斯里兰卡木薯花叶病毒TVM3分离物侵染性克隆构建及鉴定

Construction and Identification of Infectious Clone from Sri Lankan Cassava Mosaic Virus Tvm3 Isolate

【目的】斯里兰卡木薯花叶病毒(Sri Lankan cassava mosaic virus,SLCMV)是一种对世界木薯产业产生威胁的单链环状DNA病毒,为双生病毒科(Geminiviridae)菜豆金色黄花叶病毒属(Begomovirus)成员。我国木薯品种抗性不明且抗病种质资源缺乏,为进一步探究国内木薯种质资源抗花叶病的遗传背景以及SLCMV与宿主的互作机理,本研究开展SLCMV侵染性克隆构建及鉴定。【方法】以SLCMV(TVM3株系)为对象,将其1.10倍(mer)长的DNA-A和1.08倍(mer)的DNA-B组分分别构建到pCAMBIA1301载体,获得病毒侵染性克隆载体pCAMBIA1301-DNA-A(pDNA-A)和pCAMBIA1301-DNA-B(pDNA-B),利用GV3101农杆菌介导pDNA-A和pDNA-B共侵染本生烟和拟南芥。【结果】SLCMV侵染性克隆接种本生烟14 d后,系统叶可产生严重的花叶、扭曲等症状;而感染SLCMV的拟南芥叶片在18 d出现轻微的扭曲症状。提取感病本生烟和拟南芥植株新生叶片总DNA,PCR方法均检测到了病毒DNA-A和DNA-B组分。此外,本生烟的病毒感染率为100%,而拟南芥的病毒感染率仅为40%,两者差异显著(P<0.05)。【结论】本研究成功构建了SLCMV病毒侵染性克隆,且能侵染本生烟和拟南芥植物,其在本生烟中的病毒感染率高达96.67%;另外,该pDNA-A和pDNA-B侵染性克隆载体序列仅为病毒原基因组DNA-A和DNA-B大小的1.10倍(mer)和1.08倍(mer),且不含有重复的编码区序列,可为下一步探究国内木薯种质资源抗花叶病的遗传背景以及病毒致病机理研究提供重要基础。

【Objective】Sri Lankan cassava mosaic virus(SLCMV)is a circular single-stranded DNA virus,containing two components of DNA-A and DNA-B.SLCMV is a member of the genus Begomovirus in the family Geminiviridae,which is a persistent threat to the world’s cassava industry.In 2018,the SLCMV was found and reported for the first time in cassava from Fujian and Hainan provinces,China.However,the viral resistance of cassava varieties in domestic was unknown and the germplasm resources for disease resistance was lacking.To further explore the genetic background of domestic cassava germplasm resources resistance to mosaic disease and the mechanism of interaction between virus and host,SLCMV infectious clone was constructed and verified.【Method】In this study,SLCMV TVM3 strain was used as the object,and its 1.10 mer DNA-A and 1.08 mer DNA-B components were respectively constructed into pCAMBIA1301 vector,and the viral infectious clone vectors pCAMBIA1301-DNA-A(pDNA-A)and pCAMBIA1301-DNA-B(pDNA-B)were obtained.Agrobacterium tumefacien GV3101 mediated pDNA-A and pDNA-B co-infected tobacco and Arabidopsis plants.【Result】At 14 d post inoculation,the systematic leaves of tobacco plants produced severe mosaic and distortion symptoms,while the systematic leaves of Arabidopsis plants showed mild distortion at 18 d post inoculation.Total DNA was extracted from SLCMV infected tobacco and Arabidopsis plants leaves,and viral DNA-A and DNA-B components were detected by PCR method.In addition,the virus infection rate of tobacco plants was 100%,while the virus infection rate of Arabidopsis plants was only 40%,and the difference between the two group was significant(P<0.05).【Conclusion】The SLCMV infectious clone have been successfully constructed,which could infect tobacco and Arabidopsis plants,and the viral infection rate reached 96.67%in tobacco plants.Furthmore,the DNA sequences of infectious clone in pDNA-A and pDNA-B are 1.10 mer and 1.08 mer length of the original virus genomic DNA-A and DNA-B components,respectively,whichdid not contain repetitive coding region sequences.This study provides an important basis for the further explore the genetic background of domestic cassava germplasm resources resistance to mosaic disease and the pathogenesis of virus.