原核表达dsRNA抗番木瓜畸形花叶病毒的研究  

dsRNA of Prokaryotic Expression for Resistance to Papaya Leaf Distortion Mosaic Virus

作　　者：张涛[1,2] 沈文涛[2] 言普[2] 黎小瑛[2] 周鹏[1,2,3] 

机构地区：[1]海南大学农学院,海南海口570228 [2]中国热带农业科学院热带生物技术研究所,海南海口571101 [3]中国热带农业科学院分析测试中心,海南海口571101

出　　处：《热带作物学报》2014年第7期1416-1423,共8页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金(No.31171822);海南省重点科技计划(No.ZDXM20120027);海南省重大科技专项(No.ZDZX2013023-1-08)

摘　　要：番木瓜畸形花叶病毒（Papaya leaf distortion mosaic virus,PLDMV）是一种已对番木瓜种植业构成潜在威胁的新型病害,在转基因植物备受争议的背景下,利用原核表达的dsRNA来防控病毒的策略不失为一种新的选择.本文选取PLDMV CP基因全长（879 bp）,运用OZ-LIC法构建了含有pdk内含子的ihpRNA结构,将其嵌入原核表达载体pSP73中,并转化RNaseⅢ缺陷型大肠杆菌M-Jm109LacY,构建了1种能高表达dsRNA的原核表达菌株M-Jm109LacY-CP879,以终浓度为0.4 mmol/LIPTG诱导后能够稳定高效的表达CP879-dsRNA.研究共设2个处理：保护性处理与治疗性处理.分别用含有CP879-dsRNA的粗提液对这2个处理的番木瓜植株进行喷施处理,通过统计发病率、观察病症变化以及ELISA分析结果表明,保护性处理能有效抑制番木瓜畸形花叶病毒的侵染,主要表现为发病时间推迟和相对较低的发病率;治疗性处理植株的病毒积累量会在喷施后第3～12天内出现暂时性的降低.结果表明,保护效果优于治疗效果,若每月喷施2～3次dsRNA粗提液,有望能够有效地预防PLDMV的侵染.Papaya leaf distortion mosaic virus （PLDMV） is a potential threat to papaya cultivation.Nowadays,transgenic plants is highly controversial,and using the prokaryotic expressed dsRNA to control the virus could be a new choice.The full length of PLDMV CP gene （879 bp） was chosen to construct a hairpin RNA structure which contains pdk intron by OZ-LIC method.After the coding structure of hairpin RNA inserted into the prokaryotic expression plasmid of pSP73,the recombinant vector was transformed into the RNase Ⅲ-deficient Escherichia coli strain of M-Jm109LacY.Then a prokaryotic expression strain with high expression level of CP879-dsRNA was successfully constructed,named M-Jm109LacY-CP879.The recombinant E.coli strain could express CP879-dsRNA stably when induced with IPTG at 0.4 mmol/L.There are two assays in this study：a protective resistance assay and a therapeutic resistance assay.In the protective assay,CP879-dsRNA was used to spray onto the plant leaves before inoculation of PLDMV.The disease incidence,symptom change and ELISA analysis results revealed that the CP879-dsRNA could inhibit the virus＇ accumulation.In the therapeutic assay,CP879-dsRNA was used to spray onto the plants that had been inoculated with PLDMV after 25 days.ELISA analysis results showed that the virus accumulation declined temporarily during the 3th to 12th day after spraying with CP879-dsRNA.The results showed that the effect of protective treatment was better than that of therapeutic treatment.If sprayed with CP879-dsRNA two or three times a month,the plants may be able to prevent PLDMV infection.

关 键 词：番木瓜畸形花叶病毒 原核表达 DSRNA RNA沉默 

分 类 号：S667.97[农业科学—果树学]