红橘响应褐斑病菌侵染的转录组学分析  

Transcriptome Analysis of Citrus reticulata Blanco, cv. Hongjv Infected with Alternaria alternata Tangerine Pathotype

作　　者：唐科志[1] 周常勇[1] TANG KeZhi;ZHOU ChangYong(Citrus Research Institute,Southwest University,Chongqing 400712)

机构地区：[1]西南大学柑桔研究所,重庆400712

出　　处：《中国农业科学》2020年第22期4584-4600,共17页Scientia Agricultura Sinica

基　　金：国家现代农业产业技术体系专项(CARS-27)。

摘　　要：【目的】明确红橘(Citrus reticulata Blanco, cv. Hongjv)接种褐斑病致病菌——链格孢菌橘致病型(Alternaria alternata tangerine pathotype)后基因种类和表达量在转录水平的变化规律,确定红橘响应该致病型侵染的关键基因。【方法】采用链格孢菌橘致病型接种红橘离体叶片,28 h后选取感病叶片和未接种叶片提取RNA,进行转录组高通量测序,然后利用生物信息学分析,以甜橙基因组为参考,以|log2 fold change|≥1,q-value≤0.01为阈值选取感病和健康红橘叶片转录组的差异表达基因,应用GO数据库对差异表达基因(differentially expressed gene,DEG)进行功能分类,KEGG分析代谢途径,MapMan软件分析生物胁迫信号通路相关基因的表达变化。采用qRT-PCR方法对测序结果进行验证。【结果】红橘接种链格孢菌橘致病型28 h后产生大量与胁迫相关的差异表达基因,获得上调差异基因5 173个,下调差异基因6 555个。GO功能分类显示差异基因主要与蛋白结合、膜、氧化还原过程等相关。通过KEGG富集和MapMan软件分析发现,红橘在受链格孢菌橘致病型胁迫的过程中基础代谢被严重破坏。乙烯、水杨酸和生长素等寄主防御相关的植物激素信号转导途径多个基因表达出现差异,其中乙烯起主导作用,乙烯受体ETR 3个成员被不同程度激活,下游激酶和乙烯响应因子均上调,生长素大部分关键信号基因、绝大部分生长素响应因子ARF和水杨酸合成途径的基因均下调表达。同时,黄酮醇、花青素、萜类化合物和生物碱合成相关基因受该菌诱导显著变化,萜类合成中大部分基因下调,而黄酮类合成相关上调基因数量和表达趋势均强于表达下调基因,有抗虫和抑菌作用的硫代葡萄糖苷基因呈上调趋势。进一步研究发现,大量参与抗逆过程的转录因子如WRKY、bZIP、ERF、MYB、NAC被诱导激活,其中大部分WRKY和bZIP转录因子受该菌正向调控,超过【Objective】The objective of this study is to reveal the gene expression pattern at transcriptional level of tangerine(Citrus reticulata Blanco, cv. Hongjv) after inoculated with the brown spot pathogen(Alternaria alternata tangerine pathotype), and to identify the key genes of tangerine responding to the pathogen infection.【Method】Detached leaves of tangerine were inoculated with A. alternata tangerine pathotype for 28 h and used for RNA extraction and transcriptome sequencing. Sequencing results were verified by quantitative real-time PCR(qRT-PCR). Differentially expressed genes(DEGs) were screened with |log2 fold change|≥1, q-value≤0.01 as threshold and clean reads were compared with genome of sweet orange(Citrus sinensis). GO database was used to perform functional classification of DEGs, KEGG was used to analyze metabolic pathways, and MapMan software was used to analyze the expression changes of genes related to biological stress signaling pathways.【Result】A large number of DEGs related to stress were produced at 28 h post inoculation, 5 173 up-regulated and 6 555 down-regulated genes were obtained. GO functional classification showed that the DEGs were mainly related to protein binding, membrane and oxidation-reduction process. KEGG enrichment and MapMan software analysis revealed that the basic metabolism of tangerine was severely damaged during the stress of A. alternata tangerine pathotype. The expression of multiple genes in host defense-related plant hormone signal transduction pathways such as ethylene(ET), salicylic acid(SA) and auxin(AUX) varied to some extent, of which ET plays a key role. The three members of the ET receptor ETR were activated to different degrees, among which downstream kinases and ET response factors were up-regulated, whereas most key signal genes of AUX, most members of AUX response factor ARF and genes in SA synthesis pathway were all down-regulated. Meanwhile, genes related to flavonol, anthocyanins, terpenoids and alkaloid synthesis were drastically changed

关 键 词：红橘 链格孢菌橘致病型 转录组 差异表达基因 

分 类 号：S436.66[农业科学—农业昆虫与害虫防治]