进境玉米种子携带玉米褪绿斑驳病毒的检测与鉴定

 Imported maize seeds were planted in a quarantine greenhouse and two seedlings with chlorotic mottle symptoms were observed. The ELISA results showed that the two seedlings reacted positively with antibody against Maize chlorotic mottle virus (MCMV). The positive samples were further identified by RT-PCR method and the 711 bp size target bands were amplified specifically. The similarity range of nucleotide sequence of both RT-PCR product and six MCMV coat protein genes was 97%-99%. The amino acid sequence homology was 97.88%-99.89%. Based on the above results, the virus was identified as MCMV. It was the first time that MCMV was intercepted from imported maize seeds.     

藜草花叶病毒外壳蛋白基因的克隆与RT-PCR检测

 The nucleotide sequence of coat protein (cp)gene of Sowbane mosaic virus (SoMV)was determined. The cp gene of SoMV consists of 726 nucleotides and encodes a putative protein of 241 amino acid re-sidues. Sequence comparison showed that SoMV was most closely related to Rubus chlorotic mottle virus compared to other sobemoviruses. A primer pair was designed for the detection of SoMV based upon the determined cp nucleotide sequence. An expected fragment with 510 bp could be obtained from SoMV, while no specific band was observed from the healthy control. The result showed that the RT-PCR assay with the primer pair was suitable for the specific detection of SoMV.     

江西甘蔗花叶病病原的分子鉴定

 Sugarcane mosaic disease, caused by Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), Maize dwarf mosaic virus (MDMV) or Johnsongrass mosaic virus (JGMV) in Potyvirus, is one of the most important viral diseases of sugarcane. In the study, four primer pairs specific to SCMV, SrMV, MDMV and JGMV, respectively, were designed and used to detect 29 sugarcane leaf mosaic samples collected from 9 locations in Jiangxi province. The representative RT-PCR products were sequenced. The results showed that 22 samples were infected by SCMV, three by SrMV, and four were mix-infected by SCMV and SrMV. MDMV or JGMV were not identified in all samples. The result indicates that SCMV is the major pathogen of sugarcane mosaic disease in Jiangxi province, and SrMV is also a pathogen for the disease.     

同步检测为害百合种球3种检疫性病毒的多重RT-PCR方法

 The multiplex RT-PCR approach was developed for simultaneous detections of Arabis mosaic virus(ArMV),Strawberry latent ringspot virus(SLRSV)and Lily symptomless virus(LSV)from the imported lily bulbs.The results indicated that good specificity and sensitivity for simultaneous detection were obtained.The ultimate of RNA detection with three viruses mixture was 305 pg.The ultimate of RNA detection with ArMV,SLRSV and LSV were 156.0,13.4 pg and 1.12 ng respectively.This approach has potential to be used in quarantine of imports and exports.     

地高辛标记cDNA探针检测苹果茎痘病毒

 Partial sequence(314 bp) of ASPV was cloned and used as a probe labelled with digoxigenin-11dUTP. The total RNA extracted from samples with Apple stem pitting virus and a series of dilutions of plasmid with ASPV-cDNA were detected by dot blot hybridization. The results showed that the probe was sensitive and specific. The probe couldn't hybridize with total RNA of Apple stem grooving virus, Apple mosaic virus and Apple chlorotic leaf spot virus samples as well as negative control, only hybridized with that extracted from dormant shoot infected with ASPV. The sensitivity for detection of plasmid contained ASPV-cDNA was 1.64 μg.     

甘蔗花叶病毒福建分离物外壳蛋白基因的克隆及序列分析

 A fujian isolate of Sugarcane mosaic virus named SCMV-FJ was isolated from infected sugarcane. Cloning and sequence analysis of the coat protein gene of this isolate was carried out. A pair of primers was designed and synthesized based on the nucleotide sequences of coat protein genes of sugarcane mosaic viruses reported. The coat protein gene of SCMV-FJ was amplified from the extracted total RNA of the infected sugarcane by using RT-PCR, and cloned into the pMD18-T vector. The sequencing result indicated that the cloned segment included a 1137 bp open reading frame(ORF) and a 228 bp 3' untranslated region, in which the ORF comprised the whole coat protein and part of the nuclear inclusion b. The nucleotide and the deduced amino acid sequences of the coat protein gene were compared with those of the other isolates or strains of SCMV subgroup reported in GenBank. The result showed that it shares 56.8%-97.1% and 55.3%-99.4% homology in nucleotide and the putative amino acid sequences, respectively, with the highest amino acid homology of 99.4% with SCMV-D. Thus it was identified as a SCMV-D. This experiment provided a rapid, sensitive and relatively inexpensive method for RT-PCR detection of SCMV. At the same time, the cloning of SCMV-FJ coat protein gene provided the foundation for plant gene engineering against SCMV.     

侵染云南花魔芋的马铃薯Y病毒属病毒分离物的检测及分子鉴定

 YN80 was isolated from Amorphophallus rivieri Durieu showing mosaic and crinkle symptoms in Songming, Yunnan province. Flexuous filamentous particles were found in diseased leave sap and pinwheel inclusion bodies were found in the leave tissue. YN80 had positive reaction to universal antibody of Potyvirus by DAS-ELISA. 3'-terminal sequence of YN80 was cloned and sequenced. cp gene of YN80 consisted of 987 nt, encoded 328 aa (36.1 kDa). Sequence analysis showed that YN80 shared the highest identity (97.0%)with CP amino acid sequence of Dasheen mosaic virus (DsMV). These data indicated that YN80 was an isolate of DsMV. This is the first molecular identification of A. rivieri Durieu isolate of DsMV in China.     

西瓜花叶病毒抗血清制备及其应用

 西瓜花叶病毒(Watermelon mosaic virus, WMV)是危害葫芦科作物的重要病毒。制备特异性强、效价高的抗血清对快速准确检测和鉴定WMV具有重要意义。本研究将WMV外壳蛋白(Coat protein,CP)基因克隆到原核表达载体pEHISTEV获得pEHISTEV-WMV-CP。将pEHISTEV-WMV-CP转化大肠杆菌Rosetta,经IPTG诱导,成功表达出分子量约为36 kDa的蛋白,与预期WMV CP大小一致。切胶回收WMV CP,与等体积弗氏不完全佐剂充分乳化后免疫健康新西兰大白兔。Western blotting结果表明,制备的抗血清与WMV CP有反应,与同属的番木瓜环斑病毒(Papaya ringspot virus,PRSV)、马铃薯Y病毒(Potato virus Y,PVY)、小西葫芦黄花叶病毒(Zucchini yellow mosaic virus, ZYMV)和烟草花叶病毒属的黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)均无反应。酶联免疫吸附测定(PTA-ELISA)结果表明,本研究制备的WMV抗血清效价为1∶8 192,并且能够检测稀释512倍的病毒汁液。利用该抗血清对田间采集的10个RT-PCR检测为阳性的样品进行检测,全部呈现阳性。本研究利用大肠杆菌表达的CP制备的WMV抗血清具有较高的特异性和灵敏度。研究结果为WMV的快速检测和鉴定奠定了基础。     

烟草脉带花叶病毒cp基因的原核表达及抗血清制备

 利用马铃薯Y病毒属病毒的简并引物, 通过RT-PCR技术获得了云南烟草病毒分离物YND的3'端约1.7 kb片段, 序列分析证明该分离物为烟草脉带花叶病毒(Tobacco vein banding mosaic virus, TVBMV)。将TVBMVcp基因克隆到表达载体pET-22b (+)上, 在大肠杆菌BL21(DE3)中诱导表达出分子量为35.0 kD的融合蛋白。利用该融合蛋白制备了TVBMV的多克隆抗体, ELISA测定抗血清效价为1/4 096。Western blotting和DIBA分析结果表明, 获得的抗血清和原核表达的病毒蛋白及植物病汁液中的病毒均有特异性反应, 可用于TVBMV的快速检测。     

云南、福建、湖南烟区烟草花叶病主要病毒种类检测及黄瓜花叶病毒亚组鉴定

 采用抗原直接包被和双抗体夹心酶联免疫吸附测定法(ELISA)对采自云南、福建、湖南烟区烟草花叶病样品进行了病毒种类检测,利用三抗体夹心ELISA对黄瓜花叶病毒(Cucumber mosaic virus,CMV)的亚组类型进行了鉴定。在云南采集的520个花叶病样品中,烟草花叶病毒(Tobacco mosaic virus,TMV)、CMV和马铃薯Y病毒(Potato virus Y,PVY)总检出率分别为71.74%、55.01%和6.35%;在福建采集的150个花叶病样品中,TMV、CMV和PVY的总检出率分别为94%、24.66%和8.00%;在湖南采集的74个花叶病样品中,TMV、CMV和PVY的总检出率分别为58.11%、51.35%和2.70%。部分样品为2种以上病毒复合侵染。云南、福建和湖南采集的64个CMV阳性样品中,属亚组Ⅰ的样品为57个,占89.1%;属亚组Ⅱ的样品为10个,占15.6%;其中3个样品为亚组Ⅰ和亚组Ⅱ的复合侵染。     

湖南水稻上1种新矮缩病的病原研究

 Summer rice was suffered extensive damage from a new dwarf disease in Hunan Province in year 2009. In this study, the causal agent of this disease was confirmed as Southern rice black-streaked dwarf virus (SRBSDV) by nested RT-PCR, RT-PCR and PCR product sequencing.     

侵染百合的黄瓜花叶病毒亚组Ⅱ分离物cp基因克隆和序列分析

 从云南大理的东方型百合上得到黄瓜花叶病毒分离物(CMV-DL), ELISA检测初步确定为CMV亚组Ⅱ分离物, 设计并合成CMV亚组Ⅱ的特异引物, RT-PCR扩增得到1条约800 nt的特异片段, 经克隆及序列测定, 该片段长828 nt, 包含的外壳蛋白(CP)基因由657 nt组成。将该分离物的cp基因与其它14个CMV分离物进行同源性比较, 在核苷酸水平上与CMV亚组I和亚组Ⅱ的同源性分别为76.8%~78.1%和98.6%~99.2%;在氨基酸水平上与CMV亚组I和亚组Ⅱ的同源性分别为82.0%~84.3%和95.9%~100.0%。结果表明CMV-DL为CMV亚组Ⅱ成员。     

云南甘蔗花叶病病原检测及一个分离物的分子鉴定

 调查表明甘蔗花叶病在云南发生普遍。电镜检测采自云南6个蔗区主栽品种上的28个甘蔗花叶病病样(分离物),其中25个病样的病叶汁液中观察到弯曲线状的病毒粒体,病叶组织中有风轮状和卷筒状内含体;对这25个分离物进行间接ELISA检测,16个与马铃薯Y病毒属抗血清呈阳性反应,其余呈阴性反应。根据蔗区及其主栽品种的不同,挑选7个分离物进行鉴别寄主测定,结果显示不同分离物鉴别寄主范围和致病性存在明显差异,分离物HH-1有范围最广的鉴别寄主和较强的致病性。克隆并测定HH-1基因组3'末端序列,序列分析发现HH-1的外壳蛋白(CP)基因共864个核苷酸,编码287个氨基酸,与高粱花叶病毒(Sorghum mosaic virus,SrMV)余杭分离物CP氨基酸序列的同源性最高,为97.7%;因此推定HH-1属于SrMV的一个新分离物。