水稻黑条矮缩病毒基因组片段S9的cDNA克隆和全序列分析

应用RT PCR技术克隆了 3个中国水稻黑条矮缩病毒 (riceblack streakeddwarfvirus,RBSDV)分离株基因组片段S9,并测定了它们的全序列。结果表明 :RBSDV浙江分离株 (RBSDV Zj)基因组片段S9全长 190 0nt(EMBL登录号为AJ2 97430 ) ,RBSDV河北分离株 (RBSDV Heb)基因组片段S9全长 1898nt(EMBL登录号为AJ2 9742 9) ,湖北分离株S9全长 190 0nt(EMBL登录号为AJ2 9170 6 )。 3个分离株S9均含有两个开放阅读框(ORF) ,分别编码约 40kD和 2 4kD的多肽。3个中国分离株之间的核苷酸同源性高达 98.5 %～ 98.8% ,与日本分离株S9的核苷酸同源性均为 89.9%～ 90 .2 % ,而与意大利MRDVS8同源性仅为 85 .3%～ 86 .4%。我们发现ORF2十分保守 ,4个RBSDV分离株S9的ORF2同源性高达为 97.6 %～ 10 0 % ,与意大利MRDVS8的ORF2同源性也高达 94.3%。     

水稻黑条矮缩病毒基因组片段S7的cDNA克隆及全序列分析

应用RTPCR技术克隆了2个水稻黑条矮缩病毒 (rice blackstreaked dwarf virus,RBSDV)中国分离物,即浙江分离物和河北分离物的基因组片段S7,并测定了他们的全序列。结果表明：RBSDV浙江分离物(RBSDVZj)基因组片段S7全长2193nts(EMBL登录号为AJ297427),RBSDV河北分离物基因组片段S7全长2190nts(EMBL登录号为AJ297428),二者均含有两个开放阅读框(open reading frame,ORF),分别编码约41kD和36kD多肽,2个中国分离物核苷酸同源性高达99%,相应的ORF编码的多肽同源性分别为100%和94.4%,与日本RBSDV基因组片段S7核苷酸同源性为93.4%和93.8%,相应ORF编码的多肽同源性分别为98.1%(ORF1)、96.5%和97.8%(ORF2),与意大利MRDV S6核苷酸同源性为85.1%和85.3%,相应多肽同源性分别为92.3%(ORF1)、85.5%和86.8%(ORF2)。     

水稻黑条矮缩病毒第七号片段的cDNA克隆及其在大肠杆菌中的表达

运用RT-PCR技术,根据玉米粗缩病毒S6的末端序列设计引物,从玉米粗缩病感病材料中克隆得到一条2.2kb长的cDNA片段,序列分析表明,该片段全长2193bp,含两个开放阅读框,分别编码41.0kD和36.3kD的多肽。序列分析结果表明,该cDNA片段及其编码产物与水稻黑条矮缩病毒S7的cDNA序列及编码产物存在最高的相似性,推测该cDNA片段为水稻黑条矮缩病毒的S7片段,而不是玉米粗缩病毒的S6。分别将该片段的两个阅读框克隆到原核表达载体pET21d及pGEXKG中,经IPTG诱导后,两种蛋白均得到了高效的表达。表达产物回收后制备并得到了高效价的抗血清。     

水稻南方黑条矮缩病毒湖南鼎城株系S10片段的基因组序列分析

运用RT-PCR技术克隆了水稻南方黑条矮缩病毒(southern rice black-streaked dwarf virus,SRBSDV)湖南鼎城株系的基因组S10片段(SRBSDV-HuNDCS10),并对其全序列进行了测定和生物信息学分析。结果显示,SRBSDV-HuNDC S10片段全长为1797bp(登录号:JQ337964),含有1个ORF,编码557个氨基酸残基的衣壳蛋白,推测分子量约62.6kD,推测等电点为7.62,与已报道的广东、海南和云南分离物病毒的S10作比较,它们的核苷酸相似性分别为99.7%、99.0%和98.4%,氨基酸相似性分别为100.0%、99.5%和99.3%。对SRBSDV-HuNDCS10及部分Fijiviruses病毒对应片段在5’URT与3’URT存在的保守序列和互补序列进行了归纳,对其ORF编码的氨基酸序列进行了motif查找,得到该属(Fijiviruses)氨基酸序列的10个保守区段。此外,进行了糖基化位点、磷酸化位点及B细胞抗原表位预测,发现了3个可能的N端豆蔻酰基化位点,可能与病毒的侵染机制有关。     

江苏水稻黑条矮缩病毒S10的cDNA克隆序列分析

从来自江苏连云港并在本实验室保存的水稻黑条矮缩病毒接种的病株玉米中提取dsRNA,采用改进的单引物扩增技术获得了病毒基因组片段S10的cDNA克隆并测定了其全序列.结果表明S10全长1 801bp,含有一个ORF,组织结构与日本报道的RBSDV基本一致,核苷酸和推导的氨基酸序列与MRDV的相似性分别为87.5%和92.6%,与RBSDV的相似性分别为93.3%和96.4%.该研究也为病毒dsRNA克隆和序列分析奠定了基础.     

江苏水稻黑条矮缩病毒S10的cDNA克隆序列分析

从来自江苏连云港并在本实验室保存的水稻黑条矮缩病毒接种的病株玉米中提取dsRNA ,采用改进的单引物扩增技术获得了病毒基因组片段S10的cDNA克隆并测定了其全序列。结果表明S10全长 180 1bp ,含有一个ORF ,组织结构与日本报道的RBSDV基本一致 ,核苷酸和推导的氨基酸序列与MRDV的相似性分别为 87.5 %和92 .6 % ,与RBSDV的相似性分别为 93.3%和 96 .4%。该研究也为病毒dsRNA克隆和序列分析奠定了基础     

南方水稻黑条矮缩病毒快速检测

南方水稻黑条矮缩病毒(southern rice black-streaked dwarf virus,SRBSDV)引起的新水稻矮缩病近年在越南北部和我国南方各省爆发,准确快速地检测病毒是病害预测的关键。本文针对该病毒的S10序列设计了一对新的引物S10F/S10R,利用一步法RT-PCR,对2010年5~8月间湖南省下属各县送来的疑似感染该病毒引起的水稻矮缩病样本进行了检测,结果显示,新引物能有效地区分阳性样品和非阳性样品。同时对PCR产物进行了序列测定,结果表明序列均与已发表的南方水稻黑条矮缩病毒序列(登录号:EU523360和EU784840)达约99%的同源性。还在此基础上构建了系统发育树,发现SRBSDV湖南、广东和海南分离物病毒位于一个相对独立的分支。研究发现相对以往的巢式RT-PCR,本文采用的新引物及一步法RT-PCR能快速检测南方水稻黑条矮缩病毒,简化了操作步骤,缩短了检测时间,适合在SRBSDV检测和病害预测中应用。     

水稻瘤矮病毒基因组S9片段的基因结构特征

应用RT-PCR技术克隆了水稻瘤矮病毒(RGDV)中国广东信宜分离物(RGDV-C)的基因组S9片段,测定了全序列并进行了生物信息学分析。结果表明,RGDV-C S9片段全长共有1202bp(登录号AY556483),含有一个长的开放阅读框,这一开放阅读框编码一个由323个氨基酸残基组成的多肽,推测分子量约35.6kDa,与泰国分离物(RGDV-T)的全序列相比,它们的核苷酸长度相等,核苷酸同源性为98.1%,氨基酸同源性为98.5%。RGDV S9片段编码的Pns9蛋白在植物呼肠孤病毒属内未发现同源蛋白,其功能尚待确定。利用NCBI的BLAST查找与比较,发现Pns9与伯氏疏螺旋体(Borrelia burgdorferi)ATP依赖的Clp蛋白水解酶组分[ATP-dependent Clp prote-ase proteolytic component(clpP-1)]有21.8%的氨基酸序列同源性。     

水稻瘤矮病毒基因组S9片段的基因结构特征

应用RT-PCR技术克隆了水稻瘤矮病毒(RGDV)中国广东信宜分离物(RGDV-C)的基因组S9片段,测定了全序列并进行了生物信息学分析.结果表明,RGDV-C S9片段全长共有1202bp(登录号AY556483),含有一个长的开放阅读框,这一开放阅读框编码一个由323个氨基酸残基组成的多肽,推测分子量约35.6kDa,与泰国分离物(RGDV-T)的全序列相比,它们的核苷酸长度相等,核苷酸同源性为98.1%,氨基酸同源性为98.5%.RGDV S9片段编码的Pns9蛋白在植物呼肠孤病毒属内未发现同源蛋白,其功能尚待确定.利用NCBI的BLAST查找与比较,发现Pns9与伯氏疏螺旋体(Borrelia burgdorferi)ATP依赖的Clp蛋白水解酶组分[ATP-dependent Clp protease proteolytic component(clpP-1)]有21.8%的氨基酸序列同源性.     

浙江和河北发生的一种水稻、小麦、玉米矮缩病是水稻黑条矮缩病毒引起的

以浙江省的水稻黑条矮缩病和河北省的玉米粗缩病的病毒分离物为材料,对我国南北方两种病毒分离物进行了比较研究。两种病毒分离物的粒子大小形态相似,且在血清学上密切相关;二者的介体、寄主相同,并引起相同的症状,提纯两种病毒分离物的基因组片段凝胶电泳显示它们相应的基因组片段之间大小极相似。根据水稻黑条矮缩病毒（Rice black strecaked dwarf virus),RBSDV的S7和S8设计引物,利用PR－PCR技术,在两种病毒分离物中均可特异扩增到预期大小的片段,序列测定比较分析表明：它们的同源性达97．0％－98．9％,与日本RBSDV的同源性（92．2％－95．5％）高于与意大利MRDV的同源性（76．6％－88．4％）。从而认为我国南方的水稻黑条矮缩病和北方和玉米粗缩病都是同一种病毒－RBSDV引起的,也就是说我国北方的玉米粗缩病病原实际上是水稻黑条矮缩病毒,而不玉米粗缩病毒。     

I型鸭肝炎病毒VP1、3D基因克隆及其在大肠杆菌中的表达

根据GenBank中的I型鸭肝炎病毒全基因序列设计了扩增I型鸭肝炎病毒VP1、3D基因的引物, 用该特异性表达引物从I型鸭肝炎病毒cDNA模板中扩增得到目的基因VP1、3D, 用相同的限制性内切酶酶切目的基因和表达载体pET32a后构建重组表达载体, 转化宿主BL21(DE3), 用不同浓度的IPTG诱导VP1、3D基因的表达, 收集菌液进行SDS-PAGE电泳, Western-blotting分析蛋白免疫原性。结果表明, VP1、3D在大肠杆菌中表达量较高, 表达产物的分子量约为48 kD、68 kD, 并能被兔抗DHV-1血清所识别。I型鸭肝炎病毒VP1、3D蛋白在大肠杆菌中表达产物具有免疫原性。     

Expression of Rice Gall Dwarf Virus Outer Coat Protein Gene (S8) in Insect Cells

To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected b...     

Sequence Analysis of Segment 8 of Five Chinese Isolates of Rice Gall Dwarf Virus and Expression of a Main Outer Capsid Protein in Escherichia coli

The rice gall dwarf disease, caused by the Rice gall dwarf virus (RGDV) is a serious disease occurring in rice in many regions of Guangdong province. As a basis to control the disease we have studied the genomic diversity of a variety of isolates from different locations. Genome segment 8(S8), encoding a main outer capsid protein (Pns8) of RGDV five isolates (BL, CH, DQ, GZ, XY) from Guangdong province was cloned and sequenced. The results revealed that all the S8 segments of the five isolates consisted of 1 578 nucleotides and had a single open reading frame (ORF) extending for 1 301 nucleotides from nucleotide 21 which encoded a polypeptide of 426 amino acids with an estimated molecular weight of 47.4 kDa. The S8 full-length sequence and the ORF sequence shared 97.3%-98.8% and 97.3%-99.1% nucleotide sequence identities within the five Chinese isolates, and shared 94.8%-95.6% and 95.0%-96.0% identities with those of the Thailand isolate respectively. The deduced amino acid sequence of Pns8 in GZ isolate was identical to that in the Thailand isolate, while the amino acid sequence variability of Pns8 within five Chinese isolates ranged from 0.5% to 2.1%. These results indicate that the S8 segment of RGDV is highly conserved in different isolates from different locations. The S8 cDNA from the XY isolate was cloned into the plasmid vector pET-28b(+) and a fused expression protein with an apparent molecular mass of 51kDa was specifically detected in an analysis of Escherichia coli Rossetta(DE3)Ⅱcells. To our knowledge, this is the first report on analysis of the RGDV segment 8 sequence and genetic comparison of different RGDV isolates and their protein expression.     

Sequence Analysis of Segment 8 of Five Chinese Isolates of Rice Gall Dwarf Virus and Expression of a Main Outer Capsid Protein in Escherichia coli

The rice gall dwarf disease, caused by the Rice gall dwarf virus (RGDV) is a serious disease occurring in rice in many regions of Guangdong province. As a basis to control the disease we have studied the genomic diversity of a variety of isolates from different locations. Genome segment 8(S8), encoding a main outer capsid protein (Pns8) of RGDV five isolates (BL, CH, DQ, GZ, XY) from Guangdong province was cloned and sequenced. The results revealed that all the S8 segments of the five isolates consisted of 1 578 nucleotides and had a single open reading frame (ORF) extending for 1 301 nucleotides from nucleotide 21 which encoded a polypeptide of 426 amino acids with an estimated molecular weight of 47.4 kDa. The S8 full-length sequence and the ORF sequence shared 97.3%-98.8% and 97.3%-99.1% nucleotide sequence identities within the five Chinese isolates, and shared 94.8%-95.6% and 95.0%-96.0% identities with those of the Thailand isolate respectively. The deduced amino acid sequence of Pns8 in GZ isolate was identical to that in the Thailand isolate, while the amino acid sequence variability of Pns8 within five Chinese isolates ranged from 0.5% to 2.1%. These results indicate that the S8 segment of RGDV is highly conserved in different isolates from different locations. The S8 cDNA from the XY isolate was cloned into the plasmid vector pET-28b( ) and a fused expression protein with an apparent molecular mass of 51kDa was specifically detected in an analysis of Escherichia coli Rossetta(DE3)Ⅱcells. To our knowledge, this is the first report on analysis of the RGDV segment 8 sequence and genetic comparison of different RGDV isolates and their protein expression.     

抗黑条矮缩病水稻品种资源的筛选与鉴定

近年来,水稻黑条矮缩病严重影响我国水稻生产,培育高抗黑条矮缩病的水稻新品种迫在眉睫。获得抗性好的水稻资源是育种的基础。本研究通过对保存的2000份水稻地方品种连续2年设置2个试验点的黑条矮缩病抗性鉴定,初步筛选出连续2年没有发病的资源38份。对这38份中农艺性状较好的6份粳稻资源进行人工室内接虫鉴定,获得高抗水稻黑条矮缩病的水稻资源1份。与感病对照相比,水稻黑条矮缩病毒（Rice black-streaked dwarf virus, RBSDV）在抗病品种体内的表达量下降71.5倍,表明抗病品种对RBSDV在体内的复制有明显的抑制作用。本研究为水稻黑条矮缩病抗病育种和抗性基因的定位、克隆奠定了材料基础。     

通育粳1号水稻乙醇脱氢酶基因克隆与原核表达

克隆通育粳1号水稻乙醇脱氢酶(alcohol dehydrogenase,ADH)基因,并在原核系统中进行体外表达。取通育粳1号水稻幼根,提取总RNA,RT-PCR法扩增ADH基因开放阅读框架片段,双酶切后连接至pGEX-4T-1表达质粒中。将质粒转化至BL21(DE3)宿主菌中,平皿培养,挑取阳性菌落培养,提取重组质粒,酶切、电泳鉴定插入片段并测定其序列。pGEX-4T-1-ADH/BL21进行常规LB扩大培养,IPTG(1 mmol/L)作用2、3和4 h诱导表达,SDS-PAGE检测表达产物。结果显示,插入质粒中的ADH片段序列和方向正确无误,表达蛋白分子量符合预期值42 kD,表达至最大值的诱导时间为3 h。因此,该基因的成功克隆和表达为进一步研究水稻中ADH的作用和应用生物工程法大量获得ADH奠定基础。     

The cDNA Cloning and Nucleotide Sequence of the Gene Encoding Nonstrucure Protein of Rice Dwarf Virus Genome Segment 10

The results of cloning and sequencing the gene encoding nonstructure protein of the rice dwarf virus (RDV) gtnome segment 10 with polymerase chain reaction(PCR) technique were reported. The amplified PGR product was cloned into Hind Ⅱ site of plasmid pGEM3zf(-) and analysed with restriction enzymes. The physical map of the cloned fragment has been constructed, the insert is 1150 bp in length with restriction enzyme sites of Sac Ⅰ, Hind Ⅲ, NdeⅠ, BamH Ⅰ, etc. Besides, two restriction enzyme sites Bgl Ⅱ and EcoR Ⅰ have been separetely added in the 5 and 3 end of the segment in order to be cloned into plant intermediate vector in a convenient way. The fragments cleaved by the above-mentioned restriction enzymes were subcloned and the DNA sequence of full length of segment 10 was determined. In comparison with the RDV epidemic in Japan, the nucleotide sequence and deduced amino acid sequence of cloned segment 10 are 96.03% and 97.17% in homology respectively.