白粉菌侵染后的拟南芥酵母双杂交cDNA文库构建及其利用

 拟南芥广谱抗病基因RPW8对拟南芥白粉病菌、霜霉病菌和烟草花叶病毒等均具有抗性。为了深入研究其广谱抗病机制,筛选鉴定与RPW8具有直接相互作用的蛋白,我们以含有RPW8的拟南芥纯合转基因系S5为材料,接种拟南芥白粉菌系UCSC1,36 h后取样,构建了白粉菌侵染初期的拟南芥cDNA文库。为了提高文库对长片段基因5′端的覆盖率,分别使用含有oligo(dT)和oligo(dN)的接头引物反转录cDNA第一链,PCR扩增双链cDNA。将纯化后的双链cDNA与线性化载体pGADT7-Rec混合,利用同源重组技术在酵母菌株Y187中构建cDNA文库。经检测,文库转化效率为5.0×10⁶/3 μg pGADT7-Rec,滴度为2.5×10⁸ CFU·mL^-1,插入片段长度在350～2 000 bp之间,平均插入片段大小为750 bp。用RPW8.1和RPW8.2构建诱饵载体,分别获得11和12个候选互作蛋白。结果表明此cDNA文库质量较好,适用于互作蛋白的筛选。     

玉米弯孢叶斑病菌酵母双杂交cDNA 文库的构建及评价

 BD SMART technique and LD-PCR were applied to synthesize double strand cDNA(ds cDNA).The ds cDNA and pGADT7-Rec were transfered into competent yeast cell to construct yeast two-hybrid cDNA library of Curvularia lunata by homologous recombination method. The results showed that library capacitance was 2. 46 × 105 and transformation rate was 2. 13 × 105 / μg pGADT7-Rec. The plaque titer of the library was 2. 5 × 108 pfu / mL and the recombination frequency was 88. 24% . The length of inserted cDNA fragment was ranged from 0. 4 kb to 3 kb in average. This is the first time to use BD SMART technique to construct yeast two-hybrid cDNA library of C. lunata by homologous recombination.     

辣椒疫霉侵染拟南芥的酵母双杂交cDNA文库构建及其应用

正辣椒疫霉(Phytophthora capsici)是一种重要的植物病原卵菌,其寄主范围较广,可引起辣椒、番茄、黄瓜、南瓜等多种重要蔬菜作物的疫病。辣椒疫病是一种毁灭性病害,在田间发病经常造成幼苗猝倒、茎秆枯死和果实腐烂,危害相当严重,每年给全世界蔬菜产业造成的损失就高达10亿美元~([1])。辣椒疫病1918年在美国首次发生,之后在世界范围内迅速扩展。20世纪50年代我国最早在江苏     

茶树叶斑病病原菌可可毛色二孢菌的鉴定

<正>可可毛色二孢菌(Lasiodiplodia theobromae Pat. Griffon Maubl.)属葡萄座腔菌属(Botryosphaeria)病原菌的无性型,其有性型为Botryosphaeria rhodina(Berk. M.A. Curtis)Arx[1,2]。该病原菌可导致热带、亚热带地区的植物发生病害,如葡萄树干溃疡和顶梢枯死[3],春芋叶斑病[4],橄榄溃疡病[5]等。叶斑病是茶树的一类重要病害。近年来,不     

茶树叶斑病病原菌可可毛色二孢菌的鉴定

The tea leaf disease, which led to a large loss of production and decrease of quality, was found in the tea region in Huishui county, Guizhou province. Some strains were isolated from the diseased samples, and the representative strains fulfilled Koch’s postulate. Then the isolates were further identified as Lasiodiplodia theobromae based on morphological characters and phylogenetic analysis with internal transcribed spacer (ITS) and elongation factor 1-α (EF-1α) regions. The results showed that the pathogen of the leaf disease occurred in Huishui county, Guizhou province was L. theobromae.     

大豆花叶病毒诱导的应用于膜蛋白酵母双杂交的大豆cDNA文库构建

Membrane-baesd yeast two-hybrid system is an effective method for research on interaction between Soybean mosaic virus-encoded membrane-associated proteins and host factors, while the Gateway technology without the use of restriction enzyme cloning techniques is easier for construction of virus-induced host cDNA library. In this study, both membrane-based yeast two-hybrid system and Gateway^TM systems were used. With TRIZOL regent, total RNA was extracted from soybean leaves infected with soybean mosaic virus. SMV-induced soybean primary cDNA library constructed by Gateway technology was recombined into a reconstructed prey vector for membrane-based yeast two-hybrid system. The capacity and quality tests showed that the library titer was 1.7 ? 10⁶cfu/mL and the length of inserted cDNA fragments ranged from 0.5 to 2 kb. It is available for research on interaction between the virus-encoded membrane protein and host.     

大丽轮枝菌及激素处理后棉花酵母双杂交文库的构建

大丽轮枝菌是引起棉花、马铃薯等重要作物黄萎病的土传病原真菌,其分泌的胞外蛋白是侵染寄主的重要毒力因子,因此研究胞外蛋白与寄主的相互作用具有重要意义.本研究旨在构建高效的棉花酵母双杂交cDNA文库,并通过已知大丽轮枝菌效应子VCR1互作蛋白的筛选来评价构建文库的质量.分别采用大丽轮枝菌及茉莉酸甲酯、水杨酸和乙烯处理海岛棉...     

意大利首次报道毛色二孢侵染采后鳄梨引起果腐病

鳄梨(Persea americana Mill.)在意大利南部部分地区种植。2011年春季,意大利北方托里诺市(Torino)超市的鳄梨果实出现腐烂症状。病果从果柄处开始腐烂,并出现不规则软腐,病斑边缘呈黑色。病斑内部软腐,变褐。     

一种由可可毛色二孢引起的春芋新病害

正可可毛色二孢(Lasiodiplodia theobromae(Pat.) GriffonMaubl.)是由Griffon和Maublanc~([1])在对Botryodiplodia theobromae进行订正的基础上而确立,Alves等~([2])于2008年对这个种又进行了研究并补充了DNA序列信息。可可毛色二孢是一种重要的病原菌,可引起多种植物的枯萎、根腐及焦枯等病害症状~([3,4])。而假可可毛色二     

灰飞虱酵母双杂交cDNA文库的构建及分析

以灰飞虱（Laodelphax striatellus Fallen）mRNA为起始模板,利用Gateway技术构建了灰飞虱酵母双杂交cDNA文库。经过检测表明：构建的初级cDNA文库的库容量为1.85×107 cfu;扩增文库滴度为7.7×108 cfu/mL,重组率约为97%;扩增文库插入片段主要集中在1 000~1 500 bp之间。随机挑取10个克隆,经测序与GenBank数据库比对结果显示7个克隆具有同源序列,其中L2、L9为已公布的灰飞虱序列。灰飞虱酵母双杂交cDNA文库的构建为克隆全长目的基因及研究灰飞虱与其传播的水稻病毒间的互作奠定了基础。     

葡萄浆果内坏死病毒侵染的‘贝达'葡萄cDNA文库构建及其利用

 葡萄浆果内坏死病毒(grapevine berry inner necrosis virus, GINV)侵染葡萄可引起葡萄叶片表现明显的褪绿斑驳症状,其致病的分子机制,特别是病毒与寄主互作蛋白的研究尚未见报道。本研究构建了GINV侵染的‘贝达'葡萄叶片的cDNA文库,并以GINV 外壳蛋白(coat protein,CP)为诱饵筛选文库中与其互作的寄主因子。本研究构建的cDNA文库库容量达1.6×10⁷,插入片段平均大小在1.0 kb以上,质量符合cDNA文库筛库要求。筛选到了55个候选寄主蛋白与GINV CP在酵母中互作。经测序分析及回转验证,结果确定了17个寄主互作蛋白,其涉及叶绿体相关类蛋白、泛素化相关蛋白、防御相关蛋白等。本研究可为深入研究GINV致病性及其与寄主互作机制提供依据。