消除内生质粒对苏云金芽胞杆菌YBT-1463特性的影响

研究了经完全消除苏云金芽胞杆菌野生菌株ＹＢＴ－１４６３的内生质粒对该菌部分形态、遗传及生理生化特性的影响。结果表明,消除内生质粒后的无质粒突变株不形成伴胞晶体,但电转化４种供体质粒,即ｐＢＭＢ１２１、ｐＢＭＢ３０４－１Ａｂ、ｐＢＭＢＬＣ和ｐＢＭＢ９７４８的效率显著提高,转化频率最高比出发菌株提高６．８×１０^４倍,而无质粒突变株对红霉素等１０种抗生素的敏感性、对葡萄糖等１９种碳源和谷氨酸等１２种氮源的利用能力及生长性能与出发菌株无明显差异。     

苏云金芽胞杆菌菌株YBT833含不同杀虫晶体蛋白基因转化子的特性

用电脉冲方法将含有苏云金芽胞杆菌杀虫晶体蛋白基因ｃｒｙ１Ｃ的重组质粒ｐＢＭＢＬＣ转入野生菌株ＹＢＴ８３３,获得含不同杀虫晶体蛋白基因的４个转化子。质粒检测和Ｓｏｕｔｈｅｒｎ杂交证明它们均为菌株ＹＢＴ８３３含重组质粒ｐＢＭＢＬＣ的转化子。ＰＣＲ扩增表明,转化子ＹＢＴ８３３－１保留了原有的杀虫晶体蛋白基因;转化子ＹＢＴ８３３－２丢失了基因ｃｒｙ１Ａｂ;转化子ＹＢＴ８３３－３则丢失了所有的杀虫晶体蛋白基     

电脉冲法转化苏云金芽孢杆菌BMB171的研究

研究了用电脉冲法转化苏云金芽孢杆菌受体菌ＢＭＢ１７１的优化条件以及由转化导入的几类ｃｒｙ基因在ＢＭＢ１７１中的表达效果。结果表明,采用ＳＧ溶液作电脉冲缓冲液,用１０．ｏｋＶ／ｃｍ的脉冲场强和１次电脉冲（４．６ｍｓ）以及采用对数前期（ＯＤ_６５０ｎｍ为０．２～０．３）收获的受体菌,可以达到最高转化频率,其中用ｐＨＴ３１０１电转化 ＢＭＢ１７１的最高频率达 ８×１０７转化子／μｇ ＤＮＡ。转化频率随质粒ｐＨＴ３１０１浓度的增加,在５４．６９ｐｇ／ｍＬ至３．５０μｇ／ｍＬ范围内     

hBMP－2cDNA在COS细胞和小鼠肌肉中的表达

研究了骨形态发生蛋白ＢＭＰ－２ｃＤＮＡ在ＣＯＳ细胞和小鼠肌肉中的表达的情况,从ｐＳＰＳ６５ＢＭＰ－２质粒中回收ＢＭＰ－２ｃＤＮＡ,删除５＇端的非翻译序列,插入ｐＳＶＬ载体中,构建了含有ＢＭＰ－２全长编码序列的重组表达质粒ｐＳＶＬＢＭＰ－２。将表达质粒导入ＣＯＳ－７细胞中,细胞ＲＮＡ点杂交结果表明,转染ＢＭＰ－２基因的细胞内ＢＭＰ－２的ｍＲＮＡ水平明显升高;细胞培养上清的ＥＬＩＳＡ显示,转染ＢＭＰ－２ｃＤＮＡ后,细胞分泌产生的ＢＭＰ－２显著增加。小鼠实验发现,在肌肉内用注射法导入ＢＭＰ－２重组质粒后,局部组织内ＢＭＰ－２的ｍＲＮＡ转录水平也明显提高。     

高产稳产聚羟基烷酸的重组大肠杆菌的构建

重组大肠杆菌Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉＨＭＳ１７４（ｐＴＺ１８ＵＰＨＢ） 含有携带聚羟基烷酸（ＰＨＡ） 合成基因（ ｐｈａＣＡＢ）＊＊ 的质粒ｐＴＺ１８ＵＰＨＢ,是很有潜力的ＰＨＡ 生产菌,但存在着质粒不稳定和不能合成３羟基丁酸（３ＨＢ） 与３羟基戊酸（３ＨＶ） 共聚物［Ｐ（３ＨＢｃｏ３ＨＶ）］ 的缺陷。将ＲＫ２ 质粒上的ｐａｒ ＤＥ 基因引入ｐＴＺ１８ＵＰＨＢ 构成质粒ｐＪＭＣ２ ,该质粒可以在宿主Ｅ．ＣｏｌｉＨＭＳ１７４ 中稳定遗传。将培养基中的磷酸盐浓度降至１８ ｍ ｍｏｌ／Ｌ,发现Ｅ．Ｃｏｌｉ ＨＭＳ１７４（ｐＪＭＣ２） 能够以丙酸为前体合成Ｐ（３ＨＢｃｏ３ＨＶ） ,其中３ＨＶ 在共聚物中的含量为５ ％ ～８ ％ 。在５Ｌ自动发酵罐中分批补料培养Ｅ．Ｃｏｌｉ ＨＭＳ１７４（ｐＪＭＣ２） ,培养基初始磷酸盐浓度为１５ ｍ ｍｏｌ／Ｌ,３０ ｈ 后每升培养液中干菌体可达４２－５ ｇ,Ｐ（３ＨＢｃｏ３ＨＶ） 占干重的７０ ％ ,其中３ＨＶ 在共聚物中的含量为４－９ ％ 。     

hBMP—2cDNA在COS细胞和小鼠肌肉中的表达

研究了骨形态发生蛋白ＢＭＰ－２ｃＤＮＡ在ＣＯＳ细胞和小鼠肌肉中的表达的情况,从ｐＳＰＳ６５ＢＭＰ－２质粒中回收ＢＭＰ－２ｃＤＮＡ,删除５端的非翻译序列,插入ｐＳＶＬ载体中,构建了含有ＢＭＰ－２全长编码序列的重组表达质粒ｐＳＶＬＢＭＰ－２将表达质粒导入ＣＯＳ－７细胞中,细胞ＲＮＡ点杂交结果表明,转染ＢＭＰ－２基因的细胞内ＢＭＰ－２的ｍＲＮＡ水平明显升高,细胞培养上清的ＥＬＩＳＡ显示,转染ＢＭＰ－２ｃ     

苏云金芽孢杆菌的电穿孔及其工程菌的构建

本文对苏芸金芽孢杆菌 (Ｂａｃｉｌｌｕｓｔｈｕｒｉｎｇｉｅｎｓｉｓ,Ｂｔ)和蜡状芽孢杆菌 (Ｂａｃｉｌｌｕｓｃｅｒｅｕｓ,Ｂｃ)等部分菌株的电穿孔转化进行了研究 ,主要从电穿孔的供体质粒和受体菌株等方面讨论了各种因素对该方法的影响。同时利用电穿孔方法对部分Ｂｔ的高效野生株进行遗传改良 ,希望获得含有ｃｒｙ1Ａｃ和ｃｒｙ1Ｃ高效基因组合的对鳞翅目棉铃虫、甜菜夜蛾都有效的广谱工程株。利用质粒 ｐＳＢ1 40 2 (ｃｒｙ1Ｃ) ,ｐＡＭＹ(ｃｒｙ1Ａｃ) ,ｐＮＱ1 2 2 (Ｃｍｒ)对所有的供试菌株进行电穿孔转化 ,不同菌株表现出不同的转…     

niaD基因与uidA基因共转化糖化酶高产菌株Aspergillus nigerT21

从Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ Ｔ２１分离到自发性的氯酸盐抗性株,再经氮源生长试验获得硝酸盐还原酶缺陷的ｎｉａＤ突变体Ｎ４４。用含有ｎｉａＤ的质粒ｐＳＴＡ１０转化Ｎ４４,转化频率为５个／μｇ（转化子／ＤＮＡ）。转化子的Ｓｏｕｔｈｅｒｎ印迹分析表明ｎｉａＤ基因同源整合到Ｎ４４的染色体ＤＮＡ中。ｐＳＴＡ１０与含葡萄苷酸酶基因（ｕｉｄＡ）的质粒ｐＮＯＭ１０２共转化Ｎ４４,共转化频率为４０％。共转化     

青蒿转杜松烯合成酶基因发根系的培养

将已克隆的棉花杜松烯合成酶的ｃＤＮＡ（ｃａｄＣ１４）插入到植物表达载体ｐＢＩ１２１中,构建含ＣａＭＶ３５Ｓ启动子驱动下的杜松烯合成酶基因的植物表达载体ｐＢＩＣ１４。用含ｐＢＩＣ１４质粒的发根农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｒｈｉｚｏｇｅｎｅｓ）１５８３４感染青蒿（ＡｒｔｅｍｉｓｉａａｎｎｕａＬ．）叶片并诱导发根,共建立１２１个生长迅速的发根系。经浓度为２０ｍｇ／Ｌ的Ｋａｎ筛选,获得１２个抗Ｋａｎ阳性根系。ＰＣＲ和Ｓｏｕｔｈｅｒｎｂｌｏｔｉｎｇ分析表明,外源杜松烯合成酶基因已整合到青蒿基因组中,其转基因频率为３％。ＲＴＰＣＲ分析表明,外源杜松烯合成酶基因在Ｃ３７根系中,在转录水平上已有表达。     

白细胞介素-1β对骨髓基质细胞膜电位及细胞内Ca2+和pH的影响

采用荧光染料 Ｄｉ Ｂ Ａ Ｃ４（３）, Ｆｌｕｏ３／ Ａ Ｍ 和 Ｓ Ｎ Ａ Ｌ Ｆ Ｃａｌｃｅｉｎ／ Ａ Ｍ 分别标记小鼠骨髓基质细胞（ Ｂ Ｍ Ｓ Ｃ）,在激光扫描共聚焦显微镜下直接监测重组人白细胞介素１β（ Ｉ Ｌ１β）刺激后细胞膜电位,细胞内游离 Ｃａ２＋ 浓度和胞浆 ｐ Ｈ 的实时动态变化． 结果发现： Ｉ Ｌ１β加入测定体系后浓度依赖性地引起 Ｂ Ｍ Ｓ Ｃ 膜电位的迅速改变． 低浓度时发生去极化反应,高浓度时发生超极化反应． 非受体方式作用的 Ｉ Ｌ１β肽段 １６３１７１ 对膜电位无影响． Ｉ Ｌ１β不影响细胞内 Ｃａ２＋ 的浓度和胞浆ｐ Ｈ． 研究表明膜电位的变化为 Ｉ Ｌ１ 受体后早期事件,它与细胞内 Ｃａ２＋的浓度和胞浆 ｐ Ｈ 的调节无关．     

消除内生质粒对苏云金芽胞杆菌YBT-1463特性的影响

研究了经完全消除苏云金芽胞杆菌野生菌株YBT-1463的内生质粒对该菌部分形态、遗传及生理生化特性的影响。结果表明,消除内生质粒后的无质粒突变株不形成伴胞晶体,但电转化4种供体质粒,即pBMBl21、pBMB304-1Ab、pBMBLC和pBMB9748的效率显著提高,转化频率最高比出发菌株提高6.8×10 倍,而无质粒突变株对红霉素等10种抗生素的敏感性,对葡萄糖等19种碳源和谷氨酸等12种氮源的利用能力及生长性能与出发菌株无明显差异。     

苏云金芽孢杆菌质粒的检测及功能的初步研究

用改进的酶碱综合法检测了苏云金芽孢杆菌4个亚种11个野生菌株的内生质粒,获得良好的制备结果和重视性,对野生菌株YBT－1463及其无质粒突变株BMB171的形态和生理生化特性的比较研究结果表明,YBT－1463的内生质粒携带杀虫晶体蛋白基因,但不携带抗生素的抗性基因,且与该菌株对19种C源和12种N源的利用无关。     

Complete Genome Sequence of Bacillus thuringiensis Mutant Strain BMB171

Bacillus thuringiensis has been widely used as a biopesticide for a long time. Here we report the finished and annotated genome sequence of B. thuringiensis mutant strain BMB171, an acrystalliferous mutant strain with a high transformation frequency obtained and stocked in our laboratory.Bacillus thuringiensis is an insect pathogen which is widely used as a biopesticide due to its various endogenous crystal proteins and spores (12). To improve the virulence and practical effectiveness of B. thuringiensis, genetic transformation of different genes with beneficial traits is a fundamental procedure. Simultaneously, genetic transformation can facilitate functional genomic research. However, wild-type strains are not suitable to be used as recipient strains because of low transformation efficiency. This obstacle is mainly caused by the thick cell wall layer of B. thuringiensis together with multiple plasmids inside the cell, which harbor genes encoding insecticidal crystal proteins. We used the method of elevating the growth temperature and adding 0.05% sodium dodecyl sulfate to treat several parental strains and finally obtained mutant strain BMB171, with no resident plasmid, from wild-type crystalliferous strain YBT-1463 (9). The electrotransformation frequency of mutant BMB171 could reach up to 10⁷ transformants/μg DNA after optimization of the electrotransformation parameters (7), which was 4.8 × 10⁴-fold higher than that of the parental strain (8). Moreover, mutant strain BMB171 exhibited the same characteristics as YBT-1463, such as metabolic abilities and growth properties, as well as sensitivity to 10 antibiotics (8). Of course, BMB171 could produce parasporal crystals with characteristic geometric shapes through the expression of relevant cry genes carried by plasmids (7). Thus, B. thuringiensis mutant strain BMB171 has become a major recipient strain and is widely used for insecticidal crystal protein-encoding gene expression (14, 15), cell surface display (10, 13), gene function and regulation researches (2, 5), etc.The B. thuringiensis mutant strain BMB171 genome was sequenced by using a massive parallel pyrosequencing technology (454 GS-FLX). A total of 448,963 high-quality reads with an average read length of 391 bp were produced, providing about 32-fold coverage of the genome. Assembly was performed using the Newbler software of the 454 suite package (454 Life Sciences), which resulted in 193 large (defined as >500 bp) contigs. The relationship of contigs was determined by multiplex PCR, and gaps were filled through sequencing of PCR products by primer walking or shotgun sequencing with an ABI 3730 sequencer. The Phred/Phrap/Consed software package (3) was used for final sequence assembly and quality assessment. Protein-coding genes were predicted by combining the results of Glimmer 3.02 (1) and ZCURVE (4), followed by manual inspection. Both tRNA and rRNA genes were identified by tRNAscan-SE (11) and RNAmmer (6), respectively. Functional annotation was performed by searching against a protein database of the microbial genome developed in house.The 5.64-Mb genome of B. thuringiensis mutant strain BMB171 contains two replicons: a circular chromosome (5.33 Mb) encoding 5,088 open reading frames (ORFs) and a circular plasmid (0.31 Mb), which is named pBMB171, encoding 276 predicted ORFs. The G+C content of the chromosome is 35.3%, while that of the plasmid is 33.3%. The mutant strain BMB171 genome encodes 104 tRNAs and 14 rRNA operons. A previous study indicated that BMB171 is a plasmid-free mutant (9); however, our sequencing results demonstrated that a large plasmid still remains. The reason why the plasmid was not detected previously might be its large size and low copy number. We did not find any crystal protein genes in either chromosome or plasmid sequences, which was consistent with previous observations (9).In summary, the complete B. thuringiensis mutant strain BMB171 genome provides a better-defined genetic background for gene expression and regulation studies, especially crystal protein production and metabolic network construction.     

苏云金芽胞杆菌营养期杀虫蛋白基因的克隆及表达分析

选择本实验室分离的苏云金芽胞杆菌李氏亚种 (subsp. Leesis) 菌株YBT833、鲇泽亚种(subsp.Aizawai) 菌株YBT-1416和库斯塔克亚种(subsp. Kurstaki)菌株YBT1535为出发菌株,以营养期杀虫蛋白基因PCR扩增的特异片段为探针,进行总DNA酶切片段的Southern杂交定位。结果显示3株菌株的营养期杀虫蛋白基因,均位于经XbaI完全消化的4～5kb大小的DNA 片段上。将该区域DNA片段回收后克隆到pUC19载体,建立了3个较基因组文库小的亚基因组文库。通过菌落原位杂交筛选和酶切鉴定分别得到3个相应的营养期杀虫蛋白基因vip83、vip14和vip15,并对其测序。DNA序列比较发现基因vip83与已知营养期杀虫蛋白基因存在5个差异碱基。将vip83、vip14基因亚克隆到苏云金芽胞杆菌大肠杆菌穿梭载体pHT315, 分别得到重组质粒pBMB8901和pBMB8902。将它们电转化到vip^-的B.t.受体菌BMB171和4Q7,获得了相应的工程菌BMB8901-171,BMB8902-171,BMB8901-4Q7和BMB8902-4Q7。SDS-PAGE电泳检测均有88kD大小的蛋白表达。生物测定结果亦表明了,营养期杀虫蛋白Vip83和Vip14对鳞翅目棉铃虫、小菜蛾和甜菜夜蛾的三龄幼虫均有一定的杀虫活性;其中对小菜蛾的毒力最高,LC₅₀值分别为28.6,31.6,45.4和37.6μL/mL。该结果为构建高效广谱工程菌提供了实际材料和理论依据。      

苏云金芽胞杆菌拟步行甲亚种质粒复制子oril65的克隆

以苏云金芽胞杆菌拟步行甲亚种菌株（Bacillus thuringiensis subsp.tenebrionis)YBT-1765作为出发菌株,克隆了一个包含复制子的EcoRI酶切片段,大小约为11kb,称为oril65。这是国内外从此亚种中克隆到的第一个复制子,缩小到8kb左右后仍然能够复制。杂交结果显示,此复制子来源于菌株YBT-1765可以检测到的分子量最大的质粒,以此复制子构建的穿梭载体pBMB6071在不同受体菌中的稳定性差异很大,其中在以色列亚种无晶体突变株4Q7中,传40后代,稳定性100%,质粒pBMB6071与含ori1030和ori2062在库斯塔克亚种无晶体突变株BMB171中是相容的。     

Stability of plasmids in lactococci during extended incubation in growth media

The stability of plasmids in Lactococcus lactis ssp. lactis strains C2 and ML3, and L. lactis ssp. cremoris strains ML1 and SC607, was investigated by extended incubation of bacterial cells in low nutrient media under acidic conditions. Strains were grown overnight (16-18 h) in skim milk and unbuffered medium (M17-) at 32 degrees C and subsequently held at that temperature for extended periods (greater than or equal to 96 h). Lac- variants were obtained from each strain in milk and (M17-) broth. The plasmid profiles of Lac- variants when compared with their parental Lac+ strains showed loss of one or more plasmid bands. None of the Lac- mutants showed loss of smaller plasmids (less than 5 MDa) indicating that smaller plasmids in lactococci are more stable under these conditions than larger plasmids (greater than 10 MDa). Concomitant loss of the Lac+ phenotype and plasmids by the method used in the present investigation may have application for isolating mutants devoid of one or more plasmids.     

Gene clusters located on two large plasmids determine spore crystal association (SCA) in Bacillus thuringiensis subsp. finitimus strain YBT-020

Crystals in Bacillus thuringiensis are usually formed in the mother cell compartment during sporulation and are separated from the spores after mother cell lysis. In a few strains, crystals are produced inside the exosporium and are associated with the spores after sporulation. This special phenotype, named 'spore crystal association' (SCA), typically occurs in B. thuringiensis subsp. finitimus. Our aim was to identify genes determining the SCA phenotype in B. thuringiensis subsp. finitimus strain YBT-020. Plasmid conjugation experiments indicated that the SCA phenotype in this strain was tightly linked with two large plasmids (pBMB26 and pBMB28). A shuttle bacterial artificial chromosome (BAC) library of strain YBT-020 was constructed. Six fragments from BAC clones were screened from this library and discovered to cover the full length of pBMB26; four others were found to cover pBMB28. Using fragment complementation testing, two fragments, each of approximately 35 kb and located on pBMB26 and pBMB28, were observed to recover the SCA phenotype in an acrystalliferous mutant, B. thuringiensis strain BMB171. Furthermore, deletion analysis indicated that the crystal protein gene cry26Aa from pBMB26, along with five genes from pBMB28, were indispensable to the SCA phenotype. Gene disruption and frame-shift mutation analyses revealed that two of the five genes from pBMB28, which showed low similarity to crystal proteins, determined the location of crystals inside the exosporium. Gene disruption revealed that the three remaining genes, similar to spore germination genes, contributed to the stability of the SCA phenotype in strain YBT-020. Our results thus identified the genes determining the SCA phenotype in B. thuringiensis subsp. finitimus.     

Effect of low temperature on stability of theta-type plasmids in Carnobacterium maltaromaticum.

The heterologous production of useful peptides such as bacteriocins by lactic acid bacteria (LAB) has been studied for use in the biopreservation of foods. Recombinant plasmids can suffer drawbacks such as segregational instability affecting the production of these peptides in certain environments such as absence of selective pressure or low temperature. The link between growth temperature characteristics of parental strains and stability of theta-type plasmids at a low temperature was investigated. The growth of four parental strains at 4 degrees C and stability of five derivative theta-type plasmids transformed into Carnobacterium maltaromaticum UAL26 at 25 and 4 degrees C were determined. Two plasmids (pCD11 and pCaT) derived from psychrotrophic LAB and plasmid, pHW800, from Enterococcus faecium 226 with unknown growth temperature characteristics, had excellent stability when strains were grown at 4 degrees C. Plasmids (pTRKH2 and pUCB820) derived from LAB that did not grow at refrigeration temperatures were not stable at 4 degrees C. When a DNA fragment from pCD11 containing 22-bp repeats, a putative replication initiation site, and the gene for the RepA protein was inserted into pTRKH2, the resulting derivative plasmid was 100% stable at 4 degrees C.     

苏云金芽胞杆菌杀线虫伴胞晶体蛋白基因cry6Aa的克隆与表达

通过对晶体蛋白N-末端氨基酸测序,设计简并探针,从对根结线虫高毒力苏云金芽胞杆菌YBT-1518菌株中克隆到1个含有杀线虫晶体蛋白基因的片段。序列测定表明该序列含有两个ORF(orf1和orf2),其中orf1与基因cry6Aa1同源性为98%,已在GenBank上登录(Acc.NO.AF499736),并被命名为cry6Aa2。将克隆的该片段克隆到穿梭载体pHT304上,并转化苏云金芽胞杆菌无晶体突变株BMB171,重组菌株可形成米粒状伴胞晶体。生物测定表明,表达的毒素蛋白对北方根结线虫的LC₅₀为9.47μg/mL,毒力与出发菌株(10.74μg/mL)相当。     

Physiological consequences of mutations in Escherichia coli heat shock dnaK and dnaJ genes.

Mutations in the heat shock genes, dnaK and dnaJ, cause severe defects of several cellular functions. Null dnaJ and dnaKdnaJ mutations can be transduced in a restricted range of temperature. The efficiency of transformation with three unrelated plasmids, viz pACYC184, pBR322 and pSC101, is two times lower in dnaK mutants while the dnaJ mutant is characterized by slightly impaired transformation with pSC101 only. The lack of DnaJ function negatively influences the stability of pSC101 at 42 degrees C, and this plasmid cannot be stably maintained at 30 degrees C in the delta dnaKdnaJ mutant. The double deletion mutant, delta dbaKdnaJ, is characterized by impaired osmoadaptation. The galactokinase content is lower in both mutants tested compared with wild-type strains even at 30 degrees C. The efficient complementation of some of these defects by the wild-type alleles present on low-copy number plasmid was achieved.