基于黄瓜花叶病毒(CMV)基因沉默载体的构建  

Construction of Virus-induced Gene Silencing Vector Based on Cucumber mosaic virus(CMV)

作　　者：程晓东[1] 施伟[1] 杜志游[1] 廖乾生[1] 

机构地区：[1]浙江理工大学生命科学院,杭州310018

出　　处：《农业生物技术学报》2015年第12期1550-1558,共9页Journal of Agricultural Biotechnology

基　　金：浙江省自然科学项目(No.Y3080228)

摘　　要：病毒介导的基因沉默(virus-induced gene silencing,VIGS)广泛应用于植物基因功能分析。本研究以黄瓜花叶病毒(Cucumber mosaic virus,CMV)不同株系的RNA2与CMV-Fny基因组R1和R3进行组合接种,获得在本氏烟(Nicotiana benthamiana)中症状轻的病毒组合F1Tsh R2F3;将CMV-Fny基因组R1、R2、R3和Tsh2克隆到植物瞬时表达载体p CB301中,构建载体p CB301-R1R2R3。农杆菌(Agrobacterium tumefaciens)浸润接种本氏烟结果显示,p CB301-R1R2R3引起寄主症状与病毒c DNA体外转录产物侵染所产生症状一致;Tsh R2的2b基因缺失3'端的95 nt碱基(Tsh2VIGS)后病毒能系统性侵染寄主植物,但降低病毒外壳蛋白(coat protein,CP)含量;F1Tsh R2VIGS-Su351F3接种10 d后整个植株的大部分叶片出现黄色表型;q RT-PCR结果表明,p CB301-F1Tsh R2VIGS-Su351F3接种植株中的镁离子螯合镁亚基基因Sulphur m RNA含量为对照植株20%。为进一步验证该载体能否沉默植物中其他基因,将烟草中细胞自噬相关基因Beclin1基因部分序列插入载体p CB301-Tsh R2VIGS并接种于NN三生烟(N.tabacum cv.Xanthi NN)中,烟草花叶病毒(Tobacco,mosaic virus,TMV)侵染结果表明,F1Tsh R2VIGS-Beclin337F3浸润接种寄主呈现系统性坏死症状反应,而对照植株仅在TMV接种区域产生局部枯斑。本研究所获得的CMV农杆菌侵染性克隆及其VIGS载体,为构建CMV其他寄主的VIGS载体奠定基础。Virus-induced gene silencing（VIGS） has been widely used in plant gene functional analysis. In the present study, in order to obtain VIGS candidate Cucumber mosaic virus CMV strain,（CMV） pseudorecombinants between RNA2 from different CMV strains and CMV Fny strain（CMV-Fny） RNA1 and RNA3 were researched. Results of pseudo- recombinants CMV infection indicated that CMV- F1Tsh2F3 consisting of CMV-Fny genomic RNA1, RNA3 and RNA2 of CMV-Tsh R2 induced slightly mosaic symptom on tabacco（Nicotiana benthamiana）, while the other CMV pseudo- recombinants caused host plants to produce stunt or leaf curf at 7 days post-inoculation（dpi）. In order to obtain the Agrobacterium-mediated CMV infectious c DNA clones, genomic RNA1～3 of CMV was cloned into binary expression vector pCB301. Agro-infiltration results of CMV infectious clones under the control of duplicated Cauliflower mosaic virus 35 S promoter indicated that the pCB301- CMV phenotype on the host plants was identical to that induced by CMV in vitro transcripts RNAs. The seedlings of N. bethamiana could be infected by pCB301- F1Tsh2VIGSF3 systematically and expressed slightly mosaic symptom at 7 dpi, even though F1Tsh2VIGSF3 lacking 3＇ end 95 nucleotides of 2b gene. Western blot results indicated that coat protein（CP） of pCB301- F1Tsh2VIGSF3 was lower than that of pCB301-F1Tsh2F3. Chlorosis and yellowing symptom appeared on the whole N. benthamiana plants infected by pCB301-F1 Tsh R2VIGS-Su351F3 at 10 dpi, and qRT-PCR analysis showed that the magnesium chelatase subunit gene of Sulphur m RNA levle of host plants was reduced by more than 80% compared with the CMV-infected control. Chlorosis and yellowing symptom on N. benthamiana induced by inoculation of 3 or 5 passages F1 Tsh R2VIGS- Su351F3 was the same as that caused by infection with pCB301- F1 Tsh R2VIGS- Su351F3, and so CMVF1 Tsh R2VIGS- Su351F3 existed stably in the host plants. In order to confirm whether CMV- VIGS could knock down other plant functional genes, the seedlings of N.tabac

关 键 词：黄瓜花叶病毒(CMV) 农杆菌侵染性克隆 病毒诱导的基因沉默(VIGS) 

分 类 号：S432.41[农业科学—植物病理学]