侵染落葵的黄瓜花叶病毒分离物CP基因序列分析

通过间接酶联免疫法(ID-ELISA)检测到染病落葵病样中存在黄瓜花叶病毒（Cucumber Mosaic Virus,CMV）。从病叶中提取总RNA,用RT-PCR方法扩增得到657bp的CMV CP基因片断,将扩增产物与T载体连接并进行测序。用DNA MAN将得到的CP基因序列与GenBank收录的黄瓜花叶病毒两亚组部分株系或分离物的CP基因序列进行比较,结果表明该CP基因与CMV亚组Ⅰ、亚组Ⅱ之间的核苷酸序列同源性分别为91.17~95.43%和75.30~75.76%,推导氨基酸序列同源性分别为95.41~97.71%和81.28~81.74%,表明CMV-Ba与亚组Ⅰ同源关系密切。     

Coat protein-mediated resistance against an Indian isolate of the Cucumber mosaic virus subgroup IB in Nicotiana benthamiana

Coat protein (CP) -mediated resistance against an Indian isolate of the Cucumber mosaic virus (CMV) subgroup IB was demonstrated in transgenic lines of Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transformation. Out of the fourteen independently transformed lines developed, two lines were tested for resistance against CMV by challenge inoculations. The transgenic lines exhibiting complete resistance remained symptomless throughout life and showed reduced or no virus accumulation in their systemic leaves after virus challenge. These lines also showed virus resistance against two closely related strains of CMV. This is the first report of CP-mediated transgenic resistance against a CMV subgroup IB member isolated from India.     

辣椒温和斑点病毒(PMMV)外壳蛋白基因的克隆和序列测定

从本院分离的辣椒温和斑点病毒株系PMMV-QD中提取RNA.根据已有报道的外壳蛋白(CP)自行设计引物P1、P2,用RT-PCR扩增PMMV-QD的CP基因的cDNA,插入pMD 18-T载体,转化感受态受体菌XL1-B1ue,以蓝白菌落筛选转化株,用PCR检测白色菌落,确认获得了含cDNA的阳性重组子.序列测定和比较的结果表明该片段确含有PMMV的CP基因编码区,此种cDNA与国外报道的PMMV病毒株系核苷酸序列的同源性达到97.7%.     

胡椒叶片基因组DNA提取方法比较

目的：研究建立胡椒叶片中提取高质量DNA的方法。方法：采用各种CTAB法和SDS法的改良方法,提取胡椒叶片中的总DNA,并对DNA进行紫外和电泳检测。结果：改良CTAB法4和5提取的DNA经电泳检测,有一条明亮主带,且无拖尾现象,样品槽无荧光出现,说明抽出的DNA纯度较高,一致性好;测定其OD260和OD280值,并计算其比值,OD260/OD280值在1.8-2.0之间,提取率在51.667-60.000μg/g之间,获得的基因组DNA质量高。结论：改良CTAB法4和法5可从胡椒幼叶中提取高质量DNA。     

黄瓜花叶病毒衣壳蛋白基因转化辣椒研究

The plant expression vector of the coat protein(CP) gene of cucumber mosaic virus (CMV) BS strain was used to transform three kinds of pepper (Capsicum annuum) tissues (cotyledon, stem and root) by agrobacterium-mediated co-cultivation. 53%-68.4% of the total tissues (639) can be induced to be calli, but only cotyledon calli can be further regenerated to form shoots (regenerated efficiency 39.7%). 70%(42/60) of the putative transformed plants were confirmed to have CP gene in their genomes by Southern blot. The mRNAs and the CP were respectively found in 80% of transgenic plants by Northern blot and DAS-ELISA. 24 of the transgenic plants expressing CP gene of BS strain showed three kinds of resistant level (severe symptom, delay of symptomatic development, no symptom) to infection of CMV-BS and of CMV-P. However, there was distinctly higher resistance to inoculation of CMV-BS than that with CMV-P in these transgenic plants.     

Coat Protein Gene of Cymbidium mosaic virus from Vanilla fragrans in Reunion Island

Nucleotide and amino acid sequences of the coat protein (CP) of 12 isolates of Cymbidium mosaic virus from Vanilla fragrans in Reunion Island (CyMV‐R) were compared with each other and with those of previously described Asian strains. Alignment revealed that CyMV‐R isolates were highly homologous, suggesting that one strain is prevalent in Reunion. This strain also showed high homology with the Korean CyMV‐K2 and Singapore CyMV‐S2 strains, but nucleotide additions resulted in the carboxy‐terminal ends of the CP sequences differing from those of the Korean CyMV‐K1 and Singapore CyMV‐K1 strains.     

First Report of Cucumber mosaic virus Infecting Chinese Mallow in China

The virus in naturally infected, stunted Chinese mallow plants and mosaic leaves was identified as Cucumber mosaic virus (CMV). Six symptomatic plants and one symptomless plant were collected in Chongqing, China. DAS‐ELISA suggested CMV was likely associated with the diseased Chinese mallow. Double‐stranded RNA was extracted from the samples, analysed by RT‐PCR, and the coding sequences of their coat proteins (CPs) were sequenced. The results further confirmed CMV was the pathogen causing Chinese mallow stunted, mosaic disease. The isolate was named CMV‐DXC. The full sequence of CMV‐DXC CP was determined, and it had the highest nucleotide identity (99.4%) of those of CMV‐lily, CMV‐WSJ and CMV‐Hnt, respectively. Phylogenetic analysis shows that CMV‐DXC belongs to CMV subgroup II. To our knowledge, this is the first report of CMV infecting Chinese mallow in China.     

In silico Characterisation and Phylogenetic Analysis of Two Evolutionarily Conserved miRNAs (miR166 and miR171) from Black Pepper (Piper nigrum L.)

Among computationally predicted and experimentally validated plant miRNAs, several are conserved across species boundaries in the plant kingdom. In this study, a combined experimental–in silico approach was adopted for characterization of two conserved miRNAs, miR166 and miR171, from black pepper (Piper nigrum). A PCR-based detection and cloning strategy of miRNAs from tissues of black pepper was used. Conservation analysis of miR166 and miR171 along with their corresponding targets identified from P. nigrum revealed that these miRNAs are highly conserved with their counterparts in other plant species. miRNA-mediated cleavage of the conserved targets was also verified by RLM-RACE experiments. Real-time quantitative PCR revealed the differential expression patterns of these miRNAs in black pepper tissues. Our miRNA-based phylogenetic analysis of plants belonging to the Piperaceae family was in agreement with the typical paleoherb evolutionary scheme of primitive angiosperms. This method will help in the detection of evolutionarily conserved miRNAs in other plant species and provide a strategy for a novel phylogenetic reconstruction based on the evolutionary history of miRNA genes.     

侵染万寿菊的黄瓜花叶病毒外壳蛋白基因的克隆与序列分析

应用ELISA的方法检测到感染了黄瓜花叶病毒(Cucumber mosaic virus,CMV)的万寿菊。根据已报道的CMV外壳蛋白(CP)基因的保守序列设计并合成引物,提取万寿菊叶片总RNA为模板,进行cDNA合成和PCR扩增,得到约875bp的片断,与预期片断大小相符。序列分析显示:该分离物CMV-WSJ CP基因全长657bp,编码218个氨基酸,其核苷酸和氨基酸序列与CMV亚组Ⅱ的分离物有很高的同源率,分别达到98.33%～99.24%和98.63%～100%;与CMV亚组I分离物的同源率分别仅为74.43%～76.26%和77.17%～78.99%。因此,从万寿菊叶片上检测出的黄瓜花叶病毒分离物CMV-WSJ应归属于亚组Ⅱ。     

利用酵母双杂交系统筛选与黄瓜花叶病毒外壳蛋白互作的寄主蛋白

利用酵母双杂交系统,以黄瓜花叶病毒(Cucumber mosaic virus,CMV)的外壳蛋白(coat protein,CP)为诱饵,从番茄叶片c DNA文库中筛选与其互作的蛋白。结果显示,诱饵载体pBT3-SUC-CMV-CP均能在酵母细胞中正确表达,无自激活活性而且对酵母无毒性;通过对酵母双杂交文库的筛选和回转验证,共获得了98个阳性克隆,分别编码67个可能与CMV-CP相互作用的蛋白,分别参与植物防御反应、光合作用、物质转运、信号转导、能量代谢、氨基酸代谢、细胞壁的形态建成、植物的激素代谢等。本研究结果表明,CMV CP可同时调控寄主的多个代谢过程,在CMV的致病过程中有多重功能。     

Muscodor albus MOW12 an Endophyte of Piper nigrum L. (Piperaceae) Collected from North East India Produces Volatile Antimicrobials

Muscodor albus MOW12, an endophytic fungus isolated from Piper nigrum in Mawlong, Meghalaya, India, resembles some cultural and hyphal characteristics of previous isolates of Muscodor sp. In addition, it possesses about 99 % similarity in its ITS rDNA with other M. albus isolates and thus is nicely centered within the genetic tree to other Muscodor spp. This xylariaceae fungus effectively inhibits and kills certain plant pathogenic fungi by virtue of a mixture of volatile compounds that it produces. The majority of these compounds were identified by gas chromatography/mass spectrometry as small molecular weight esters, alcohols, and acids. The main ester components of this isolate of M. albus in its volatile mixture are acetic acid, ethyl ester; propanoic acid, 2-methyl-, methyl ester and acetic acid, 2-methylpropyl ester. This appears to be the first report of any M. albus strain from India.     

Occurrence of symptomless source of Piper yellow mottle virus in black pepper (Piper nigrum L.) varieties and a wild Piper species

Symptomless nature of Piper yellow mottle virus (PYMoV) infection in three varieties of black pepper (Piper nigrum) (Panniyur 1, Panniyur 5 and Panchami) and a wild species of Piper (Piper colubrinum) was confirmed by polymerase chain reaction (PCR) using PYMoV specific primers. The virus could be transmitted from these PYMoV-infected symptomless plants onto symptom producing black pepper cv. Karimunda through mealybug vector, Ferrisia virgata and by graft transmission. About 20–50% seedlings showed typical symptoms of the PYMoV at 30 days after mealybug inoculations while it was 75–94% at 90 days after inoculation. PCR test of the inoculated seedlings confirmed the presence of PYMoV in 50–64%, 76–100% and 80–100% of plants in 30, 60 and 90 days after inoculation, respectively. Similarly, 50–66%, 91–100% and 100% of graft-transmitted plants showed typical symptoms of the disease at 30, 60 and 90 days after grafting. PCR test of the graft-transmitted plants showed 100% PYMoV infection at 60 days after grafting. The results clearly demonstrated the existence of PYMoV-infected symptomless plants that can act as source for secondary spread of the virus in the field.     

Expression of coat protein gene of Cucumber mosaic virus (CMV-subgroup IA) Gladiolus isolate in Nicotiana tabacum

The coat protein (CP)-mediated resistance against Cucumber mosaic virus (CMV) subgroup IA was developed in transgenic lines of Nicotiana tabacum cv. Petit Havana using Agrobacterium tumefaciens-mediated transformation. Ten independently transformed lines have developed, four of which were tested for resistance against CMV using virus challenge inoculations. The transgenic lines exhibiting complete resistance remained healthy and symptomless in their life span and showed reduced or no virus accumulation in their systemic leaves after virus challenge inoculation. These transgenic lines also showed resistance against CMV strains which are not closely related to CMV-Gladiolus strains. This is the first report of CP-mediated transgenic resistance against a CMV subgroup IA member isolated from India showing resistance to all CMV strains occurring in the same vicinity.