【作者】 兰玉菲；

【导师】 李向东；

【作者基本信息】 山东农业大学 ， 植物病理学， 2009， 博士

【摘要】 马铃薯Y病毒(Potato virus Y,PVY)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)和马铃薯X病毒(Potato virus X,PVX)是烟草上的三大主要病毒病。病毒间的复合侵染非常普遍,往往给烟草生产造成严重损失。本研究利用适体和小干扰RNA 2种不同的策略获得了同时抗2种或3种病毒的转基因烟草。烟草脉带花叶病毒(Tobacco vein banding mosaic virus,TVBMV)属于马铃薯Y病毒属(Potyvirus)病毒,近几年在我国烟草上的发生有上升和蔓延的趋势。本研究获得了TVBMV YND分离物的全基因组序列,利用原核表达方法成功表达了TVBMV 10个蛋白中除了P3蛋白之外的9个,并利用镍柱对部分蛋白进行了纯化,同时制备了TVBMV CP的抗血清。主要结果如下:将编码一个九肽(与CMV CP互作)和PVY HC-Pro N端83个氨基酸(与HC-Pro蛋白自身互作)的基因片段(C-P)与GFP基因(GFP)同时连接到pMD18-T载体,通过定点突变获得了3个突变体。将C-P-GFP及其3个突变体分别连接到植物表达载体pROKⅡ上,农杆菌共浸润后接种实验筛选到最有效的抗性诱导载体。将此载体通过农杆菌介导法成功转化烟草K326,分子检测获得了15棵转基因烟草。抗病性实验表明获得抗PVY的转基因烟草1棵,抗CMV的植株3棵,同时抗PVY和CMV的植株1棵。首次利用适体策略获得了同时抗PVY和CMV的转基因烟草。利用软件筛选到能够有效切割PVX和CMV CP基因的siRNA以及PVY HC-Pro基因中能够有效形成siRNA的区域,将筛选片段连接构建反向重复的植物表达载体。农杆菌介导法转化烟草K326,分子检测证明成功获得了22棵转基因烟草。抗病性分析表明获得了2棵抗PVY、8棵抗CMV和6棵抗PVX的转基因烟草,同时抗3种病毒的转基因烟草1棵。ELISA和实时荧光RT-PCR检测结果表明抗病的转基因植株中病毒含量均明显低于感病对照,说明筛选的siRNA能够特异性地降解病毒的mRNA,有效抑制病毒的复制。利用针对马铃薯Y病毒属病毒的简并或特异引物,通过RT-PCR方法扩增得到TVBMV云南分离物YND的全基因组序列。除了3′-端poly(A)外,YND基因组全序列包括9 570个核苷酸,含有一个大的开放阅读框,编码一个由3 079个氨基酸组成的多聚蛋白,分子量约为348.6 kDa。该分离物的NIb/CP切割位点为Q/N,这在马铃薯Y病毒属病毒中非常少见。另外,HC-Pro中含有比较罕见的参与蚜传的RITC基序。将TVBMV CP基因克隆到表达载体pET-22b(+)上,在大肠杆菌BL21 (DE3)中诱导表达出分子量为35.0 kDa的融合蛋白。利用该融合蛋白制备了TVBMV的多克隆抗体,ELISA测定抗血清效价为1/4 096。Western blotting和DIBA分析结果表明,获得的抗血清和原核表达的病毒蛋白及植物病汁液中的病毒均有特异性反应,可用于TVBMV的快速检测。同时在大肠杆菌中成功表达了TVBMV 10个蛋白中除了P3蛋白之外的9个,并利用镍柱对部分蛋白进行了纯化。可溶性分析结果表明HC-Pro、NIa-VPg和CP蛋白中可溶性蛋白较多,完全能够满足纯化要求,而其它蛋白破碎后可溶性蛋白占总蛋白的比例均低于10%,需要先变性后复性进行纯化。利用生物信息学软件对TVBMV编码蛋白的结构进行了预测,结果表明6K2和P3蛋白为跨膜蛋白。以上研究结果为研究蛋白晶体结构和功能奠定了基础。更多还原

【Abstract】 Potato virus Y (PVY), Cucumber mosaic virus (CMV) and Potato virus X (PVX) are the most important viruses in the tobacco plants. The mixed infection of viruses is very common, and it caused more severe losses in tobacco production. In this paper, we obtained multiple virus resistant transgenic plants using two different strategies: aptamer and siRNA. Tobacco vein banding mosaic virus (TVBMV) is one species of the genus Potyvirus. The losses caused by TVBMV had been increasing during the recent years. In this paper, we obtained the complete genomic sequence of TVBMV from Yunnan China and have nine proteins of TVBMV expressed in the E. coli BL21 (DE3), and then prepared the antiserum against TVBMV CP. The detailed results read as follows:The random nonapeptides specifically binding to the CP of CMV, the N-terminal 83 amino acids of the HC-Pro interacted with PVY HC-Pro and GFP genes were simultaneously cloned to the pMD18-T vector, resulting in the plasmid pMD18 C-P-GFP. Three mutants were obtained by site-directed mutagenesis. We cloned C-P-GFP and these three mutants to the plant expression vector pROKⅡand screened by agroinfiltration the most efficient resistance inducing vector, which was introduced into tobacco plants (K326) via agrobacterium tumefaciens-mediated transformation. After tissue culture, we obtained 15 regeneration plants that were PCR-positive. After inoculation, one regeneration plant showed resistance to PVY, three to CMV, and one to both PVY and CMV. We first obtained multiple virus resistance transgenic tobacco plants using aptamer strategy.The most effective siRNA and region degraded PVX CP gene, CMV CP gene and PVY HC-Pro gene were screened using software and then constructed the reverse repeat plant expression vector. The vector was introduced into tobacco plants (K326) via agrobacterium tumefaciens-mediated transformation. Among the 22 regeneration plants, two were resistant to PVY, eight resistant to CMV, six resistant to PVX, and one resistant to PVY, CMV and PVX together. ELISA and real-time RT-PCR showed that virus accumulation in transgenic plants was lower than that in the susceptible control. This indicated that screened effective siRNA could degrade virus mRNA specifically and restrain virus replication.The complete genomic sequence of TVBMV-YND, an isolate from Yunnan, was determined by sequencing overlapping cDNA fragments obtained by RT-PCR with degenerate and/or specific primers. The genome is composed of 9,570 nucleotides (nt) excluding the 3′-terminal poly(A) tail and contains one single open reading frame of 9,240 nt encoding a large polyprotein of 3,079 amino acids with predicted Mr of 348.6 kDa. TVBMV-YND had a rare Q/N cleavage site for NIb/CP and uncommon RITC motif in HC-Pro that is crucial for aphid transmission of potyviruses.The CP gene of TVBMV YND was cloned into expression vector pET22b(+) and transferred into E.coli BL21(DE3). SDS-PAGE showed TVBMV CP gene was expressed as a 35.0 kDa fusion protein to high level when induced with IPTG. Antiserum against TVBMV CP obtained with the fusion protein, and resulting titer was 1:4 096 by ELISA. Western blotting and dot-immunobinding assay (DIBA) results showed that the acquired antiserum could specifically react with both TVBMV CP expressed in E.coli and extracts from diseased plants. Nine, except P3, of 10 TVBMV proteins were successfully expressed in E.coli. Solubility analysis showed that the soluble protein in the HC-Pro, NIa-VPg and CP proteins were suitable for purification, while others weren’t because the soluble parts were lower than 10%. Structures prediction of TVBMV proteins using bioinformatics software showed that 6K2 and P3 were transmembrane proteins while others were not. All the results are useful to the crystalization and structure/function researches of TVBMV proteins.更多还原