突变靶基因xylE转基因小鼠

本研究以ｐＥＳｎｘ穿梭质粒为载体,选用恶臭假单孢杆菌ＴＯＬ质粒上的ｘｙｌＥ基因作为突变靶基因组构建的重组构件,经微注射导入ＩＣＲ小鼠５４９枚受精卵雄原核,将存活的３５２枚卵移入２４只假孕鼠输卵管内,７只妊娠,共产仔４１只,其中７只死胎,出生后死亡４只,３０只存活。注射卵存活率为６４％（３５２／５４９）,出生率为１１．６％（４１／３５２）。存活鼠经ＰＣＲ－Ｓｏｕｔｈｅｒｎ检测,整合率为５７％（１７／３０）。在整合阳性鼠中选择了杂交信号较强的２只当代公鼠,通过回收载体和转化试验,结果表明整合基因完整,具有转化的功能,由此,建立了突变靶基因ｘｙｌＥ转基因小鼠模型。     

嗜热脂肪芽孢杆菌CU21中有关质粒pFDX163的整合和游离状态的一种新的相转变现象

pFDX1是带有外源基因xyIE的重组质粒。从嗜热脂肪芽孢杆菌CU21(pFDX1)出发,提高培养温度后选择卡那霉素抗性突变菌落,得到一个变异株CU21-163。该菌株含有突变质粒pFDX163,它由一个来自宿主基因组的2.0kb的H片段插入pFDX1而构成。pFDX163可通过H片段的同源重组而整合到染色体上。CU21-163包含y、w两类细胞,二者的xyIE基因表达量有明显差异。这两类细胞在分裂过程中呈现一种新的相转变现象,即y细胞的后代中经常出现少数w细胞,w细胞的后代中经常出现少数y细胞。对CU21-163的不同细胞群体的总DNA样品中游离质粒与整合质粒的含量进行了测定,由此推断y细胞含有游离质粒和整合质粒,w细胞只含整合质粒。     

xylE基因用于监测根瘤菌在土壤中存活的研究

通过细菌接合将质粒ｐＬＶ１０１６（含ｘｖｌＥ基因）转移至ＲｈｉｚｏｂｉｕｍｆｒｅｄｉｉＱＢ１１３０和Ｒｈｉｚｏｂｉｕｍｌｅｇｕｍｉｎｏｓａｒｕｍｂｖ．ｖｉｃｉａｅＢ４０,ｘｙｌＥ基因在根瘤菌内表达活性较高．质粒ｐＬＶ１０１６携带的ＸｙｌＥ基因用于监测根瘤菌在灭菌和未灭菌土壤中的存活,结果表明,在灭菌土壤中含质粒与不含质粒菌株存活菌数量之间无显著差异（Ｐ＜０．０５）,大接种量有利于细菌的生长与繁殖,Ｂ４０（ｐＬＶ１０１６）质粒丢失比例很低．以低于１０６ＣＦＵ·ｇ－１干土浓度接种时,ＱＢ１１３０（ｐＬＶ１０１６）质粒丢失率随接种浓度的降低而增大．未灭菌土壤中生物因素抑制了释放菌株的生长,大接种量有利于细菌存活．以低浓度（１０６ＣＦＵ·ｇ－１干土）接种时,ＱＢ１１３０（ｐＬＶ１０１６）质粒丢失比例高于Ｂ４０（ｐＬＶ１０１６）．     

邻苯二酚2,3—双加氧酶在大肠杆菌的表达与定域

本文在大肠杆菌/枮草芽孢杆菌间的穿梭质粒pTG 402的基础上构建了几个新的带有显色标志基闲xylE的表达质粒,摸索了该基因所编码的邻苯二酚2,3-双加氧酶(CatO_2ase)的表达条件,分析了该酶一级结构与二级结构的亲水性和疏水性,测定了它在大肠杆菌中的产量与分布。结果表明,CatO_2ase与各质粒的表达量不等,表达量高低与培养时间、宿主菌及诱导与否等影响因素有关;表达后有部分酶可在胞外测出,但大部分仍定域于膜内,亲、疏水性分析示该酶不具分泌性蛋白的显著特点。因该酶易于检测和定量,可作为一种选择性标记和监测指示系统在基因工程中推广应用,同时亦为用基因工程菌消除芳烃类化合物的污染提供了理论依据。     

Rapid method for the detection of genetically engineered microorganisms by polymerase chain reaction from soil and sediments

A rapid and sensitive method for the detection of genetically engineered microorganisms in soil and sediments has been devised by in vitro amplification of the target DNAs by a polymerase chain reaction. A cloned catechol 2,3-dioxygenase gene located on the recombinant plasmid pOH101 was transferred to Pseudomonas putida MMB2442 by triparental crossing and used as a target organism. For the polymerase chain reaction from soil and sediment samples, the template DNA was released from a 100-mg soil sample. Bacterial seeded soil samples were washed with Tris-EDTA buffer (pH 8.0) and treated with a detergent lysis solution at 100°C. After addition of 1% polyvinylpolypyrrolidine solution, the samples were boiled for 5 min. Supernatant containing nucleic acid was purified with a PCR purification kit. The purified DNA was subjected to polymerase chain reaction, using two specific primers designed for the amplification of catechol 2,3-dioxygenase gene sequences. The detection limit was 10² cells per gram of soil. This method is rapid and obviates the need for lengthy DNA purification from soil samples. Received 28 February 1997/ Accepted in revised form 23 November 1997     

Detection and quantification of degradative genes in soils contaminated by toluene

Abstract: A method based on the polymerase chain reaction (PCR) was developed for a rapid and specific detection of toluene degradative genes in soil. The xylE gene coding for catechol 2,3-dioxygenase was chosen as a target gene. The detection threshold was evaluated in microcosms using a sterilized standard soil inoculated with various amounts of a degradative strain of Pseudomonas putida (mX). The extracted DNA was used as a template to amplify the xylE gene. PCR followed by hybridization with an internal probe allowed us to detect 10² bacteria per g of soil. In polluted soils, quantification of target DNA by competitive PCR was compared with enumeration of degradative microflora. This molecular method appeared to be rapid, sensitive and more suitable than the microbiological approach to estimate the biodegradative potential of a polluted soil.     

变铅青链霉菌TK24 secE启动子表达特性的研究

通过PCR扩增得到变铅青链霉菌（Streptomyces lividans)TK24 secE基因上游496bp的片段,其序列与S.coelicolor secE启动子序列同源性为99.8%。将该序列克隆到以儿茶酚加氧酶基因（xylE)为报告基因的链霉菌启动子探测质粒pIJ4083上,并转化S.lividans TK24原生质体,获得了重组菌株S.lividans [pIJ4083-secE]。S.lividans[pIJ4083-secE]菌株发酵结果表明,secE启动子为强启动子,活性与vsi基因启动子相当。secE启动子的表达在对数生长期达到高峰,平台期下降;28℃发酵培养时secE启动子活性远高于37℃发酵培养;比较了不同发酵培养基Phage,NB和CM中secE启动子的活性;实验结果还表明培养基中葡萄糖含量对secE启动子的表达有抑制作用。     

xyLE基因在嗜热脂肪芽孢杆菌中的表达

以嗜热脂肪芽孢杆菌CU21的表达型质粒pFDC11为载体,插入来自恶臭假单孢菌的编码邻苯二酚2,3-双加氧酶(CatO_2ase)的xylE基因,构建成重组质粒pFDX1转化入CU21受体,在48℃培养时得到了表达产物,表明常温细菌的xylE基因可以在高温菌启动子的带动下在高温菌宿主中表达。采用提高培养温度后选择卡那毒素抗性突变的方法,得到了在55℃及6O℃的CatO_2ase表达量明显提高的变异菌株CU21-161。本文同时报道一种用完整细胞悬浮液测定CatO_2ase活力的方法。     

Three types of phenol and p-cresol catabolism in phenol- and p-cresol-degrading bacteria isolated from river water continuously polluted with phenolic compounds

A total of 39 phenol- and p-cresol-degraders isolated from the river water continuously polluted with phenolic compounds of oil shale leachate were studied. Species identification by BIOLOG GN analysis revealed 21 strains of Pseudomonas fluorescens (4, 8 and 9 of biotypes A, C and G, respectively), 12 of Pseudomonas mendocina, four of Pseudomonas putida biotype A1, one of Pseudomonas corrugata and one of Acinetobacter genospecies 15. Computer-assisted analysis of rep-PCR fingerprints clustered the strains into groups with good concordance with the BIOLOG GN data. Three main catabolic types of degradation of phenol and p-cresol were revealed. Type I, or meta-meta type (15 strains), was characterized by meta cleavage of catechol by catechol 2,3-dioxygenase (C23O) during the growth on phenol and p-cresol. These strains carried C23O genes which gave PCR products with specific xylE-gene primers. Type II, or ortho-ortho type (13 strains), was characterized by the degradation of phenol through ortho fission of catechol by catechol 1,2-dioxygenase (C12O) and p-cresol via ortho cleavage of protocatechuic acid by protocatechuate 3,4-dioxygenase (PC34O). These strains carried phenol monooxygenase gene which gave PCR products with pheA-gene primers. Type III, or meta-ortho type (11 strains), was characterized by the degradation of phenol by C23O and p-cresol via the protocatechuate ortho pathway by the induction of PC34O and this carried C23O genes which gave PCR products with C23O-gene primers, but not with specific xylE-gene primers. In type III strains phenol also induced the p-cresol protocatechuate pathway, as revealed by the induction of p-cresol methylhydroxylase. These results demonstrate multiplicity of catabolic types of degradation of phenol and p-cresol and the existence of characteristic assemblages of species and specific genotypes among the strains isolated from the polluted river water.