胶孢炭疽菌侵染不同抗性葡萄叶片的转录组学分析  

Transcriptomic analysis of different resistant leaves infected by Colletotrichum gloeosporioides in grape

作　　者：沈才琦 孙磊[1] 张颖[1] 姜建福[1] 刘崇怀[1] 樊秀彩[1] SHEN Caiqi;SUN Lei;ZHANG Ying;JIANG Jianfu;LIU Chonghuai;FAN Xiucai(Zhengzhou Fruit Research Institute,Chinese Academy of Agricultural Sciences,Zhengzhou 450009,Henan,China)

机构地区：[1]中国农业科学院郑州果树研究所,郑州450009

出　　处：《果树学报》2022年第5期730-742,共13页Journal of Fruit Science

基　　金：国家现代农业产业技术体系建设专项(CARS-29-yc-1);中国农业科学院科技创新工程(CAAS-ASTIP-2019-ZFRI);国家园艺种质资源库运行服务(NHGRC2021-NH00-2);郑州市重大科技创新专项(2020CXZX0082)。

摘　　要：【目的】通过转录组测序筛选葡萄由胶孢炭疽菌侵染引起的差异表达基因,以进一步分析葡萄抗炭疽病分子机制。【方法】用胶孢炭疽菌侵染不同抗性葡萄的叶片,以清水处理叶片为对照,设置0、24、48、72 h 4个时期取样。采用RNA-seq分析两者间的差异表达基因,挑选出抗病候选基因,并利用qRT-PCR进行验证。【结果】共对42个样品进行转录组测序,每个样品平均数据量为6.65 Gb。KEGG富集分析中差异表达基因主要集中在植物与病原体互作、类黄酮生物合成和MAPK信号途径;转录因子家族分析从MYB、AP2-EREBP、bHLH、NAC和WRKY家族中筛选出12个在抗病株系中高表达的差异表达基因;WGCNA得到抗病相关模块MEdarkgreen。综合各项分析,挑选出9个差异表达基因进行q RT-PCR验证,其中8个基因的表达趋势同转录组测序结果一致。【结论】获得了葡萄响应胶孢炭疽菌侵染的差异表达基因,显著富集在植物与病原体相互作用、类黄酮生物合成和MAPK信号途径等3个通路,并筛选出8个抗病候选基因,包括MLO蛋白,WRKY、ERF转录因子等。【Objective】Grape anthracnose is a fungal disease caused by Colletotrichum gloeosporioides.It is one of the four major diseases of grapes due to its wide distribution and great harm.Grape anthracnose has a great negative impact on the yield of grape berries.At present,there are many researches on the prevention and control methods of grape anthracnose,but there are few reports on grape anthracnose disease resistance genes.In this study,RNA-seq was used to screen the differentially expressed genes(DEGs)caused by C.gloeosporioides in grapes to further analyze the molecular mechanism of grape anthracnose resistance.【Methods】The experimental materials were collected from the Zhengzhou Grape Garden of the National Fruit Species Fund of the Zhengzhou Fruit Research Institute,Chinese Academy of Agricultural Sciences.The anthracnose-susceptible progeny 7-2-6 strains and the anthracnose-resistant progeny 7-1-8 strains selected from the hybrid progeny through resistance identification were used as the research materials;the C.gloeosporioides were inoculated on the PDA medium and placed in a thermotank at a constant temperature of 28℃for two to three weeks,and the leaves were inoculated by the method of bacterial cake inoculationand placed in a 28℃incubator under moisturizing conditions.Simultaneously,the leaves sprayed with pure water on the stab wound were used as controls.The leaves were collected at 0,24,48 and 72 h after inoculation.Three biological replicates were set for samples at all periods.They were frozen by liquid nitrogen and stored in a refrigerator at-80℃for later use.The high-throughput second-generation sequencing technology RNA-seq was used for sequencing,and the grape genome 12×PN40024 was used as the reference gene for comparison.Bowtie2 clean reads were aligned to the reference sequence to calculate the gene alignment rate,and RSEM was used to calculate the expression level of genes and transcripts.DEGseq algorithm detected DEGs,and selected genes with a multiple of more than twice the di

关 键 词：葡萄炭疽病 转录组 胶孢炭疽菌 差异表达基因 

分 类 号：S663.1[农业科学—果树学]