csi-miR399响应柑橘溃疡病菌侵染的表达模式及其抗病性分析  

Expression Pattern of csi-miR399 in Response to Xanthomonas citri subsp.citri Infection and Its Disease Resistance Analysis

作　　者：王兆昊 郭兴茹 张乐欢 何永睿 陈善春 姚利晓 WANG ZhaoHao;GUO XingRu;ZHANG LeHuan;HE YongRui;CHEN ShanChun;YAO LiXiao(Citrus Research Institute,Southwest University/National Citrus Engineering Technology Research Center/National Center for Citrus Varieties Improvement,Chongqing 400712)

机构地区：[1]西南大学柑桔研究所/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：《中国农业科学》2023年第8期1484-1493,共10页Scientia Agricultura Sinica

基　　金：国家重点研发计划(2021YFD1400800,2021YFD1600800);国家现代农业(柑橘)产业技术体系(CARS-26)。

摘　　要：【目的】明确csi-miR399响应柑橘溃疡病菌(Xanthomonas citri subsp.citri,Xcc)侵染的表达模式,筛选其靶基因,进而分析csi-miR399与寄主Xcc抗性的相关性,为柑橘溃疡病抗性种质的创制打下基础。【方法】分别以柑橘溃疡病抗性品种四季橘(Citrus microcarpa)和感病品种纽荷尔脐橙(Citrus sinensis)为试材,通过茎环qPCR方法分析csi-miR399在叶片离体注射Xcc 1、3和5 d时的表达量变化,明确抗/感品种中csi-miR399响应Xcc侵染的表达模式;利用在线软件psRNATarget预测csi-miR399的靶基因,并通过qPCR分析候选靶基因在接种Xcc柑橘叶片和瞬时表达csi-miR399叶片中表达量的变化;克隆csi-miR399前体基因序列,通过同源重组方法构建病毒表达载体pCLBV202-MIR399,根癌农杆菌介导的真空浸润法接种尤力克柠檬(Citrus limon),通过qPCR分析csi-miR399表达量;进而采用离体叶片针刺法接种Xcc,观察发病症状,统计病情指数,分析csi-miR399过表达对Xcc抗性的影响。【结果】接种Xcc后,csi-miR399在抗病品种四季橘中的表达呈现先下降再上升的趋势,而在感病品种纽荷尔脐橙中的表达呈持续下降趋势。接种Xcc 5 d时,csi-miR399在四季橘与纽荷尔脐橙中的表达量分别是健康对照的4.64倍和7.61%,初步表明csi-miR399与柑橘的溃疡病抗性相关。从13个预测靶基因中筛选鉴定了csi-miR399的3个靶基因Cs2g06030、Cs7g03830、Cs8g18800,分别编码泛素偶联酶PHO2、未知蛋白和漆酶。利用构建的病毒表达载体pCLBV202-MIR399获得过表达csi-miR399的柠檬植株(Y37、Y41和Y57),与空载体对照pCLBV202接种植株(L35)相比,Y37、Y41和Y57中csi-miR399表达量显著增加,接种Xcc后的溃疡病病斑面积显著减小,病情指数显著降低(P<0.01),表明csi-miR399过表达显著提高了柠檬的溃疡病抗性。【结论】csi-miR399与柑橘的溃疡病抗性密切相关,过表达csi-miR399显著提高柑橘对溃疡病的抗性,可应用于柑橘抗溃疡病的【Objective】The objective of this study is to identify the expression pattern of csi-miR399 in response to the infection of citrus canker bacteria(Xanthomonas citri subsp.citri,Xcc),screen its target genes,analyze the correlation between csi-miR399 and Xcc resistance in host plants,and to lay a foundation for the creation of citrus canker resistant germplasms.【Method】In order to clarify the expression pattern of csi-miR399 in response to Xcc infection,Xcc-resistant variety Calamondin(Citrus microcarpa)Xcc-sensitive variety Newhall Navel Orange(Citrus sinensis)were used as materials,and changes in the relative expression of csi-miR399 were analyzed by stem-loop qPCR after their leaves were injected with Xcc at 1,3 and 5 d in vitro.The online software psRNATarget was used to predict the target genes of csi-miR399,which were further confirmed by qPCR in citrus leaves infected with Xcc and transiently over-expressed with csi-miR399.The viral expression vector pCLBV202-MIR399 was constructed by in-fusion cloning through csi-miR399 precursor sequence being inserted into pCLBV202,and transferred into Eureka Lemon(Citrus limon)by Agrobacterium tumefaciens-mediated vacuum infiltration.The lemon over-expressed with csi-miR399 was evaluated for resistance against Xcc through being stab-inoculated with the pathogen and investigated disease index.【Result】After inoculation with Xcc,the expression of csi-miR399 in Calamondin showed a downward trend and then an upward trend,while that in Newhall Navel Orange continued to decrease.At 5 d,the expression of csi-miR399 in Calamondin and Newhall Navel Orange was 4.64 times and 7.61%as its expression in healthy leaves,respectively,preliminary indicating that csi-miR399 was related to citrus canker resistance.Thirteen predicted target genes were screened from citrus genome.Three of them were confirmed because of the opposite expression trends with csi-miR399,which were Cs2g06030(PHO2),Cs7g03830(unknown protein),and Cs8g18800(laccase).Three lemon strains(Y37,Y41 and Y57)with

关 键 词：csi-miR399 柑橘溃疡病 靶基因 柑橘病毒表达载体 生物胁迫 

分 类 号：S436.66[农业科学—农业昆虫与害虫防治]