电脉冲穿孔法将苏云金杆菌δ－内毒素基因导人野生型芽孢杆菌

利用电脉冲穿孔法将带有苏云金杆菌毒蛋白基因的穿梭质粒导人几株野生型芽孢杆菌中。它们是野生型的蜡状芽孢杆菌、短芽孢杆菌和枯草芽孢杆菌。通过观察在新霉素和氨苄青霉素平板上长出的抗性菌落数t计算出转化效率为10¹一10¹转化子/μgDNA。从转化子中分离到的质粒DNA大小及其用HindⅢ酶切的片段与原始质粒DNA相同,毒性测试表明重组转化子对烟青虫六天的致死率达90—100％。     

枯草芽孢杆菌感受态细胞的制备及质粒转化方法研究

为便于枯草芽孢杆菌工业化生产应用,对Spizizen创立的枯草芽孢杆菌DNA转化方法进行改进.用GMI和GMII溶液处理枯草芽孢杆菌野生型菌株BS501a、营养缺陷型突变株DBl342和非营养缺陷型突变株WB800,用改进的方法制备感受态细胞,用7.5kb质粒pSBPTQ进行转化,并研究RNA、酵母粉、水解酪蛋白、培养方法对枯草芽孢杆菌质粒转化的影响.结果表明,该方法适用于不同基因型枯草芽孢杆菌的质粒转化,营养缺陷型突变株DBl342的转化率为750 CFU/μg/DNA,非营养缺陷型突变株WB800转化率为1 070 CFU/xg DNA,野生型菌株BS501a转化率为270 CFU/μg/DNA.根据影响转化效率的因素,推测在该方法中,枯草芽孢杆菌质粒转化原理:一定生物量的枯草芽孢杆菌在外界营养条件和钙、镁离子作用下,细胞壁和细胞膜形成缺陷,使外源DNA转入枯草芽孢杆菌细胞内.     

球形芽孢杆菌Mtx1杀蚊毒素在苏云金芽孢杆菌以色列亚种中的表达*

将来源于球形芽孢杆菌SSII 1的mtx1毒素基因克隆至穿梭载体 pBU4上 ,得到mtx1插入方向相反的重组质粒 pMT9和pMT4。含有 pMT9和 pMT4的大肠杆菌转化子能表达产生Mtx1毒素 ,发酵液对敏感和抗性致倦库蚊幼虫具有中度毒杀作用 ;含有pMT9和pMT4的苏云金芽孢杆菌转化子B pMT9和B pMT4在营养体生长阶段对敏感蚊幼和抗性幼虫也具有毒性 ,毒力与野生型SSII 1相当 ,而不同转化子在芽孢形成期的毒力因插入的mtx1基因转录方向不同而表现出差异 ,其中B pM     

环状芽孢杆菌α—羟基—γ—氨丁酰酰化酶基因的克隆和表达

本文报道了以质粒pUB110为载体,以枯草芽孢杆菌(Bacillus subtilis 168)作为受体菌,对丁酰苷菌素产生菌(Bacillus circulans NRRL-B3312)总DNA进行了鸟枪克隆,在所获得的转化子中,No.733转化子经薄层层析,生物显迹和质谱分析表明,它具有将卡那霉素A生物转化成为丁胺卡那霉素的能力,说明该转化子所含重组质粒pUBC733的插入片段中含有a-羟基-r-氨丁酰(HABA)酰化酶基因,HABA酰化酶基因已经在枯草芽孢杆菌中获得了克隆和表达。该重组质粒分子量为7.3kb,插入片段为2.8kb,经Southern分子杂交确证此片段确来源于环状芽孢杆菌,已构建了该质粒限制性内切酶图谱。     

黄地老虎颗粒体病毒DNA片段在枯草芽孢杆菌中的克隆

EcoRI酶解的AsGV-XJ DNA和质粒pUB 110 DNA,经T4 DNA连接酶连接后,转化枯草芽孢杆菌BR 151感受态细胞,涂布在加有新霉素(5,ug／ml)的完全培养基上。用琼脂糖凝胶电泳快速法检测抗新霉素转化子中的重组质粒。在388个转化子中得到12个较PuB110 DNA分子量大的重组质粒。所有重组质粒均可与”PdcTP标记的Fco RI酶解AsGV—XJDNA片段的探针进行分子杂交。通过琼脂糖凝胶电泳对重组质粒中插入AsGV一XJ DNA 片段进行了测定。     

球形芽孢杆菌Mtx1杀蚊毒素在苏云金芽孢杆菌以色列亚种中的表达

将来源于球形芽孢杆菌SSII-1的mtx1毒素基因克隆至穿梭载体pBU4上,得到mtx1插入方向相反的重组质粒pMT9和pMT4.含有pMT9和pMT4的大肠杆菌转化子能表达产生Mtx1毒素,发酵液对敏感和抗性致倦库蚊幼虫具有中度毒杀作用;含有pMT9和pMT4的苏云金芽孢杆菌转化子B-pMT9和B-pMT4在营养体生长阶段对敏感蚊幼和抗性幼虫也具有毒性,毒力与野生型SSII-1相当,而不同转化子在芽孢形成期的毒力因插入的mtx1基因转录方向不同而表现出差异,其中B-pMT4对目标蚊幼毒力极低(LC50＞100mg/mL),而B-pMT9对蚊幼虫具有毒性(LC50=2.49mg/mL).     

地衣芽孢杆菌感受态细胞的形成及高效电转化

芽孢杆菌在营养缺乏的饥饿状态下,细胞易产生感受态因子,处于生长芽孢时期的芽孢杆菌更容易产生感受态。基于此原则利用芽孢杆菌极限营养培养基通过体外处理诱导使地衣芽孢杆菌产生感受态性能,同时调整参数,建立了感受态细胞对质粒pAPR的高效电转化方法。当质粒DNA浓度为1．5μg/ml、转化时电压为1750V的时候,可以得到261个转化子,经鉴定均为阳性克隆子。而常规电转化的最高仅为20个转化子。为以芽孢杆菌为宿主进行高效电转化提供了模型,也为建立适合工业应用的分泌型表达载体的构建打下了一定基础。     

地衣芽孢杆菌原生质体电转化方法的研究

为了解决地衣芽孢杆菌(Bacillus licheniformis)工业菌株难以转化的问题,将原生质体制备、电穿孔和原生质体再生技术相结合,建立了一种地衣芽孢杆菌原生质体电击转化方法。在对菌体生长状态、溶菌酶作用时间、电转电压、渗透压保护剂等条件进行优化后,试验了将不同类型的表达载体,即游离型质粒p GJ103(3.3kb)和整合型质粒p AX01(9.3kb),分别转入两株地衣芽孢杆菌工业生产菌株B.licheniformis CICC 10181和B.licheniformis CICC 20204中。实验结果显示,对数生长期后期的菌体酶解40min后制备的地衣芽孢杆菌原生质体得率为96%,再生率达25%以上。原生质体与质粒DNA在最适电压0.6k V/mm下电击转化,并以0.5mol/L山梨醇或甘露醇作为渗透压保护剂进行再生培养后,最终游离型质粒的转化率可达0.88×102~1.1×102CFU/μg p GJ103,整合型质粒的转化率达到0.45×102~0.52×102CFU/μg p AX01。该方法为地衣芽孢杆菌野生工业菌株的遗传改造提供了一种新的、高效的转化手段。     

嗜热脂肪芽孢杆菌质粒的分离

从堆肥和污泥中分离到一批抗药性高温细菌,经电泳检查,发现6株高温细菌细胞中有质粒存在。其中,嗜热脂肪芽孢杆菌T653的细胞DNA提取液电泳图谱上,有三条非染色体DNA条带,用电镜直接观察,证明它们是T653细胞中的三个质粒。测得两个较小质粒的分子量分别为3.6×10~6和45×10~6道尔顿。研究了嗜热脂肪芽孢杆的T653的温度生长条件与其细胞中质粒的关系。T653细胞中三个质粒的明确功能有待进一步探讨。     

多粘芽孢杆菌质粒的研究——Ⅱ.多粘芽孢杆菌原生质体形成、再生与转化

多粘芽孢杆菌P250-2完整菌体不能作为质粒DNA的转化受体,但其质粒消除菌株P_0250-5制成的原生质体,可接受多粘芽孢杆菌的pBD2502及枯草杆菌的pUB110质粒DNA转化。在高渗蔗糖再生培养基上,原生质体再生率为20％左右,形成率在95％以上。在含新霉素(10μg/ml)、青霉素(25μg/ml)、四环素(12.5μg/ml)的高渗蔗糖再生培养基上分别获得了转化子。多粘芽孢杆菌的转化频率为3.29×10~(-3),枯草杆菌为4.4×10~(-4)。转化子的形态表现、生化特性和抗菌谱与给体菌株一致,表明多粘芽孢杆菌株间及多粘芽孢杆菌和枯草杆菌种间的质粒可以进行转化。     

Electroporation: application to human lymphoid cell lines for stable introduction of a transactivator gene of human T-cell leukemia virus type I.

Conditions were developed for stable introduction of foreign DNA into human lymphoid cell lines by electroporation. To introduce stably the p40 gene of human T-cell leukemia virus type I (HTLV-I) into the human lymphoid cell line Jurkat, the p40 expressing plasmid, pMAXRHneo-1, which carries the neo resistant gene, was transfected into Jurkat cells at a voltage of 2500 V and capacitance of 21.7 microF, and stable transformants were screened for neo (G418) resistance. The frequency of transformants was more than one per 2 x 10(5) cells used initially. Clones that were resistant to G418 were shown to have the p40 gene integrated into the host genome and to express mRNA and protein from the introduced plasmid. Expression of p40 in the transformed Jurkat cells was also confirmed by testing the trans-activating effect of HTLV-I enhancer by p40. High frequencies of stable transformations of 10(-4) to 10(-6) were also reproducibly obtained by electroporation of the human T cell lines HSB-2 and TALL-1, a human B cell line Raji, a human monocytic cell line U937, and a human erythroleukemia cell line K562. These results demonstrate that electroporation is a very efficient method for introducing foreign DNA into human lymphoid cell lines.     

'Integron'-bearing vectors: a method suitable for stable chromosomal integration in highly restrictive corynebacteria.

A pBR322-derived plasmid (pCGL107) that carries the Corynebacterium melassecola ATCC17965 analogue of Escherichia coli gdhA gene (encoding glutamate dehydrogenase), was introduced into the related strain, Brevibacterium lactofermentum CGL2002, by electroporation and integrated into its chromosome by homologous recombination. However, pCGL107 cannot integrate into C. melassecola, since the host restriction prevents successful electroporation by E. coli-modified DNA. Nevertheless, B. lactofermentum-modified replicative plasmid DNA can be transformed by electroporation into C. melassecola; thus pCGL519-2, a shuttle plasmid that carries the C. melassecola analogue of E. coli gltA (encoding citrate synthase), was extracted from the former host and electroporated into the latter. Rare restriction sites conveniently placed in pCGL519-2 were used to recover a replicon-less cartridge called 'integron', that contains a selectable marker and gtlA within a single fragment. Integron prepared from pCGL519-2 DNA which had been extracted from C. melassecola, and thus, was capable of eluding the C. melassecola restriction barrier(s), was successfully electroporated into this host. The molecular analysis of the resulting transformants suggests that they result from the integration of a single circular integron molecule by homologous recombination between the gltA regions of the host genome and the integron. These transformants were stable for 30 generations in the absence of selection.     

双功能枯草杆菌诱导型高效表达分泌载体的构建与鉴定

利用大肠杆菌质粒pSP72和枯草杆菌质粒pUB18共整合得到双功能克隆载体pSB。在pSB多克隆位点依次引入枯草杆菌果聚糖蔗糖酶基因启动子-信号肽序列sacBp.s.、地衣芽孢杆菌淀粉酶基因终止子序列α-amyT和短小芽孢杆菌增强子基因degQ,最终构建了双功能枯草杆菌诱导型高效表达分泌载体pSBPTQ。将VasostatinⅠ基因作为靶基因检测sacBp.s.、α-amyT和degQ在pSBPTQ进行外源基因表达时的功能,结果表明,在蔗糖诱导下,sacB启动子有效启动了Vasostatin I基因的表达和分泌,α-amy T提高了VasostatinⅠ基因的转录效率,而degQ明显增强了VasostatinⅠ基因的表达水平。VasostatinⅠ基因在蔗糖诱导下成功表达并分泌到枯草杆菌细胞外,蛋白质分泌效率达到90%左右。质粒稳定性试验结果表明,经过40个世代之后,质粒pSBPTQ在枯草杆菌DB1342中仍旧保持在83%以上。     

粟长蠕孢菌的原生质体转化

本文报道了利用具有潮霉素抗性标记的质粒(pAN7-1)对粟长蠕孢菌原生质体进行转化的结果。经pAN7-1质粒DNA转化处理的粟长蠕孢菌原生质体在含潮霉素(200μg/ml)的选择性培养基上出现两类转化子。一类是正常转化子,其转化率为2个转化子/μg DNA;另一类是流产转化子,其产生频率为500—600个转化子/μg DNA。DNA杂交分析结果表明,在正常转化子中质粒DNA以首尾相接、重复排列的形式整合入受体菌染色体DNA。初筛获得的转化子多数以异核状态存在,经单孢分离纯化后可通过有丝分裂稳定传代。     

Improved method for electroporation of Staphylococcus aureus

We have developed a significantly improved method for the electroporation of plasmid DNA into Staphylococcus aureus. The highest transformation efficiency achieved with this procedure was 4.0 x 10(8) transformants per microgram of plasmid pSK265 DNA. This represents a 530-fold improvement over the previously reported optimum efficiency of 7.5 x 10(5) transformants per microgram of plasmid DNA after electroporation of S. aureus cells [9]. Identical results were obtained when electrocompetent cells, which had been stored frozen at -80 degrees C, were used. The improved efficiency is due primarily to the use of a modified medium (designated as B2 medium) and secondarily to the use of 0.1-cm cuvettes. Several other plasmids (pI258, pMH109, and pSK270) were also electrotransformed into competent cells using our procedure, and for each plasmid, the transformation efficiency was significantly reduced compared to that observed when pSK265 DNA was used. With respect to plasmid pI258, the transformation efficiency was 3500-fold higher than that reported previously for transformation of this plasmid into S. aureus RN4220 [9]. The optimized electroporation procedure was less successful in transforming other staphylococci. Electrocompetent cells of S. aureus ATCC 29213 and S. epidermidis ATCC 12228 produced 5.5 x 10(5) and 5 x 10(3) transformants per microgram of pSK265 DNA, respectively.     

生孢噬纤维粘菌基因文库的构建和纤维素酶基因的克隆

将生孢噬纤维粘菌（ＳｐｏｒｏｃｙｔｏｐｈａｇａＢ２９）染色体用ＰｓｔＩ部分酶切后,连接到大肠杆菌（Ｅ．ｃｏｌｉ）质粒载体ｐＵＣ８上,然后转化Ｅ．ｃｏｌｉＪＭ８３,从而建立了Ｂ２９的基因文库,并筛选一个含有内切葡聚糖纤维素酶（ＣＭＣａｓｅ）的阳性克隆．从此阳性克隆中提取质粒再转化ＪＭ８３,发现所有的氨苄青霉素抗性（Ａｐｒ）转化子都具有ＣＭＣａｓｅ酶活性,证明在大肠杆菌中克隆到一个Ｂ２９的内切葡聚糖酶基因．     

Use of electroporation to construct isogenic mutants of Haemophilus ducreyi.

Little is known about the genetics of Haemophilus ducreyi, the etiologic agent of chancroid. To develop a method for constructing isogenic mutants of this organism that could be utilized in pathogenesis-related studies, electroporation techniques were evaluated as a means of introducing DNA into this organism. Electroporation of the plasmid shuttle vector pLS88 into H. ducreyi yielded approximately 10(6) antibiotic-resistant transformants per microgram of plasmid DNA. Studies of the feasibility of moving mutated genes into H. ducreyi were initiated by using NotI linker insertion and mini-Tn10kan mutagenesis techniques to introduce insertion mutations into cloned H. ducreyi genes encoding cell envelope antigens. In the former case, a gene encoding chloramphenicol acetyltransferase was then inserted into the NotI linker site created in the cloned H. ducreyi gene. The recombinant Escherichia coli strains containing these mutated plasmids no longer expressed the homologous H. ducreyi cell envelope antigens, as evidenced by their lack of reactivity with monoclonal antibody probes for these H. ducreyi proteins. Subsequent electroporation of both circular and linearized forms of plasmids carrying these mutated H. ducreyi genes into the homologous wild-type strain of H. ducreyi yielded antibiotic-resistant transformants which also lacked reactivity with the cell envelope antigen-specific monoclonal antibodies. Southern blot analysis confirmed that homologous recombination had occurred in these monoclonal antibody-unreactive transformants, resulting in the replacement of the wild-type allele with the mutated allele. Allelic exchange was most efficient when linear DNA molecules were used for electroporation. These results indicate that electroporation methods can be utilized to construct isogenic mutants of H. ducreyi.     

Transformation of the medicinal basidiomycete Trametes versicolor to hygromycin B resistance by restriction enzyme mediated integration

Trametes versicolor, a white-rot basidiomycete, degrades cellulose and lignin as well as many recalcitrant chemicals. There have been many reports about the cloning of laccase and peroxidase genes of T. versicolor which are involved in lignin degradation. In order to analyze a gene function and introduce foreign genes into an organism, genetic transformation is required. Here we have successfully transformed T. versicolor to hygromycin B resistance using pAN 7-1 plasmid by restriction enzyme mediated integration and have obtained many mutants in peroxidase activity and growing patterns. The transformation frequency was 25-50 transformants (microg plasmid DNA)(-1). The transformants were quite stable after 10 consecutive transfers in non-selectable medium.     

Electrotransformation of Chlorella vulgaris

Using hygromycin B resistance as a marker for selection, we have established the conditions required for the transformation of Chlorella vulgaris. The exponentially grown C. vulgaris cells were transformed by electroporation with plasmid pIG121-Hm, and transformants were selected with hygromycin B at a concentration of 50 μg/ml. Cell extracts prepared from the late-log cultures of the transformants exhibited glucuronidase activities as conferred by the gus gene on pIG121-Hm. The maintenance of plasmid in the algal cells seemed to be transient as many cultures derived from the hygromycin B-resistant colonies gradually lost the hygromycin resistance upon prolonged growth. The result of Southern blotting of the genomic DNAs prepared from transformant cultures exhibiting persistent hygromycin resistance showed that integration of part of the plasmid DNA into the host chromosome had taken place. Received: 19 December 1997 / Revision received: 5 October 1998 / Accepted: 27 October 1998     

Gene rescue in plants by direct gene transfer of total genomic DNA into protoplasts.

To study the possibility of gene rescue in plants by direct gene transfer we chose the Arabidopsis mutant GH50 as a source of donor DNA. GH50 is tolerant of chlorsulfuron, a herbicide of the sulfonylurea class. Tobacco protoplasts were cotransfected with genomic DNA and the plasmid pHP23 which confers kanamycin resistance. A high frequency of cointegration of the plasmid and the genomic DNA was expected, which would allow the tagging of the plant selectable trait with the plasmid DNA. After transfection by electroporation the protoplasts were cultivated on regeneration medium supplemented with either chlorsulfuron or kanamycin as a selective agent. Selection on kanamycin yielded resistant calluses at an absolute transformation frequency (ATF) of 0.8 x 10(-3). Selection on chlorsulfuron yielded resistant calluses at an ATF of 4.7 x 10(-6). When a selection on chlorsulfuron was subsequently applied to the kanamycin resistant calluses, 8% of them showed resistance to this herbicide. Southern analysis carried out on the herbicide resistant transformants detected the presence of the herbicide resistance gene of Arabidopsis into the genome of the transformed tobacco. Segregation analysis showed the presence of the resistance gene and the marker gene in the progeny of the five analysed transformants. 3 transformants showed evidence of genetic linkage between the two genes. In addition we show that using the same technique a kanamycin resistance gene from a transgenic tobacco could be transferred into sugar beet protoplasts at a frequency of 0.17% of the transformants.