黄瓜花叶病毒香蕉株系衣壳蛋白转基因烟草的研究

构了侵染香蕉的黄瓜花叶病毒两个株系的衣壳蛋白基因的植物表达载体,并通过农杆菌共培养法的基因枪法,分别将两个株系的ＣＰ基因转化入了两种烟草植株。其ＣＰ基因转化频率及植株再生率研究结果表明,农杆菌共培养法比基因枪法,土耳其烟比本生烟,农杆菌１：１０倍稀释液比培养原液和１：１００倍稀释液,具有更高的转化频率和植株再生能力。Ｓｏｕｔｈｅｍ ｂｌｏｔ,ＰＣＲ－Ｓｏｕｔｈｅｍ ｂｏｌｔ检测ＣＰ基因整合研究结果     

杏鲍菇抗烟草花叶病毒蛋白的筛选

采用离子交换层析和凝胶层析方法,从杏鲍菇干样中分离得到多个蛋白组分,经枯斑寄主检测,发现多个蛋白组分都有抗烟草花叶病毒(TMV)的活性,对TMV的抑制率均在70%以上,高者可达99%。其中xb68Ab已得到了纯化,分子量约为23.7kD,在心叶烟和苋色藜上它对TMV侵染的抑制率分别达到99.43%和98.9%。     

对烟草花叶病毒外壳蛋白亚基因组RNA生成机制的进一步研究

用原生质体瞬间表达的方法证明,在烟草细胞中ＴＭＶ外壳蛋白亚基因组ＲＮＡ的生成,不是通过核酸酶在基因组ＲＮＡ上直接切割,也不是通过在生成负链ＲＮＡ时预成性终止,再从这种负链ＲＮＡ终止部位起始产生的,ＴＭＶ外壳蛋白亚基因组ＲＮＡ,是ＴＭＶ识别利用负链ＲＮＡ中间的亚基因组启动子后产生的,这个启动子位于外壳蛋白读码框５′端２５６碱基范围之内,但大于４８个碱基,虽然ＴＭＶ能利用单链的负链ＲＮＡ上的亚基因组启     

黄瓜花叶病毒衣壳蛋白存在于被其侵染的烟草叶绿体中

用原生质体法制备出高纯度的完整叶绿体经SDS-PAGE电泳,银染后,发现黄瓜花叶病毒(CMV)侵染的烟草病叶叶绿体蛋白质图谱和健叶叶绿体相比,多出一条染色较弱的迁移率与CMV衣壳蛋白质相同的带,经Western转移,用CMV游离衣壳蛋白亚基抗血清进行斑点酶联(Immunoblot)检测,证明这条带就是CMV衣壳蛋白质。健康叶绿体中加入去掉叶绿体的病叶汁液而制备出的叶绿体中无CMV衣壳蛋白质,说明这不是在叶绿体提纯过程中得到的假象,即衣壳蛋白质存在于被CMV侵染的完整叶片叶绿体中。这个结果否认了以往报道的CMV衣壳蛋白质不存在于烟草叶绿体中的结论。另外还发现,叶绿体中的衣壳蛋白质浓度与叶片症状严重程度呈正相关。     

烟草花叶病毒运动蛋白基因的cDNA克隆、序列测定及植物转化

植物病毒侵染宿主植物的一个重要过程是通过它在宿主体内的转移和传播,产生病害。植物病毒在宿主体内的转移主要有两种方式,一种是通过植物维管组织进行的系统转移,另一种是植物病毒在宿主细胞之间的转移,这种转移是通过植物细胞的胞间连丝实现的。实验表明,病毒自身编码的一种蛋白参与了这个转移过程,对烟草花叶病毒(TMV)而言,这种蛋白就是分子量为30kDa的运动蛋白。     

烟草花叶病毒和番茄花叶病毒在含N基因烟草上的症状差异是由运动蛋白基因决定的

烟草花叶病毒(TMV)和番茄花叶病毒(ToMV)是烟草花叶病毒属中关系最为密切的病毒, 但它们在含N基因烟草上产生的枯斑大小有明显的差异. 比较了TMV, ToMV及用ToMV运动蛋白基因(MP)精确置换TMV MP后获得的重组病毒T/OMP在不同寄主上的症状差异, 发现T/OMP在含N基因烟草上产生的枯斑大小与ToMV相似. 分析比较TMV, ToMV和T/OMP外壳蛋白和MP在植物体内的积累水平, 发现三者之间没有明显的差异, 而TMV和T/OMP在原生质体中的复制水平也没有差异. 比较TMV, ToMV和T/OMP接种后烟草体内防御相关酶(PAL, POD和PPO)的活性变化, 结果T/OMP和TMV所诱导酶的变化趋势基本一致, 而与ToMV有所差异, 因此认为MP基因功能的差异决定了TMV和ToMV在N基因烟草上枯斑的大小.     

一种食用菌提取物y3对烟草花叶病毒的钝化作用及其机制

测定了从食用菌毛头鬼伞(Coprinus comatus)中提纯的y3蛋白对烟草花叶病毒(Tobacco mosaic virus,TMV)的钝化作用,结果表明y3对TMV有较强的体外钝化作用,在心叶烟枯斑寄主上对TMV的抑制中浓度约为2．0μg／mL;y3在pH9．0时较稳定;TMV与y3混合后刚RNase处理,测得侵染率为61．74％,比未用RNase处理的对照降低了38．26％,说明y3具有一定的体外脱衣壳作用;另外电镜观察发现y3可使部分TMV粒体发生裂解,变短。     

转望江南核糖体失活蛋白基因cassin烟草及其对烟草花叶病毒的抗性

通过构建植物表达载体,由农杆菌介导,将望江南核糖体失活蛋白基因cassin转入烟草。PCR和Southern blot杂交结果证明：外源基因已经以单拷贝整合到烟草基因组内,并且在后代发生遗传分离。RT—PCR和Northern blot杂交结果显示：外源基因可以正常转录。用不同浓度的TMV机械摩擦接种转基因T1、T2代各3个自交株系,以非转基因烟草为阴性对照,实验结果表明转基因烟草对TMV表现出不同程度的抗性。     

烟草花叶病毒运动蛋白cDNA的克隆及融合蛋白的表达

从烟草花叶病毒(TMV)中提取总RNA,通过反转录PCR (RTPCR) 扩增得到其运动蛋白(MP)的基因,将扩增产物克隆到pMD18T载体上。DNA序列分析表明,所得到的运动蛋白的基因全长为807bp (GenBank接受号AY300161), 与已发表TMV序列（GenBank登陆号为NC-001367）和同属的番茄花叶病毒（ToMV, GenBank登陆号为NC-002692相比核苷酸的同源性分别为98.0％和80.9％,氨基酸的同源性分别为99.1％和80.0％。 将目的片段亚克隆到表达载体pET30a上,并在大肠杆菌JM109中诱导表达,诱导9h 后,融合蛋白表达量最大。诱导后的工程菌超声后经SDSPAGE检测,融合蛋白以可溶形式存在。     

Properties of the Coat Protein of a New Tobacco Mosaic Virus Coat Protein ts-Mutant

Amino acid substitutions in a majority of tobacco mosaic virus (TMV) coat protein (CP) ts-mutants have previously been mapped to the same region of the CP molecule tertiary structure, located at a distance of about 70 Å from TMV virion axis. In the present work some properties of a new TMV CP ts-mutant ts21-66 (two substitutions I21 T and D66 G, both in the 70-Å region) were studied. Thermal inactivation characteristics, sedimentation properties, circular dichroism spectra, and modification by a lysine-specific reagent, trinitrobenzensulfonic acid, of ts21–66 CP were compared with those of wild-type (U1) TMV CP. It is concluded that the 70-Å region represents the most labile portion of the TMV CP molecule. Partial disordering of this region in the mutant CP at permissive temperatures leads to loss of the capacity to form two-layer aggregates of the cylindrical type, while further disordering induced by mild heating results also in the loss of the ability to form ordered helical aggregates.     

大蒜花叶病毒外壳蛋白基因cDNA的克隆和序列分析

我们从自然发病的大蒜中分离得到了大蒜花叶病毒。以其基因组ＲＮＡ为模板合成了３＇末端部分ｃＤＮＡ。从中选出一批插入片段在２．０ｋｂ以上的重组克隆,经Ｎｏｒｔｈｅｒｎ点杂交分析证实了所选克隆与基因组ＲＮＡ同源。通过对若干个克隆的插入片段两端部分序列的测定,选出一个克隆ｐＧＭ４９５,其插入片段的长度约为２．４ｋｂ,３′末端存有一个Ｐｏｌｙ（Ａ）结构,它应包含了编码该病毒外壳蛋白全部序列。序列测定的结果表明,这个ｃＤＮＡ片段全长为２３７９ｂｐ,其中含有与酶切图谱分析结果相符的ＥｅｏＲＩ、ＰｓｔＩ及ＢａｍＨＩ酶切位点。第一个终止密码子ＴＡＡ与３′ｇ末端相距２６４ｂｐ,我们根据碱基序列推定的氨基酸序列与其它已发表的Ｐｏｔｙｖｉｒｕｓ的外壳蛋白氨基酸序列以及外壳蛋白翻译后加工的蛋白酶专一切点相比较后推测,编码该病毒外壳蛋白序列可能起始于３′末端上游的１１７０ｂｐ处,共编码３０２个氨基酸,其分子量为３６ｋＤ,略大于ＳＤＳ－ＰＡＧＥ所测定的３３ｋＤ,非编码区域长２６４ｂｐ,富含ＡＴ,并有多个终止密码子的存在。趾３′末端３２～２７ｂｐ处有一个ＡＡＴＡＡ序列。     

Macroscopic Aggregation of Tobacco Mosaic Virus Coat Protein

The relationship between processes of thermal denaturation and heat-induced aggregation of tobacco mosaic virus (TMV) coat protein (CP) was studied. Judging from differential scanning calorimetry melting curves, TMV CP in the form of a trimer–pentamer mixture (4S-protein) has very low thermal stability, with a transition temperature at about 40°C. Thermally denatured TMV CP displayed high propensity for large (macroscopic) aggregate formation. TMV CP macroscopic aggregation was strongly dependent on the protein concentration and solution ionic strength. By varying phosphate buffer molarity, it was possible to merge or to separate the denaturation and aggregation processes. Using far-UV CD spectroscopy, it was found that on thermal denaturation TMV CP subunits are converted into an intermediate that retains about half of its initial -helix content and possesses high heat stability. We suppose that this stable thermal denaturation intermediate is directly responsible for the formation of TMV CP macroscopic aggregates.     

应用核酶体外切割马铃薯卷叶病毒外壳蛋白基因的研究

针对马铃薯卷叶病毒外壳蛋白基因第356～358位点“GUC”．设计、合成了一种“锤头状”核酶。将核酶基因克隆在体外转录载体PSPT19的SP6启动子下游;同时将PLRVCPcDNA亚克隆在体外转录载体pSPT18的SP6启动子下游。利用SP6RNA聚合酶分别体外转录,获得核酶分子和靶RNA序列。在41℃保温进行核酶切割反应,检测到预期大小且被切开的两个RNA短片段。     

烟草环斑病毒外壳蛋白基因的原核表达及抗血清的制备

烟草环斑病毒(Tobaccoringspotvirus,TRSV)是我国二类进境检疫危险性有害生物,对农业生产危害较大。本研究依据TRSV外壳蛋白基因cp序列设计合成了2条引物,通过RT-PCR扩增得到长约1500bp的目的片段。将目的片段与质粒pET-22b( )连接,构建了含TRSVcp基因的融合蛋白原核表达载体pETRSV-CP。序列分析表明,TRSV-SD1的cp基因全长1548bp,编码515个氨基酸与GenBank中其它TRSV分离物cp基因相比,核苷酸及推导的氨基酸序列同源性为90.7%~94.6%。将pETRSV-CP转入大肠杆菌,诱导表达。SDS-PAGE结果显示,表达的TRSVCP融合蛋白的相对分子质量约为58kDa。以此融合蛋白制备的抗血清的效价为1/1024,抗血清与TRSV具有良好的特异性反应。     

黄瓜花叶病毒衣壳蛋白基因转化辣椒研究

The plant expression vector of the coat protein(CP) gene of cucumber mosaic virus (CMV) BS strain was used to transform three kinds of pepper (Capsicum annuum) tissues (cotyledon, stem and root) by agrobacterium-mediated co-cultivation. 53%-68.4% of the total tissues (639) can be induced to be calli, but only cotyledon calli can be further regenerated to form shoots (regenerated efficiency 39.7%). 70%(42/60) of the putative transformed plants were confirmed to have CP gene in their genomes by Southern blot. The mRNAs and the CP were respectively found in 80% of transgenic plants by Northern blot and DAS-ELISA. 24 of the transgenic plants expressing CP gene of BS strain showed three kinds of resistant level (severe symptom, delay of symptomatic development, no symptom) to infection of CMV-BS and of CMV-P. However, there was distinctly higher resistance to inoculation of CMV-BS than that with CMV-P in these transgenic plants.     

Demonstration of the Specific Involvement of Coat Protein in Tobacco Mosaic Virus (TMV) Cross Protection using a TMV Coat Protein Mutant

The DT-1G mutant of tobacco mosaic virus (TMV) which has no coat protein was used to study the specific involvement of coat protein in TMV cross protection in N. sylvestris. Leaves of N. sylvestris previously inoculated with the mutantor the common strain of TMV were challenged with either turnip mosaic virus (TuMV) or a strain of TMV (TMV-N). Both TuMV and TMV-N produce necrotic lesions on N. sylvestris. About one-half as many lesions were produced by TuMV and TMV-N on leaves, inoculated with the DT-1G mutant compared with lesions produced by the same inoculum on control leaves. When leaves of N. sylvestris previously inoculated with the common strain of TMV were challenged with either TuMV or TMV-N, TuMV produced about one-half as many lesions as on control leaves whereas TMV-N produced about one-tenth as many lesions as on control leaves. A high level of non-specific resistance was induced by the mutant without coat protein, but it did not specifically protect against TMV.     

TMV在不同水体与温度条件下的灭活动力学

了解植物病毒在不同水体与温度条件下的灭活规律具有重要的理论与实际意义.本文以典型植物病毒烟草花叶病毒(TMV)为模型,比较了其在不同温度条件下,在闽江水、自来水、生活污水、微孔滤膜过滤除菌污水及超纯水中的灭活动力学.结果显示,温度是导致TMV灭活的重要因素,水温升高,病毒灭活速率加快;此外,某些水质因子也影响TMV的灭活效率,其中可溶性盐的存在及其含量对TMV的灭活会因所处的环境不同而异;某些微生物或代谢产物对植物病毒TMV具有灭活作用,而能生化降解的有机质加速TMV灭活可能是通过促进水体中的微生物增殖而起作用.