用斑点免疫结合法检测转化的烟草植株中的TMV—CP和CMV—CP

1982年,Hawkes 等人仿照分子生物学中点杂交(dot hybridization)的方法发展了斑点免疫结合测试技术(DIBA)用于检测单克隆抗体。本实验就 DIBA 应用在检测转基因烟草。烟草用发根农杆菌 Ri 质粒介导,将烟草花叶病病毒外壳蛋白基因(TMV—CP)和黄瓜花叶病毒外壳蛋白基因(CMV—CP)导入烟草的叶片外植体中,通过对外植体的培养,得到愈伤组织,从愈伤组织进而获得了再生植株,对再生植株进行检测导入的外壳蛋白基因的表达量。     

玉米矮花叶病毒外壳蛋白基因的克隆研究

玉米(Zea mays L.)矮花叶病在国内外广泛发生,且在玉米生产中造成了重大损失.通过RT-PCR法从具有典型的玉米矮花叶病症状的玉米叶片中克隆了外壳蛋白(Coat protein,CP)基因,测序和同源性比较表明所克隆的CP基因来自玉米矮花叶病毒(Maize dwarf mosaic virus,MDMV)B株系,全长920个碱基对,开放阅读框编码219个氨基酸,该基因可进一步用于玉米抗矮花叶病的转基因研究,以获得生产应用的抗病材料.     

番茄真核翻译起始因子4E基因RNA干涉及其抗病毒特性研究

植物真核翻译起始因子4E（eIF4E）在蛋白质翻译过程中发挥重要作用．最近研究表明eIF4E在参与植物病毒互作,影响病毒在寄主中复制和侵染过程．文中通过RNA干涉调控番茄eIF4E表达,鉴定eIF4E在植物病毒互作中的作用．根据数据库假设性一致序列（TC171564,TC170275）分别克隆了番茄“中蔬五号”SleIF4E基因及其异构体基因SleIF（iso）4E．构建了SleIF4ERNAi抑制表达载体pSL4D,通过农杆菌介导的方法导入番茄品种“中蔬五号”基因组．PCR检测和Southern blot结果表明,目标基因已整合到番茄基因组中．通过半定量RT-PCR对转基因番茄进行eIF4E表达分析,表明pSL4D转化番茄中eIF4E得到不同程度抑制．转基因植株分别接种PVY和CMV两种病毒,病毒ELISA测定和病毒RNA积累量分析表明,pSL4D转化植株相对于对照组均获得不同程度的病毒抗性,番茄eIF4E参与PVY的复制或侵染寄主过程．就不同病毒而言,RNAi对PVY抗性的调控效果比CMV调控效果好,抑制PIF4E表达可提高植物对PVY的抗性．     

大麦黄矮病毒外壳蛋白基因和运动蛋白基因酵母表达载体的构建

根据已知的大麦黄矮病毒GPV株系的外壳蛋白(Coat Protein CP)和移动蛋白(Movement Protein MP)基因序列合成了CP，MP基因的上下游引物，通过PCR扩增获得目的片段，经过Sal I和Rst I酶切、连接、转化、重组质粒的酶切鉴定及基因测序，构建了酵母表达载体pGBKT7-GPV-CP和pGBKT7-GPV-MP，用于在酵母双杂交分析中表达诱饵融合蛋白，为进一步筛选小麦cDNA文库内与大麦黄矮病毒相互作用的寄主因子、克隆寄主因子，推测其种类和功能打下基础。     

侵染新疆辣椒CMV的外壳蛋白基因的克隆和序列分析

从表现黄化的辣椒病株上获得分离物XJ-P,用1对CMV基因引物进行RT-PCR,扩增得到了774bp的特异性核苷酸片段。将PCR产物克隆到pMD18-T载体中再转化到大肠杆菌TOP10。经限制性内切酶酶切鉴定,重组克隆中含有与PCR产物大小相同的774bp的插入片段。cDNA全序列分析表明,所扩增出的774bpCMV基因,含1个657bp开放阅读框架（ORF）,编码218个氨基酸组成的蛋白,所克隆的基因包含完整的CMVCP基因。所测得的XJ-P分离物其CP基因与CMV亚组Ⅰ、亚组Ⅱ各株系之间的核苷酸同源性分别为90.84%-94.22%和74.37%-76.52%,氨基酸同源性分别为94.04%-98.17%和80.37%-82.19%,因此将该分离物鉴定为黄瓜花叶病毒亚组Ⅰ。     

长叶车前花叶病毒上海株(RMVsh)外壳蛋白基因的克隆、序列分析及原核表达

长叶车前花叶病毒上海分离株（RMVsh)是从上海郊区的青菜（Brassica chinensis)上分离鉴定的，以提纯的病毒为材料，SDS－酚法纯化的基因组RNA作为模板，通过RT－PCR方法克服了该病毒的外壳蛋白基因（CP），DNA序列测定结果表明，外壳蛋白基因全长474个碱基，编码157个氨基酸，将CP基因插入原核表达载体pBAD/His-C中，转化E.coli Top10后，诱导表达，经聚丙烯酰胺凝胶电泳分析呈－特异性的蛋白条带，Western blot检测表明，表达产物与RMVsh抗血清呈阳性反应，RMVsh CP基因的核苷酸序列及氨基酸序列与烟草花叶病毒属中其他能侵染十字花科作物的成员相比，同源率分别为83．5％－98．9％和87．9％－99．4％，并讨论了RMVsh的分类地位为烟草花叶病毒属侵染十字花科植物2个亚组中的第一亚组的代表。     

新疆加工番茄条斑坏死病原黄瓜花叶病毒外壳蛋白基因的克隆和序列分析

从表现花叶、畸形以及坏死症状的加工番茄病株上获得黄瓜花叶病毒分离物，用1对CMV CP基因引物进行RT-PCR，扩增得到了776bp的特异性核苷酸片段。将PCR产物插入到克隆载体PUCm-T中转化大肠杆菌DHSa。经限制酶酶切鉴定，重组克隆中含有与PCR产物大小相同的776bp的插入片段。cDNA全序列分析表明，重组克隆含1个开放阅读框架（ORF），长度为657nt，编码218个氨基酸组成的蛋白。与国内外报道的CMV株系序列比较，所测得的CMV新疆分离物与CMV亚组Ⅰ的核苷酸同源性高达91．0％-98．9％．而与CMV亚组Ⅱ的核苷酸同源性仅在76．4％-78．2％，所测得的CMV新疆分离物与CMV亚组Ⅰ的氨基酸同源性高达94．2％-98．6％；而与CMV亚组Ⅱ的核苷酸同源性仅在79．9％～83．5％．CMV亚组Ⅰ的株系较CMV亚组Ⅱ株系同源关系更密切，所以CMV新疆分离物应该归属于CMV亚组Ⅰ。     

番茄中一个抗病基因功能的研究

植物中最大的一类抗病基因(R)编码的蛋白质含有卷曲螺旋结构(coiled-coil,CC)、核苷酸结合位点(nucleotide-binding site,NBS)和富含亮氨基酸的重复序列(leucine-rich repeat,LRR)结构域。R基因以抗病著称。文章通过酵母双杂筛选方式获得了DDI3基因,该基因与番茄中重要功能基因DDB1具有相互作用,而又含有CC-NBS-LRR结构,预测具有R基因抗病性,参与植物抗病。研究中构建了DDI3过表达载体并通过根瘤农杆菌介导转化到野生型番茄中,筛选获得DDI3高表达的阳性转基因株系。通过病菌接种实验,观察植株发病情况和统计细菌菌落数,结果发现,转基因植株的抗病性明显高于野生型番茄。DDI3基因对提高番茄的抗病性有很高的价值,为采用基因工程方法改良番茄植株抗病性做出新的尝试,同时又与番茄DDB1基因具有相互作用,对研究DDB1基因在参与植物抗病途径中的功能研究提供新的方向。     

侵染半夏的黄瓜花叶病毒RNA3全序列分析及侵染性克隆构建

对从我国甘肃省天南星科植物半夏上获得的黄瓜花叶病毒分离物（CMV-PGs）的RNA3进行全长克隆和序列分析及侵染性克隆构建．结果表明：CMV—PGsRNA3序列全长2179nt,与CMV—Fny株系RNA3的同源性为83．1％．选取已报道CMV株系的38个RNA3全长序列,根据CP核苷酸序列进行同源性比较,系统进化树分析表明：CMV-PGs属于亚组ⅠB,但是相对独立．进一步构建CMV—PGsRNA3的侵染性克隆,通过体外转录获得具有侵染活性的RNA转录本,将CMV—PGsRNA3的侵染性转录本与CMV—FnyRNA1和RNA2的侵染性转录本混合接种烟草,成功获得假重组体F1F2P3．     

Delayed resistance to Cucumber mosaic virus mediated by 3’UTR-derived hairpin RNA

RNA silencing has been shown to function in the plant antivirus defense response, leading to viral RNA degradation induced by vsiRNA-containing RISC cleavage activity. Cucumber mosaic virus （CMV） 3′UTR sequences share a high conservation of nucleotide sequence and secondary structures that are important for CMV replication. Here, in an attempt to simultaneously target the multiple genomic and subgenomic RNAs of CMV for degradation, CMV 3′UTR were used to design hairpin RNA （hpRNA） to transform tobacco （Xanthi. nc） so as to constitutively produce viral siRNAs. Most of the transgenic plants expressing CMV Q strain （Q-CMV, subgroup Ⅱ strain） RNA3 3′UTR-derived hpRNA showed delayed resistance to Q-CMV infection and exhibited recovery phenotypes. Compared with Q-CMV-inoculated leaves, the upper leaves showed weak or no disease symptoms and a reduced accumulation level of viral RNAs. Together with transient assays, our results indicate that the 3′UTR-derived siRNAs were biologically active in targeting viral RNA for degradation. Recovery resistance in transgenic plants was also observed against subgroup IB strain SD-CMV infection, indicating a broad-spectrum anti-CMV effect of the 3′UTR-based antiviral silencing. Northern blot assays indicated that there was no strong correlation between the degree of resistance and the accumulation level of 3′UTR-derived siRNAs, suggesting that to target a highly structured RNA, such as the CMV 3′UTR, the quantity of siRNAs may not be the only determinant of silencing efficiency. Target RNA secondary structures may also affect target accessibility, siRNA-containing RISC-target recognition and the consequent antiviral effect.     

Functional analysis of DNA 4 coding region from a Chinese Zhangzhou isolate of banana bunchy top virus

The DNA 4 coding region of banana bunchy top virus from a Chinese Zhangzhou isolate (BBTV-ZZ) is cloned by PCR. The sequencing analysis shows that it is 351 nucleotides long and it putatively encodes a protein of 116 amino acids. On the basis of a plant binary vector pBin438, the plant expression vector pBBTV-4B harboring the BBTV-ZZ DNA 4 coding region has been constructed and then transferred to tobacco (Nicotiana tobacum cv. Xanthi nc) by a Agrobacterium-mediated procedure. Under insect-free condition, movement-defective mutant of CMV-Fny strain (CMV-Fny-△MP) is mechanically inoculated on the lower leaves of transgenic plants. Systemic symptoms with different degrees of severity are developed in the upper uninoculated leaves of transgenic plants at 12 days postinoculation (dpi), while no symptoms can be seen in the uninoculated leaves of untransformed plants at any time. Accumulation of CMV-Fny is detected on the upper uninoculated leaves of transgenic plants, but is not on that of untransformed plants by indirect double antibody sandwich enzyme-link immunosorbent assay (DAS-ELISA). The results reveal that transgenic plants have acquired the property of cell-to-cell movement and systemic spread of CMV-Fny-△MP. This suggests that the protein encoded by BBTV-ZZ DNA 4 might have function of viral movement protein.     

黄瓜花叶病毒研究进展

介绍了近年来对黄瓜花叶病毒(CMV)在生物学特性、血清学特性、基因组研究、卫星RNA以及该病毒的防治等方面的最新研究进展.此外,由于黄瓜花叶病毒株系众多,其株系的划分和分类对研究致病机理和各个株系之间的关系具有重要的作用,重点介绍了根据生物学、血清学以及最新的分子生物学研究结果对黄瓜花叶病毒株系的不同分类方法.     

加工番茄CMV与ToMV的ELISA检测及其相关性分析

用针对CMV和ToMV的单克隆抗体对田间表现病毒病症状的137个加工番茄自然病株进行了间接ELISA检测,检测结果表明,CMV和ToMV在加工番茄上的侵染率分别为83．2％和61．3％,并且2种病毒常复合侵染,复合侵染率达58．4％。用Eviews3．0软件借助加权最小二乘法（WLS）分析CMV和ToMV在病株上的带毒量,结果表明,2种病毒在病株上的积累是相互影响的,互为正相关关系。     

Obtaining transgenic rice resistant to rice fungal blast disease by controlled cell death strategy

The strategy of the two-component system,composed of Barnase and Barstar which encode RNase and a specific inhibitor to the RNase respectively, is adopted to obtain transgenic rice resistant to rice fungal blast disease. In this study, two chimeric promoters, induced by rice blast fungus pathogen (Magnaporthe grisea), are fused with Barnase respectively to construct two plant expression vectors, pWBNBS and pPBNBS together with the Barstar driven by CaMV 35S promoter. The resistance of the transgenic rice lines to rice blast fungus disease and rice blight disease are evaluated. The results show that (1) the expression of Barnase is induced in rice leaves when inoculated with the spores of Magnaporthe grisea; (2) the induced expression level of Barnase surpasses the level of Barstar, which elicits a similar hypersensitive response (HR) in the leaves, and the transgenic plant shows high resistance to the rice fungal blast disease; and (3) transgenic rice plants also show obvious resistance to rice bacterial blight disease. Taken together, these results suggest that the transgenic rice plants harboring this two-component system acquire relatively broad spectrum resistance against pathogens, especially high resistance to rice fungal pathogen.     

The pathogenicity on legumes of Cucumber mosaic virus was determined by 243 nucleotides on 2a polymerase gene of viral RNA2

We had isolated and identified two Cucumber mosaic virus (CMV) isolates, the CMV red bean (CMV-RB)isolate and the CMV pea (CMV-P1) isolate. CMV-RBinduces necrotic local lesions on inoculated leaves of broad bean, pea, cowpea and bean, and could not infect these hosts systemically. However, CMV-P1 was able to infect these legumes systemically. To study the difference of pathogenicity. on the legumes induced by these two CMV isolates, the full-length infectious cDNA clones of CMV-Fny, which induced similar symptoms as CMV-RB in the four legumes,were used. The 243 nucleotides fragment, which encodes highly conserved GDD amino acid motif on 2a replicase gene of CMV-Fny RNA2, was replaced with that of CMV-P1. The constructed chimeric virus FP could infect these legumes systemically. The exchange of this region changes the virus symptoms on the legumes, indicating that this 243 nucleotides fragment has major effect on pathogenicity of CMV on the legumes.``     

The pathogenicity on legumes of Cucumber mosaic virus was determined by 243 nucleotides on 2a polymerase gene of viral RNA2

We had isolated and identified two Cucumber mosaic virus (CMV) isolates, the CMV red bean (CMV-RB) isolate and the CMV pea (CMV-P1) isolate. CMV-RB induces necrotic local lesions on inoculated leaves of broad bean, pea, cowpea and bean, and could not infect these hosts systemically. However, CMV-P1 was able to infect these legumes systemically. To study the difference of pathogenicity on the legumes induced by these two CMV isolates, the full-length infectious cDNA clones of CMV-Fny, which induced similar symptoms as CMV-RB in the four legumes, were used. The 243 nucleotides fragment, which encodes highly conserved GDD amino acid motif on 2a replicase gene of CMV-Fny RNA2, was replaced with that of CMV-P1. The constructed chimeric virus FP could infect these legumes systemically. The exchange of this region changes the virus symptoms on the legumes, indicating that this 243 nucleotides fragment has major effect on pathogenicity of CMV on the legumes.     

北疆地区辣椒病毒病毒源种类分离鉴定的初报

1990～1992年,从石河子、昌吉、阜康、乌鲁木齐及哈密等地采集了不同症状的辣椒病毒病标样75份。通过指示植物3次单斑分离,纯化后获8个病毒分离物,编号分别为P—91—5、P—91—7、P—91—8、P—91—10、P—89—2、P—89—27、P—90—3及P—90—4。上述分离物经病毒的寄生范围、鉴别寄主反应、传毒介体及传播方式、体外稳定性、电镜粒体观察、血清学反应及理化性质的测定,鉴定出6种病毒:黄瓜花叶病毒(CMV)占28％、烟草花叶病毒(TMV)占24.7％、蚕豆萎蔫病毒(BBWV)占5.0％、马铃薯X病毒(PVX)占4.3％、马铃薯Y病毒(PVY)占2.7％、烟草花叶病毒组一新成员病毒(暂定为C_aMV)占21.3％。还有两种未知病毒分别占13.2％及3％。研究表明,在北疆地区辣椒病毒病的主要毒源是CMV、TMV及C_aMV,但对辣椒危害性最大的是TMV及BBWV,导致辣椒萎蔫、坏死及顶枯。     

Generation of transgenic wheat resistant to wheat yellow mosaic virus and identification of gene silence induced by virus infection

The plasmid containing the promoter Act1, the coat protein (cp) gene of wheat yellow mosaic virus (WYMV) and the selectable bar gene, was delivered via particle bombardment, directly into immature embryos of a wheat cultivars. PCR and PCR-RFLP were employed to screen the existence of the cp gene in T0 and T1 generations. Seeds from the positive T1 plants were sowed in fields heavily contaminated with WYMV to detect their resistance. In field trial of virus infection, one of the transgenic wheat lines, P8-T2, exhibited highly disease-resistance. Western blot and RT-PCR analysis showed that the expression level of cp gene in the resistant transgenic line was reduced greatly compared to those susceptible to WYMV infection. This provided evidence to presume that the resistance obtained by the transgenic wheat line was stimulated by the mechanism of the virus induced gene silencing.