360 and Csa7G420890,and the decreased expression of Csa4G015850 can induce downy mildew resistance in resistant cucumber varieties.The increased expression of Csa4G016360,Csa7G420890 and Csa7G425940,and the decreased expression of Csa2G008000 and Csa3G684170 can induce the resistance to powdery mildew in resistant cucumber varieties,while the increased expression of Csa7G420890 can simultaneously induce disease resistance to downy mildew and powdery mildew in resistant cucumber varieties.