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.     

水稻条纹病毒NS2基因原核表达产物多克隆抗体制备及受侵染水稻和灰飞虱体内NS2检测

 The NS2 gene of Rice stripe virus (RSV) was amplified by RT-PCR, cloned into pGEM-T vector and sequenced. The NS2 gene was inserted into prokaryotic expression vector pET32a to produce recombinant plasmid pET32a-NS2. The recombinant plasmid was introduced into Escherichia coli strain BL21 (DE3) pLysS. SDS-PAGE and Western blot analysis confirmed that NS2 fusion protein was expressed after induction by IPTG. The recombinant NS2 protein was purified with Ni²⁺-NTA agarose affinity chromatography and the polyclonal antibody against NS2 protein was raised in rabbit. NS2 protein was successfully detected in small brown planthopper (Laodelphax striatellus) at 1:1 600 dilution of the total protein of single planthopper and in infected rice (Oryza sativa) at 1:800 dilution of 10 mg leave by dot immunobinding assay using the polyclonal antibody.     

甘蔗黄叶病毒复制酶基因的克隆及序列分析

 The gene of RNA-dependent RNA polymerase (RdRP) was cloned by RT-PCR from Sugarcane yellow leaf virus-Fuzhou isolate (CHN-FJ1) and then cloned into pMD18-T vector. The sequence showed that the fragment comprised 1 212 nucleotides including part gene of ORF1 and ORF2. The ORF2 was involved the RdRP gene consisted of 995 nucleotides and encoded putative protein of 331 amino acids. Compared the nucleo-tide sequence and encoded putative protein of CHN-FJ1 with the other isolates from different countries, they shared the homology above 92.0%. Phylogenetic tree suggested that the sixteen isolates were classified into four types according to the amino acid sequence of the RdRP. One of the groups contained CHN-FJ1 and the other isolates from China, American, Brazil, Australia and Colombia;however, there was the closest relation between CHN-FJ1 and BRA-YL1 isolate from Brazil.     

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.     

马铃薯疮痂病菌致病相关基因的克隆及表达

 A pathogenic-related gene nec1 was cloned in Streptomyces scabies CPS-1, potato scab strain. Analysis results showed that the length of open reading frame(ORF) for nec1 gene was 666 bp, and the GC content was 54.2%. Sequence alignment indicated that a 650 bp up-stream sequence shared 91% similarity with IS 256 family transposase nucleotide sequences by BLASTn searches against GenBank. The segments obtained by PCR amplification were digested by enzymes SphⅠand SacⅠ, and linked to the expression vector pIJ702. The recombinants were transformed into nonpathogenicity strain Streptomyces lividans 66 TK24. Bioas-say results suggested that the transformants possessed the same symptoms as pathogenic strain on potato tuber slices and radish seedlings, which implied that nec1 gene was associated with the pathogenicity of S. scabies CPS-1.     

小麦蓝矮病植原体转运蛋白secY基因片段序列分析

 Wheat blue dwarf(WBD) is a disease caused by phytoplasma and only reported from China. A fragment about 1.3 kb in protein translocation gene, secY was amplified by PCR from the total DNA of di-seased wheat sample with primer pair secYF/secYR, which was designed based on secY gene sequence of known 16SrI group members. Nucleotide acid sequence analysis of amplified fragment indicated that the length was 1 240 bp. A phylogenetic tree based on secY gene sequences was constructed and showed that wheat blue dwarf phytoplasma was clustered into the Candidatus Phytoplasma asteris, subgroup 16SrI-C. Wheat blue dwarf phytoplasma showed high homology with clover phyllody phytoplasma strains based on sequence comparison and phylogenetic analysis.     

广东番茄上检测到Tospovirus病毒

 Some tomato samples possibly infected by tospovirus in Guangdong were detected with indirect ELISA and RT-PCR. The results showed that the virus infected tomato did not react with the antiserum of Tomato spotted wilt virus (TSWV), but about 500 bp fragment of RT-PCR shared 83%-84% nucleotide identities with N gene of those reported tospoviruses. The phylogenetic tree of the N gene fragment compared with those of other tospoviruses indicated that the virus infected tomato was belonged to Tospovirus.     

草莓镶脉病毒ORF Ⅱ基因的克隆、原核表达与抗血清制备

 用CTAB法从感染草莓镶脉病毒（Strawberry vein banding virus, SVBV）的草莓叶片中提取总DNA,设计特异性引物扩增SVBV中国分离物ORF Ⅱ基因,克隆并测序。结果表明,SVBV中国分离物ORF Ⅱ基因全长486 nts,编码161个氨基酸。将其与SVBV美国分离物以及花椰菜花叶病毒属其他成员的ORF Ⅱ基因相比较,结果表明,SVBV中国分离物 ORF Ⅱ基因与SVBV美国分离物的核苷酸序列相似性最高,达91.2%。将SVBV ORF Ⅱ基因插入原核表达载体pET32a（+）,重组质粒pET-ORF Ⅱ转化大肠杆菌BL21（DE3）。经IPTG诱导及Ni²⁺-NTA亲和柱纯化,获得分子质量约为38 kDa的融合蛋白。以此纯化的融合蛋白为抗原免疫家兔,可以制备出高效价的抗血清。Western blot分析表明,制备的抗血清能与重组融合蛋白发生特异性反应。利用抗血清进行ELISA测定,能够检测出感病草莓植株病汁液中SVBV ORF Ⅱ基因表达的蛋白。     

草莓镶脉病毒ORF I基因的克隆、原核表达及抗血清制备

The total DNA was extracted from strawberry leaves infected with Strawberry vein banding virus (SVBV) by CTAB method. Specific primer pairs were designed to amplify the gene ORF I of SVBV-Shenyang isolate by PCR, gene ORF I was cloned into modified prokaryotic expression vector pET-32a (+)-GST, the recombinant plasmid pET-ORF I was transformed into E. coli DH5α, then the positive clones were screened and sequenced. The recombinant plasmid was extracted and transformed into Escherichia coli BL2l (DE3), the fusion protein with an approximate molecular weight of 56 kDa was obtained with IPTG induction and Ni²⁺-NTA affinity column purification. The purified fusion protein was used to immunize the rabbits to prepare the specific antiserum. The result of ELISA showed that the titer of the prepared antiserum is up to 1:520 000, Western blot analysis indicated that the prepared antiserum could reacted specifically with purified recombinant fusion protein. Not only the expression of P1 protein in SVBV infected-strawberry leaves, but also the expression of P1 protein in P1-infiltrated Nicotiana benthamiana leaves could be detected, using 2 000 times diluted antiserum.     

草莓镶脉病毒ORF I基因的克隆、原核表达及抗血清制备

The total DNA was extracted from strawberry leaves infected with Strawberry vein banding virus (SVBV) by CTAB method. Specific primer pairs were designed to amplify the gene ORF I of SVBV-Shenyang isolate by PCR, gene ORF I was cloned into modified prokaryotic expression vector pET-32a (+)-GST, the recombinant plasmid pET-ORF I was transformed into E. coli DH5α, then the positive clones were screened and sequenced. The recombinant plasmid was extracted and transformed into Escherichia coli BL2l (DE3), the fusion protein with an approximate molecular weight of 56 kDa was obtained with IPTG induction and Ni²⁺-NTA affinity column purification. The purified fusion protein was used to immunize the rabbits to prepare the specific antiserum. The result of ELISA showed that the titer of the prepared antiserum is up to 1:520 000, Western blot analysis indicated that the prepared antiserum could reacted specifically with purified recombinant fusion protein. Not only the expression of P1 protein in SVBV infected-strawberry leaves, but also the expression of P1 protein in P1-infiltrated Nicotiana benthamiana leaves could be detected, using 2 000 times diluted antiserum.     

大麦黄矮病毒GPV株系ORF4基因在大肠杆菌中的表达及特异抗血清的制备

 根据已报道的大麦黄矮病毒GPV株系(BYDV-GPV)相关基因序列设计合成引物,利用RT-PCR方法获得ORF4基因,并将其克隆到原核表达载体pET-5a中。经IPTG诱导、SDS-PAGE分析,结果表明:ORF4基因在大肠杆菌BL21(DE3) pLysS中获得了高效表达,分子量为17 kDa。以回收的表达产物为抗原免疫家兔,制备了BYDV-GPV 17kDa蛋白的特异性抗血清。Western blot检测结果,制备的抗血清可用于检测BYDV-GPV侵染后在燕麦体内表达的17 kDa蛋白。     

烟草丛顶病毒ORF1蛋白的多克隆抗体制备及应用

PCR扩增烟草丛顶病毒(Tobacco bushy top virus,TBTV)的ORF1序列并克隆到原核表达载体pEHISTEV中,转化大肠杆菌Rosetta菌株经IPTG诱导表达TBTV ORF1蛋白。利用切胶纯化的ORF1蛋白免疫新西兰大耳白兔制备并获得抗血清,间接ELISA检测效价为1:24 3000。经抗原亲和纯化从抗血清中得到特异性和灵敏度俱佳的ORF1多克隆抗体。Western blot分析显示,TBTV ORF1多克隆抗体既可以检测田间发病的烟草丛顶病样品中ORF1蛋白,也可检测在体内和体外翻译体系中的TBTV ORF1蛋白的表达。另外发现ORF2蛋白以ORF1延长蛋白的形式存在,根据ORF1和ORF2的重叠情况及潜在的七核苷酸滑动序列和下游的稳定二级结构,推测此ORF1延长蛋白是移码翻译产物。     

泡桐丛枝植原体pPaWBNy-1-ORF5编码蛋白的原核表达和抗体制备

 利用含泡桐丛枝植原体质粒pPaWBNy-1的3.0 kb片段的克隆为模板,PCR扩增pPaWBNy-1-ORF5的亲水性肽段编码区。目的片段连接到原核表达载体pET28a(+),重组质粒pET28a-ORF5-5转化大肠杆菌(Escherichia coli)Rosseta (DE3)感受态细胞,菌液处在对数生长期时(OD₆₀₀=0.52),添加IPTG诱导5 h后收集菌体,提取蛋白并进行聚丙烯酰胺凝胶电泳。用六聚组氨酸标签抗体进行Western blot检测,发现分子量约为15 kDa的含多聚组氨酸标签的目的蛋白得到表达。切胶回收融合蛋白,免疫德国大白兔以制备抗血清,间接ELISA法测定抗血清的效价约为1∶8 100。用制备的ORF5编码蛋白的抗血清进行Western blot分析,结果在带菌的介体昆虫茶翅蝽(Halyomorpha halys)中检测到大小约为17 kDa的特异蛋白条带,但在健康和感病泡桐(Paulownia sp.)及无菌茶翅蝽中均未检测到,由此推测pPaWBNy-1-ORF5蛋白与介体昆虫传播植原体有关。     

葡萄A病毒外壳蛋白原核表达及抗血清制备

 葡萄A病毒（Grapevine virus A,GVA）为线性病毒科（Flexiviridae）葡萄病毒属（Vitivirus）的代表种,是葡萄皱木复合病（rugose wood complex disease）的重要病原之一,可引起葡萄嫁接成活率下降、春季萌芽延迟、生长减弱甚至衰退死亡等危害\[1,2\]。GVA为线状单链RNA病毒,基因组共编码5个开放阅读框（ORF1\|5）,其中ORF4 编码外壳蛋白（coat protein, CP）,是病毒粒子包裹和系统移动所必需的功能蛋白\[3,4\]。GVA自然寄主为葡萄,机械摩擦可侵染本氏烟等草本寄主\[2\],由于嫁接和无性繁殖材料调运等因素造成该病毒远距离传播,目前在世界多个国家和地区均有发生。     

香蕉条斑病毒LAMP快速检测方法的建立

 环介导等温扩增（loop-mediated isothermal amplification,LAMP）是一种特异、灵敏、快速的新型基因检测技术。本研究以香蕉条斑病毒（Banana streak virus,BSV）ORF3保守区域为基础针对6个特定区域设计并筛选了4条LAMP扩增引物,通过对LAMP反应中MgSO₄、dNTPs、Betaine等主要试剂浓度进行优化,建立了香蕉BSV的LAMP检测方法,63℃反应90 min后通过在反应产物中添加SYBR Green Ⅰ染料后颜色的变化,肉眼即可判断检测结果。LAMP具有极高的检测特异性和灵敏性,其检测下限约为3.2 ng·μL^-1,是PCR检测灵敏度的25倍,能快速、准确地对疑似样品进行检测,本研究对华南地区部分疑似样品的检测结果显示LAMP阳性检出率比PCR检出率高。本文建立的BSV LAMP检测方法是对BSV检测方法的拓展和延伸,为香蕉病毒的快速检测提供技术保障。     

应用TaqMan real-time PCR研究BSV在带毒香蕉组培苗中传递

由香蕉线条病毒(Banana streak virus,BSV)引起的香蕉线条病,严重影响了香蕉产量及种质资源交流。本研究根据BSV ORF Ⅲ基因保守序列设计引物和探针,建立了BSV的实时荧光定量PCR检测方法。该法检测BSV时,与黄瓜花叶病毒(Cucumber mosaic virus,CMV)和香蕉束顶病毒(Banana bunchy top virus,BBTV)无交叉反应;不论是检测质粒DNA还是香蕉病株总DNA,其灵敏度都比普通PCR高,检测质粒DNA的灵敏度比普通PCR高100倍,检测香蕉病株总DNA的灵敏度比普通PCR高10倍,且具有良好的重复性。利用建立的实时荧光定量PCR方法检测我国主栽的不同香蕉品种‘大蕉’、‘粉蕉’和‘粤优抗一号’香蕉组培苗中BSV的浓度,发现不同品种BSV传递规律不同。‘大蕉’第3代、第9代组培分化芽中BSV含量较原代显著减少;‘粉蕉’中BSV含量随继代数增加表现为先增加后减少;‘粤优抗一号’中BSV含量随继代数增加呈增加趋势。BSV在‘粉蕉’、‘大蕉’和‘粤优抗一号’第10代到第12代组培苗中以顶部第1片或(及)第2片叶中含量最高。直接结合PCR分析初步表明不同品种在原代和组培至第12代,其植株中的BSV均为游离状态。本文通过对带毒香蕉组培苗中BSV的含量监测,明确了BSV在不同品种带毒香蕉组培苗中的传递和分布规律,为研究BSV与寄主互作及BSV检测提供了理论依据。     

甘蔗花叶病毒VPg蛋白的原核表达及其在玉米中的免疫定位

 利用RT-PCR方法从感染甘蔗花叶病毒北京分离株(SCMV-BJ)的玉米叶片中扩增得到VPg基因,将VPg基因连接到原核表达载体pET-28a上。获得的重组子转化大肠杆菌BL21(DE3)后,用IPTG进行诱导表达。SDS-PAGE分析表明,VPg在大肠杆菌中获得了高效表达,产生的融合蛋白分子量为30 kD。将融合蛋白纯化后免疫兔子获得了特异性较高的抗血清。ACP-ELISA测定抗血清的效价为1/4096。利用该抗血清对健康玉米和病毒侵染的玉米茎、叶脉和叶片进行免疫金标记,结果表明在茎和叶脉的韧皮部筛管的细胞壁处有金颗粒。