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 DNAβsequences of whitefly-transmitted geminiviruses (WTGs). The complete nucleotide sequences of DNAβof Y5 and Y8 (TLCV DNAβ) were determined. Y5 DNAβcomprises 1333 nucleotides encoding 8 predicted ORFs with 4 ORFs in virion-sense DNA and 4 ORFs in complementary-sense DNA; Y8 DNAβconsists of 1338 nucleotides encoding 7 predicted ORFs with 4 ORFs in virion-sense DNA and 3 ORFs in complementary-sense DNA. TLCV DNAβhas 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 DNAβshared 85% sequence homology with Y8 DNAβ, and both Y5 DNAβand Y8 DNAβhad relatively low sequence identity (51%-65%) with the reported DNAβmolecules associated with Ageratum yellow vein virus and Cotton leaf curl virus. The immunotrapping PCR and whitefly transmission tests showed that DNAβmolecule could be encapsidated in virus particle and transmitted by Bemisia tabaci. This is the first report of DNAβassociated with WTGs in China.     

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).     

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.     

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.     

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.     

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.     

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.     

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。     

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.     

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）。     

一个新水稻低温敏感叶色突变体tcm11的鉴定及基因定位

对粳稻"嘉花1号"经~(60)Coγ诱变处理获得的稳定遗传低温敏感叶色突变体tcm11(thermo-sensitive chloroplast mutant 11)进行了表型鉴定与遗传分析.在20℃条件下,该突变体三叶期之前幼苗均表现为黄色,光合色素含量明显下降,叶绿体发育不完整,从第4叶开始逐渐转为浅黄绿色直至最后死亡.而在32℃条件下,其表型与野生型相比没有明显差异,具有低温敏感属性.通过对培矮64S与tcm11杂交的F_2代分离群体进行遗传分析,发现该低温敏感突变体性状是受单个隐性核基因(tcm11)控制,利用图位克隆技术对tcm11进行定位,将其定位在第11号染色体的InDel分子标记ID13252与SSR分子标记MM1361之间一个约1 566 kb的区域内.这也为后续的研究奠定了基础.     

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…     

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.     

鸡蛋花鞘锈菌重寄生真菌的种类鉴定及其寄主范围测定

鉴定鸡蛋花鞘锈菌Coleosporium plumeriae重寄生真菌的种类,明确其寄主范围,为鸡蛋花锈病及其他植物锈病的生物防治提供依据。通过分离和纯化鸡蛋花锈病叶背夏孢子堆上的重寄生真菌,经单孢纯化获得12个单孢菌株,其菌落及分生孢子特征一致,菌落白色,分生孢子近圆形或卵圆形,单胞,无色,呈头状聚生在分生孢子梗顶端。选取菌株PR-CPH1作为代表,分析3个片段的核苷酸序列,PR-CPH1的ITS(MW505984)、SSU(MW505988)和LSU(MW505987)序列与Simplicillium subtropicum的ITS(AB603990)、SSU(LC496895)和LSU(LC496880)序列的一致性分别为99.00%、98.11%和98.11%。系统发育树分析发现,菌株PR-CPH1的ITS-SSU-LSU联合序列与S.subtropicum(菌株号：JCM18180)聚在同一个分支。结合形态特征和ITS、SSU、LSU的序列分析,鉴定鸡蛋花鞘锈菌重寄生真菌为S.subtropicum。测定S.subtropicum寄主范围的结果显示,在8种植物锈菌中,菌株PR...     

一个新的水稻幼苗黄叶突变体的遗传分析及其基因的初步定位

在60Coγ射线辐照的水稻突变体库中,发现了一个以粳稻品种日本晴为遗传背景的幼苗叶色黄化突变体syl11(seedling yellow leaf 11).与野生型相比,突变体幼苗第二和第三叶表现黄色,在其完全展开之前叶片自其顶端开始转绿,长到四叶期其叶色恢复正常;并且该突变体syl11幼苗黄色叶片光合色素含量明显下降.遗传分析表明,该突变体的遗传性状由1对隐性核基因控制.本研究以培矮64S/syl11的F2代突变型植株作为定位群体,应用微卫星(SSR)分子标记以及新发展的InDel分子标记,将基因syl11定位在水稻第11号染色体长臂上的RM26652和处于着丝粒附近的ID11974分子标记之间,其遗传距离分别为0.5 cM和0.7 cM.     

3种蓑藓属(Macromitrium)植物DNA C-值测定——兼论苔藓DNA C-值研究的生物学意义

分别以小立碗藓(Physcomitrella patens)和水稻(日本晴亚种)(Oryza sativa spp.japonica)为标样,用流式细胞术分别测定了中华蓑藓(Macromirium cavaleriei)、钝叶蓑藓(M.japonicum)和缺齿蓑藓(M.gymnostomum)3种藓类植物的DNA C-值,由此建立了应用流式细胞术测定蓑藓属植物DNA C-值的方法,讨论了影响藓类植物DNA C-值测定结果的因素.最后讨论了苔藓植物DNA C-值变异式样、系统发育和分类学意义,以及今后有关藓类植物DNA C-值研究中值得关注的几个方向.     

Genetic analysis and gene mapping of a narrow leaf mutant in rice ( Oryza sativa L.)

A narrow leaf mutant was obtained after T-DNA transformation conducted on a rice variety Zhonghua 11. Several abnormal morphological characteristics, including semi-dwarf, delayed flowering time, narrow and inward rolling leaves, and lower seed-setting, were observed. The rate of net photosynthesis (under saturate light) of flag leaves in the mutant was significantly lower than that of the wild type. Moreover, the leaf transpiration rate and stomatal conductance in the mutant flag leaf were lower than those of the wild type at the grain filling stage. It was found that the mutant phenotype was not caused by the T-DNA insertion. Genetic analysis showed that the mutant was controlled by a single recessive gene, designated as nal3(t). A genetic linkage map was constructed using a large F₂ mapping population derived from a cross between nal3(t) and an indica variety Longtefu B with 6 polymorphic markers on chromosome 12 identified from 366 SSR markers by the BAS method. Gene nal3(t) was mapped between the markers RM7018 and RM3331. Fine mapping of nal3(t) locus was conducted with 22 newly developed STS markers based on the sequence diversity around the region harboring nal3(t) between Nipponbare and 93–11, and nal3(t) was finally mapped to a 136-kb region between the STS markers NS10 and RH12-8. Supported by National High Technology Research and Development Program of China (863 Program) (Grant No. 2006AA10A102), National Natural Science Foundation of China (Grant No. 30600349) and Natural Science Foundation of Zhejiang Province (Grant No. Y306149)     

Genome organization and phylogenetic tree analysis of Garlic virus E, a new member of genus Allexivirus

The complete sequence of an Allexivirus isolated from garlic plants in Yuhang City, Zhejiang Province, China had been determined. The single-strand, positive RNA genome was 8451 nucleotides in length excluding poly(A) tail. The genome organization of this virus was similar to that of the other Allexiviruses but only with 62.8%–64.8% nucleotide acid identities. The amino acid sequences of proteins encoded by ORF1-6 shared 67.6%–78.5%, 55.4%–66.2%, 56.7%–66.4%,40.3%–55.6%,66.3%–79.7%and 52.2%–68.8% identities with those of the others respectively. The homology range between it and the other Allexiviruses was similar to that between the other distinct species in this genus. A more comprehensive comparison using all available CP amino acid sequences showed that it shared only 63.9%–79.8% amino acids identical with the others. Therefore, it had been considered as a new member of the genus, named as garlic virus E (GarV-E). Phylogenetic analysis confirmed GarV-E as a distinct member and the correct names and classification of some members of genus Allexivirus were also discussed.     

西昌某奶牛场乳房炎病原菌的分离鉴定及药敏试验

目的 为了确定导致西昌市某奶牛场引起乳房炎的病原。方法 无菌采集患病奶牛乳样2份进行细菌分离培养、形态学观察、生化鉴定、溶血酵素基因（khe基因）和16S rRNA基因鉴定、小鼠致病性试验以及药敏试验。结果 （1）分离到1株菌，该菌为革兰氏阴性的粗短杆菌，形态学观察与生化特性均与肺炎克雷伯菌相符合；（2）khe和16S rRNA基因序列与GenBank中肺炎克雷伯菌的同源性分别为99.6%~99.8%和99.7%~100%，因此，将分离菌确定为肺炎克雷伯菌，并将其命名为XC1分离菌株；（3）小鼠致病性试验结果显示，4只接种小鼠均在接种第3天全部死亡，表明XC1分离菌株具有较强的毒力；（4）药敏试验结果显示，XC1分离菌株对恩诺沙星、诺氟沙星、头孢比肟和头孢他啶高度敏感，对妥布霉素、庆大霉素和新霉素中度敏感，对红霉素、氨苄西林和阿莫西林耐药。结论 该奶牛场发生的乳房炎与肺炎克雷伯菌感染有关，且XC1分离菌株对临床常用的抗生素存在不同程度的耐药性。