马铃薯Y病毒HC-Pro中心区域在病毒协生作用中的主导地位

利用PCR方法获得了马铃薯病毒中国株系(PVY-C)HC-Pro基因的5个缺失突变体,构建了相应的植物表达载体。通过土壤农杆菌(Agrobacterium tumefaciens)介导法转化了烟草品种K₃₂₆(Nicotina tabacum cv.K₃₂₆)。PCR和Southern blot分析证明了HCPro基因及其缺失突变体已整合到烟草基因组中,Western blot表明它们在转基因烟草中得到了表达。侵染性试验发现HCPro中心区域介导转基因烟草中PVY-C和黄瓜花叶病毒(CMV)、PVYC和马铃薯X病毒(PVX)之间的协生作用,从而明确了PVY-C HC-Pro中心区域为病毒协生作用的功能区域。     

马铃薯Y病毒蚜传辅助成分介导PVX/PVY协生作用

构建了马铃薯Y病毒中国株系（PVY－C）蚜传辅助成分（HC－Pro）基因的正义、反义和缺失三种植物表达载体,通过农杆菌介导法转化烟草品种NC89。Southern blot分析表明,HC－Pro基因及其突变体已经整合到烟草染色体中,Western blot分析证明,正义HC－Pro基因及其缺失突变体在转基因烟草中有表达产物,攻毒试验结果表明,转正义,HC－Pro基因及其缺失突变体不仅能够提高T1转基因烟草中PVY－C的病毒积累和致病,而且对异源病毒PVX具有同样的作用,而转反义HC－Pro基因烟草对PVY－C和PVX的致病性无影响,因此,PVY－C HC－Pro基因介导PVX／PVY的协作作用。     

马铃薯Y病毒属病毒HC—Pro蛋白功能研究进展

马铃薯Ｙ病毒属病毒基因组编码的ＨＣ－Ｐｒｏ蛋白（蚜传辅助因子）具有多种功能,在病毒生活中史各个环节中起重要作用。ＨＣ－Ｐｒｏ蛋白具有蛋白酶活性,作为蚜传辅助因子参与病毒蚜传过程,调节病毒中宿主体内的转移,并在病毒复制、宿主症状表达及增强异源病毒复制等方面发挥作用。本文对ＨＣ－Ｐｒｏ蛋白的既定、预测功能作一综述。深入了解ＨＣ－Ｐｒｏ蛋白的功能不仅在理论上有助于明确病毒生活周期,而且在实践上也可根据其     

马铃薯Y病毒蚜传辅助因子促进马铃薯X病毒长距离运输

采用PCR和定点突变法,对马铃薯Y病毒中国株系（Chyinese strain of potato Ypotyvirus,PVY-C)蚜传辅助成分（helper component proteinase,HC-Pro)基因中心区域的CCCT基序和PTK基序进行定点改造,获得了4种突变体。然后将突变体砍降到植物表达载体pBin438中,所得到的重组体通过根癌土壤杆菌（Agrobacterium tumefaciens(Smith et Townsend)Conn）介导法转了烟草（Nicotiana tabacum L.cv.K326).Southern blotting和Western blotting分析表明4种突变体已经成功整合到烟草的基因组中,并在蛋白水平上得到了表达。马铃薯X病毒（potato X potexvirus,PVX)对转基因烟草的攻毒实验表明,4种突变体均使PVY－C HYC－Prog严重丧失了促进PVX病毒粒子在寄主体内积累和提高PVX致病性的功能,说明CCCT、PTK基序为PVY－C HYC－Pro介导PVX／PVY协生作用所必需。同时证明了HC-Pro具有增强PVX在寄主体内长距离运输的功能。     

马铃薯Y病毒属病毒HC-Pro蛋白功能研究进展

马铃薯Ｙ病毒属病毒基因组编码的ＨＣ－Ｐｒｏ 蛋白（蚜传辅助因子）具有多种功能,在病毒生活史各个环节中起重要作用。ＨＣ－Ｐｒｏ 蛋白具有蛋白酶活性,作为蚜传辅助因子参与病毒蚜传过程,调节病毒在宿主体内的转移,并在病毒复制、宿主症状表达及增强异源病毒复制等方面发挥作用。本文对ＨＣ－Ｐｒｏ 蛋白的既定、预测功能作一综述。深入了解ＨＣ－Ｐｒｏ 蛋白的功能不仅在理论上有助于明确病毒生活周期,而且在实践上也可根据其特性设计抗病毒的新策略。     

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…     

植物病毒协生作用及其分子机理

植物病毒协生作用分布广,是造成农作物减产的重要原因之一,抗病毒转基因植物中协生作用的出现,严重限制了基因工程植物的商品化生产。本文对植物病毒协生作用的类型和特点、协生作用中病毒与病毒、病毒与环境间的相互作用及其分子机制进行了阐述。     

马铃薯Y病毒复制酶基因的克隆和序列分析

马铃薯Ｙ病毒复制酶基因的克隆和序列分析彭学贤,项瑜,刘俊君,莽克强（中国科学院微生物研究所,北京１００００８０）ＭｏｌｅｃｕｌａｒｃｌｏｎｉｎｇａｎｄｓｅｑｕｅｎｃｅａｎａｌｙｓｉｓｏｆＰＶＹＮＩｂｇｅｎｅ￥ＰｅｎＸｕｅｘｉａｎ;ＸｉａｎｇＹｕ;Ｌｉ...     

马铃薯Y病毒pipo基因的分子变异及结构特征分析

为揭示马铃薯Y病毒(Potato virus Y, PVY) pipo基因的分子变异和结构特征, 文章根据文献报道的马铃薯Y病毒属(Potyvirus) pipo 基因保守区序列设计一对简并引物, 从感染PVY的马铃薯病叶中克隆获得pipo基因的cDNA全长序列, 分析其核苷酸序列和氨基酸序列的特征, 并基于氨基酸序列使用贝叶斯法重建了Potyvirus的系统发育树。结果显示：20个PVY分离物成功扩增出预期大小(约235 bp)的特异性片段, 其核苷酸序列与已报道的其它PVY 株系的pipo基因核苷酸序列一致性均在92%以上; 5′端均含有典型的G1-2A6-7 基序(motif), 无碱基插入/缺失, 所有的核苷酸变异都是碱基置换, 共发现13个多态性位点, 其中4个简约信息位点, 9个单一变异位点, 表明该基因高度保守, 但不同分离物也存在一定的分子变异; PIPO蛋白理论等电点11.26~11.62, 无信号肽和跨膜区, 是可溶的亲水性蛋白; 整个蛋白含有3个保守区, 其中位于10~59aa的基序最为保守。该蛋白主要定位于线粒体中, 可能是线粒体导肽。系统发育分析结果显示, 源于PVY不同株系优先相聚成簇, 而向日葵褪绿斑驳病毒(Sunflower chlorotic mottle virus, SuCMoV)与辣椒重花叶病毒(Pepper severe mosaic virus, PepSMV)的亲缘关系较PVY相比更近, 与前人的结果相一致, 表明PIPO蛋白可以作为研究Potyvirus系统发育关系的新的分子标记。     

植物病毒协生作用及其分子机理

植物病毒协生作用分布广,是造成农作物减产的重要原因之一.抗病毒转基因植物中协生作用的出现,严重限制了基因工程植物的商品化生产.本文对植物病毒协生作用的类型和特点、协生作用中病毒与病毒、病毒与环境间的相互作用及其分子机制进行了阐述.     

Potato Y Potyvirus Helper Component Proteinase Enhances Long-distance Movement of Potato X Potexvirus

To mutagenize two conserved CCCT and PTK motifs in the central domain of Chinese strain of potato Y potyvirus (PVY-C) helper component proteinase (HC-Pro), four mutants of HC-Pro gene were obtained by PCR and site-directed mutagenesis, and then were inserted into the constitutive expression vector pBin438. Leaves from tobacco ( Nicotiana tabacum L. cv. K326) were transformed with these four plant expression plasmids by Agrobacterium -mediated transformation, respectively. Southern and Western blotting analyses showed that these four mutants were integrated into tobacco genomic DNA and could express the corresponding proteins in most of the transgenic plants. The challenge of transgenic plants with potato X potexvirus (PVX) revealed that the expression products of PVY-C HC-Pro mutants in transgenic plants greatly abolished functions of HC-Pro in enhancing the accumulation and pathogenicity of PVX, indicating that CCCT and PTK motifs of HC-Pro were required for PVX/PVY synergism. Meanwhile, the results demonstrated that PVY-C HC-Pro had a function in accelerating the long-distance movement of PVX in these transgenic plants for the first time.     

马铃薯Y病毒NIb复制酶基因在转录水平介导的相对广谱抗病性

在大肠杆菌中表达了马铃薯Ｙ病毒中国分离物（ＰＶＹ－Ｃ）复制酶ＮＩｂ基因,并制备了其抗血清。利用ＰＣＲ定点突变方法使ＮＩｂ基因移码－１位,构建了移码－１位ＮＩｂ基因（ＵＮ）的植物表达载体。通过土壤农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ ＬＢＡ４４０４）介导转化烟草ＮＣ８９,获得５１株再生植株。对再生植株的分子检测结果表明,转基因烟草中检测到ＵＮ基因相应的ＲＮＡ转录产物,３推测该基     

Potato virus Y NIa protease activity is not sufficient for elicitation of Ry-mediated disease resistance in potato

Ry confers extreme resistance (ER) to all strains of potato virus Y (PVY). In previous work, we have shown that the protease domain of the nuclear inclusion a protease (NIaPro) from PVY is the elicitor of the Ry-mediated resistance and that integrity of the protease active site is required for the elicitation of the resistance response. Two possibilities arise from these results: first, the structure of the active protease has elicitor activity; second, NIa-mediated proteolysis is required to elicit the resistance response. To resolve these possibilities, the NIaPro from PVY was randomly mutagenised and the clones obtained were screened for elicitation of cell death as an indicator of resistance and proteolytic activity. We did not find any mutants that had retained the ability to elicit cell death but had lost protease activity, as measured by processing of the NIa cleavage site in the viral genome. This was consistent with the idea that protease activity is necessary for elicitor activity. However, protease activity was not sufficient because we found three elicitor-defective mutants in which there was a high level of protease activity in this assay.     

Comparison of resistance to potyviruses within Solanaceae: infection of potatoes with tobacco etch potyvirus and peppers with potato A and Y potyviruses

The reaction of several cultivated potato varieties (Solarium tuberosum L.) to three strains of tobacco etch potyvirus (TEV-F, TEV-Mex21 and TEV-ATCC) and the reaction of several pepper lines (Capsicum annuum L. and C. chinense L.) to two strains of potato Y potyvirus (PVY^O and PVY^N) and one strain of potato A potyvirus (PVA-M) was tested. The potato varieties included in this study carried resistance genes against PVY, PVA and potato V potyvirus, but all were susceptible to TEV and developed mottle and mosaic symptoms. TEV was readily transmitted by mechanical inoculation from tobacco and potato to potato, whereas transmission from pepper to potato occurred infrequently. TEV was transmitted through potato tubers, and from pepper to potato plants by aphids. Lack of detectable systemic infection following graft-inoculation indicated extreme resistance to PVY^O and PVA in several pepper lines. No pepper line was systemically infected with PVY^N following mechanical inoculation (graft-inoculation was not carried out with PVY^N). The development of necrotic lesions following mechanical and graft-inoculation indicated hypersensitive response to PVY^O in several pepper lines which resembled the resistance responses to these potyvirus strains in potato. Results of this study together with previous work indicate that C. annuum cv. Avelar is resistant to four potyviruses [PVY, PVA, pepper mottle potyvirus (PepMoV) and some isolates of TEV]; C. annuum cv. Criollo de Morelos and C. chinense PI 152225 and PI 159236 are resistant to three potyviruses (PVY, PepMoV and PVA; and PVY, PepMoV and TEV, respectively); C. annuum 9093–1 and 92016–1 are resistant to PVY and PepMoV; and C. annuum cv. Jupiter and C. annuum cv. RNaky are resistant to PVY^N and PVA.     

Tobacco plants transformed with the potato virus YN coat protein gene are protected against different PVY isolates and against aphid-mediated infection

Tobacco plant lines transformed with the coat protein (CP) gene of the tobacco veinal necrosis strain of potato virus Y (PVY^N), and previously shown to be protected against mechanical inoculation with the virus, have now been tested for specificity and protection against virus infection mediated by viruliferous aphids. To determine the specificity of virus protection, two transgenic tobacco lines, A30 and A80, were challenged with several isolates of distinct PVY strains (PVY^N, PVY^O and PVY^C) by mechanical inoculation. Clear levels of protection against the PVY^O-isolates tested were maintained in the transgenic plants, although these levels were slightly lower than the protection against the homologous PVY^N strain from which the CP gene was derived. Interestingly, no protection against mechanical virus inoculation with the Gladblaadje isolate of PVY^C could be observed. To assess the levels of protection against aphid-mediated virus infection, two transgenic plant lines, A30 and D25, showing respective levels of protection of 95 and 80% against mechanical virus inoculation, were challenged using PVY^N viruliferousMyzus persicae. Virus inoculation using six aphids per plant, resulted in similar levels of protection in both transgenic lines as found previously for mechanical inoculation. Protection was maintained in both lines, even when as many as 60 viruliferous aphids were used per plant in the inoculation experiments.