复合亚氯酸钠灭活烟草花叶病毒的机理分析  

Inactivation mechanism of tobacco mosaic virus with composite sodium chlorite

作　　者：杨明明 陈泽鹏[2] 邓海滨 沈会芳[4] 杨祁云[4] 林壁润[4] 阮小蕾[1] YANG Mingming;CHEN Zepeng;DENG Haibin;SHEN Huifang;YANG Qiyun;LIN Birun;RUAN Xiaolei(Guangdong Key Laboratory of Microbial Signals and Disease Control,College of Plant Protection,South China Agricultural University,Guangzhou 510642,China;Tobacco Leaf Management Office of Guangdong Provincial Tobacco Corporation of CNTC,Guangzhou 510610,China;Guangdong Institute of Tobacco Science,Shaoguan 512026,Guangdong,China;Guangdong Provincial Key Laboratory of High Technique for Plant Protection,Plant Protection Research Institute,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China)

机构地区：[1]华南农业大学植物保护学院广东省微生物信号与作物病害防控重点实验室,广州市510642 [2]中国烟草总公司广东省公司烟叶管理处,广州市510610 [3]广东省烟草科学研究所,广东省韶关市512026 [4]广东省农业科学院植物保护研究所广东省植物保护新技术重点实验室,广州市510640

出　　处：《烟草科技》2022年第10期1-9,共9页Tobacco Science & Technology

基　　金：广东省烟草专卖局(公司)科技项目“广东省烟草病虫害绿色防控”(粤烟科专项201701)。

摘　　要：为揭示复合亚氯酸钠(500 mg/L)杀灭烟草花叶病毒(Tobacco mosaic virus,TMV)的机理,采用荧光定量RT-PCR检测药剂处理后的病毒CP基因和Rep基因表达量,并利用间接酶联免疫吸附法测定了TMV CP的破坏程度;同时接种心叶烟,通过枯斑数统计分析了TMV的侵染活性;利用大片段步移RT-PCR检测TMV相关基因片段的损伤,并使用透射电镜观察药剂处理后病毒粒子被破坏的情况。结果显示:①药剂处理30 min可以完全抑制TMV CP基因的表达,处理60 min可完全抑制TMV Rep基因的表达。此时,TMV CP被完全破坏,病毒也完全丧失了对枯斑寄主心叶烟的侵染能力。②药剂处理30 min时,TMV的183kDa-1、183kDa-2、183kDa-3、183kDa-4、54kDa、CP&MP基因编码区等6个区域中的183kDa-2、183kDa-4和CP&MP区域首先被损伤,但接种叶叶脉和接种叶叶柄上的这3个区域可以正常扩增。处理45 min时,接种叶叶脉、接种叶叶柄和接种叶上部叶片的183kDa-1、183kDa-3和54kDa区域可以正常扩增,而其他3个区域不能正常扩增;对照叶、茎、接种叶下部叶片的6个区域均不能正常扩增。处理60 min时,各部位的基因组片段扩增结果均呈阴性。因此,可以确定药剂处理导致的CP损伤是造成病毒侵染性丧失的主要因素,核酸损伤与病毒失活无关。③透射电镜观察发现,药剂处理1.5 h后病毒粒体完全断裂成碎片状。To study the mechanism for treating tobacco mosaic virus(TMV)with compound sodium chlorite(500 mg/L),the expression levels of the virus CP gene and rep gene were detected by fluorescence quantitative RT-PCR,and the damage degree of TMV CP was determined by indirect enzyme-linked immunosorbent assay.After inoculating TMV to Nicotiana glutinosa,the infection activity of TMV was analyzed by counting the number of withered spots.The damages of corresponding TMV gene fragments were detected by large fragment step-by-step RT-PCR,and the destruction of virus particles was observed by transmission electron microscope.The results showed that:1)The expression of TMV CP gene was completely inhibited by the 30-min drug treatment and the expression of TMV rep gene was completely inhibited by the 60-min drug treatment.After the treatment,TMV CP was completely destroyed,and the virus also completely lost its ability to infect the host Nicotiana glutinosa.2)After the 30-min drug treatment,183kDa-2,183kDa-4 and coding regions of CP&MP among six regions(183kDa-1,183kDa-2,183kDa-3,183kDa-4,54kDa and the coding regions of CP&MP genes)of TMV were damaged first,but the amplifications of the three regions in leaf vein and petiole of inoculated tobacco were normal.After treating for 45 min,the amplifications of the 183kDa-1,183kDa-3and 54kDa regions of leaf veins,petioles and upper leaves were normal,while those of the other three gene regions were not.The amplifications of the six gene regions of the control leaf,stem and lower leaf of inoculated tobacco were abnormal.After treating for 60 min,the amplifications of all the regions were negative.Therefore,it could be determined that CP damage caused by the drug treatment was the main factor leading to the disappearance of virus infectivity,and nucleic acid damage was not related to virus inactivation.3)Transmission electron microscopy indicated that the virus particles were completely broken into fragments after 1.5 h drug treatment.

关 键 词：烟草花叶病毒(TMV) 复合亚氯酸钠 荧光定量RT-PCR 枯斑 大片段步移RT-PCR 

分 类 号：S432.41[农业科学—植物病理学]