新疆番茄黄化曲叶病毒与烟粉虱隐种的区域分布检测

由TYLCV(Tomato yellow leaf curl virus)引起的番茄黄化曲叶病危害严重。为明确该病毒在新疆的发生与分布,本研究利用TYLCV特异引物对采集自新疆南、北和东疆温室及露地的229株表现黄化曲叶症状的番茄样品通过PCR检测其带毒率;为进一步明确TYLCV传毒介体烟粉虱的隐种类型及带毒情况,利用烟粉虱和TYLCV特异引物对番茄上采集的543头烟粉虱进行了MED和MEAM1隐种及带毒率的PCR检测。结果表明:170个病株检测到TYLCV,病株带毒率为74. 2%;烟粉虱MED隐种和MEAM1隐种在新疆均有分布,检出率分别为82. 7%和17. 3%,说明MED隐种是新疆烟粉虱优势种群;烟粉虱MED隐种和MEAM1隐种均可携带TYLCV,带毒率分别为60. 8%和4. 3%,说明MED隐种带毒率远高于MEAM1隐种的带毒率。本研究明确了TYLCV可侵染新疆番茄,且在温室及露地番茄上均主要由烟粉虱优势种群MED隐种携带和传播。     

番茄黄化曲叶病毒病抗性材料的筛选鉴定

 为评估新培育番茄栽培品种对番茄黄化曲叶病毒（tomato yellow leaf curl virus,TYLCV）的抗性水平,更全面地了解这些品种的抗病性差异,以金棚1 号和金曼为感病对照,采用带毒烟粉虱自然传毒方式,对各品种的发病时间、发病率及病情指数等参数进行比较,结合PCR 及ELISA 对TYLCV 的检测结果,综合分析了18 个番茄品种对番茄黄化曲叶病毒的抗病性。结果表明,不同品种对TYLCV 的抗性差异较大。金棚1 号和金曼两份材料发病率都达到100%,病情指数在65.2 以上,属于典型的感病品种;秋光15-6、PC-88 和秋光69 号3 份材料发病率和病情指数均为0,属于抗性较高的品种;其他13 份材料的发病率和病情指数分别在5.6％～45.2%和2.6～18.0 之间,分别表现了不同程度的抗、耐病水平。     

陕西地区烟粉虱生物型的鉴定及其危害分析

为探明陕西地区烟粉虱的危害及主要生物型,实地调查了陕西地区主要农作物上烟粉虱的危害状况与发生程度,并利用mt DNACOI基因测序方法,对陕西地区烟粉虱的生物型组成进行了分析鉴定。同源性分析结果表明,B型和Q型烟粉虱是陕西地区烟粉虱种群的主要生物型,其中B型占所测总样本的78.85%,表明陕西省烟粉虱主要生物型仍是B型,但Q型种群比例在增长,不排除有取代B型成为陕西地区烟粉虱主要危害类型的趋势。在陕西地区烟粉虱主要危害的植物是茄科的烟草、甜椒、番茄以及葫芦科的黄瓜、西葫芦,而十字花科植物普遍受害较轻,茄科的马铃薯和豆科的豇豆、豌豆受害最轻。     

番茄黄化曲叶病毒侵染性克隆的构建

为研究番茄黄化曲叶病毒(TYLCV)的基因功能及相关致病机理,构建了2种TYLCV的侵染性克隆。将两端含有间隔序列(intergenic sequence,IR)的TYLCV全基因组片段分别克隆到质粒p UC57和p CAMBIA2300上得到侵染性隆p TYLCV1.X和p CB2300-1.X。利用质粒p TYLCV1.X直接注射法与p CB2300-1.X的农杆菌浸润法分别接种番茄植株。结果显示:侵染性克隆p TYLCV1.X在注射番茄6周后出现系统症状,侵染效率为23.1%-42.1%;农杆菌浸润法接种的植株4周后观察到明显的病害症状,侵染效率为87.5%-100%。PCR检测结果证明2种侵染性克隆均可在番茄体内产生完整的病毒基因组DNA,实时定量PCR测定病毒浓度可达102个/番茄细胞。以上结果表明,含有2个IR的TYLCV基因组载体具有侵染活性。     

烟草双生病毒RaMoV在烟粉虱中的检测

苎麻花叶病毒(Ramie mosaic virus,RaMoV)是常见由烟粉虱传播的双生病毒(Geminivirus)。用特异性引物进行PCR检测,结果发现,B型烟粉虱(Bemisia tabaci)能够在烟草上传播该双生病毒。     

中国番茄黄化曲叶病毒（TYLCV—CHI）外壳蛋白基因在大肠杆菌中的表达及抗血

经PCR获得了中国番茄黄化曲叶病毒（TYLCV－CHI）外壳蛋白（CP）基因，并克隆到pGEM-7Zf( ) 载体上，序列分析表明，TYLCV－CHI感染番茄或烟草时，CP基因的核苷酸序列发生变化，并且在烟草上随感染时间的延长，突变频率增高。连接到含有编码tRNAarg4基因argU的表达载体pSBET后，TYLCV－CHI CP基因在大肠杆菌中以包含体形式得到大量表达（约33ku)。以表达的蛋白作抗原免疫家兔，制备了抗TYLCV－CHI CP的抗血清。ELISA实验表明，该血清与抗原特异反应，血清效价为TYLCV－CHI CP的抗血清。ELISA实验表明，该血清与抗原特异反应，血清效价为1：3000。Western印迹实验还表明，该血清除了与感染TYLCV－CHI的番茄、烟草有特异免疫反应外，还与感染南瓜曲叶病、烟草曲叶病的叶片有免疫反应，表明这些病毒之间有明显的 血清学关系。     

微卫星位点BEM06与BEM23鉴别烟粉虱B型与Q型的有效性

烟粉虱Bemisia tabaci(Gennadius)B型与Q型是烟粉虱复合种中入侵性较强、分布较广的2种生物型,当前在许多地区混合发生。这2种生物型的快速鉴别对其种群动态调查及入侵生态学研究具有重要价值。为了验证微卫星位点BEM06与BEM23鉴别B型与Q型烟粉虱的有效性,本研究分析了这2个微卫星位点的等位基因在国内外17个B型、4个Q型、3个非B/Q型烟粉虱种群的分布特点。结果表明:这2个微卫星位点的联合使用可鉴别B型与Q型烟粉虱,但是无法有效地将B型、Q型与一些非B/Q型烟粉虱某些个体区分开来。结果提示:利用微卫星位点BEM06与BEM23鉴别B型与Q型烟粉虱具有一定的局限性,尤其是田间烟粉虱存在其他生物型时需要慎重使用这种鉴别方法。     

侵染加工番茄的BBWV2的分子鉴定及田间检测

利用蚕豆病毒属的兼并引物对23个加工番茄病株进行RT—PCR检测,其中19株检测出被该属病毒侵染。从中选取4个病株的RTPCR产物进行克隆和测序,将所得序列经过基因库检索发现,4个序列均与蚕豆萎蔫病毒2（BBWV2）基因组RNA1的序列同源性最高。然后对病毒分离物XJ14-1基因组进行进一步的克隆和测序,获得RNA1组分3659个核苷酸,RNA2组分1300个核苷酸,序列比对结果显示,它们与BBWV2的RNAl和RNA2具有高的序列同源性,分别为76．6％～94％和76．9％～94．1％,而与BBWVl的RNA1和RNA2的序列相似性都很低。因此分子鉴定结果表明,新疆加工番茄受到BBWV2的侵染。利用BBWV2的单克隆抗体对田间不同品种（品系）加工番茄病株进行ELISA检测,结果显示田间病株带毒率达到53．3％,并且供试20品种（品系）均可被BBWV2侵染,该数据表明,BBWV2在田间发生普遍。     

感染番茄抗病品种的番茄黄化曲叶病毒的基因变异分析

近几年来,生产中发现一些番茄抗病品种在不同环境条件下或应用几年后出现感病现象。为了解不同抗病品种中番茄黄化曲叶病毒(tomato yellow leafcurl virus,TYLCV)基因变异的情况,对5个感染TYLCV的番茄抗病品种进行了TYLCV全长基因克隆和序列测定。扩增结果显示,5个样品携带的TYLCV基因组长度均为2 781 bp,且均编码6个功能蛋白。基因组序列比较发现,这5个分离物与TYLCV-Israel株系同源性达到99%以上;通过功能蛋白比对发现,复制增强因子AC3蛋白存在变异,同世界各地报道的TYLCV-Israel株系典型分离物的AC3蛋白存在7处氨基酸差异的位点。分析结果表明,这五个病毒分离物均属于TYLCV-Israel株系,其AC3蛋白的氨基酸序列变异程度均不显著,并没有产生新的病毒株系。     

感染番茄抗病品种的番茄黄化曲叶病毒的基因变异

近几年来,生产中发现一些番茄抗病品种在不同环境条件下或应用几年后出现感病现象。为了解不同抗病品种中番茄黄化曲叶病毒(tomato yellow leafcurl virus,TYLCV)基因变异的情况,对5个感染TYLCV的番茄抗病品种进行了TYLCV全长基因克隆和序列测定。扩增结果显示,5个样品携带的TYLCV基因组长度均为2 781 bp,且均编码6个功能蛋白。基因组序列比较发现,这5个分离物与TYLCV-Israel株系同源性达到99%以上;通过功能蛋白比对发现,复制增强因子AC3蛋白存在变异,同世界各地报道的TYLCV-Israel株系典型分离物的AC3蛋白存在7处氨基酸差异的位点。分析结果表明,这五个病毒分离物均属于TYLCV-Israel株系,其AC3蛋白的氨基酸序列变异程度均不显著,并没有产生新的病毒株系。     

番茄黄化曲叶病毒自然种群的分子变异和进化研究

研究目的：创新要点：研究方法：重要结论：2006年我国上海首次发现番茄黄化曲叶病毒（TYLCV），随后TYLCV迅速蔓延至全国13个省份和自治区。本研究分析了2006至2010年期间TYLCV在我国首发地上海市的分子变异规律。本研究持续五年追踪田间TYLCV，分析TYLCV的全长基因组序列、分子变异及种群遗传结构，为防控TYLCV提供理论依据。2006至2010年从上海采集了26个TYLCV的分离物，利用高保真性的滚环扩增技术获得TYLCV分离物的全长基因组。应用MEGA5等生物信息学软件分析TYLCV的分子变异。TYLCV自然种群具有与RNA病毒相似的突变率，以基因间隔区的分子变异最大，平均突变率为4．81×10-3（见图2和表2）。TYLCV的大部分基因都处于负向选择，但包含在c1开放阅读框内的C4，却与c1承受着不同的选择压而处于正向选择（见图3和表6）。     

Identification of three major Bemisia tabaci biotypes in China based on morphological and DNA polymorphisms

The sweet potato whitefly, Bemisia tabaci, is a complex species, and many of its biotypes are important agricultural pests. The B and Q biotypes are invasive and coexist with the native Cv biotype on vegetable and ornamental crops in China. In this study, these three biotypes were identified based on their morphological characteristics, RAPD–PCR analysis, and DNA sequences of the COI gene. The anterior and posterior wax fringes of the B, Q, and Cv biotypes significantly differed from each other. Based on this morphological characteristic, the three biotypes can easily be distinguished in greenhouses and fields. Genomic DNA RAPD–PCR band patterns also revealed differences between these biotypes using the H16 primer. The B biotype has three bright DNA bands between 250 and 600 bp, the Q biotype only has one bright band at 300 bp, while the Cv biotype has no band between 250 and 500 bp. Both the Cv and Q biotypes have two bright bands at 750 and 1000 bp while the B biotype has only one band at 1000 bp. Based on the COI gene, the genetic identity between B and Cv, B and Q, and Q and CV was 85.8%, 94.7%, and 86.0%, respectively. The MP tree indicated that the phylogenetic relationship between the B and Q biotypes is much closer than that between the B and Cv or the Q and Cv biotypes. The uses of the morphological, RAPD–PCR, and DNA sequencing methods in biotype identification of B. tabaci are discussed.     

There is the second virus that causes tobacco leaf curl disease (not TbLCV-CHI) in the field

Sequence analysis of virus isolation DNA of tobacco leaf curl disease shows that there is the second geminivirus (not Chinese Tobacco Leaf Curl Virus, TbLCV-CHI) that causes tobacco leaf curl disease in the field in the Guangxi Zhuang Autonomous Region, China. This virus DNA-A contains 2 734 nt. Large intergenic region (LIR) contains 269 nt, the virus sense strand contains 2 open reading frames (ORFs): AV1 (115 aa) and AV2 (coat protein gene, CP, 256 aa), and the complementary sense strand contains 4 ORFs: AC1 (replicase gene, 361 aa), AC2 (transactivator, 134 aa), AC3 (134 aa) and AC4 (97 aa). The virus belongs to one kind of subgroup III geminiviruses from old world, and could be the Chinese tomato yellow leaf curl virus (TYLCV-CHI).     

Modification of a viral satellite DNA-based gene silencing vector and its application to leaf or flower color change in Petunia hybrida

Gene silencing conserved in plants and animals is mediated by mechanisms that involve sequence- specific RNA degradation[1,2]. Gene silencing has been proven to play an important role in the study of gene function. Recently, a procedure known as virus ind…     

Discovery and demonstration of small circular DNA molecules derived from Chinese tomato yellow leaf curl virus

Tomato yetlow leaf curl viruses betong to Begomoviruses of geminiviruses. In this work, we first found and demonstrated that the small circular DNA molecules were derived from Chinese tomato yetlow leaf curl viruses (TYLCV-CHI). These small circular DNA molecules are about 1,3 kb, which are half the full-length of TYLCV-CHI DNA A. It was shown by sequence determination and analysis that there was unknown-origin sequence insertion in the middle of the small molecules. These sequences of unknown-origin were neither homologous to DNA A nor to DNA B, and were formed by recombination of virus DNA and plant DNA. Although various defective molecules contained different unknown-origin sequence insertion, all the molecules contained the intergenic region and part of the AC1 (Rep) gene. But they did not contain full ORF.     

利用番茄U3snRNA基因上游启动区构建植物表达载体及对烟草的转化

利用番茄U3snRNA基因上游启动区和ACC合成酶反义RNA-核酶嵌合基因DNA片段,构建含U3snRNA基因上游启动区-ACC合成酶的反义RNA-核酶嵌合序列的表达载体,重组于植物双元表达载体pGA643中,得到pGU3R.用三亲融合法导入农杆菌LBA4404中,采用叶盘法转化烟草,诱导再生小植株,获得了卡那霉素的抗性植株.提取抗性植株总DNA,通过PCR、PCRSouthern杂交检测并分别用启动区序列和ACC合成酶的反义RNA-核酶嵌合序列作探针,通过Southern杂交检测,已筛选出整合有外源基因的转化植株.为进一步研究U3snRNA上游启动区增强反义RNA-核酶基因的表达奠定了基础.     

Identification of a nanovirus-like DNA molecule associated with Tobacco curly shoot virus isolates containing satellite DNA

A circular single-stranded DNA molecule, designated DNA1, was identified from Tobacco curly shoot virus (TbCSV) isolates Y35 and Y115 containing satellite DNAβ using abutting primers based on the two reported DNA1 sequences of whitefly-transmitted geminiviruses, while DNA1 molecule was not found in TbCSV isolates Y1 and Y121 without DNAβ. The immunotrapping PCR test showed that DNA1 could be encapsidated in virus particles. Southern blot further confirmed that DNA1 molecules were only associated with TbCSV isolates (Y35 and Y115) containing DNAβ. Sequences of Y35 and Y115 DNA1 comprise 1367 and 1368 nucleotides, respectively, each having a conserved ORF encoding nanovirus-like replication-associated protein (Rep). A low nucleotide sequence identity was found between DNA1 molecules and their cognate DNA-As. Y35 and Y115 DNA1 shared 92% overall nucleotide sequence identity and 96% amino acid sequence identity for Rep, while 69%～79% overall nucleotide sequence identity and 87%～90% amino acid sequence identity were found when compared with two reported DNA1 molecules associated with Ageratum yellow vein virus and Cotton leaf curl Multon virus. Sequence analysis showed that DNA1 was less related to nanovirus DNA.     

Identification of a novel DNA molecule associated with Tobacco leaf curl virus

A circular DNA molecule, designated as DNAβ, was identified in tobacco plants infected with Tobacco leaf curl virus (TLCV) isolates Y5 and Y8 by PCR using primers based on the conserved region of the two reported DNAβ sequences of whitefly-transmitted geminiviruses (WTGs). The complete nucleotide sequences of DNAβ of Y5 and Y8 (TLCV DNAβ) were determined. Y5 DNAβ comprises 1333 nucleotides encoding 8 predicted ORFs with 4 ORFs in virion-sense DNA and 4 ORFs in complementary-sense DNA; Y8 DNAβ consists of 1338 nucleotides encoding 7 predicted ORFs with 4 ORFs in virion-sense DNA and 3 ORFs in complementary-sense DNA. TLCV DNAβ has little sequence homology to DNA-A of TLCV., except that it shares conserved TAATATTAC loop sequence with TLCV DNA-A. Sequence comparison showed that Y5 DNAβ shared 85% sequence homology with Y8 DNAβ, and both Y5 DNAβ and Y8 DNAβ had relatively low sequence identity (51%–65%) with the reported DNAβ molecules associated with Ageratum yellow vein virus and Cotton leaf curl virus. The immunotrapping PCR and whitefly transmission tests showed that DNAβ molecule could be encapsidated in virus particle and transmitted by Bemisia tabaci. This is the first report of DNAβ associated with WTGs in China.