葡萄卷叶伴随病毒Ⅲ外壳蛋白基因的克隆

根据美国报道的葡萄卷叶伴随病毒Ⅲ（ＣＬＲａＶ－３）ＣＰ基因序列,设计并合成一对引物,用ＩＣ－ＲＴ－ＰＣＲ对ＧＬＲａＶ－３进行检测,琼脂糖凝胶电泳表明其大小与预计相符。将其ＣＰ基因克隆到ｐＧＥＭ－３ｚｆ（＋）中,经双酶切和序列分析表明所克隆的是ＧＬＲａＶ－３ ＣＰ基因,它与美国分离物相应序列同源性为９４．１％。     

马铃薯卷叶病毒外壳蛋白基因的合成,分子克隆和全序列分析

以分离自马铃薯主栽品种“紫花白”的马铃薯卷叶病毒(PLRV)分离物的RNA为模板,用人工合成的引物,用反转录和随后PCR扩增的方法合成了PLRV外壳蛋白(CP)基因的cDNA,并克隆于pUC19中。进一步用限制酶切和核苷酸序列分析表明,合成的cDNA由627个核苷酸组成(包括起始和终止密码),序列中有Hinc Ⅱ和BamH Ⅰ两个酶切位点,与国外报道一致。和国外的4个PLRV分离物CP基因序列对比结果,具有高度同源性,其同源率达99.0—99.7％。     

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。     

葡萄卷叶病毒的纯化,血清学研究及其在脱毒组培苗检测中的应用

将具有典型葡萄卷叶病(Grapevine leafroll diseas,GLRD)症状的葡萄组织,经差速和硫酸铯—蔗糖密度梯度离心,提纯了GLRV,并制备了兔抗血清。电镜下可观察到长度从600～2000nm的线形病毒颗粒,其中以1400nm左右为主。免疫电镜结果表明线形病毒颗粒能被美国的NY-1分离株抗血清(Ⅲ型)所修饰。在间接ELISA中提纯制品与GLRV的Ⅲ、Ⅳ、Ⅱ型抗血清均能产生免疫反应。与Ⅲ型抗血清产生较强的免疫反应,Ⅳ型次之,Ⅱ型最弱。在SDS-免疫双扩散实验中病组织韧皮部粗提液与GLRV的Ⅲ,Ⅳ、Ⅱ型抗血清均产生免疫沉淀线。从而推测我国葡萄园内的葡萄卷叶病很可能由2种或3种卷叶病毒感染所致.采用A蛋白夹心酶联免疫吸附试验(PAS-ELISA)检测葡萄试管苗,Ⅲ型抗血清和自制抗血清的平行测试结果基本相符,共获得11个生食葡萄和10个山葡萄品种的脱葡萄卷叶病毒和扇叶病毒的组培苗,扩繁后田间试种表现出良好的农艺性状。     

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...     

马铃薯卷叶病毒的提纯

本文提出了一个应用液氮冷冻,一步提取,蔗糖垫层差速离心,Sephadex G-200柱层析以及蔗糖密度梯度离心法纯化马铃薯卷叶病毒的程序,改进后的马铃薯卷叶病毒提纯方法,使病毒产量达到1.18mg/kg酸浆组织,病毒提取物纯度比差速离心者更高,20％蔗糖垫层差速离心能够更加有效地去除宿主细胞成份,纯化病毒的OD260/280,260/240比值分别达到1.77和1.43。     

葡萄卷叶病毒的纯化、血清学研究及其在脱毒组培苗检测中的应用

将具有典型葡萄卷叶病(Grapevine leafroll diseas,GLRD)症状的葡萄组织,经差速和硫酸铯—蔗糖密度梯度离心,提纯了GLRV,并制备了兔抗血清。电镜下可观察到长度从600～2000nm的线形病毒颗粒,其中以1400nm左右为主。免疫电镜结果表明线形病毒颗粒能被美国的NY-1分离株抗血清(Ⅲ型)所修饰。在间接ELISA中提纯制品与GLRV的Ⅲ、Ⅳ、Ⅱ型抗血清均能产生免疫反应。与Ⅲ型抗血清产生较强的免疫反应,Ⅳ型次之,Ⅱ型最弱。在SDS-免疫双扩散实验中病组织韧皮部粗提液与GLRV的Ⅲ,Ⅳ、Ⅱ型抗血清均产生免疫沉淀线。从而推测我国葡萄园内的葡萄卷叶病很可能由2种或3种卷叶病毒感染所致.采用A蛋白夹心酶联免疫吸附试验(PAS-ELISA)检测葡萄试管苗,Ⅲ型抗血清和自制抗血清的平行测试结果基本相符,共获得11个生食葡萄和10个山葡萄品种的脱葡萄卷叶病毒和扇叶病毒的组培苗,扩繁后田间试种表现出良好的农艺性状。     

葡萄扇叶病毒外壳蛋白基因的克隆,序列分析及其在大肠杆菌中…

通过ＲＴ－ＰＣＲ方法把葡萄扇叶病毒（ＧＦＬＶ）外壳蛋白基因（ＣＰ ｇｅｎｅ）分成两部分扩增,扩增产物克隆入ｐｇＥＭ－５Ｚｆ（＋）载体,并通过ＢｇｌⅡ位点连接成一完整的外壳蛋白基因,通过序列分析测得全长外壳蛋白基因为１５１２ｂｐ,编码５０４个ＡＡ＇ｓ与国外株系ＧＦＬＶ－Ｆ１３相比,核苷酸同源性为８８．４％,氨基酸同源性为９５．８％。并且这一外壳蛋白基因在大肠杆菌Ｅ． ｃｏｌｉＤＨ－５α中得到了表达。     

Use of Optimised PCR Methods for the Detection of GLRaV3: A Closterovirus Associated with Grapevine Leafroll in Tunisian Grapevine Plants

We report a modification and optimisation of a previously published procedure (Minafra and Hadidi, 1994) for the detection of GLRaV3 in infected grapevine plants. GLRaV3 RNA was successfully detected not only in total crude nucleic acid extracts of infected grapevine tissues but also in viruliferous mealybug extracts by IC-RT-PCR. This detection was rapid, sensitive and specific without occurrence of any background. A comparative ELISA, RT-PCR and IC-RT-PCR assays were carried out and revealed the greater sensitivity and specificity of PCR techniques.     

Serological and molecular properties of isolates of Bean common mosaic virus-BCMV from Khorasan Razavi province

During growing season, 2011–2012, selected bean plant samples with symptoms of mosaic, vein clearing, leaf rolling, were collected from different part of Khorasan Razavi province of north-eastern Iran. To identify the virus, leaf samples were tested serologically by DAS-ELISA and tissue blot, using specific BCMV polyclonal antibody. Elisa positive samples were rechecked by RT-PCR and IC-RT-PCR using set of primers directed to the coat protein gene which were designed to detect and characterise the viral species. Similarly, the BCMV primers amplified product of approximately 373?bp in 1% agarose gel electrophoresis. In phylogenic analysis, A and B groups were formed for 12 Iranian isolate comparing with 18 BCMV isolates from GenBank. Iranian isolates were classified into three clusters indicating to have more homology 99.3% with Mexican Isolates. There are also indications of some diversities among Iranian BCMV isolates.     

盐酸胍诱导CP43和CP47变性的太赫兹时域光谱技术研究

太赫兹时域光谱技术(THz-TDS)是近年来新兴的一种研究分子构型状态的技术. 把这项技术运用到两种光合膜蛋白CP43和CP47的研究上, 研究结果表明, 运用太赫兹时域光谱技术能有效地把具有相似结构的蛋白质区分开来, 这项技术也是监测蛋白质变性的一种有力工具. 在盐酸胍(GuHCl)作用下, CP47和游离叶绿素a(Chl a)的太赫兹(THz)振幅谱中都出现一个明显的位于1.8 THz处的峰. 我们认为这个峰来自于叶绿素a和盐酸胍的相互作用. 和CP43相比, CP47中的叶绿素a更易于和盐酸胍发生作用.     

RNA oligoprobe capture RT-PCR, a sensitive method for the detection ofGrapevine fanleaf virus in Tunisian grapevines

A simple, sensitive and specific RNA capture method is described for the detection of Grapevine fanleaf virus (GFLV) in infected grapevines. This method consists of hybridizing GFLV-RNAs to oligoprobes immobilized on nylon membranes, followed by RT-PCR amplification of targeted viral sequences. The RNA oligoprobe capture RT-PCR method is 10-fold more sensitive than IC-RT-PCR. The efficiency of the RNA oligoprobe capture RT-PCR and the reuse of immobilized oligoprobe membranes without loss of efficiency could make this procedure suitable for the routine diagnosis of GFLV in grapevines.