利用WGCNA鉴定谷子内源脱落酸响应禾生指梗霉胁迫的共表达基因

Identification of Co-Expression Genes Related to Endogenous Abscisic Acid in Response to the Stress of Sclerospora graminicola by WGCNA in Foxtail Millet

【目的】脱落酸(ABA)作为一类逆境激素,在植物生长发育、生物胁迫和非生物胁迫中发挥着重要作用。脱落酸受体蛋白PYR/PYL/PCAR及SNF1相关的蛋白激酶(SnRK2)是介导脱落酸信号转导的重要调控因子。本研究通过预测脱落酸及其信号转导途径中关键基因在谷子白发病致病菌禾生指梗霉(Sclerospora graminicola)中的调控作用,为谷子内源脱落酸响应禾生指梗霉侵染的互作研究提供参考。【方法】通过对禾生指梗霉侵染的晋谷21号谷子进行转录组测序和脱落酸含量测定,基于谷子全基因组对脱落酸信号转导通路上的PYL和SnRK2家族基因进行鉴定、分析,利用测定的转录组构建加权基因共表达网络(WGCNA),并与禾生指梗霉侵染引起的寄主内源脱落酸含量进行关联,预测脱落酸及其下游信号转导基因PYL和SnRK2在谷子与禾生指梗霉互作调控中的关键核心基因;利用qRT-PCR技术对候选基因进行验证。【结果】谷子中存在禾本科中较为保守的PYL和SnRK2家族基因各11个,且在PYL和SnRK2家族基因的启动子上均预测到脱落酸响应元件。在禾生指梗霉侵染后,寄主内源脱落酸在第一、第二时期大量积累,含量显著高于对照组,分别为22.50和18.08 ng·mL^-1,而在第三、第四和第五时期脱落酸含量下降,低于对照组。在基因共表达网络分析中,利用18535个基因共构建了34个基因共表达模块。通过对脱落酸含量和PYL、SnRK2家族基因的关联分析,预测到MEpaleturquoise和MEbrown模块为核心候选模块。利用GO功能富集和模块关键基因的挖掘共预测到1个PYL家族基因Seita.1G030500和2个SnRK2家族基因Seita.2G394500、Seita.3G03200,以及3个核心基因Seita.4G105600、Seita.6G218100和Seita.9G138400,共6个基因可能在脱落酸及其信号转导调控过程中参与谷子与禾生指梗霉的互作。对预测到的3个核心基因在水稻和拟南芥数据中进行比对,鉴定到Seita.4G105600为转导蛋白/WD40重复超家族蛋白、Seita.6G218100为WRKY57转录因子、Seita.9G138400为TIFY转录因子。qRT-PCR分析表明Seita.2G394500、Seita.4G105600和Seita.6G218100基因在谷子白发病早期表达均上调。【结论】谷子在受到禾生指梗霉侵染后脱落酸会在体内大量积累,预测到1个PYL家族基因、2个SnRK2家族基因、2个转录因子基因和1个WD40家族蛋白基因参与谷子内源脱落酸响应禾生指梗霉侵染过程。qRT-PCR结果表明1个SnRK2家族基因、1个WD40家族蛋白基因和1个WRKY57转录因子基因共3个基因可能在谷子脱落酸响应禾生指梗霉侵染过程中发挥重要作用。

【Objective】Abscisic acid(ABA),as a stress hormone,plays an important role in plant growth and development,biological and abiotic stresses.The ABA receptor protein PYR/PYL/PCAR and SNF1-related protein kinase(SnRK2)are important regulatory factors that mediate ABA signaling.The objective of this study is to predict the regulatory roles of ABA and the key genes in its signaling pathway in foxtail millet(Setaria italica)downy mildew caused by Sclerospora graminicola,and to provide a reference for the research of endogenous ABA in S.italica in response to the infection of S.graminicola.【Method】S.italica variety Jingu 21 infected by S.graminicola was used for transcriptome sequencing and ABA content measurement,and the PYL and Sn RK2 family genes in the ABA signaling pathway were identified and analyzed based on the whole genome sequence of S.italica.A weighted gene co-expression network analysis(WGCNA)was constructed using the transcriptome,and it was associated with the content of host endogenous ABA in the context of S.graminicola infection to predict the key genes in the interaction between S.italica and S.graminicola regulated by ABA and the PYL and SnRK2 genes in its downstream signaling.The candidate genes were validated by qRT-PCR.【Result】There were 11 PYL and 11 Sn RK2 family genes in S.italica,which were relatively conservative in the Gramineae,and ABA responsive elements were predicted in the promoters of PYL and SnRK2 family genes.The endogenous ABA in the host accumulated at the first and second stages after S.graminicola infection,and its content(22.50 and 18.08 ng·mL^-1,respectively)was significantly higher than that of the control group.However,the content of ABA decreased at the third,fourth,and fifth stages,which was lower than that of the control group.For the WGCNA,a total of 34 gene co-expression modules were constructed by using 18535 genes.The MEpaleturquoise and MEbrown modules were predicted as the core candidate modules through the association analysis of ABA content and PYL,SnRK2 family genes.GO function enrichment and module key gene mining revealed that one PYL family gene(Seita.1 G030500),two SnRK2 family genes(Seita.2 G394500 and Seita.3 G03200),and three core genes(Seita.4 G105600,Seita.6 G218100,and Seita.9 G138400)might be involved in the interaction between S.italica and S.graminicola during the regulation of ABA and its signal transduction.After comparing three predicted core genes with the reference genes in Oryza sativa and Arabidopsis thaliana databases,Seita.4 G105600 was identified as the transducin/WD40 repeat-like superfamily protein,Seita.6 G218100 as the WRKY57 transcription factor,and Seita.9 G138400 as the TIFY transcription factor.qRT-PCR analysis showed that Seita.2 G394500,Seita.4 G105600,and Seita.6 G218100 genes were up-regulated at the early stage of S.graminicola infection.【Conclusion】The ABA accumulates in S.italica infected by S.graminicola.One PYL family gene,two SnRK2 family genes,two transcription factor genes,and one WD40 family protein gene were predicted as the key genes related to the response to S.graminicola infection.qRT-PCR results showed that one SnRK2 gene,one WD40 family protein gene,and one WRKY57 transcription factor gene may play an important role in the response of ABA in S.italica to the infection of S.graminicola.