基于VIGS的柑橘黄化脉明病毒防控技术研究  

Research on Prevention and Control Technology of Citrus Yellow Vein Clearing Virus Based on VIGS

作　　者：李楚欣 宋晨虎 周金环 李佳欣 王新亮 田旭斌 宋震 LI ChuXin;SONG ChenHu;ZHOU JinHuan;LI JiaXin;WANG XinLiang;TIAN XuBin;SONG Zhen(Citrus Research Institute,Southwest University/National Citrus Engineering Research Center,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,重庆400712

出　　处：《中国农业科学》2024年第22期4473-4482,共10页Scientia Agricultura Sinica

基　　金：重庆市技术创新与应用发展重点项目(CSTB2024TIAD-KPX0063);国家现代农业(柑橘)产业技术体系(CARS-26-05B)。

摘　　要：【目的】柑橘黄化脉明病毒(citrus yellow vein clearing virus,CYVCV)是一种严重危害柑橘产业的新发病毒,目前尚无有效的治疗药剂,主要通过使用无病毒苗木和严格防控传播虫媒进行防控。本研究旨在探索基于病毒介导的基因沉默(virus-induced gene silencing,VIGS)的CYVCV防控技术,以期获得抗病毒“疫苗”,为柑橘病毒病防控提供新思路。【方法】基于实验室前期构建的VIGS载体pCLBV201,针对CYVCV基因组ORF1(open reading frame 1)、ORF6、CP(coat protein)和TGB(triple gene block)的保守区域设计、构建一系列重组载体,通过农杆菌介导的真空浸润接种尤力克柠檬并进行RT-PCR检测。获得pCLBV201-ORF1、pCLBV201-ORF6、pCLBV201-CP、pCLBV201-TGB阳性植株后,通过农杆菌介导的注射法接种CYVCV侵染性克隆,并以pCLBV201空载接种植株为对照,开展RT-qPCR、Western blot检测、症状观察和病情指数统计,从而明确不同VIGS重组载体对CYVCV的防控效果。【结果】构建了系列pCLBV201重组载体,接种后的RT-PCR检测结果表明,分别获得p CLBV201-ORF1、pCLBV201-ORF6、pCLBV201-CP、pCLBV201-TGB阳性植株多株;注射接种CYVCV侵染性克隆,7、14、28、70和150 dpi(days post infection)的RT-qPCR检测结果显示,pCLBV201-CP和pCLBV201-TGB处理组的CYVCV相对表达量均较对照显著降低;70和150 dpi Western blot检测结果表明,pCLBV201-CP和pCLBV201-TGB处理组的CP蛋白表达量显著下降;症状观察显示,对照组表现为严重的脉明、叶片扭曲等CYVCV侵染典型症状,pCLBV201-CP处理组植株的症状轻微,pCLBV201-TGB处理组植株不表现明显症状;70和150 dpi病情指数统计显示,pCLBV201-CP和pCLBV201-TGB的病情指数最低,分别为17.6、41.2和15.6、29.1,而对照组为52.1、80.0,差异明显。【结论】研发了基于VIGS的CYVCV防控技术,明确了pCLBV201-CP、pCLBV201-TGB能够显著降低病毒滴度、减轻病毒引起症状,有用作CYVCV防控“疫苗”的潜力。【Objective】Citrus yellow vein clearing virus(CYVCV)is a novel virus that poses a significant threat to the citrus industry.Currently,there is no effective therapeutic agent.The primary strategies for prevention and management involve utilizing virus-free seedlings and implementing stringent control measures against insect vectors.The objective of this study is to investigate the technology of virus-induced gene silencing(VIGS)as a means of developing antiviral“vaccines”for CYVCV,and to offer innovative approaches for the prevention and management of citrus viral diseases.【Method】Based on the previously constructed VIGS vector pCLBV201 in the laboratory, a series of recombinant vectors were designed and developed to target the conserved regions of the CYVCV genome, specifically open reading frame 1 (ORF1), open reading frame 6 (ORF6), coat protein (CP), and triple gene block (TGB). Agrobacterium-mediated vacuum infiltration was inoculated on Eureka lemon, followed by RT-PCR detection. After acquiring several positive plants including pCLBV201-ORF1, pCLBV201-ORF6, pCLBV201-CP, and pCLBV201-TGB, CYVCV infectious clones were inoculated via Agrobacterium-mediated injection, with plants infiltrated with the pCLBV201 empty vector serving as controls. Subsequent RT-qPCR, Western blot analysis, symptom observation, and disease index statistics were conducted to elucidate the preventive and control effects of the various VIGS recombinant vectors on CYVCV. 【Result】A series of recombinant vectors of pCLBV201 were constructed, and the results of RT-PCR following inoculation indicated that multiple positive plants for pCLBV201-ORF1, pCLBV201-ORF6, pCLBV201-CP, and pCLBV201-TGB were obtained, respectively. CYVCV infectious clones were inoculated by injection. The results of RT-qPCR at 7, 14, 28, 70, and 150 dpi (days post infection) showed that the relative expression of CYVCV in the pCLBV201-CP and pCLBV201-TGB treatment groups was significantly lower than that in the control group. Western blot analyses conduc

关 键 词：柑橘黄化脉明病毒 pCLBV201 病毒介导的基因沉默 RNA干扰 

分 类 号：S436.66[农业科学—农业昆虫与害虫防治]