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1.
刺芹侧耳(P1eurotus eryngii)rDNA的IGS2多样性分析   总被引：5，自引：0，他引：5  
对8个刺芹侧耳（Pfeumfus eryngit）菌株进行了转录单位间隔区2（IGS2）分析,结果表明不同菌株的存在长度异质性和数量多态性。应用BsuRⅠ、Hin6Ⅰ、HpaⅡ和RsaⅠ限制性内切酶,对IGS2扩增产物进行酶切（IGS2-RFLP）,不同菌株的酶切位点不同,电泳后产生多样性丰富的图谱,成为菌株特有的DNA指纹,实验应用的4个限制性内切酶中的任何一个都可有效地将供试菌株加以鉴别。这一方法的结果重复性和稳定性良好,IGS2-RFLP有望作为刺芹侧耳菌株鉴定和鉴别的分子标记。  相似文献   

2.
为弄清海南薯蓣茎腐病病原,采用离体致病性测定、形态学和分子生物学鉴定等方法,对其进行了研究。从海南临高、海口等薯蓣生产基地采集病样89份,纯化出分离物28个。经致病性试验证实,分离物lp3—1、b16-5、bd6-4和lp2—5为薯蓣茎腐病病原菌。经形态学鉴定,4个分离物均为镰刀菌（Fusariumsp．）。利用真菌18SrRNA基因引物、镰刀菌特异性引物和轮枝镰刀菌特异引物进行PCR扩增,能扩增出510bp大小的镰刀菌DNA片段,不能扩增出轮枝镰刀菌DNA片段。序列分析表明：分子生物学和形态学鉴定结果一致,且镰刀菌Fu3／Fu4区序列分析能进一步确定薯蓣茎腐病是由尖孢镰刀菌薯蓣专化型（Fusariumoxysporumfsp．dioscorea）侵染引起的一类植物真菌病害。  相似文献   

3.
克隆了位于自交不亲和型甘蓝(Brassica oleracea)S位点受体激酶基因(SRK)上第一编码区中包含两个CCGG位点的目的片段,通过甲基化敏感限制性内切酶-PCR法,利用对甲基化敏感性不同的Msp Ⅰ和HpaⅡ分别对柱头乳突和花药基因组DNA及其PCR产物进行交叉组合式的酶切与电泳,首次对SRK基因编码区的特定DNA区段进行了甲基化分析.使用相同的SRK基因特异性引物时,柱头乳突和花药基因组DNA作为模板均可以扩增出清晰目标谱带,且目标谱带经Msp Ⅰ/Hpa Ⅱ完全酶切后可产生预期的谱带类型;而经Msp Ⅰ/Hpa Ⅱ完全酶切后的此二基因组DNA再作为模板进行PCR,均无特异性的目标谱带扩出.这些结果初步表明,自交不亲和型甘蓝花粉的SRK基因可能不存在甲基化封闭.  相似文献   

4.
我国南方部分稻区稻瘟病菌的群体结构   总被引：6，自引：0，他引：6  
用重复序列探针ＭＧＲ－５８６，与限制性内切酶ＥｃｏＲＩ组合，分析了我国南方部分稻区８６个稻瘟病菌株的限制性片段长度多态性（ＲＦＬＰｓ）依其ＭＧＲ－ＤＮＡ指纹的相似性，结合病菌的致病型测定，将表现为２８个不同致病型的８６个稻瘟病菌株区分为１８个系谱，每个系谱的寄主范围有限，且显示出不同栽培稻区内稻瘟病菌的群体结构有明显差别。本研究通过分析一稻区内稻瘟病菌的群体遗传结构，用分子生态学的方法探索病流行的  相似文献   

5.
红壤荒草地氨氧化细菌富集液16SrDNA文库的RFLP分析   总被引：6，自引：1，他引：6  
分析红壤荒草地富集液中氨氧化细菌的种群组成,选取氨氧化细菌16S rDNA特异性引物序列,利用PCR技术对从富集液中抽提的细菌总DNA进行扩增,并建立了氨氧化细菌特异性的16S rDNA文库。用酶HhaⅠ和RsaⅠ对该文库特异性片段进行了限制性酶切片断长度多态性分析(Restriction fragment lengthpolymorphism,RFLP),随机挑选的35个特异性克隆片段被分成3个不同的RFLP类型,其中优势型占了所有分析克隆子的94%,另两个型各占3%。从每个RFLP类型中挑取一定的转化子进行测序,测序结果经GenBank检索,发现在该富集液体系文库中存在大量亚硝化单胞菌属(Nitrosomonas)细菌序列,由此推测红壤荒草地中存在氨氧化细菌,Nitrosomonas属细菌能在富集条件下成为优势菌。  相似文献   

6.
土壤微生物DNA木聚糖酶基因多样性的研究   总被引：2，自引：0，他引：2       下载免费PDF全文
采用国产硅胶Gk254（60型）代替进口Glass bead的改良方法抽提土壤微生物DNA，然后设计了一对扩增木聚糖酶基因片段的新简并引物，对抽提的土壤微生物DNA进行PCR扩增。扩增片段连接pMD18T载体，转化大肠杆菌，重组片段通过酶切进行RFLP分析后，测序分析得到10个木聚糖酶基因片段。对所得片段翻译的氨基酸序列进行BLAST分析表明有8个片段与来自放线菌的木聚糖酶具有较高的同源性，2个与假单胞菌的木聚糖酶具有较高的同源性。所得10个木聚糖酶片段的氨基酸序列同源性比较显示，第27个氨基酸均为天冬酰胺（N），暗示这些来自土壤微生物DNA的基因片段编码耐碱的木聚糖酶。通过构建系统进化树，发现扩增的木聚糖酶片段之间的相似性均在70％以上。  相似文献   

7.
为了明确2010年食源性疾病监测中分离的肠道沙门菌O：4（B）菌群的PFGE分子型别,探讨其多态性及其与分子流行病学关系,我们将分离获得的29株O：4（B）血清型沙门菌进一步鉴定培养,挑取单个菌落增菌,供试菌株基因组DNA用限制性内切酶xbaI消化酶切后进行脉冲场凝胶电泳分型,所得结果用Bionumerics5．1软件进行聚类分析。实验结果表明,根据电泳指纹图谱,可将29株肠道沙门菌O：4（B）菌群分为24个PFGE型别,菌株间的相似值在56.31％～100％之间,同一血清型别的沙门菌有多个PFGE型别。脉冲场凝胶电泳对O：4（B）群沙门菌有较高的分型能力,可有效的应用于食物中沙门菌溯源分析及分子流行病学研有。  相似文献   

8.
用限制性片段长度多态性（Restriction fragment length polymorphism, RFLP）方法研究了在农业废物堆肥一次发酵和二次发酵期间添加黄孢原毛平革菌 （ P. chrysosporium ）对微生物多样性的影响。结果表明,在不同发酵期接种P. chrysosporium对堆肥进程的影响不同。3种典型的限制性内切酶Alu Ⅰ ,Ho Ⅲ和TaqⅠ在分析堆肥细菌微生物多样性的灵敏性上,Hae Ⅲ效果最好,AluⅠ次之,TaqⅠ则并不很适用于分析堆肥细菌微生物多样性。接种黄孢对堆肥细菌群落的影响非常显著且迅速,二次接种可起到巩固一次接种效果的作用。  相似文献   

9.
利用差减链式反应构建白菜基因组代表性文库   总被引：1，自引：0，他引：1  
构建多态性比例高的基因组代表性文库是提高芯片和测序基础上的标记技术效率的关键。通过差减链式(differential subtractive chain, DSC) 反应,去除同一物种内的不同栽培种群间不具多态性的片段,能够提高基因组代表性文库中多态性片段的比例。研究利用白菜类作物（Brassica campestris） 8个栽培种群的材料构建tester DNA样品,利用水菜(B.campestris ssp. nipposinica) DNA作为driver样品。这两个DNA样品经EcoRⅠ/Mse Ⅰ酶切,酶切产物连接不同的接头。3遍DSC反应后,将反应产物连接到pDONR201载体中,构建基因组复杂性降低文库。分2批随机取其中20个单克隆和95个单克隆,分别进行Southern斑点杂交,结果分别显示9个和98个多态性片段,其比例分别为45%和61%,较之前关于多态性芯片技术的相关研究中15%～17%的多态性克隆比例大大提高。  相似文献   

10.
番茄果实中NEVER—RIPE基因的克隆及其反义表达载体的构建   总被引：6，自引：0，他引：6  
从粉红期番茄果实中提取总RNA，根据Genebank中NEVER－RIPE（即NR基因）cDNA序列，设计合成特异性引物，用反转录－聚合酶链式反应（RT－PCR）进行cDNA扩增，获得一个715bp左右的扩增产物，克隆于pGEM-T easy载体，测序确定其正确性。将该扩增产物反向克隆到植物表达载体pBI121中，通过PCR及限制性内切酶酶切鉴定重组质粒。将重组表达质粒导入根癌农杆菌LBA4404中，PCR及限制性内切酶酶切确定质粒已被导入。  相似文献   

11.
The DNA sequence diversity of Sardina pilchardus (Walbaum, 1792) and some closely related species of Clupeomorpha was investigated using the mitochondrial DNA gene encoding cytochrome b. The nucleotide sequences of complete and partial mtDNA cytochrome b were determined in numerous specimens. Sequence divergence between species and genera was evenly distributed in the cytochrome b gene but rather high compared to reports for other fish species. Phylogenetic analyses on complete cytochrome b were used to study the relationships among the considered species. S. pilchardus was easily differentiated, showing a genetic distance of 0.25 with respect to Clupeidae species and 0.26 with respect to the other species. A species-specific short fragment (<150 bp) was isolated by polymerase chain reaction (PCR) using primers designed for Clupeomorpha. A rapid and reliable PCR method using restriction fragment length polymorphism (RFLP) with two restriction enzymes (MnlI/HinfI) was optimized for unambiguous differentiation of S. pilchardus from the other species tested (raw and canned products).  相似文献   

12.
Using peptides as energy sources, H2 as electron donor, thiosulfate as electron acceptors, we isolated, from four ricefield soils originating from France and the Philippines, 52 strains of anaerobes, among which 18 reduced thiosulfate but not sulfate. These 18 strains were strict proteolytic asaccharolytic anaerobes producing H2S when grown on thiosulfate + H2. They exhibited the same restriction fragment length polymorphism (RFLP) profile (11 restriction enzymes tested). Partial sequencing of the 16S rDNA showed that they belonged to the genus Clostridium and were phylogenetically related to C. subterminale. DNA–DNA hybridization of a representative strain with the closest C. subterminale strain (DSM 6970T) yielded a value of 68.9%. Previous counts of thiosulfate reducers unable to reduce sulfate (TSRnSR) in ricefield soils, their identification as Clostridium strains, and the known ubiquity of this genus in such soils indicate that TSRnSR of the genus Clostridium may play a significant role in S cycling in some wetland soils.  相似文献   

13.
The molecular identification of several strains of Campylobacter jejuni and Campylobacter coli involved in foodborne disease was carried out by investigating the restriction profiles of their chromosomal DNA and by DNA/DNA hybridization. Cleavage with EcoRV allowed the visualization of a 3 kb DNA fragment characteristic of C. jejuni, whereas restriction with ClaI allowed the identification of a 9.3 kb DNA fragment, also characteristic of C. jejuni, and a DNA duplet of 9.5-10 kb, specific to C. coli.Restriction analysis with enzyme BglII allowed the visualization of DNA fragments of 3.5, 4, and 6.7 kb, characteristic of C. jejuni. C. jejuni subsp. doylei strains investigated shared a higher genetic homology among themselves-as determined by DNA/DNA hybridization-than with C. jejuni subsp. jejuni. A DNA probe, initially designed by Korolik et al. (Korolik, V.; Coloe, P. J.; Krishnapillai, V. J. Gen. Microbiol. 1988, 134, 521-529), including a DNA fragment encoding an antigenic membrane protein of 31.5 kDa in C. jejuni, when used as probe, allowed the specific identification of all strains of C. jejuni through the detection of strong hybridization signals in two BglII DNA fragments of 2.3 and 2.5 kb, which were not observed in C. coli. Cleavage of chromosomal DNA with BglII-either alone or coupled with probing assays with specific probes-proved to be a valuable tool for the speciation of Campylobacter isolates involved in foodborne disease.  相似文献   

14.
随着分子生物技术的发展,不可培养微生物多样性研究的难题得到了解决。肠道微生物处于特殊的生态环境条件下,分子生物学技术的应用使得肠道微生物多样性的研究进入了一个崭新的阶段。本文主要介绍了基于16S rRNA基因片段的一些肠道微生物研究工作中常用的分子生物学分析方法,主要包括变性梯度凝胶电泳(DGGE),温度梯度凝胶电泳(TGGE),单链构象多态性(SSCP),限制性片段长度多态性(RFLP),放大片断长度多态性(AFLP)和随机扩增多态性DNA(RAPD)等指纹图谱技术。  相似文献   

15.
EMS诱导小麦品种烟农15突变体的鉴定和EST-SSR分析   总被引：4，自引：2，他引：2  
用EMS对小麦品种烟农15进行诱变处理,以构建突变体库、创造小麦新种质,为小麦功能基因研究和小麦遗传改良提供基础材料。经过M2代筛选和M3代鉴定,得到11个农艺性状发生明显变异的突变系,其中籽粒大小和株高2个性状的变异幅度最大。11个突变系均有复合性状突变出现,将其分为3类突变表型:8个大粒、高秆突变系;2个半矮秆突变系;1个高秆、多蘖突变系。用715个EST-SSR引物对受体烟农15和4个M3突变系进行了分析,共有14个引物对在受体和突变系间能扩增出差异条带。其中12个引物对扩增结果的差异表现为条带的有无;2个引物对表现为扩增出长度不同的差异条带。  相似文献   

16.
To investigate the influence of papaya ringspot virus resistant transgenic papaya on soil microorganisms, upper (0-15 cm) and lower layers (15-30 cm) of soil samples were collected around transgenic papaya planting area and nontransgenic papaya planting area and from soils in which plants had not been grown. The moisture content, pH value, total organic carbon content, and total nitrogen content were not significantly different among groups. The populations of total count, fungi, and actinomycete were highest in upper layer soils around transgenic papaya planting area and lowest in lower layer soils in which plants had not been grown. The microbial populations were all higher in upper layer of soils. Amplified fragment length polymorphism, amplified ribosomal DNA restriction analysis, terminal restriction fragment length polymorphism, and denaturing gradient gel electrophoresis analyses indicated that the similarity of soil microorganisms of upper layer soils around transgenic papaya planting area and around nontransgenic papaya planting area was >80%. A similar result was observed in lower layer soils. Thus, planting transgenic papayas does have a limited impact on soil microorganisms.  相似文献   

17.
Analysis of restriction fragment length polymorphism (RFLP) profiles of a 464 bp amplicon obtained from the mitochondrial cytochrome b gene was used to differentiate between several different fish species. The method was tested by a collaborative study in which 12 European laboratories participated to ascertain whether the method was reproducible. Each laboratory was required to identify 10 unknown samples by comparison with RFLP profiles from authentic species. From a total of 120 tests performed, unknown samples were correctly identified in 96% of cases. Further work attempting to use the method to analyze mixed and processed fish samples was also performed. In all cases the species contained within mixed samples were correctly identified, indicating the efficacy of the method for detecting fraudulent substitution of fish species in food products.  相似文献   

18.
In the present study a technique was developed with the aim of guaranteeing the composition and security of fish meals, since it allows verification of whether these meals contain land animal remains. The method is based on polymerase chain reaction (PCR) and length polymorphism, followed by a restriction fragment length polymorphism (RFLP). Specific primers for every species were designed and calibrated, generating exclusively a PCR product with a specific size when DNA for each species was present in the sample. This technique allows the detection of land animal remains in fish meals, specifically cow, chicken, pig, horse, sheep, and goat. The identity of the PCR products can be confirmed by RFLP analysis using only one restriction enzyme. The selected restrictase generated one characteristic restriction profile for every species included in this study. The detection limit of this method was calculated by using mixtures of fish meals in different proportions and meal that exclusively contained remains of one of these land species studied. The analytical strategy herein proposed was applied to fish and meat meals, giving good results, both in the analyzed standards and in commercial samples.  相似文献   

19.
The European chicken meat market is characterized by numerous quality marks: "Label de Qualité Wallon" in Belgium, "Label Rouge" in France, denominations of geographical origin, organic agriculture, etc. Most of those certified productions have specifications requiring the use of slow-growing chicken strains. The amplified fragment length polymorphism (AFLP) technique has been used to search molecular markers able to discriminate slow-growing chicken strains from fast-growing ones and to authenticate certified products. Two pairs of restriction enzymes (EcoRI/MseI and EcoRI/TaqI) and 121 selective primer combinations were tested on individual DNA samples from chicken products essentially in carcass form that were ascribed as belonging to either slow- or fast-growing strains. Within the resulting fingerprints, two fragments were identified as type-strains specific markers. One primer combination gives a band (333 bp) that is specific for slow-growing chickens, and another primer pair generates a band (372 bp) that was found to be characteristic of fast-growing chickens. The two markers were isolated, cloned, and sequenced. The effectiveness and the specificity of the two interesting determinants were assessed on individuals of two well-known strains (ISA 657 and Cobb 500) and on commercialized products coming from various origins.  相似文献   

20.
为了建立一套基于DNA分子标记技术快速鉴定榆黄蘑菌株的有效方法,本研究通过对生产上常用的16个榆黄蘑菌株进行SRAP多态性分析,从榆黄蘑2号菌株中扩增获得了一个片段长为537bp的特异片段,将其克隆、测序并设计引物,成功转化为稳定的SCAR标记。试验结果表明,利用该特异SCAR标记,能在1d时间内准确鉴定出榆黄蘑2号菌株。由此可见,SCAR分子标记是一种快速、稳定、准确鉴定榆黄蘑菌株的新方法。  相似文献   

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