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1.
用成团肠杆菌(Emterobacter agglomerans)T7(pEA9::Km)为供体菌,以E.a.339(pEA9^-)为受体菌在天然土壤中进行接合实验,测定各种营养物质对重组质粒pEA9::Km同源接合转移频率的影响。实验发现,如果在土壤中加入生理盐水或无机盐培养基,就观察不到细菌数量的增加和质粒的转移;只有加入碳源物质后才检测到细菌数量的增加和质粒的接合转移,接合转移频率的大小位于1  相似文献   

2.
抗砷载体的构建及在氧化亚铁硫杆菌中的表达   总被引:5,自引:2,他引:5  
利用DNA体外重组技术,将抗砷质粒pUM3经HindⅢ酶切后得到的4.3kb抗砷片段克隆到有广泛寄主的IncQ质粒pJRD215的HindⅢ位点上,构建了一个新的抗砷质粒pSDX3。砷抗性研究表明,在大肠杆菌中,pSDX3的抗砷水平与pUM3相近。将pSDX3通过接合的方式引入氧化亚铁硫杆菌Tf-59中并得到了表达,与对照相比,含质粒pSDX3的Tf-59抗砷水平有了很大的提高。  相似文献   

3.
将从自杀性重组质粒pDB30上分离的3.6kb含卡那霉素抗性基因和发光酶基因(luxAB)的BglⅡ酶切片段,克隆到小Mr的高拷贝质粒pIJ2925上,构成新的质粒,pHNLX1011;然后将pHNLX1011经BglⅡ部分酶切,酶连在含蔗糖酶基因(sacB)的自杀性重组质粒载体pMH1701上,载体分别采用BglⅡ酶切和BamHⅠ酶切.连接转化后共得到4种重组质粒类型的菌株,它们具有不同发光活性;将它们分别与快生型大豆根瘤菌HN01进行三亲本杂交,发现它们的转座频率均较pDB30高.对经转座标记后的根瘤菌进行发光活性检测,只有pHNC3质粒所转座的根瘤菌浇具有较强发光活性.所得发光根瘤菌衍生菌株能在野大豆和栽培大豆黑农39根际形成正常根瘤,能用X光片记录下根瘤的发光活性.  相似文献   

4.
分别在北京和江苏省连云港市对携带Btcry基因的荧光假单胞菌工程菌进行了田间残留与扩散的追踪检测 .对越冬前后的试验地和保护地土壤样品进行抗生素抗性平板分离 ,能检测到极少量的具有与出发菌株相同抗性的荧光假单胞菌菌落 ,但没有发现工程菌株的残留与扩散 .对江苏试验地样品还进行了工程菌株质粒卡那霉素和壮观霉素抗性标记基因的抗性菌落分离 ,绝大多数样品中都能分离到包括荧光假单胞菌在内的抗性菌落 ,土样中菌密度n(cfu) =10 4 ~ 10 5g-1,但进行Btcry基因PCR -RFLP检测时没有从样品中得到特异扩增产物 .研究结果表明 :工程菌株抗性标记基因在自然界广泛存在 ,工程菌在株环境中没有残留和扩散 ,具有良好的生物安全性 .表 4参 8  相似文献   

5.
苏云金芽孢杆菌无质粒突变株BMB171的转化和表达性能   总被引:4,自引:1,他引:3  
研究了苏云金芽孢杆菌无质粒突变株BMB171的转化性能和表达cry1Ac和cry1Ca基因的性能.用pHT3101、pBMB305、pBMB1736、pBMB671、pBTL1和pHV1249等6种外源质粒电转化无质粒突变株BMB171,其转化频率分别是Bt4Q7、Bt4D10和Bti.IPS.78/11等3种常用受体菌相应最高转化频率的1000、6.7、12.5、66.7、3500和2倍,其每μgDNA的最高转化子数达107.导入BMB171的外源质粒的稳定性与其复制子类型和质粒大小有关.无质粒突变株BMB171表达cry1Ac基因的表达量高于对照受体菌Bt4Q7,略低于Bt4D10和Bti.IPS.78/11,而BMB171表达cry1Ca基因的表达量高于这3种受体菌;对小菜蛾3龄幼虫和甜菜夜蛾初孵幼虫的毒力测定结果表明,BMB171表达cry1Ac基因的杀虫毒力高于Bt4Q7和Bt4D10,略低于Bti.IPS.78/1;而表达cry1Ca基因的毒力高于这3种受体菌  相似文献   

6.
苏云金芽孢 力无质粒突变株BMB171的转化和表达性能   总被引:7,自引:0,他引:7  
研究了苏云金芽孢杆菌无质粒突变株BMB171的转化性能和表达cry1Ac和cry1Ca基因的性能,用pHT3101、pBMB3305、pBMB1736、pBMB671、pBTL-1和pHV1249等6种外源质粒电转化无质粒突变株BMB171,其转化频率分别是Bt-4Q7、Bt-4D10和Bti.78/11等3种常用体菌相应最高转化频率的1000、6.7、12.5、66.7、3500和2倍,其每μg  相似文献   

7.
邻苯二酚1,2-双加氧酶(C12O)基因的定位、克隆和表达   总被引:3,自引:0,他引:3  
射性同位素标记tfdC基因片段作探针,Southernblot杂交定位L1菌株的邻苯二酚1,2双加氧酶基因位于PstⅠ的I片段和BamHⅠ的M和N片段.低熔点琼脂糖回收PstⅠ的I段,直接克隆至表达载体pKT230上,获得重组子pKT230p.重组子转化不具开环酶活性的甲胺磷降解菌P2,获得高效的C12O酶活  相似文献   

8.
用分离获得的嗜热厌氧纤维素降解细菌 Clostridium sp . E V A4 菌株进行了直接转化纤维素产生乙醇的动力学、发酵最适条件及其影响因子的研究.结果表明, E V A4 菌株在p H6 .2 ~8 .9 ,θ45 ℃~60 ℃的范围内能直接转化纤维素滤纸产生乙醇,最适p H 为7 .5 ~8 .0 ,最适θ为55 ~60 ℃. E V A4 菌株能利用纤维素滤纸,纤维素粉 Whatman C F I I,微晶纤维素,纤维素粉 M N300 和未经处理的玉米秆芯,甘蔗渣,水稻秸秆产生乙醇.乙醇浓度 ,纤维素降解率和培养基还原糖浓度均随培养时间延长而增大.不同的纤维素材料、p H、θ、底物浓度、酵母粉含量、振荡、培养气相、外加 O2 和乙醇等条件均能影响 E V A4 菌株转化纤维素为乙醇的能力.最适条件下 E V A4 菌株利用1 % 纤维素滤纸培养120 h 产乙醇浓度为1 123 mg/ L,纤维素降解率为59 %  相似文献   

9.
二亲本杂交实验通常要求受体菌不能携带有与质粒上相同的抗性.本次实验中受体菌铜绿假单胞菌(Pseudo-monas aeruginosa)和重组质粒pHN102都带有四环素(Tc)抗性.考虑到质粒pHN102导入受体菌后至少带有两个Tc抗性基因片段,转移接合子的抗性会增强,所以提高四环素浓度至20μg/mL,并利用pHN102上携带的luxAB发光酶基因标记受体菌筛选转移接合子,并设立对照菌株.通过抽提质粒、琼脂糖凝胶电泳检测,确定pHN102成功转入P.aeruginosa中.转移接合子经4次转接后,质粒仍可以稳定存在且能够稳定表达.P.aeruginosa(pHN102)的发光动力学曲线表明,加入底物20min后,发光强度趋于稳定.经液体培养稀释后进行发光度和活菌数测定,发现二者呈显著的线性关系(r=0.994).图4表2参6  相似文献   

10.
对从玉米茎内分离的18株固氮菌进行了回接分离试验,经多碳源无氮培养基、菌落形态、颜色与REP-PCR相结合的方法,筛选、鉴定出了两株具有较高乙炔还原活性和能在玉米体内定殖的菌株R1和R6。把携带gfp标记基因的质粒pKK223-GFP和nifH-gfp的质粒pMGFP2.1分别转化R1和R6,得到携带gfp,且菌落带绿色荧光的转化子R1A和R6A,以及携带nifH-gfp的转化子R1B和R6B。在限菌条件下对R1A和R6A,在玉米根内的定殖以及R1B和R6B中nifH-gfp融合基因的表达进行了研究,结果表明了R1A和R6A主要定殖在根的皮层细胞、胞间隙、中柱细胞和导管内,有时在活的细胞内也可检测到,R1B和R6B中的融合基因因受玉米根内营养以及其它与固氮酶基因表达相关因素的限制,很难在玉米根内表达,只有在添加了1‰蔗糖后,nifH-gfp才能在根内表达。  相似文献   

11.
从同一植株不同根瘤分离40株紫云英根瘤菌,所有菌株对10种抗生素的抗药性测定表明,该群体分为22个抗药类群,质粒检测显示所有公离株都含有质粒,质粒数1~4条,用快生型大豆根瘤菌USDA205质粒作参考,估测质粒Mr分布范围为83~226MU.根据图谱分析表明,该菌群可分为6个不同质粒型.各类型质粒通过与Dig-nodABC和Dig-nifHDK杂交,结果显示带有1条质粒的菌株其共生基因定位在染色体上.带有2条或2条以上质粒的菌株各拥有1条共生质粒,共生质粒Mr范围有差异,大约为117~220MU.研究结果也显示,不同质粒型的菌株其共生效应存在明显差异,其中第6质粒型的菌株共生固氮率最强,第1质粒型菌株共生固氮率较低.共生固氮能力最强的第6质粒型菌株,只占总菌数7.5%.  相似文献   

12.
pRKZ3 is a non-conjugative IncQ plasmid, while pKANJ7 is a conjugative IncX plasmid. The optimal mating time of pKANJ7 varied under different conditions. Both of the two transferable ARPs had little impact on the growth of their hosts. A relatively high level of fitness cost was observed for pKANJ7. The fitness cost of ARPs depended on their hosts. Plasmid-mediated antibiotic resistance genes (ARGs) have recently become a more prominent concern in the global environment. However, the prevalence of aminoglycoside resistance plasmids in the livestock industry is under reported. In this study, two transferable aminoglycoside resistance plasmids, pRKZ3 and pKANJ7, isolated from pig and chicken manure, were characterized. Results showed that pRKZ3 (8236 bp) is a non-conjugative IncQ plasmid and contains genes encoding for plasmid replication and stabilization (repA, repB and repC), mobilization (mob), and antibiotic resistance (arr-3 and aacA). pKANJ7 (30142 bp) is a conjugative IncX plasmid which codes for a type IV secretion system (T4SS). Conjugative transfer experiments showed that the optimal mating time of pKANJ7 was 8 h under the starvation condition, but the number of tranconjugants increased with time under the nutrient condition. Statistical analysis indicated that the two plasmids had little impact on the growth of their hosts, but a relatively high level of fitness cost due to pKANJ7 was observed. We also found that the fitness cost of plasmids depended on their hosts. Compared with pKANJ7, the relative fitness cost index of pRKZ3 varied within a narrow range during the 10 days of competition. The low level of fitness cost of pRKZ3 might contribute to the persistence of the plasmid in the environment. Our study provides new information for understanding the characterizations of antibiotic resistance plasmids (ARPs) in manure sources and helps to clarify the transfer and persistence of ARPs in the environment following the application of manure.  相似文献   

13.
Transport of engineered antibiotic resistance plasmids in porous media has been reported to potentially cause significant spreading of antibiotic resistance in the environment. In this work, transport of an indigenous resistance plasmid pK5 in porous media was investigated through packed column experiments. At identical ionic strengths in CaCl2 solutions, the breakthroughs of pK5 from soil columns were very close to those from quartz sand columns, indicating that transport of pK5 in quartz sand and soil was similar. A similarity in transport behavior was also found between pK5 and an engineered plasmid pBR322 that has approximately the same number of base pairs as pK5. The influence of surfactants, a major group of constituents in soil solutions, was examined using an engineered plasmid pcDNA3.1(+)/myc-His A. The impact of an anionic surfactant, sodium dodecyl sulfate (SDS), was negligible at concentrations up to 200 mg·L–1. Cetyltrimethyl ammonium bromide (CTAB), a cationic surfactant, was found to significantly enhance plasmid adsorption at high concentrations. However, at environmentally relevant concentrations (<1 mg·L–1), the effect of this surfactant was also minimal. The negligible impact of surfactants and the similarity between the transport of engineered and indigenous plasmids indicate that under environmentally relevant conditions, indigenous plasmids in soil also have the potential to transport over long distances and lead to the spreading of antibiotic resistance.
  相似文献   

14.
以湖北洪湖公离的快生型大豆根瘤菌为材料,考察了它们的内源质粒在培养过程及共生过程中的稳定性.结果表明,所有供试菌株的质粒在培养条件下比较稳定,传代50次后没有明显变异.对其中2个菌株与4种不同的大豆宿主共生过程中质粒的稳定性研究,发现HA12-1-12的共生质粒和隐性质粒高度构定,HA7-2-2的质粒却发生了不同程度的变异.质粒检测的结果及RFLP分析表明,HA7-2-2共生质粒的稳定性高于隐性质粒,而且共生质粒上携带的nodDABC和nifHDK基因相当保守,不易受宿主的影响而产生变异.盆栽试验的结果也说明,虽然HA7-2-2的内源质粒在共生过程中发生了遗传重排,但它的共生性状没有明显的变异.  相似文献   

15.
为促进高GC含量基因在荧光假单胞菌(Pseudomonas fluorescens)中表达效果更加理想、操作更加简便,本研究首先采用不依赖基因序列和连接反应的克隆(Sequence and ligation independent cloning,SLIC)方法将载体pCIBhis上与复制相关的序列和标记基因片段构建成克隆载体pCIBS1.然后优化荧光假单胞菌转化方法,用电转化法将pCIBS1导入荧光假单胞菌BL915中,随后又将T7和tac基因启动子分别插入pCIBS1中,成功构建了表达载体pCIBS3和pCIBS2.研究发现载体pCIBS1在大肠杆菌和荧光假单胞菌中均较为稳定,并且将绿色荧光蛋白基因插入表达载体中,在大肠杆菌BL21(DE3)中获得表达,验证了表达载体功能.本研究构建的表达载体和建立的荧光假单胞菌BL915电转化方法,为高GC含量基因在荧光假单胞菌中的表达奠定了基础.  相似文献   

16.
绿色荧光蛋白基因标记内生枯草芽孢杆菌   总被引:14,自引:0,他引:14  
用枯草芽孢杆菌168菌株rpsD基因的启动子替换质粒pGFP4412中蜡状芽孢杆菌4412启动子,从而构建了能在枯草芽孢杆菌中表达绿色荧光蛋白基因gfpmut3a的载体pS4GFP,将其导入具有内生、防病、促生作用的野生型枯草芽孢杆菌BS-2菌株中,筛选获得遗传稳定性好且具有良好发光表型的标记菌株BS-2-gfp.该标记菌株在小白菜体内的定殖研究结果表明,该菌株能够在小白菜根际及根、茎、叶内定殖和传导,接菌50d后仍能在其体内分离到标记菌株.图5参17  相似文献   

17.
This research work was conducted in Uppanar estuary to ascertain the role of plasmids in the antibiotic resistance of bacteria. Water and sediment samples were collected for a period of three months. When tested against 20 antibiotics 22 MAR strains were isolated from the samples, which were found resistant to 5-13 antibiotics. They belong to 7 genera and 10 species. Gram-negative bacteria namely Neisseria mucosa, N. sicca, Branhamella catarrhalis, Klebsiella ozaenae, Citrobacterintermedius, Pseudomonas fluorescens and Enterobacter aerogenes were isolated. Gram-positive bacteria were of Bacillus subtilis, B. megaterium and Micrococcus luteus. When plasmid curing was done using acredine orange, the resistance against penicillin-G, ampicillin, tetracycline, amoxycillin, kanamycin, and chloramphenicol were totally lost in all strains, which confirmed the role of plasmid in these strains against antibiotics. Ten strains belong to different species were selected for the plasmid isolation and electrophoresis was done. Presence of plasmids in all strains was confirmed and the molecular weight was in the range of 2850 to 3170 bp. The study revealed that MAR strains are common in Uppanar estuary and they are plasmid mediated. This environment is seemed to be deteriorating at an alarming rate.  相似文献   

18.
水环境中耐热大肠菌群的抗生素耐药性与质粒谱研究   总被引:5,自引:0,他引:5  
用滤膜法、mFC培养基从5种水体中分离出疑似耐热大肠菌162株,用API微生物分析系统鉴定到种,以Kirby-Bauer法分析其对人畜常用10种抗生素的耐药性,碱裂解法小量制备各菌株质粒DNA做质粒谱分析.结果表明,埃希氏大肠杆菌为优势菌,占分离菌总数的96.3%.除分离自泉水的3株外,其它菌株都对3种及3种以上抗生素耐药,多重耐药率为98.1%.不同水体分离菌株对氧氟沙星、诺氟沙星、庆大霉素、丁胺卡那霉素、链霉素的耐药率有显著性差异(P<0.005).92株菌(56.8%)提取到大小为0.90~158.83kb、数量为1~6个的质粒,有81种质粒谱型.70株(43.2%)未提取到质粒的细菌中有67株为多重耐药.具有相同质粒谱型的菌株耐药谱都不相同.未发现质粒谱与抗生素耐药性间有明显相关性.图1表3参15  相似文献   

19.
利用双农杆菌/双质粒的共转化系统获得了无选择标记转pepc基因的水稻植株.采用两个农杆菌EHA105转化粳稻品种台粳9号幼胚诱导的胚性愈伤组织,其中一个农杆菌内的质粒表达载体的T-DNA区仅含有目的基因pepc,另一个的T-DNA区含有选择标记基因hpt和GUS报告基因.获得抗性愈伤的转化率为9.2%.85.3%的抗性愈伤分化成T0代株系,其中78.8%的T0代植株为GUS阳性转基因植株.PCR分析表明,在78个T0代GUS阳性植株(来源于23个抗性愈伤组织)中共有35个植株(来源于7个抗性愈伤组织)含有pepc基因,即在23个GUS阳性株系中发生共转化株系的频率为30.4%.7个共转化的株系中有5个株系自交产生后代植株中含有无标记转pepc基因植株,其比例为71.4%.无标记的转pepc基因水稻植株中PEPC活性平均比对照提高8.8倍;与非转基因对照植株相比,转pepc基因水稻植株表现出更强的光合能力.图5表3参26  相似文献   

20.
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