黄龙病菌侵染晚锦橙叶脉和根早期病原蛋白组学的比较分析  

Proteomics analysis of early pathogens after infection of Candidatus Liberibacter asiaticus with Jincheng Orange(Citrus sinensis Osbeck)

作　　者：赵珂[1] 谢竹 杜美霞 龙俊宏 何永睿[1] 陈善春[1] 邹修平[1] ZHAO Ke;XIE Zhu;DU Meixia;LONG Junhong;HE Yongrui;CHEN Shanchun;ZOU Xiuping(Citrus Research Institute,Southwest University/Chinese Academy of Agricultural Sciences/National Citrus Engineering Research Center/National Center for Citrus Varieties Improvement,Chongqing 400712,China)

机构地区：[1]西南大学·中国农业科学院柑桔研究所·国家柑桔工程技术研究中心·国家柑桔品种改良中心,重庆400712

出　　处：《果树学报》2021年第4期485-496,共12页Journal of Fruit Science

基　　金：国家自然科学基金(31972393);科技部国家重点研发计划专项(2018YFD0201500);国家现代农业产业技术体系建设专项资金(CARS-26);广西创新驱动发展专项资金项目:耐(抗)黄龙病柑橘种质资源收集、相关抗性基因挖掘及新种质创制(桂科AA18118046-6)。

摘　　要：【目的】黄龙病(Huanglongbing,HLB)是世界柑橘生产上最具毁灭性的病害,由于黄龙病病原菌无法离体培养,其关键的致病机制仍不清楚。为此,通过蛋白组学分析探究黄龙病菌侵染晚锦橙早期时病原蛋白的表达情况。【方法】利用叶圆片嫁接技术对晚锦橙(Citrus sinensis Osbeck)进行嫁接传毒,在传毒2个月时,对感病后的根和叶脉组织进行蛋白组测序,以黄龙病亚洲种(Candidatus Liberibacter asiaticus,CLas)基因组为参考,对测序结果进行注释,并以感病叶脉为对照,筛选病原菌差异表达蛋白,最后利用qRT-PCR对显著性差异表达蛋白质进行验证。【结果】共鉴定出38个病原蛋白,其中有6个显著性表达差异蛋白;GO功能注释揭示这些病原蛋白主要与核酸结合和物质转运活性相关;qRT-PCR结果显示,6个显著差异蛋白在根部组织中的表达水平显著高于叶片组织;其中ABC转运系统重要组成成分CLasMalE(ACT56611.1)转运蛋白基因的表达倍数高达595倍。【结论】在黄龙病菌侵染柑橘根和叶的早期,与病原菌物质运输和核酸代谢相关基因的表达差异明显,暗示其在黄龙病菌侵染寄主中起着重要作用,为黄龙病菌致病机制研究提供了基因资源和理论基础。【Objective】Huanglongbing(HLB)is the most devastating disease in citrus production throughout the world.Because Candidatus Liberibacter asiaticus(CLas),the most virulent pathogen of HLB identified at present,cannot be cultured in vitro,its main pathogenic mechanism is still unrevealed.Therefore,proteomics analysis was used to explore the expression of pathogenic proteins during the early CLas infection stage in Citrus sinensis Osbeck.【Methods】In this study,the leafdisc grafting was used to infect the healthy Jincheng Orange(Citrus sinensis Osbeck)to transmit the CLas.Two months later,the CLas was detected by PCR,then the infected roots and leaf veins were collected for iTRAQ proteome Sequencing.The sequencing result was annotated while the genome of the CLas was used as a reference,and the infected leaf veins were used as control to screen the proteins of the CLas which expressed differently.Finally,qRT-PCR was used to verify those significantly expressed differently proteins.【Results】A total of 38 pathogenic proteins were identified,13 pathogenic proteins were up-regulated and 14 pathogenic proteins were down-regulated while there were 7 pathogenic proteins with unknown functions.The proteome of the infected roots was screened,the infected leaf veins were used as control,the difference fold value more than 1.5 times were used as a significant up-regulation and less than 1/1.5 as a significant down-regulation standard.As a result,there were 2 proteins(ACT56611.1.ACT66821.4)were significantly up-regulated,and 4 proteins were significantly downregulated(ACT56726.1,ACT56912.1,ACT57006.1,ACT57090.1).Gene ontology(GO)analysis was performed on the 38 identified pathogenic proteins.The results showed that pathogenic proteins were mainly involved in 10 biological processes,but were mainly related to nucleic acid binding and material transport activities.All pathogenic proteins were involved in 10 molecular functions and there were 22 related to the cellular biosynthetic process,accounting for 57.9%of the total

关 键 词：柑橘黄龙病 晚锦橙 蛋白组 QRT-PCR ABCtransporter MalE 

分 类 号：S666[农业科学—果树学]