烟草环斑病毒PCR产物的克隆及部分序列分析

将ＴＲＳＶ的ＰＣＲ产物与ｐＧＥＴ－ＴＥａｓｙＶｅｃｔｏｒ连接,克隆到大肠杆菌ＴＧ１中,得到白色菌落,重组质粒通过酶切鉴定、ＰＣＲ扩增和部分序列分析,表明ＴＲＳＶ的ＰＣＲ产物确实插入了质粒,并已克隆到大肠杆菌中,测序列２４０个碱基,与资料显示的序列相比较,同源性达９２．５％,可用于解决病毒检疫应用中阳性对照有扩散危险的疑难问题。     

RT-PCR检测李坏死环斑病毒的研究

为了从植物组织中快速检测李坏死环斑病毒９ＰＮＲＳＶ）,根据该病毒ＲＮＡ３序列设计引物,对感病和健康组织总ＲＮＡ进行ＲＴ－ＰＣＲ,结果从感病组织中扩增出了大约４５０ｂｐ的目的片段,而健康组织中无此扩增带。将此ＰＣＲ产物连接到ｐＧＥＭ－Ｔ－ｅａｓｙ载体也能转化大肠杆菌ＤＨ５５α菌株,得到了含有目的片段的重组子。并采用双脱氧终止法进行序列配美国报道的李坏死环斑病毒ＲＮＡ３序列对应部分核苷酸基本一致,这表     

黄瓜花叶病毒运动蛋白基因及其缺失突变体在大肠杆菌中的表达和表达产物的纯化

利用ＣＭＶＦｎｙ 株系ＲＮＡ３ 全长ｃＤＮＡ 克隆, 构建了运动蛋白（ＭＰ） 基因５′端缺失突变体和３′端缺失突变体的原核表达载体。ＳＤＳ－ ＰＡＧＥ 分析表明, 经ＩＰＴＧ 诱导, ＭＰ 基因及其２ 种缺失突变体均能在大肠杆菌ＢＬ２１（ＤＥ３） ｐＬｙｓＳ中高效表达。利用分离包含体的方法, 提纯了全长的及Ｃ 端缺失的ＭＰ。光密度扫描分析表明, 提纯产物的纯度达９６ ．６ ％ 。     

苏云金芽孢杆菌cry218基因改造

通过合成特异性的寡核苷酸，利用定点突变和ＰＣＲ扩增技术，在苏云金芽孢杆菌ｃｒｙ２１８杀虫晶体蛋白基因的编码区上游－１４４ｂｐ和下游２４２ｂｐ处分别引入ＢａｍＨＩ和ＨｉｎｄⅢ位点，以利于该基因克隆到其它表达载体中。当修饰后的ｃｒｙ２１８基因克隆到表达载体和穿梭载体后，均能表达出对小菜蛾有毒的１３０ｋＤａ蛋白质     

EB-82灭蚜菌对蔷薇长管蚜的毒力及温湿度对其的影响

ＥＢ－８２灭蚜菌对蔷薇长管蚜的毒力及温湿度对其的影响马江龙厚茹（中国农科院生物防治研究所,北京１０００８１）ＴＯＸＩＣＩＴＹＯＦＥＢ┐８２ＡＰＨＩＤＩＣＩＤＥＯＮＭＡＣＲＯＳＩＰＨＵＭＲＯＳＡＥＡＮＤＴＨＥＩＮＦＬＵＥＮＣＥＯＦＴＥＭＰＥＲＡＴＵＲＥ...     

1993年全国小麦秆锈菌种群动态分析

１９９３年我国小麦秆锈病发生较轻，从８个省１８个区县中的４７个品种上采集到的９５个标样中分离到菌株３３６个，鉴定出２１Ｃ３ＣＫＲ、２１Ｃ３ＣＫＨ、２１Ｃ３ＣＴＲ、２１Ｃ３ＣＴＨ、２１Ｃ３ＣＦＲ、２１Ｃ３ＣＦＨ、３４Ｃ１ＭＫＲ、３４Ｃ１ＭＫＨ和３４Ｃ２ＭＫＲ等９个致病类型；２１Ｃ３ＣＫＲ的出现频率为６５．２％，居于首位，２１Ｃ３ＣＤＨ为１４．５％，２１Ｃ３ＣＴＲ为１０．７％，２１Ｃ３ＣＦＲ为４．２％     

南方菜豆花叶病毒(SBMV)两典型株系特异cDNA和RNA探针的制备及应用

南方菜豆花叶病毒（ＳＢＭＶ）主要有２株系：菜豆株系（ＳＢＭＶ－Ｂ）和豇豆株系（ＳＢＭＶ－Ｃ）,血清学方法不能予以区分。根据已报道的核酸序列设计了２对特异引物,进行ＲＴ－ＰＣＲ扩增并克隆到载体Ｂｌｕｅｓｃｒｉｐｔ中,序列测定予以证实后利用随机引物法合成放射性ｃＤＮＡ探针,再进行体外转译合成地高辛ＵＴＰ标记的ＲＮＡ探针。２种探针均可分别特异地检测Ｎｏｒｔｈｅｒｎｂｌｏｔ膜上的ＳＢＭＶ－Ｂ和ＳＢＭＶ－Ｃ。ＲＮＡ探针检测ＳＢＭＶ－Ｂ和ＳＢＭＶ－Ｃ灵敏度分别为：０．１μｇ和０．０１μｇ。     

杏（Prunus armeniaca L.）的茎尖培养研究

９个杏品种的茎尖培养结果表明,初代培养基因ＭＳ＋０．２ｍｇ／ｌＫＴ＋０．２５ｍｇ／ｌ２,４,－Ｄ＋０．２５ｍｇ／ｌＮＡＡ＋３％蔗糖,增殖培养基为ＭＳ＋１．５ｍｇ／ｌＢＡ＋０．１ｍｇ／ｌＩＡＡ＋２．５％山梨醇,加长生长培养基为ＭＳ（１／２Ｎ）＋２．２ｍｇ／ｌＺＴ＋３％蔗糖,诱导生根培养基为１／２ＭＳ＋０．５ｍｇ／ｌＩＢＡ＋２％蔗糖,在相同培养条件下,不同品种的茎尖增重速率不同,从大到小依次为Ｔｙｒｎ     

球形芽孢杆菌C3-41和2362菌株的发酵特性和毒力比较

球形芽孢杆菌Ｃ３－４１和２３６２菌株的发酵特性和毒力比较①袁志明刘娥英蔡全信张用梅（中国科学院武汉病毒研究所,武汉４３００７１）ＣＯＭＰＡＲＩＳＯＮＳＯＦＴＨＥＦＥＲＭＥＮＴＡＴＩＯＮＰＲＯＰＥＲＴＹＡＮＤＴＯＸＩＣＩＴＹＯＦＢＡＣＩＬＬＵＳＳＰＨＡ...     

应用聚合酶链反应技术鉴定印度腥黑穗病菌

用一对专化于印度腥黑穗病菌的引物Ｔ１１７Ｍ１（５＇－ＴＣＣＣＣＴＴＧ－ＧＡＴＣＡＧＡＡＣＧＴＡ－３＇）和Ｔ１１７Ｍ２（５＇－ＡＧＡＡＧＴＣＴＡＡＣＴＣＣＣＣＣＣＴＣＴ－３＇）可特异地扩增印度腥黑穗病菌产生一段８２５ｂｐ的产物，而稻粒黑粉病菌则不能被扩增。实验还表明，用聚合酶链反应（ＰＣＲ）方法检测灵敏度可达到１００个未萌发的冬孢子，这为进口粮印度腥黑穗病菌的检疫提供了有力工具。     

应用基因枪法获得抗大麦黄矮病毒转基因小麦

 以我国特有的大麦黄矮病毒GPV株系的外壳蛋白(CP)基因为材料,设计合成了分别含有Act启动子或Emu启动子的植物表达载体pPPI2、pPPI3和pPPI5。采用基因枪法分别转化小麦幼胚和愈伤组织。诱导成苗后进行PCR检测,T₀代阳性率为18%。对转基因苗的后代进行进一步检测,部分转基因苗阳性株至T₃代PCR检测阳性率为100%,PCR结果CP探针杂交呈阳性反应,序列测定结果与GPV CP基因序列一致,表明GPV CP基因已整合到小麦基因组中。室内抗病性鉴定结果,虽然转基因植株全部发病,但对大麦黄矮病毒GPV株系具有一定的延迟发病作用。     

Bt菌株FB的生物学特性及其cry、vip基因型鉴定

菌株FB是1株对小菜蛾Plutella xylostella幼虫具有高毒力的苏云金芽胞杆菌Bacillus thuringiensis （Bt）。本研究通过扫描电子显微镜、大质粒电泳、总蛋白SDS-PAGE及菌株生长特征观察的方式研究了菌株FB特征,克隆得到了基因cry1Ia、cry1Ea、cry1Ab、cry2Ab和vip3Aa全长,依据全基因组测序结果得到了1个cry8基因部分片段,首次在Bt菌株中同时发现基因cry1类和cry8类,这五种基因推导的氨基酸序列与已知基因序列相比,最高相似性分别为99%、98%、99%、100%、99%,而cry8半长基因与已知基因仅为63%。生测结果表明,蛋白Cry1Ia、Cry1Ea和Cry1Ab对小菜蛾幼虫具有较高杀虫活性。     

Novel PCR primers for detection of genetically diverse virulent <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> biovar 1 strains

Novel PCR primers were developed to amplify a 243-bp fragment of an intergenic region between gene 5 and tms2 on the T-DNA of Agrobacterium tumefaciens biovar 1. These primers exhibit 100% positive correlation with strain virulence, 100% negative correlation with avirulence, and did not generate extraneous bands, thus facilitating robust real-time PCR detection.     

表达dsRNA的细菌提取液可抑制黄瓜花叶病毒对烟草的侵染

 利用RT-PCR分别克隆了CMV P3613株系的RNA2片段、MP(movement protein)基因片段及CMV AN株系的CP(coat protein)基因片段。以CP基因为中间间隔序列,分别构建了含有RNA2片段和MP基因反向重复片段的原核表达载体。体外转录试验表明:两个载体转录后都能形成预期大小的dsRNA。经过IPTG诱导,在大肠杆菌HT115(DE3)菌株中可表达产生预期大小的核酸片段,经DNase和RNaseA消化处理,证实为dsRNA。将表达病毒基因dsRNA的细菌超声破碎后处理烟草,进行保护和治疗试验,结果表明:表达CMV MP基因和RNA2片段dsRNA的细菌破碎液能够诱导烟草对CMV产生抗性。接种病毒60d后,保护效果试验病株率分别为45%和60%,治疗效果试验病株率分别为75%和85%,而其他对照发病率均为100%。本研究结果证明了利用RNA沉默的原理,构建具有反向重复序列的原核表达载体,用细菌表达dsRNA的粗提取物可防治CMV对烟草的侵染。     

Analysis of Variable Short-sequence DNA Repeats on the 29 kb Plasmid of Erwinia amylovora Strains

The fire blight pathogen Erwinia amylovora has been specifically and sensitively detected by PCR assays with primers derived from plasmid pEa29. The amplified fragment of approximately 1kb can vary in length for individual strains, easily seen in a digest with restriction enzymes Sau3A or HpaII. DNA fragments from this variable region were cloned and DNA sequence analysis revealed short-sequence DNA repeat (SSR) motifs which were reiterated to various extents. The SSR units consisted of eight nucleotides (ATTACAGA), and terminated with ATTA which is part of an SSR. The shortest repetition consisted of four units and the longest one in Austrian E. amylovora strains was 15 units. The number of SSR units was remarkably stable during propagation of strains, but was occasionally changed when a strain was stressed by exposure to antibiotics, copper sulphate or storage at low temperature. Changes in the SSR number could be due to adjustment in bacterial fitness to environmental pressure. We designed oligonucleotide PCR primers from DNA sequences adjacent to the SSR region of pEA29 for rapid analysis of SSR length variations. With this PCR assay, more than 130 strains were classified into at least 11 types based on the number of repeats. E. amylovora strains isolated in Germany carried mostly six repeats in pEA29, which never changed under laboratory conditions. E. amylovora strains from Hungary and the Netherlands were quite divergent for the SSRs and further changes were sometimes observed after plating on agar medium. Homology search of nucleotide sequence data libraries revealed similarities of the SSR motif to partition functions of low copy number plasmids. Amino acid homology searches showed similarity of the deduced amino acid sequence in the ORF adjacent to the SSR motif to replication proteins of plasmids. The SSR may play a role in regulation of plasmid replication and partition as assumed for iterons.     

安徽桑黄花型萎缩病植原体16S rDNA序列分析及分子检测

 Mulberry yellow dwarf(MYD)disease is an quarantine disease and the causal agent is a phytoplasma.Two pairs of published universal primer, P1/P7 and Rm16F2/Rm16R1, based on the 16S-23S rDNA sequence of phytoplasma and total DNA extracted from infected mulberry tissues were employed for PCR and nested-PCR detection.The results revealed that a phytoplasma-specific 1 830 bp fragment with a G+C content of 46.01% was sequenced(GenBank accession No.GQ249410).The sequence shared 99.7% and 99.8% identity with aster yellows, the representatiive phytoplasma in 16SrI group, and mulberry dwarf phytoplasma classified into subgroup B in 16SrI group and named as the MYD phytoplasma strain Anhui(MYD-Anh).A phylogenetic tree based on 16S rDNA sequences was constructed and showed that MYD-Anh was clustered into 16SrI group.Identity of 16S rDNA sequence between MYD-Anh and mulberry yellow dwarf phytoplasma strain Zhenjiang(MD-zj) was nearly 100%, and they might belong to the same strain.Nested-PCR was used to detect the pathogenic phytoplasma from the differential tissues of mulberry infected with MYD-Anh.The results showed that a phytoplasma-specific 1.4 kb fragment was amplified with total DNA extracted from bark and vein.Nested-PCR was more sensitive than PCR for detecting MYD phytoplasma.     

检疫性疫霉DNA条形码筛选

 疫霉是世界关注的一类植物病原真菌。以疫霉属80个种122个菌株为研究材料,以ITS、CO1、EF-1α和β-tubulin 4个基因片段为候选DNA条形码,分析表明ITS、CO1基因的PCR扩增和测序成功率最高,分别为100%和96.7%;ITS、CO1和β-tubulin存在明显的条码间隔,但种间、种内距离频率分布存在较小重叠;种间、种内遗传距离Wilcoxon秩和检验结果认为各基因对种内遗传距离的效力相等,对种间遗传距离的区分能力为ITS>CO1>β-tubulin。CO1基因和ITS片段可同时作为11种检疫性疫霉的首选DNA条形码,β-tubulin基因可作为辅助DNA条形码。     

臭矢菜丛枝病植原体的分子鉴定研究

 本实验采用DAPI荧光显微镜、PCR、克隆和测序等技术,对海南臭矢菜丛枝病样进行了检测和鉴定。以染病臭矢菜总DNA为模板应用3对植原体特异性引物进行PCR扩增,获得PCR产物为16S rDNA(1 430 bp)、16S-23S rDNA(358bp)、rp DNA(1 294 bp)。应用DNA回收试剂盒获得了3个PCR扩增片断的纯化产物,并克隆到DH5α大肠杆菌中测序。应用DNAMAN和MEGA软件对获得的序列与NCBI数据库中植原体序列进行同源性分析和构建系统发育树。结果显示臭矢菜丛枝病植原体与花生丛枝病植原体序列同源性最高,16S rDNA的序列同源性为99.9%,16S-23S rDNA高达100%,rp为99.7%,因而将臭矢菜丛枝病植原体归为花生丛枝组(16SrⅡ),根据16S rDNA的RFLP分析,将其归为16SrⅡ-A亚组。     

Detection of Fusarium oxysporum f.sp. vasinfectum in cotton tissue by polymerase chain reaction

A polymerase chain reaction assay was developed for the detection of Fusarium oxysporum f.sp. vasinfectum (FOV), a serious wilt pathogen of cotton in many parts of the world. Based on small nucleotide differences in internal transcribed spacer sequences between 18S, 5.8S and 28S ribosomal DNAs, primers Fov1 (5'-CCCCTGTGAACATACCTTACT-3') and Fov 2 (5'-ACCAGTAACGAGGGTTTTACT-3') were selected. These primers unambiguously amplified a 400-bp DNA fragment of all the FOV isolates tested (from Angola, Brazil, China and the USA) but did not amplify any other isolates of mycoflora associated with cotton, such as F. moniliforme , Verticillium albo-atrum , V. dahliae , Aspergillus sp., F. oxysporum , F. sambucinum or F. solani . A control PCR assay was developed employing the universal primer pair ITS1 and ITS2 which amplified a fragment of approximately 220 bp from all isolates tested. This control assay demonstrated that all fungal DNAs were readily amplifiable, thus confirming that the lack of amplification with Fov1 and Fov2 primers was a result of primer specificity and not of other possible causes, such as DNA degradation or the presence of PCR inhibitors. The assay was effective on samples from the stems, leaves, roots and calli, and from plant tissues both with and without symptoms. This detection system proved to be accurate and sensitive and could aid not only diagnosis but also disease monitoring and forecasting.