Molecular characterization of<Emphasis Type="Italic">Tomato yellow leaf curl China</Emphasis> virus and its satellite DNA isolated from tobacco

Virus isolates, Y8, Y36 and Y38, were obtained from tobacco plants showing leaf curl symptoms in Honghe,Yunnan Province. In reactions with 14 monoclonal antibodies raised against Begomoviras particles, Y8, Y36 and Y38 had similar antigenic reaction in TAS-ELISA as Tomato yellow leaf carl China virus (TYLCCNV). The complete DNA-A nueleotide sequences of Y8, Y36 and Y38 were determined and they contain 2727, 2730 and 2730 nueleotides, respectively. Each of the DNA-A sequences has a typical Begomovirus genome organization encoding 60RFs with 20RFs[AVI(CP) and AV2] in virion-sense DNA and 40RFs (AC1 to AC4) in complementary-sense DNA. Comparisons with total DNA-A, intergenie region and deduced amino acid sequences of individual ORFs show that Y8, Y36 and Y38 are isolates of TYLCCNV. Satellite DNA molecules (DNAβ) were found to be associated with Y8, Y36 and Y38, which consist of 1338, 1339 and 1338 nucleotides, respectively. Comparisons show that these DNAβ molecules share 98%--99% sequence identities on nucleotide level and have a common ORF (designated C1) encoding 126 amino acids on the complementary strand.     

Regulatory effect of cotton leaf curl virus AC2 on virion sense promoter

The AC2 gene of cotton leaf curl virus (CLCuV) was obtained by polymerase chain reaction (PCR) . The total DNA of the CLCuV infected tomato leaves was used as template, and the amplified DNA fragment was inserted into a cloning vector. Transient expression vectors were constructed by inserting the AC2 gene into downstream region of CaMV 35S promoter. These constructs were delivered into tobacco and cotton leaf cells for transient expression by particle bombardment. The results indicated that the virion sense promoter was activated by AC2 and its activity increased remarkably. However, the activity of transactivated virion sense promoter was still lower than that of the complementary sense promoter. The expression pattern of transactivated virion sense promoter was similar to that of the complementary sense promoter, namely with high activity in both mesophyll and vascular tissues. The possibility of application of AC2 in plant genetic manipulation was also explored.     

AC2 and AC4 proteins of <Emphasis Type="Italic">Tomato yellow leaf curl China virus</Emphasis> and <Emphasis Type="Italic">Tobacco curly shoot virus</Emphasis> mediate suppression of RNA silencing

Tomato yellow leaf curl China virus Y10 isolate (TYLCCNV-Y10) alone could systemically infect host plants such as Nicotiana benthamiana without symptoms. In contrast, Tobacco curly shoot virus Y35 isolate (TbCSV-Y35) alone induces leaf curl symptoms in N. benthamiana. When inoculated into transgenic N. benthamiana plants expressing GFP gene (line 16c), TYLCCNV-Y10 neither reverses the established GFP silencing nor blocks the onset of GFP silencing. In contrast, TbCSV-Y35 can partially reverse the established GFP silencing and block the onset of GFP silencing in new leaves. In the patch co-infiltration assays, the AC2 and AC4 proteins of TYLCCNV-Y10 and TbCSV-Y35 could suppress local GFP silencing and delay systemic GFP silencing, suggesting that they are suppressors of RNA silencing. Comparison of the accumulation levels of GFP mRNA in the co-infiltration patches showed that Y10 AC2 and Y35 AC2 proteins had similar efficiency for suppression of RNA silencing. However, Y35 AC4 protein functioned as a stronger suppressor of RNA silencing than Y10 AC4 protein. There-fore, the pathogenicity difference between TbCSV-Y35 and TYLCCNV-Y10 may be related to the functional difference in their AC4 proteins.     

Malvastrum yellow vein virus,a new Begomovirus species associated with satellite DNA molecule

Virus isolate Y47 was obtained from Malvastrum coromandelianum showing yellow vein symptom in Honghe, Yunnan Province. The complete nudcotide sequence of DNA-A was determined, it contains 2731 nuclcotides,having typical genomic organiTation of a begomovirns, encoding 6ORFs with 2ORFs [AVI(CP) and AV2] in virionsense DNA and 40RFs (ACl-AC4) in complementary-sense DNA. Comparisons show that the total DNA-A of Y47 has the highest sequence identity (77%) with that of Okra yellow vein mosaic virus-[201] (AJ002451), while less than 76% identities are found when compared with other begomoviruses. The molecular data show that virus isolate Y47 is a distinct begomovirns species, for which the name Maivastrum yellow vein vorus is proposed. Satellite DNA molecule (Y47β) was found to be associated with Y47 using the primers (beta01 and beta02) specific for DNAβ Y47β consists of 1348 nuclcotides, with a functional ORF (CI) in complemen-tary-sense DNA.Y47β has 62%--67% sequence identity with DNAβ molecule associated with Cotton leaf curl Muitan virus or Cotton leaf curl Rajasthan virus, while lower than 46% sequence identities are found when compared with other reported DNA[~ molecules. Relationship dendrograms show that DNAβ molecules are co-evolved with their help begomoviruses.     

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.     

<Emphasis Type="Italic">Malvastrum yellow vein virus</Emphasis>, a new<Emphasis Type="Italic">Begomovirus</Emphasis> species associated with satellite DNA molecule

Virus isolate Y47 was obtained fromMalvastrum coromandelianum showing yellow vein symptom in Honghe, Yunnan Province. The complete nucleotide sequence of DNA-A was determined, it contains 2731 nucleotides, having typical genomic organization of a begomovirus, encoding 6 ORFs with 2 ORFs [AV1(CP) and AV2] in virionsense DNA and 4 ORFs (AC1–AC4) in complementarysense DNA. Comparisons show that the total DNA-A of Y47 has the highest sequence identity (77%) with that ofOkra yellow vein mosaic virus- [201] (AJ002451), while less than 76% identities are found when compared with other begomoviruses. The molecular data show that virus isolate Y47 is a distinct begomovirus species, for which the nameMalvastrum yellow vein virus is proposed. Satellite DNA molecule (Y47β) was found to be associated with Y47 using the primers (beta01 and beta02) specific for DNAβ. Y47β consists of 1348 nucleotides, with a functional ORF (C1) in complementary-sense DNA. Y47β has 62%–67% sequence identity with DNAβ molecule associated withCotton leaf curl Multan virus orCotton leaf curl Rajasthan virus, while lower than 46% sequence identities are found when compared with other reported DNAβ molecules. Relationship dendrograms show that DNAβ molecules are co-evolved with their help begomoviruses.     

Tobacco curly shoot virus iso-lated in Yunnan is a distinct species of Begomovirus

Virus isolate Y1 was obtained from tobacco showing curly shoot symptoms in Baoshan, Yunnan Province. Whitefly transmission test and virion morphology observation showed that it is a begomovirus. In reactions with 14 monoclonal antibodies raised against begomoviruses, Y1 was readily differentiated from begomoviruses reported in China, Pakistan and India. The complete nucleotide sequence of DNA-A was determined, it contains 2746 nucleotides, with two ORFs in virion-sense DNA and four ORFs in complementary-sense DNA. Comparisons with total DNA-A, intergenic region and deduced amino acid sequences of individual ORFs showed that Y1 is a distinct Begomovirus species, for which the name Tobacco curly shoot virus (TCSV) is proposed. The total DNA-A of TCSV is most closely related to that of Tomato leaf curl virus from India (85% sequence identity). In contrast, the deduced coat protein of TCSV is most like that of Cotton leaf curl virus 72b isolate from Pakistan (98% amino acid sequence identity).     

Identification of a bi-directional promoter from Tomato yellow leaf curl China virus

A bi-directional promoter of Tomato yellow leaf curl China virus (TYLCCNV) was obtained with the total DNA from TYLCCNV isolate Y10 infected tobacco leaves as a template. Plant expression vectors were constructed by fusing the amplified DNA fragment with the gus gene and nopaline terminator in different orientations. The vectors containing promoter fragments were transferred into leaf cells and plant stems of Nicotiana benthamiana by Agrobacterium-mediated method. Transient expression results showed that both the complementary and virion-sense promoters could drive the gus gene to express, and the GUS activity of the complementary-sense promoter was stronger than that of the virion-sense. Co-expression of the vector containing βC1 gene of TYLCCNV DNAβ with the vector containing a bi-directional promoter revealed that the βC1 protein has no impact on expression of either the virion- or the complementary-sense promoter.     

Function of A-Rich region in DNAβ associated with Tomato yellow leaf curl China virus

A novel type of circular single-stranded satellite DNA, known as DNAβ, was recently characterized and demonstrated to be associated with monopartite begomoviruses. DNAβ was essential for induction of characteristic symptoms in plants. DNAβ has three structural features: an 115 bp highly conserved region, tiC/gene and A-Rich region. The in-frame ATG mutation of βC1 gene of Tomato yellow leaf curl China virus isolate Y10 (TYLCCNV-TY10) DNAβ demonstrated that βC1 gene is required for leaf curl symptom. Here, the function of A-Rich region in TYLCCNV-Y10 DNAβ was identified. When A-Rich region was deleted, the A-Rich deleted mutant was still capable of replication and systemic infection in plant, indicating that A-Rich region is not required for trans-replication of DNAβ. The immunotrapping-PCR demonstrated that A-Rich deleted mutant could be encapsidated in the coat protein encoded by TYLCCNV-Y10 DNA-A, suggesting that A-Rich region is not related with DNAβ encapsidation. However, the A-Rich region deleted mutant caused milder symptom.     

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.     

吐鲁番温室番茄病株及传毒烟粉虱的双生病毒检测与鉴定

为了明确吐鲁番地区温室番茄黄化曲叶病的病毒种类及传毒烟粉虱的生物型及携带病毒的情况,本研究利用番茄黄化曲叶病毒的特异引物通过PCR检测和DNA测序,对温室采集的43个番茄病株进行了病毒种类的分子鉴定;利用烟粉虱特异引物和番茄黄化曲叶病毒特异性引物通过PCR检测和DNA测序,对鄯善县温室采集的180头烟粉虱进行了生物型鉴定和带毒检测,并构建了双重PCR检测方法。结果显示:吐鲁番市和鄯善县温室发生的番茄黄化曲叶病的病毒种类为番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV),病株带毒率为39.5%。鄯善县温室发生的烟粉虱主要为Q型烟粉虱,带毒率为67.8%,是主要的传毒介体。本研究建立的双重PCR检测技术,能快速有效地确定Q型烟粉虱的生物型和携带TYLCV的情况。     

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。     

Coat protein promoter from cotton leaf curl virus is not a tissue-specifically expressed promoter

Geminivirus is a kind of single-stranded DNA virus. Experimental results from tomato golden mosaic virus (TGMV) showed that expression pattern of coat protein gene (cp) promoter was phloem specifically expressed. In this note, the studies oncp promoter of cotton leaf curl virus (CLCuV) which is found and identified recently suggest that the promoter is not phloem specifically expressed. The expressing activity ofgus gene driven by the promoter exists not only in phloem but also in mesophyll tissues and root tip meristem. Transient expression suggests thatcp promoter transactivated by AC2 shows expressing activity in mesophyll and vascular tissue of leaf vein.     

Complete nucleotide sequence and genomic organization of Periplaneta fuliginosa densonucleosis virus

We have cloned the replicative form of thePeriplaneta fuliginosa densonucleosis virus (PfDNV) genome and determined its complete sequence. The sequence has 5 454 nucleotides (nt), the genome consists of an internal unique sequence flanked by inverted terminal repeats (201 nt). The first 122 nt at the 5′ end and the terminal 122 nt at the 3′ end of both plus and minus strands can fold into a typical hairpin structure. The genome contains seven major open reading frames (ORFs). The plus strand has 4 ORFs occupying the 5′ half of the plus strand, whereas the others span the 5′ half of the minus strand. Two potential promoters were found at map units (m.u.) 3 and 97. Computer analysis of sequence homologies with other parvoviruses suggests that the plus strand ofPf DNV encodes very likely the nonstructural proteins and the minus strand probably encodes the structural proteins.     

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）。     

Tobacco curly shoot virus isolated in Yunnan is a distinct species of Begomovirus

Virus isolate Y1 was obtained from tobacco showing curly shoot symptoms in Baoshan, Yunnan Province. Whitefly transmission test and virion morphology observation showed that it is a begomovirus. In reactions with 14 monoclonal antibodies raised against begomoviruses, Y1 was readily differentiated from begomoviruses reported in China, Pakistan and India. The complete nucleotide sequence of DNA-A was determined, it contains 2746 nucleotides, with two ORFs in virion-sense DNA and four ORFs in complementary-sense DNA. Comparisons with total DNA-A, intergenic region and deduced amino acid sequences of individual ORFs showed that Yl is a distinct Begomovirus species, for which the name Tobacco curly shoot virus (TCSV) is proposed. The total DNA-A of TCSV is most closely related to that of Tomato leaf curl virus from India (85% sequence identity). In contrast, the deduced coat protein of TCSV is most like that of Cotton leaf curl virus 72b isolate from Pakistan (98% amino acid sequence identity).     

转乙肝病毒表面抗原基因烟草发根的获得

构建了乙肝病毒表面抗原基因（HBsAg）植物表达载体,通过冻融法将HBsAg 基因转入到发根农杆菌LBA1314中,采用叶盘法将HBsAg基因导入到烟草中,获得了转基因烟草发根.对转基因烟草发根的GUS检测结果表明：转HBsAg基因烟草发根可以染成蓝色,而非转基因烟草的根没有染色反应.这说明gus基因在转HBsAg基因烟草发根获得了表达.     

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。     

转SOD基因烟草的光合特性初探

 以转基因Fe-SOD,Mn-SOD高表达、转基因Mn-SOD反义低表达烟草及非转基因品系为供试材料,比较CO₂光强对烟草光合作用和蒸腾作用的影响及不同叶位叶片的光合差异.在相同条件下,Mn-SOD高表达烟草CO₂补偿点最高(105μmol·mol^-1),Mn-SOD低表达烟草最低(78μmol·mol^-1),CO₂浓度对非转基因烟草蒸腾作用的影响比对其他三个转基因品系的影响要强.Mn-SOD低表达烟草的光补偿点最高(45μmol·m^-2·s^-1),而Mn-SOD高表达烟草最低(25μmol·m^-2·s^-1),并且强光对非转基因烟草的光合作用抑制大,转基因品系对强光具有较强的耐受力.同一叶位叶片,转基因烟草的光合速率、蒸腾速率更强.但转基因SOD高表达烟草在整体上并没有表现出明显的光合作用优势.     

JD病毒基因组片段的克隆和序列分析

扩增并克隆了ＪＤ病毒的ｇａｇ 基因片段（位于３００ｎｔ～５６４ｎｔ,编码基质蛋白ＭＡ）和ｐｏｌ基因片段（位于３４６７ｎｔ～３６９１ｎｔ,编码反转录酶ＲＴ）,测定其序列并同ＢＩＶ、ＢＦＶ 和ＢＬＶ 的相应基因进行比较．所建立的方法能够特异性扩增ＪＤＶ序列,适用于ＪＤＶ的检测