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1.
从海南根瘤菌(Rhizobium hainanense)166提取总DNA，用EcoR I酶进行完全酶切和电泳，然后用苜蓿中华根瘤菌(Sinorhizobium meliloti)042B的nodABC DNA片段作探针进行Southern杂交，发现1条含有nodABC基因DNA片段的阳性条带，大小为2．1-2．5kb。通过电泳回收该DNA片段，用pUCl8作载体连接，并转化大肠杆菌(Eschedchia coli)DH5α，从而构建了含有nodABC的部分基因文库。提取该文库的质粒与nodABC探针做菌落杂交，筛选到nodABC的阳性克隆，称为pUER12。将含有nodABC的DNA片段与EcoR I酶切的pBBR-MCS-5连接，转化大肠杆菌DH5α，获得转化子pBER12。用三亲本杂交方法，把pBERl12转入苜蓿中华根瘤菌AK1657，将其接合子进行结瘤实验，验证了pBER12具有nodABC基因的功能。然后将含有166 nodABC片段的DNA与EcoR I酶切的pGEM-7Z( )连接、测序和进行同源性比较，发现166 nodABC编码的蛋白与苜蓿中华根瘤菌具有高度的同源性。  相似文献   

2.
从海南根瘤菌（Rhizobium hainanense)I66提取总DNA，用XbaI完全酶切，然后用苜蓿中华根瘤菌（Sinorhizobium meliloti)042B的nodDDNA片段作探针进行Southern杂交，杂现2条含有nodD基因DNA征段的阳性条带，分别为4.0和6.0kb，电泳回收含有4.0-6.0kb的DNA片段，用pUC18作载体连接，并转化E.coliDH5α，构建含有nodD的部分基因文库，提取该文库的质粒与nodD探针做点杂交，筛选到nodD的阳性克隆，称为pUOD75。再将pUOD75的nodD片段克隆到pBBR-MCS-5,构建成pBOD75。用两亲本杂交，把pBOD75转入豌豆根瘤菌蚕豆生物型（Rhizobium eguminosarum bv.viciae)根瘤菌I66nodD基因在毛地黄黄酮诱导下得到表达，说明其表达产物可能具有结瘤调节功能。  相似文献   

3.
将来自Bacillus thuringiensis subsp.kurstaki的Bt CRy I A(c) 杀虫基因通过综合质粒载体pBC601，整合到棉花（Gossypium hirsutum)优势内生细菌Bacillus cereus(Bc9002)的染色体上，得到的工程菌对棉铃虫（Helicoverpa armigera Hb.)有杀虫活性。此综合质粒含有能在营养期表达Bt cry I A(c) 基因的强启动子，cry I A(c)杀虫基因，四环素抗性标记基因tet^r 8.0kb的EcoR I-Nco I B.cereus染色体片段。将综合质粒通过电击导入B.cereus(Bc9002)中，综合质粒因含B.cereus染色体片段，可与B.cereus染色体发生同源重组，从而将Bt cry I A(c)基因整合到B.cereus的染色体上。通过对转化子的DNA酶切分析，PCR扩增，SDS－PAGE凝胶电泳检测，ELISA检测，电镜观察，毒力测定。结果表明Bt cry I A(c)基因已经整合到内生细胞Bc9002的染色体上，并可高效表达。  相似文献   

4.
植物激活蛋白Ap36在苏云金芽胞杆菌的表达及抗病作用   总被引：1，自引：0，他引：1  
以含有植物激活蛋白(Activator protein)基因ap36的质粒pMYE为摸板,设计一对特异引物,通过PCR反应扩增出激活蛋白基因开放读码框序列,使用XbaI和SphI双切PCR产物,插入到用这两酶双切的pBMB982-304质粒slh motif的下游,构建重组表达质粒pBMB0588,转化苏云金芽胞杆菌无质粒突变株BMB171。结果表明,目标蛋白以融合蛋白的形式成功表达。为探讨表达植物激活蛋白的重组菌是否能够诱导植物获得抗性,用重组菌在28℃培养24小时发酵液原液处理番茄幼苗和土豆片。结果显示,发酵液处理90min后,番茄叶片过氧化物酶(POD)和脯氨酸含量较对照分别提高了57.14%和131.59%;4d后苯丙氨酸解氨酶(PAL)含量较对照也有所提高;经重组菌发酵液处理的马铃薯片,对马铃薯软腐病表现出很强的抗病性。  相似文献   

5.
广宿主稳定表达载体pQMV和pGMP的构建   总被引：3，自引：0，他引：3  
以Eschedchia coli高效表达载体pET-29a为基础，将广宿主载体pUCP19中稳定的假单胞菌(Pseudomonas)质粒DNA复制子和自行分离克隆的荧光假单胞菌(P.fluorescens)P303组成型表达启动子PP303插入其中，分别获得了重组载体pQMV(4．6kb)和pGMP(5．6kb)。它们均含有假单胞菌和大肠杆菌复制子、荧光假单胞菌内源强启动子，以及包含8～11个限制性内切酶多克隆酶切位点；此外，载体pGMP还含有绿色荧光蛋白基因gfp作为辅助检测标记。连续培养和继代培养测定，这两种载体在荧光假单胞菌中的稳定性均为100％(120h)。  相似文献   

6.
通过功能互补法从Frankia菌株At4的基因文库中筛选到可互补豌豆根瘤菌nodD基因功能的pAt2GX和pAt3GX,把pAt2GX和pAt3GX重新导入tmdD突变体中,接种豌豆苗,可观察到nodD突变体恢复了结瘤功能。扫描电镜观察、根瘤内生菌抗性标记检测及内生菌重组质粒的酶切分析均表明nadD突变体恢复结瘤功能是山于带有外源质粒pAt2GX和pAt3GX所致,这就证明了pAt2GX和pAt3GX带有类nodD基因功能的DNA片段。酶切分析及DNA-DNA杂交实验结果说明pAt2GX和pAt3GX不属于同—克隆,但这两个质粒的DNA之间有同源区。  相似文献   

7.
从北京奶山羊白细胞中提取高分子量基因组DNA,用Sau3A部分酶切,连接到经BamHI-EcoRI酶切过的λEMBL3载体上,构建了6.7x106plus的基因组文库.荧光标记牛βlg5'端0.5kb片段作探针,原位杂交得到4个阳性斑A、B、C和D.三轮杂交后,经PCR产物序列分析和物理图谱分析,发现克隆D(10.6kb)包括完整的β乳球蛋白基因,其5'侧翼序列长为3.8kb,3'侧翼序列长为2.2kb.将克隆D5'和3'调控区亚克隆,发现5'侧翼序列包含βlg核心启动子,3'侧翼序列具有MAR序列.比较北京奶山羊与绵羊β乳球蛋白基因的5'侧翼序列和3'侧翼序列,发现它们的同源性分别是90%、89%.结果分析表明,克隆D为β乳球蛋白的编码基因.  相似文献   

8.
低毒病毒／板栗疫病菌系统是一个具有独特优点的、崭新的研究病毒与宿主相互作用的模型系统。以同源重组为基础的基因打靶是研究基因功能和病毒与宿主相互作用分子机制的重要手段。为了研究在板栗疫病菌同源重组遗传操作中同源片段大小对同源重组频率的影响，利用一段5．4kb板栗疫病菌染色体DNA序列，构建了以潮霉素抗性基因为遗传转化筛选标记的3个同源双交换片段以及3个同源单交换质粒，分别转化板栗疫病菌EPl55原生质体，检测筛选同源重组转化子并计算发生同源重组频率。结果表明，在板栗疫病菌中3个同源双交换片段FHA1（1．7～2.0kb）、FHA2（1.0～1.2kb）和FHA3（0.5kb）的同源重组频率分别为3．73％、2。06％和0；3个同源单交换质粒pFHl（1．76kb）、pFH2（1．23kb）和pFH3（O．54kb）的同源重组频率分别为0.95％、0和0。这些结果为板栗疫病菌同源重组遗传操作中同源片段大小的设计提供了依据。  相似文献   

9.
采用＾３２Ｐ标记的巴西固氮螺菌Ａｚｏｓｐｉｒｉｌｌｕｍ ｂｒａｓｉｌｅｎｓｅ Ｓｐ７ ｎｉｆＨ ＤＮＡ探针，对不同限制性内切酶完全酶解的粪产碱菌Ａｌｃａｌｉｇｅｎｅｓ ｆａｅｃａｌｉｓ Ａ１５０１总ＤＮＡ进行Ｓｏｕｔｈｅｒｎ杂交分析，证明在粪产碱菌中存在与ｎｉｆＨ同源的ＤＮＡ序列。在不同ＮＨ４＾＋浓度下测定ｎｉｆＨ－ｌａｃＺ融合基因在粪产碱菌结合子中的β－半乳糖苷酶活性，结果证明该融合基因的表达受  相似文献   

10.
供试菌株Alcaligenes faecalis A15,Enterobacter cloacae EnSs.E·cl-oacae E26,Klebsiella planticola DWUL2.K·oxytoca NG13和Pseudomonassacharophila均能还原2,3,5-氯化三苯基四氮唑(TTC),说明它们含有吸氢酶。以Rhizobium japonicum的hup基因为探针进行杂交的结果表明。除E·oloacae E26和K·oxytoca NG13外,其它菌株的DNA与hup探针具有同源性。A·faecalis A15 hup基因位于染色体上‘E·cloacae EnSs则位于较大的一个质粒上。A·faecalis A15和E·Cloacae EnSs的nif基因与hup基因位于同一复制子上。hup基因不具必然的保守性,因此异源hup基因探针不一定都适宜于探测hup基因。  相似文献   

11.
Since the 18th of April 2004, two new regulations, EC/1829/2003 on genetically modified food and feed products and EC/1830/2003 on traceability and labeling of GMOs, are in force in the EU. This new, comprehensive regulatory framework emphasizes the need of an adequate tracing system. Unique identifiers, such as the transgene genome junction region or a specific rearrangement within the transgene DNA, should form the basis of such a tracing system. In this study, we describe the development of event-specific tracing systems for transgenic maize lines Bt11, Bt176, and GA21 and for canola event GT73. Molecular characterization of the transgene loci enabled us to clone an event-specific sequence into a plasmid vector, to be used as a marker, and to develop line-specific primers. Primer specificity was tested through qualitative PCRs and dissociation curve analysis in SYBR Green I real-time PCRs. The primers were then combined with event-specific TaqMan probes in quantitative real-time PCRs. Calibration curves were set up both with genomic DNA samples and the newly synthesized plasmid DNA markers. It is shown that cloned plasmid GMO target sequences are perfectly suitable as unique identifiers and quantitative calibrators. Together with an event-specific primer pair and a highly specific TaqMan probe, the plasmid markers form crucial components of a unique and straighforward tracing system for Bt11, Bt176, and GA21 maize and GT73 canola events.  相似文献   

12.
摘要: 利用PCR方法从家蚕（Bombyx mori）总DNA中克隆到细胞质肌动蛋白基因（cytoplasmic actin）启动子片段，序列分析表明，该片段中含有典型的组成型表达的细胞质肌动蛋白基因A3（BmA3）调控序列：SRE元件（serum response element）和ActE1元件(active element 1)。该序列的核苷酸序列与来自欧洲的一个家蚕品种的序列同源性为94%。通过多次重组，将A3启动子片段(不含信号肽序列)与转座子 piggyBac的转座酶编码区融合构建成辅助质粒；将其(含信号肽序列和部分编码区)与报告基因多管水母绿色荧光蛋白基因gfp融合，插入到人工合成的转座子piggyBac两反向重复末端序列之间，进而构建成基于转座子piggyBac的转基因载体。研究中构建的转座子转基因载体仅6.4 kb，并在两反向重复末端序列之间设计了1个多克隆位点区，便于目的基因的插入。  相似文献   

13.
WB9是我国分离自武夷山的对多种重要农业害虫具有高毒力的苏云金芽孢杆菌(Bacillus thuringiensis,简称Bt)菌株,经PCR-RFLP鉴定含有cry2Ac基因。根据cry2基因序列设计引物,以WB9质粒为模板扩增cry2Ac全长基因,与大肠杆菌(Escherichia coli)克隆载体pMD18-T连接获得含有cry2Ac全长基因的重组质粒pMD2Ac并测序。该基因在GenBank上登录号为DQ361267,被Bt国际命名委员会正式命名为cry2Ac4。通过亚克隆方法将cry2Ac4基因插入穿梭表达载体pHT315获得重组表达质粒pHT2Ac,将其转化大肠杆菌SCS110和Bt无晶体突变株HD73 Cry-,得到的工程菌能正常表达70 kD蛋白,形成方形晶体。生物测定结果表明,cry2Ac4基因表达产物对桔小实蝇(Bactrocera dorsalis Hendel)幼虫具有显著的毒杀作用,但对小菜蛾(Plutella xylostella)和致倦库蚊(Culex fatlgans)幼虫基本没有效果。  相似文献   

14.
Horizontal gene transfer is useful for enhancing bioremediation through gene bioaugmentation. However, factors affecting transfer of degradative plasmids have not been systematically addressed. To this end, plasmid transfer experiments were performed using a TOL-like plasmid carrying the gene encoding for catechol 2,3-dioxygenase (C23O) between two soil bacteria under different conditions. Transfer frequency increased with air temperature in the range of 10–35 °C and reached 6 × 10−4 transconjugants per donor cell at 35 °C. The transfer frequency detected at soil depth 5–10 cm was significantly higher (p < 0.05) compared with other depths. Addition of 5–75% LB in the microbial inoculum promoted plasmid transfer frequencies. Addition of phenol to the experimental system resulted in significantly higher transfer frequency (p < 0.05) compared with no addition. Transfer frequency heat-moisture in loam was significantly higher (p < 0.05) than in other soils. The highest transfer frequency was found in the experiment containing tomato seedlings, with up to about 1.3 × 10−3 transconjugants per donor cell. Corn and wheat seedlings also led to significantly higher transfer frequencies (p < 0.05) compared with no plants. Furthermore, C23O activities of transconjugants formed under different conditions were measured, as a surrogate measure of the activity of transconjugant. Transfer temperature, soil and plant types had a minor influence on activities of transconjugants. Topsoil (0–5 cm) transconjugants expressed C23O more efficiently under normal incubation condition, but less efficiently when soils incubated with excessive LB medium concentrations, and in the absence of phenol in soil. These results suggested that transfer temperature, soil depth, dilutions of LB broth, phenol content, and soil and plant types had important effects on transfer of the TOL-like plasmid in soil, and some factors also affected activities of transconjugants.  相似文献   

15.
以拟南芥cDNA为模板,用PCR扩增出GAPB的基因全长,然后将GAPB基因片段连接到PET28a(+)载体上,构建重组质粒并转化大肠杆菌DH5α,经菌落PCR和酶切鉴定筛选出阳性克隆,测序正确后,再将阳性克隆的质粒转化大肠杆菌BL21(DE3)。结果表明:成功构建了原核表达载体PET28a(+)-GAPB,为后续的GAPB融合蛋白的表达以及纯化奠定了基础。  相似文献   

16.
STAT3基因转染牛乳腺上皮细胞及生物学特性观察   总被引：1，自引：0，他引：1  
应用PCR技术从含有人STAT3基因cDNA的质粒pOTB7中扩增STAT3基因片段。将其克隆入真核表达载体pEGFP-C1中,构建重组表达质粒。利用脂质体介导法将重组表达质粒转染原代牛乳腺上皮细胞(Bovine Mammary Epithelial cells)。G418筛选阳性克隆,RT-PCR检测STAT3基因在牛乳腺上皮细胞中的表达情况,并通过流式细胞术(Flow cytometry)检测转染细胞的增殖能力、周期以及DNA含量。结果表明,转染24h后大约16%的细胞被导入含STAT3基因的重组表达质粒。在mRNA水平证实细胞内有STAT3基因的高表达。导入STAT3基因后,细胞增殖能力增强,染色体倍数发生变化,细胞的增殖寿命较对照细胞延长了13代,表明导入STAT3基因后,能延长体外培养的牛乳腺上皮细胞的寿命。  相似文献   

17.
《Applied soil ecology》1999,11(1):79-90
Escherichia coli K12 strain (J5-3/RP4) persisted in sandy loam for more than 70 days when incubated at 10°C or 4°C. It decreased to below the level of detection within 20 days when incubated at 25°C. No loss of multi-resistance plasmid RP4 from the E. coli cells was detected during incubation in soil. There was a positive relation between the bacterial inoculum size and the following increase of the protozoan numbers in the soil. When soil microcosms were amended with an eukaryotic inhibitor, the period of survival was increased. These observations indicate a direct involvement of protozoa in the decline of E. coli in soil. Transfer of plasmid RP4 from E. coli donor bacteria to indigenous bacteria in soil was detected already 24 h after addition of the E. coli K12 donor strain. The efficiency of transfer during the first 48 h was approximately 10−6 transconjugants per donor. Inhibition of protozoan predation increased the number of transconjugants appearing in the soil, but the transfer efficiency per donor was not affected by the decreased predation. No transfer could be detected when the donor strain was washed and resuspended in saline before addition to the soil, but transconjugants were detected in this experiment when nutrients (LB) were supplemented after two days of incubation. Plasmid RP4 was maintained in the transconjugant soil bacteria throughout the experiment. The data presented here indicate that the indigenous bacteria in soil may serve as a sink for plasmidborne traits.  相似文献   

18.
亲环蛋白（cyclophilin,CyP）是一类广泛存在于原核和真核生物体内的胞溶性蛋白,是刚地弓形虫（Toxoplasmngondii）速殖子的主要成分,能够诱导产生IL-12和IFN-γ,在控制弓形虫急性感染过程中起重要作用。本研究根据GenBank发表的TgCyP基因序列,设计并合成一对包含BamHI和EcoRI酶切位点的引物,以cDNA为模板,应用PCR技术扩增TgCyP基因。PCR产物连接到pMD18-T克隆载体。用限制性内切酶BamHI和EcoRI双酶切克隆载体,将TgCyP目的基因克隆到载体pVAXI,构建真核表达质粒pVAXl-TgCyP。将真核表达质粒转染Hela细胞,利用问接免疫荧光检测其在Hela细胞内的表达情况。结果表明扩增的TgCyP基因与GenBank上相应基因序列（U04633．1）的一致性达100％,构建的真核表达质粒pVAX1-TgCyP能在转染的Hela细胞中表达,其表达产物与刚地弓形虫阳性血清具有免疫反应性。本研究表明Tg-CyP有望作为弓形虫疫苗的候选抗原,将为进一步研究该质粒的动物免疫试验奠定基础。  相似文献   

19.
 Plasmid transfer between introduced strains of Pseudomonas stutzeri JM302 (pLV1013), Pseudomonas putida PAW340 (pLV1017), Pseudomonas aeruginosa PAO5 (RP4), and Enterobacter cloacae MF10 (RP4), all of them harbouring genetically modified or naturally occurring plasmids and bacteria indigenous to oak leaf litter, soddy-podzolic soil or vermicompost was monitored using non-sterile laboratory microcosms inhabited by the millipede Pachyiulus flavipes, the woodlouse Armadillidium vulgare, or the earthworms Aporrectodea caliginosa and Eisenia fetida, respectively. Plasmid transfer from genetically modified Streptococcus faecalis JH2-2 (pAMβ1) to Bacillus thuringiensis var. israelensis 1-5 under similar conditions was also followed. A recombinant conjugative plasmid (pLV1017) encoding resistance to antibiotics and expressing catechol 2,3-dioxygenase was transferred into two facultatively anaerobic gram-negative bacteria, isolated from the excrement of A. vulgare. Presumed transconjugants of the facultatively anerobic gram-negative bacteria given above were isolated from the leaves and soil (four strains) and the excrement of the invertebrates (11 strains). They carried the same plasmids and exhibited the same enzymatic activity as the respective inoculant strains; for most of them, however, DNA fingerprints were slightly different. Transfer from S. faecalis JH2-2 to B. thuringiensis var. israelensis of a naturally occurring plasmid, pAMb1, encoding resistance to erythromycin was observed in vermicompost. Received: 18 November 1997  相似文献   

20.
A novel type of quantitative competitive polymerase chain reaction (QC-PCR) system for the detection and quantification of the Roundup Ready soybean (RRS) was developed. This system was designed based on the advantage of a fully validated real-time PCR method used for the quantification of RRS in Japan. A plasmid was constructed as a competitor plasmid for the detection and quantification of genetically modified soy, RRS. The plasmid contained the construct-specific sequence of RRS and the taxon-specific sequence of lectin1 (Le1), and both had 21 bp oligonucleotide insertion in the sequences. The plasmid DNA was used as a reference molecule instead of ground seeds, which enabled us to precisely and stably adjust the copy number of targets. The present study demonstrated that the novel plasmid-based QC-PCR method could be a simple and feasible alternative to the real-time PCR method used for the quantification of genetically modified organism contents.  相似文献   

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