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1.
将大肠杆菌HB101嗜碱转化子中质粒pGCA所携带的嗜碱基因亚克隆至双元载体pBI121质粒中,构建了植物表达载体pLGC重组质粒。用其转化大肠杆菌HB101获得了能在碱性和卡那霉素抗性平板上生长的转化子,再通过三亲交配法将亚克隆质粒pLGC转化进农杆菌LBA4404,又获得能在碱性平板和卡那霉素及利福平双抗平板上生长的转化子,Southern杂交结果表明HB101转化子亚克隆质粒pLGC是由来自  相似文献   

2.
耐热芽孢杆菌E2菌株纤维素酶基因克隆的研究   总被引:5,自引:0,他引:5  
官家发  江明 《遗传学报》1995,22(4):322-328
用质粒pBR322作载体,大肠杆菌E.coli DH5αF’作受体,克隆到耐热孢杆菌E2菌株的羧甲基纤维素酶基因。重组质粒PBG3从产生CMCase的转化子中分离得到。克隆到的CMCase基因位于4.0kbHindⅠⅠⅠ片段上。功能状态CMCase基因被亚克隆到2.4kbDNA片段上。  相似文献   

3.
陈炜  何秉旺 《微生物学报》1997,37(4):270-275
用PCR方法扩增短芽孢杆菌α-乙酰乳酸脱羧酶基因,引入原核表达载体pBV220中,得到重组质pALD1.pALD1的ALDC基因在大肠杆菌中高效表达,每毫升发酵液产酶80单位以上,双原始菌株提高200余倍。SDS-PAGE蛋白质分析表明,大肠杆菌DH5α表达的ALDC占细胞总蛋白量的40%以上。研究了重组质粒稳定性,大肠杆菌DH5α和HB101分别大无选择压力下30℃连续培养50代以上,41℃诱导  相似文献   

4.
具有真细菌基因启动子活性的盐生盐杆菌质粒DNA片段   总被引:5,自引:1,他引:4  
孙广秀  江爱民 《遗传学报》1997,24(4):380-384
利用大肠杆菌启动子探测质粒pKK232-8为载体、用两组限制性内切酶BamHI-SalI和HindⅢ-SalI分别消化盐生盐杆菌J7(Halobacteriumhalobium)的质粒pHH205,在体外进行重组,转化E.coliHB101感受态细胞,在含氨苄青霉素和氯霉素的选择平板上筛选转化子,并从随机挑选的20株转化子中,获得抗氯霉素水平达到110μg/ml的转化子T1和T2,所含重组质粒分别被命名为pJH和pJB。经限制性酶切分析及杂交分析表明,pJH质粒上插入了一段来源于pHH205质粒的DNA片段,其大小为800bp左右。通过重新转化实验进一步表明,该DNA片段在大肠杆菌中具有启动子功能,从而证明,在古细菌(盐生盐杆菌)的质粒DNA中存在具有真细菌(大肠杆菌)基因启动子活性的DNA片段。  相似文献   

5.
利用从真养产碱杆菌(Alcaligeneseutrophus)H16染色体DNA中克隆到的催化聚β羟基丁酸酯(polyβhydroxybutyrate,PHB)生物合成的两个关键酶基因:依赖NADPH的乙酰乙酰CoA还原酶基因(phbB)以及PHB合酶基因(phbC),同时利用大肠杆菌高效表达载体pKK2233,构建了嵌合有phbB和phbC基因的表达载体pKCB,转入大肠杆菌JM109,通过显微镜观察及气相色谱分析检测PHB的合成。在确证克隆基因正确的基础上构建了马铃薯块茎特异性表达载体pPSAGB(含phbB基因)、pBIBGC(含phbC基因)和pPSAGCB(含phbB和phbC双基因),转化5个马铃薯品种,经检测获得20个转基因阳性株系。  相似文献   

6.
转基因培育抗除草剂水稻   总被引:14,自引:1,他引:13  
吴爱忠  唐克轩  潘俊松 《遗传学报》2000,27(11):992-998
以pAHC20(含Bar基因)和pWRG1515(含GUS基因和潮霉素抗性基因)以及含Bar基因和雪莲凝集素(GNA)基因的pCAMBIA3300 RG为供体DNA,选用水稻品系87203、上农香糯及鄂宜105的成熟胚诱导出的愈伤组织及微不定芽为受体材料,分别采用基因枪和根癌农杆菌(LBA4404,含pAL4404)导入法进行基因转化;经抗性筛选、GUS检测和PCR分析。结果表明,外源基因已通过基  相似文献   

7.
Ti质粒介导的磷酸烯醇式丙酮酸羧化酶cDNA转化烟草植株   总被引:1,自引:0,他引:1  
将玉米C4-磷酸烯醇式丙酮酸羧化酶(PEP羧化酶)cDNA亚克隆至穿梭质粒pBin19,通过在杆菌Ti质粒(LBA4404)介导的时圆片共培养法将其转入C3植物烟草中。在获得的抗性转化植株中,80%具有较强的NPTⅡ报道基因表达。Southern杂交表明C4-PEP羧化酶cDNA已被整合到了烟草核基因组中。  相似文献   

8.
绿色荧光蛋白基因在青蒿转基因芽中的表达   总被引:5,自引:1,他引:4  
将改良的绿色荧光蛋白(GFP)基因,插入到植物表达载体中,构建双CaMV35S启动子驱动下的植物表达载体pBIGFP,在Kam浓度为20mg/L的筛选培养基上,用含有pBIFP质粒的根癌农杆菌LBA4404感染青蒿叶片,获得5个抗Kan阳性丛生芽系。Southern blotting分析表明,外源GFP基因已整合到青蒿转基因芽G-1系的基因组中。在OLYMPUS-BH2型荧光显微镜下,观察到转基因  相似文献   

9.
嗜麦芽假单胞菌酪氨酸酶基因在大肠杆菌中的克隆与表达   总被引:6,自引:0,他引:6  
王戈林  沈萍 《遗传学报》1999,26(3):274-279
酪氨酸酶基因(mel)编码的酪氨酸酶是合成黑色素的关键酶。用鸟枪法分离嗜麦芽假单胞菌的mel基因:以pUC18为载体,E.coli HB101为受体菌,在加有一定量的Amp和L-tyr的酪素平板上筛选到分泌可溶性黑色素的转化子,所含重组质粒pWSY约700bp的外源DNA片段上携有mel基因,该片段无BamHI、HindIII、EcoRI、BclI等酶的识别位点。Southern杂交证实此片段确实  相似文献   

10.
枯草杆菌启动子—信号肽序列的克隆及序列分析   总被引:1,自引:1,他引:0  
利用含红霉素抗生基因和缺启动子-信号肽序列的氨苄青霉素抗性基因的双功能质粒pGPB14为探针载体,克隆了枯草杆菌的启动子-信号肽序列并对克隆的片段进行序列分析。枯草杆菌染色体DNA经Sau3A酶解后与BanHI酶切的质粒pGPB14连接,转化大肠杆菌C600,筛选抗氨苄青霉素及抗红霉素的转化子,从双抗性转化子中提取重组质粒并经酶切分析,显示克隆的DNA片段在0.27-1.5kb之间。用Sanger  相似文献   

11.
A gene coding for a xylanase activity of alkalophilic Aeromonas sp. no. 212 (ATCC 31085) was cloned in Escherichia coli HB101 with pBR322. Plasmid pAX1 was isolated from transformants producing xylanase, and the xylanase gene was located in a 6.0 kb Hind III fragment. The pAX1-encoded xylanase activity in E. coli HB101 was about 80 times higher than that of xylanase L in alkalophilic Aeromonas sp. no. 212. About 40% of the enzyme activity was observed in the periplasmic space of E. coli HB101. The pAX1-encoded xylanase had the same enzymic properties as those of xylanase L produced by alkalophilic Aeromonas sp. no. 212, but its molecular weight was lower (135 000 vs 145 000, as estimated by SDS polyacrylamide gel electrophoresis).  相似文献   

12.
We wish to report the initial characterization of a recombinant clone containing the BamHI methylase gene. Genomic chromosomal DNA purified from Bacillus amyloliquefaciens was partially cleaved with HindIII, fractionated by size, and cloned into pSP64. Plasmid DNA from this library was challenged with BamHI endonuclease and transformed into Escherichia coli HB101. A recombinant plasmid pBamM6.5 and a subclone pBamM2.5 were shown to contain the BamHI methylase gene based on three independent observations. Both plasmids were found to be resistant to BamHI endonuclease cleavage, and chromosomal DNA isolated from E. coli HB101 cells harboring either of the plasmids pBamM6.5 or pBamM2.5 was resistant to cleavage by BamHI endonuclease. In addition, DNA isolated from lambda phage passaged through E. coli HB101 containing either plasmid was also resistant to BamHI cleavage. Expression of the BamHI methylase gene is dependent on orientation in pSP64. In these clones preliminary evidence indicates that methylase gene expression may be under the direction of the plasmid encoded LacZ promoter.  相似文献   

13.
We constructed a new type of cloning vector, pERISH2, that transforms Escherichia coli HB101 only when a foreign DNA fragment is ligated into the cloning site of the plasmid vector. Plasmid pERISH2 carries the rcsB gene which is derived from the chromosome of E. coli HB101 and is involved in the regulation of colanic acid production. When E. coli HB101 is transformed by this vector carrying the intact rcsB gene, the gene product RcsB blocks bacterial growth. However, if the rcsB gene is inactivated by the insertion of a foreign DNA fragment, this recombinant plasmid no longer inhibits the growth of E. coli HB101. Although E. coli HB101 is not stably transformed by pERISH2, E. coli K-12 strains such as JM109 and C600 can harbor this vector. Therefore, pERISH2 can be amplified in JM109 and be prepared from this strain in a large quantity using conventional methods. A chromosomal gene library of Klebsiella pneumoniae is constructed easily and efficiently by the utilization of this new cloning vector.  相似文献   

14.
Pseudomonas cepacia 4G9 utilizes 2-tridecanone as its sole carbon source and has been shown to be resistant to a variety of antibiotics. To ascertain whether any of these characteristics were plasmid mediated, Escherichia coli HB101 was transformed with plasmid DNA isolated from Pseudomonas cepacia 4G9. No 2-tridecanone-utilizing transformants were obtained. Tetracycline (Tc)- and ampicillin (Ap)- resistant transformants were obtained at a low frequency. Plasmid deoxyribonucleic acid from antibiotic-resistant E. coli HB101 transformants had molecular weights of 2.9 x 10(6) for pJW2 Tcr and 5.4 x 10(6) for pJW3 Apr as determined by electron microscopy. Electron microscopy of plasmid deoxyribonucleic acid from P. cepacia 4G9 revealed a single plasmid species, pJW1 of 1.78 x 10(6). Tetracycline resistance in both P. cepacia 4G9 and E. coli HB101(pJW2) was inducible, whereas ampicillin resistance in P. cepacia 4G9 was constitutive. The level of ampicillin resistance coded by pJW3 was lower in P. cepacia 4G9 than in the transformant E. coli HB101(pJW3).  相似文献   

15.
将苏云金芽胞杆菌以色列亚种的杀蚊晶体蛋白基因cry11A亚克隆到大肠杆菌-蓝藻的穿梭质粒载体pRL25C,然后用三亲本杂交的方法将重组质粒转移到一种具有固氮能力且可被蚊幼虫吞食的鱼腥藻(Anabaena)PCC7120中。Southernblot及Westernblot分析表明cry11A基因在鱼腥藻PCC7120中得以克隆和表达,但生物测定未能检测到转基因鱼腥藻对库蚊(Culex)的毒性,可能是因为带有苏云金芽胞杆菌自身启动子的Cry11A基因在鱼腥藻PCC7120中表达量不够高的缘故。  相似文献   

16.
The recombinant shuttle vector pSV2gpt was introduced into V79 Chinese hamster cells, and stable transformants expressing the Escherichia coli gpt gene were selected. Two transformants carrying tandem duplications of the plasmid at a single site were identified and fused to simian COS-1 cells. Plasmid DNA recovered from the heterokaryons was used to transform a Gpt- derivative of E. coli HB101, and the relative frequency of plasmids carrying a mutation in the gpt gene was determined. The high frequency of Gpt- plasmids (ca. 1%) was similar to that observed when plasmid was recovered from COS-1 cells which had been transfected with pSV2gpt. Most of the mutant plasmids had rearrangements in the region containing the gpt gene.  相似文献   

17.
苎麻CCoAOMT基因cDNA反义转化模式烟草'WS38'   总被引:1,自引:0,他引:1  
苎麻咖啡酰辅酶A氧甲基转移酶(CCoAOMT)是其木质素合成过程的一种关键酶,运用克隆的该酶基因cDNA及植物表达载体pBI121、pWM101,分别构建了35S启动子控制的苎麻CCoAOMT基因反义cDNA基因质粒(pBI121-antiBnCCoAOMT)和cDNA全长表达质粒(pWM101-BnCCoAOMT),并通过根癌农杆菌介导法将其转化至模式烟草WS38,获得了转基因烟草.对转基因植株进行分子分析和组织学初步研究表明,转反义RNA基因植株叶柄木质素含量较野生烟草或转正义基因烟草叶柄木质素含量降低.说明运用反义RNA技术对CCoAOMT基因的表达进行基因工程调控,一定程度上可以对木质素的合成产生干扰,为获得低木质素或木质素组分改良的苎麻基因工程奠定基础.  相似文献   

18.
A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 micrograms of chloramphenicol per ml in both E. coli and C. perfringens. Following shuttle vector construction in E. coli, plasmid pAK201 was transformed into E. coli HB101 and C. perfringens ATCC 3624A, using intact cell electroporation. The transformation frequencies were 10(6) and 10(4) transformants per microgram of DNA in E. coli and C. perfringens, respectively. Restriction enzyme analysis of the chimera isolated from transformants of both microorganisms suggested that the plasmids were identical. Reciprocal transformation experiments in E. coli and C. perfringens indicated no difference in transformation frequency. Plasmid pAK201 was stable in C. perfringens following repeated transfer in the absence of chloramphenicol pressure. The restriction map of plasmid pAK201 shows six unique cut sites which should be useful for future genetic analysis and C. perfringens gene library construction.  相似文献   

19.
A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 micrograms of chloramphenicol per ml in both E. coli and C. perfringens. Following shuttle vector construction in E. coli, plasmid pAK201 was transformed into E. coli HB101 and C. perfringens ATCC 3624A, using intact cell electroporation. The transformation frequencies were 10(6) and 10(4) transformants per microgram of DNA in E. coli and C. perfringens, respectively. Restriction enzyme analysis of the chimera isolated from transformants of both microorganisms suggested that the plasmids were identical. Reciprocal transformation experiments in E. coli and C. perfringens indicated no difference in transformation frequency. Plasmid pAK201 was stable in C. perfringens following repeated transfer in the absence of chloramphenicol pressure. The restriction map of plasmid pAK201 shows six unique cut sites which should be useful for future genetic analysis and C. perfringens gene library construction.  相似文献   

20.
A 4.84-kilobase-pair plasmid was isolated from Proteus vulgaris (ATCC 13315) and cloned into the plasmid vector pBR322. Plasmid pBR322 contains substrate sites for the restriction endonucleases PvuI and PvuII. The recombinant plasmids were resistant to in vitro cleavage by PvuII but not PvuI endonuclease and were found to cause production of PvuII endonuclease or methylase activity or both in Escherichia coli HB101. The approximate endonuclease and methylase gene boundaries were determined through subcloning, Bal 31 resection, insertional inactivation, DNA-dependent translation, and partial DNA sequencing. The two genes are adjacent and appear to be divergently transcribed. Most E. coli strains tested were poorly transformed by the recombinant plasmids, and this was shown by subcloning and insertional inactivation to be due to the PvuII methylase gene. At a low frequency, stable methylase-producing transformants of a methylase-sensitive strain were obtained, and efficiently transformed cell mutants were isolated from them.  相似文献   

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