利用VIGS防控烟草马铃薯Y病毒的研究

发布时间：2019-06-27 10:55

【摘要】：马铃薯Y病毒(potato virus Y,PVY)是马铃薯Y病毒组的典型成员,主要依靠烟蚜、桃蚜及人为机械性损伤在烟草上传播和扩散。多年来,由该病毒引起烟草病毒病在我国烟草种植区多有发生,该病危害严重、防治困难,对我国烟草生产造成极大经济损失。随着RNA沉默技术的发展,为马铃薯Y病毒病在烟草上的防治提供全新的途径。本研究以马铃薯Y病毒脉坏死株系(PVYN)为研究对象,设计带有酶切位点的特异性引物,通过PCR扩增PVY、的CP和HC-pro2个基因片段。以PVY、的CP.HC-pro基因为目的基因,烟草脆裂病毒(TRV)改造载体PTV00为病毒载体,通过BamHI/KphI2个限制性内切酶构建PTV00-CP、PTV00-HC-pro重组载体,使用电击法将重组载体转入农杆菌GV3101中。通过PCR、酶切、测序等验证目的片段已转入农杆菌GV3101中,被试烟苗剪叶后使用农杆菌菌液喷施,通过剪叶后造成的伤口将重组表达载体导入烟草中,诱导烟草V1GS。通过人工接种PVY病毒,调查被试烟草抗病性实验证明无论在实验条件下还是自然条件下,都能有效的降解PVY CP、HC-pro基因表达,从而表现出抗马铃薯Y病毒。在自然条件下,设置8个处理、共760株G80烟草,抗性试验表明导入CP基因的烟草对PVY防治效果为83.11%,导入HC-pro基因的烟草对PVY防治效果为86.83%,同时导入CP和HC-pro基因的烟草对PVY防治效果为78.34%。
[Abstract]:Potato Y virus (potato virus Y (PVY) is a typical member of potato Y virus group, which mainly relies on tobacco aphids, peach aphids and artificial mechanical damage to spread and spread in tobacco. For many years, tobacco virus disease caused by this virus has occurred in tobacco growing areas of our country, which has serious harm and difficult prevention and control, and has caused great economic losses to tobacco production in our country. With the development of RNA silencing technology, a new way to control potato Y virus disease in tobacco is provided. In this study, potato Y virus pulse necrosis strain (PVYN) was used as the research object, and specific primers with enzyme digestion sites were designed to amplify the CP and HC-pro2 gene fragments of PVY, by PCR. Using CP.HC-pro gene of PVY, as target gene, tobacco embrittlement virus (TRV) modified vector PTV00 as virus vector, PTV00-CP,PTV00-HC-pro recombinant vector was constructed by BamHI/KphI2 restriction endonuclease, and the recombinant vector was transferred into Agrobacterium tumefaciens GV3101 by electric shock method. The target fragment was transferred into Agrobacterium tumefaciens GV3101 by PCR, enzyme digestion and sequencing. The tested tobacco seedlings were cut and sprayed with Agrobacterium tumefaciens solution. The recombinant expression vector was introduced into tobacco through the wound caused by leaf cutting, and tobacco V1GS was induced. Through artificial vaccination of PVY virus, the experimental results of tobacco disease resistance showed that the expression of PVY CP,HC-pro gene could be effectively degraded under both experimental and natural conditions, thus showing resistance to potato Y virus. Under natural conditions, 760 strains of G80 tobacco were treated with 8 treatments. The resistance test showed that the control effect of CP gene on PVY, the control effect of HC-pro gene on PVY was 86.83%, and the control effect of CP and HC-pro gene on PVY was 78.34%.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S435.72

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