大麦黄矮病毒GAV株系外壳蛋白基因在大肠杆菌中的高效表达

大麦黄矮病毒 (ＢＹＤＶｓ)是一种由蚜虫专化性传播的循回非增殖型单链正义ＲＮＡ病毒 ,是黄症病毒属 (Ｌｕｔｅｏｖｉｒｕｓ)的代表成员。ＢＹＤＶＧＡＶ是我国的主流株系之一 ,它与美国的ＢＹＤＶＭＡＶ有较强的血清学关系 ,但美国的ＭＡＶ仅由麦长管蚜传播 ,而我国的ＧＡＶ除麦长管蚜传播外 ,还可由麦二叉蚜传播[1,2 ] 。近年来 ,有关麦蚜传播病毒专化性的研究已成为ＢＹＤＶ的研究热点[3 ] 。ＢＹＤＶ病毒的传播涉及病毒粒子表面成分与蚜虫体内专化性受体的相互作用 ,ＢＹＤＶ病毒粒子表面主要成分是ＣＰ ,另外一个次要成分是通读蛋白 (…     

黑龙江省及长春市烟草病毒病的种类鉴定

１９９１－１９９３年在黑龙江省主要烟区的１１个县及长春市采样,得１２９个毒株。经鉴别寄主测定,抗血清反应（板式酶联法或斑点酶联法）及电镜或免疫电镜观察,有ＴＭＶ（４３．４％）,ＰＶＹ（１７．８％）,ＣＭＶ（３．９％）,ＴＲＶ（０．８％）,ＴＳＷＶ等病毒,ＴＭＶ与ＰＶＹ混合侵染的占１０．１％,ＰＶＹ与其它病毒混合侵染的占１１．６％,另有５个标样为马铃薯Ｙ病毒组成员,１０个为未知。     

双价外壳蛋白基因植物表达载体的构建及马铃薯转基因植株的鉴定

在克隆了马铃薯Ｘ病毒（ＰＶＸ）、马铃薯Ｙ 病毒（ＰＶＹ）和马铃薯卷叶病毒（ＰＬＲＶ）的外壳蛋白基因的基础上,构建同时包含ＰＶＸ和ＰＶＹ 与ＰＶＹ 和ＰＬＲＶ 两个外壳蛋白基因植物表达框架的表达载体,通过农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ）介导转化烟草（Ｎｉｃｏｔｉａｎａｔａｂａｃｕｍ ）和生产上常用的几个马铃薯（Ｓｏｌａｎｕｍ ｔｕｂｅｒｏｓｕｍ ）优良品种：“Ｆａｖｏｒｉｔａ”、“虎头”、“克４”。经ＰＣＲ检测证明外源基因已整合到植物的染色体上,得到批量转基因植株。在转ＰＶＸ＋ＰＶＹ 外壳蛋白基因的烟草上接种ＰＶＸ （５ μｇ／ｍ Ｌ）、ＰＶＹ（２０ μｇ／ｍ Ｌ）病毒,得到有一定抗性的植株     

表达马铃薯X病毒和Y病毒双价外壳蛋白基因马铃薯植株的抗病性

表达马铃薯Ｘ病毒（ＰＶＸ）和马铃薯Ｘ病毒（ＰＶＹ）双价外壳蛋白（ＣＰ）基因的马铃薯虎头和克新４号,用机械摩擦同时接种ＰＶＸ和ＰＶＹ后,通过症状观察,植株中ＰＶＸ和ＰＶＹ的ＥＬＩＳＡ检测结果表明,转基因头虎头和克新４号的多数株系的平均病毒含量均明显低于未转基因的对照植株,不同时期病毒测定结果表明,许多株系病毒积累缓慢,延迟发病,说明转ＰＶＸ,ＰＶＹ双价ＣＰ基因的马铃薯,对ＰＶＸ和ＰＶＹ复合侵染发生不     

转PVY外壳蛋白基因马铃薯及其田间实验

报道了将马铃薯Ｙ 病毒（ＰＶＹ）中国分离株的外壳蛋白基因通过农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍ ｅｆａ－ｃｉｅｎｓ）介导转入马铃薯（Ｓｏｌａｎｕｍ ｔｕｂｅｒｏｓｕｍ Ｌ．）的生产品种“Ｆａｖｏｒｉｔａ”、“虎头”和“克４”,在获得大量再生植株的基础上经过ＰＣＲ检测和Ｓｏｕｔｈｅｒｎ 杂交证明,大部分株系中ＰＶＹ 外壳蛋白基因的表达框架已完整整合到马铃薯的染色体上。人工接种ＰＶＹ 病毒（２０ ｍ ｇ／Ｌ）后一些转基因株系对ＰＶＹ 病毒的侵染表现较强的抗性,同时其单株结薯数和平均薯重有所增加。在田间实验中,转基因马铃薯植株生长良好而且部分转基因株系产量高于未转基因的脱毒马铃薯,从这些株系中有希望得到抗病性好而且高产的马铃薯品系     

马铃薯卷叶病毒复制酶基因5端克隆及序列分析

马铃薯卷叶病毒（Ｐｏｔａｔｏｌｅａｆｒｏｌｖｉｒｕｓ,ＰＬＲＶ）是正链ＲＮＡ病毒,属黄化病毒组（Ｌｕｔｅｏｖｉｒｕｓ）〔１〕,严格虫传,分布广泛,难以控制,侵染马铃薯能引起大面积减产,给马铃薯生产造成巨大损失。ＰＬＲＶ基因组全长６．０ｋｂ,有６个读...     

植物病原真菌毒素的研究现状

近几年来,关于寄主专化性毒素(Host-Specific Toxin,HST)的作用机制及分子生物学方面的研究有了新进展;在14种寄主专化性毒素中,以Alternaria属的病原菌产生的专化性毒素的研究更为深入。非寄主专化性毒素(Non-Host-Specific Toxin,NHST)的研究着重在毒素产生的条件、生物活性测定、抗性鉴定以及检测被感染植物体的毒素含量等。本文综述了一些能产生专化性毒素和非专化性毒素的植物病原真菌,有链格孢(Alternaria)、镰孢(Fusarium)、尾孢(Cercospora)、轮枝孢(Verticillium)、梨孢(Pyricularia)、疫霉(Phytophthora)、长蠕孢(Helminthosporium)、黑团孢(Periconia)和核盘菌(sclerotinia)等属的病原菌。从所发表的文献表明真菌毒素在植物病害发展中起着重要作用。     

大麦黄矮病毒分子生物学的研究进展

大麦黄矮病毒（ＢａｒｌｅｙＹｅｌｏｗＤｗａｒｆＶｉｒｕｓ,ＢＹＤＶ）是黄矮病毒组（Ｌｕｔｅｏｖｉｒｕｓ）中的一员。它只能通过蚜虫传播,广泛流行于北美、欧洲、东亚的大麦产区。大麦黄矮病主要引起大麦矮化,抑制分蘖,减少穗数,造成不孕以至不能结实。除大麦外...     

马铃薯Y病毒外壳蛋白基因在转基因马铃薯中的表达

ＰＶＹ是马铃薯Ｙ病毒组的典型成员,主要感染马铃薯、番茄、辣椒和烟草等。近年来,利用植物基因工程手段获得了不少抗病毒转基因工程植物,为培育抗病毒作物新品种提供了新途径［１］。病毒外壳蛋白基因导入并使之在植物中表达可获得抗相应病毒的转基因植物,已在烟草、番茄、马铃薯、苜蓿、黄瓜和番木瓜等植物中获得成功［１～３］。本室已成功地对在我国流行的ＰＶＹＮ株系外壳蛋白基因进行了克隆及序列测定［４］,在此基础上,我们构建了植物表达中间载体,通过土壤农杆菌介导的叶盘法转化马铃薯,获得了大量转基因植株。分子检测证明…     

棉蚜对寄主的选择及寄主专化型研究

采用叶片选择法,生命表及EPG技术研究了棉蚜对寄主植物的选择和专化性,结果表明,棉花上生长的棉蚜对棉花,西葫芦和西瓜叶片均具有强选择性,而对黄瓜和南瓜选择性弱,西瓜,南瓜和黄瓜上生长的棉蚜对其原寄主选择性强,而对棉花选择性弱,棉花上的棉蚜转接到黄瓜和南瓜上,其存活率和繁殖力极低,棉蚜的取食行为在黄瓜和马铃薯,黄瓜和棉花之间存在明显的寄主专化型,黄瓜与棉花上的棉蚜相互转接均难成功,而黄瓜和马铃薯上的棉蚜转移具有不对称性。     

Host-Specific Involvement of the HC Protein in the Long-Distance Movement of Potyviruses

Plum pox virus (PPV) is a member of the Potyvirus genus that, in nature, infects trees of the Prunus genus. Although PPV infects systemically several species of the Nicotiana genus, such as N. clevelandii and N. benthamiana, and replicates in the inoculated leaves of N. tabacum, it is unable to infect systemically the last host. The long-distance movement defect of PPV was corrected in transgenic tobacco plants expressing the 5"-terminal region of the genome of tobacco etch virus (TEV), a potyvirus that infects systemically tobacco. The fact that PPV was unable to move to upper noninoculated leaves in tobacco plants transformed with the same TEV transgene, but with a mutation in the HC protein (HC-Pro)-coding sequences, identifies the multifunctional HC-Pro as the complementing factor, and strongly suggests that a defect in an HC-Pro activity is responsible for the long-distance movement defect of PPV in tobacco. Whereas PPV HC-Pro strongly intensifies the symptoms caused by potato virus X (PVX) in the PPV systemic hosts N. clevelandii and N. benthamiana, it has no apparent effect on PVX pathogenicity in tobacco, supporting the hypothesis that long-distance movement and pathogenicity enhancement are related activities of the potyviral HC proteins. The movement defect of PPV in tobacco could also be complemented by cucumber mosaic virus in a mixed infection, demonstrating that at least some components of the long-distance machinery of the potyviruses are not strictly virus specific. A general conclusion of this work is that the HC-Pro might be a relevant factor for controlling the host range of the potyviruses.     

The use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants

As the processing mechanism of all known potyviruses involves the activity of cysteine proteinases, we asked whether constitutive expression of a rice cysteine proteinase inhibitor gene could induce resistance against two important potyviruses, tobacco etch virus (TEV) and potato virus Y (PVY), in transgenic tobacco plants. Tobacco lines expressing the foreign gene at varying levels were examined for resistance against TEV and PVY infection. There was a clear, direct correlation between the level of oryzacystatin message, inhibition of papain (a cysteine proteinase), and resistance to TEV and PVY in all lines tested. The inhibitor was ineffective against tobacco mosaic virus (TMV) infection because processing of this virus does not involve cysteine proteinases. These results show that plant cystatins can be used against different potyviruses and potentially also against other viruses, whose replication involves cysteine proteinase activity.     

Field resistance of transgenic burley tobacco lines and hybrids expressing the tobacco vein mottling virus coat protein gene

Fifty transgenic lines expressing the tobacco vein mottling virus (TVMV) coat protein (CP) gene in five genetic backgrounds were evaluated under field conditions for response to mechanic inoculation with TVMV, tobacco etch virus (TEV) and potato virus Y (PVY). TVMV CP transgenic lines conferred resistance to TVMV, TEV and PVY under field conditions. Combining two strategies, coat protein-mediated resistance (CPMR) coupled with an endogenous resistance gene (Virgin A Mutant, VAM) significantly extended the range and magnitude of virus resistance and provided a potential valuable new source of protection against potyviruses. CP transgenic lines lacking the VAM gene had high resistance to TEV, medium resistance to PVY, and a recovery phenotype to TVMV. A series of hybrids involving transgenic lines were generated and tested under field conditions for response to virus inoculation. One copy of TVMV-CP gene presented in lines homozygous for the VAM gene provided effective resistance to all three potyviruses. These studies also suggested that selection of a suitable recipient genotype was critical and that field evaluation was necessary in order to select elite resistant transgenic lines. Engineering viral CP genes into genotypes possessing some level of virus resistance could be critical to achieve an effective level of resistance.     

Comparison of the substrate specificity of two potyvirus proteases

The substrate specificity of the nuclear inclusion protein a (NIa) proteolytic enzymes from two potyviruses, the tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV), was compared using oligopeptide substrates. Mutations were introduced into TEV protease in an effort to identify key determinants of substrate specificity. The specificity of the mutant enzymes was assessed by using peptides with complementary substitutions. The crystal structure of TEV protease and a homology model of TVMV protease were used to interpret the kinetic data. A comparison of the two structures and the experimental data suggested that the differences in the specificity of the two enzymes may be mainly due to the variation in their S4 and S3 binding subsites. Two key residues predicted to be important for these differences were replaced in TEV protease with the corresponding residues of TVMV protease. Kinetic analyses of the mutants confirmed that these residues play a role in the specificity of the two enzymes. Additional residues in the substrate-binding subsites of TEV protease were also mutated in an effort to alter the specificity of the enzyme.     

Formation of plant RNA virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein.

The mechanisms that direct positive-stranded RNA virus replication complexes to plant and animal cellular membranes are poorly understood. We describe a specific interaction between a replication protein of an RNA plant virus and membranes in vitro and in live cells. The tobacco etch virus (TEV) 6 kDa protein associated with membranes as an integral protein via a central 19 amino acid hydrophobic domain. In the presence or absence of other viral proteins, fluorescent fusion proteins containing the 6 kDa protein associated with large vesicular compartments derived from the endoplasmic reticulum (ER). Infection by TEV was associated with a collapse of the ER network into a series of discrete aggregated structures. Viral RNA replication complexes from infected cells were also associated with ER-like membranes. Targeting of TEV RNA replication complexes to membranous sites of replication is proposed to involve post-translational interactions between the 6 kDa protein and the ER.     

A fluorogenic substrate as quantitative in vivo reporter to determine protein expression and folding of tobacco etch virus protease in Escherichia coli

Quantitative and folding reporters are adequate tools to optimize recombinant protein expression in various host organisms, including Escherichia coli. To determine the yield of soluble active protease from the tobacco etch virus (TEV), we developed a single-molecule assay based on the fluorogenic substrate ANA-QS-MCA. This substrate consists of a 10 amino acid peptide (ENLYFQSGTK) containing the proteolytic cleavage sequence of the TEV protease. The peptide works as a linker N-terminally tagged with a fluorescent donor group (7-Methoxycoumarin-4-yl)acetyl (MCA) and C-terminally tagged with the acceptor group 5-Amino-2-nitrobenzoic acid (ANA). Fluorescence can be observed after specific cleavage of the substrate at the Gln-Ser bond by active TEV protease. Purified His-tagged TEV protease was used for in vitro analysis. Through determination of proteolytic activity in living E. coli cells and through application of Confocal Laser-Scanning-Microscopy we demonstrate that the peptide is well suited to in vivo expression analysis. This provides an effective tool to monitor the accumulation of active recombinant TEV protease in crude extracts and intact cells.