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。     

苏云金芽孢杆菌无晶体突变株的逐级升温筛选及其转化性能

逐级从４２ ℃到４４ ℃和４６ ℃升温培养、并用０－０５ ％ ＳＤＳ 处理苏云金芽孢杆菌ＹＢＴ１４６３ ,获得了一系列内生质粒被部分或完全消除的无晶体（Ｃｒｙ －） 突变株,对４ 种Ｃｒｙ － 突变株的转化性能及导入的外源质粒的稳定性进行了研究。用限量培养基和４２ ℃培养筛选到Ｃｒｙ － 突变株后,升温至４４ ℃,从Ｃｒｙ － 突变株得到内生质粒被进一步消除的突变株;然后升温至４６ ℃来培养其中突变株ＢＭＢ１７０ ,并用０－０５ ％ 的ＳＤＳ进行处理,最终筛选到１ 株无质粒突变株ＢＭＢ１７１ 。用ｐＨＴ３１０１ 、ｐＢＭＢ１７３６ 、ｐＢＴＬ１ 和ｐＨＶ１２４９ 等４ 种外源质粒进行的转化及稳定性研究表明,转化频率的大小及导入质粒的稳定性与用作受体菌的Ｃｒｙ － 突变株携有的内生质粒数之间呈现一定的相关性,Ｃｒｙ－ 突变株的转化频率显著高于出发菌株,其中ＢＭＢ１７１ 的转化频率最高达１０７ 转化子／μｇ ＤＮＡ,且所导入的外源质粒的稳定性也高于其它Ｃｒｙ － 突变株及出发菌株ＹＢＴ１４６３ 。     

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。     

苏云金芽胞杆菌大质粒pBMB28的克隆

苏云金芽胞杆菌幕虫亚种YBT-020具有典型的晶胞粘连表型。在前期的研究中,通过质粒消除实验,推测晶胞粘连现象与YBT-020内生质粒pBMB28有关。为了定位质粒pBMB28上控制晶胞粘连表型的基因,首先对质粒pBMB28进行克隆。利用穿梭载体pEMB0557,成功构建了苏云金芽胞杆菌YBT-020的基因组人工染色体(BAC)文库。前期的研究表明晶体蛋白基因cry28Aa定位在质粒pBMB28上,根据cry28Aa基因序列设计引物,从文库中筛选到含有cry28Aa的重组质粒pBMB231。镜检和SDS-PAGE证明质粒pBMB231转化无晶体突变株BMB171形成的重组子BMB231可以产生Cry28Aa晶体蛋白,但不能恢复晶胞粘连表型。对重组质粒pBMB231的插入片段末端序列测定并设计引物筛选文库,通过染色体步移方式得到4个可以重叠覆盖质粒pBMB28不同区域的克隆子,从而克隆了该质粒。对这4个克隆子末端测序和酶切分析,测算出该质粒的大小约为140 kb。进一步确定应用基因组BAC文库以及重叠片段筛选的方法,可以快速有效的克隆苏云金芽胞杆菌大质粒。     

含质粒复制起始区ori44的苏云金芽胞杆菌解离载体的构建

将苏云金芽胞杆菌转座子Tn4430的解离酶识别位点res分别插入克隆载体pRSET B和pUC19得到质粒pBMB1201和pBMB1202。这两个质粒分别经BamHI/Hin dⅢ和EcoRI/HindⅢ双酶切回收含res位点的小DNA片段,与穿梭载体pHT3101经EcoRI/HindⅢ双酶切后加收的含大肠杆菌复制起始区、氨苄青霉素抗性基因和红霉素抗性基因的3．3kb片段连接,获得重组质粒pBMB1203。封闭pBMB1203两res位点外的BamHI和EcoRI位点后,得到解离载体pBMB1204。将来源于苏云金芽胞杆菌库斯塔克亚种YBT－1520的质粒复制起始区ori44片段插入pBMB1204的两res位点之间,得到解离穿梭载体pBMB1205。该解离载体插入壮观霉素抗性基因后电转化无晶体突变株,在辅助质粒所提供的解离酶作用下可发生解离消除抗性基因,解离频率为100％,解离后的质粒稳定性为93％。利用解离穿梭载体pBMB1205可在用抗性筛选到转化子后特定消除抗性标记基因和其它非苏云金芽胞杆菌DNA片段。     

苏云金芽胞杆菌拟步行甲亚种质粒复制子ori165的克隆

以苏云金芽胞杆菌拟步行甲亚种菌株 (Bacillusthuringiensissubsp.tenebrionis)YBT 1 76 5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 1 1kb ,称为ori1 6 5。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 1 76 5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pBMB6 0 71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 1 0 0 %。质粒pBMB6 0 71与含ori1 0 3 0和ori2 0 6 2在库斯塔克亚种无晶体突变株BMB1 71中是相容的     

苏云金芽胞杆菌幕虫亚种晶胞粘连现象与晶体蛋白基因cry26Aa所在质粒有关

苏云金芽胞杆菌幕虫亚种T02菌株的伴胞晶体在芽胞外壁内侧形成,呈现晶胞粘连的现象。在此菌株中克隆了cry26 Aa和cry28 Aa两个基因,并对晶胞粘连现象与质粒的相关性做了系统研究。通过消除幕虫亚种T02菌株的质粒,得到了仅消除cry26 Aa所在质粒的菌株BMB1151和无质粒的菌株BMB1152。通过穿梭载体将cry26 Aa和cry28 Aa两个基因分别和同时转化无质粒突变株BMB1152并表达,形成的晶体与芽胞独立存在不能粘连,表明在幕虫亚种染色体背景下仅仅cry的表达不能形成晶胞粘连现象,从而推断晶胞粘连现象可能与幕虫亚种两个基因所在的质粒有关;进一步的研究发现将cry26 Aa在仅消除cry26 Aa所在质粒的突变株BMB1151中表达,形成的晶体与芽胞也分别独立存在不能粘连,从而进一步推断幕虫亚种晶胞粘连现象与cry26 Aa所在质粒有关。     

含质粒复制起始区ori44的苏云金芽胞杆菌解离载体的构建

将苏云金芽胞杆菌转座子Tn4430的解离酶识别位点res分别插入克隆载体pRSET B和pUC19得到质粒pBMB1201和pBMB1202。这两个质粒分别经BamHI/HindⅢ和EcoRI/HindⅢ双酶切回收含res位点的小DNA片段,与穿梭载体pHT3101经EcoRI/HindⅢ双酶切后回收的含大肠杆菌复制起始区、氨苄青霉素抗性基因和红霉素抗性基因的3.3kb片段连接,获得重组质粒pBMB1203。封闭pBMB1203两res位点外的BamHI和EcoRI位点后,得到解离载体pBMB1204。将来源于苏云金芽胞杆菌库斯塔克亚种YBT-1520的质粒复制起始区ori44片段插入pBMB1204的两res位点之间,得到解离穿梭载体pBMB1205。该解离载体插入壮观霉素抗性基因后电转化无晶体突变株,在辅助质粒所提供的解离酶作用下可发生解离消除抗性基因,解离频率为100%,解离后的质粒稳定性为93%。利用解离穿梭载体pBMB1205可在用抗性筛选到转化子后特定消除抗性标记基因和其它非苏云金芽胞杆菌DNA片段。     

利用苏云金芽胞杆菌转座子Tn4430构建含cry1Ac10基因的解离载体

将cry1Ac10基因和苏云金芽胞杆菌的复制起始区连 接在一起,并在其两侧按相同方向各连接一个来自苏云金芽胞杆菌转座子Tn4430的解离位点,构成转移单位。再将革兰氏阳性细菌的抗性标记基因和大肠杆菌克隆载体pUC19与之连接,获得含cry1Ac10基因的解离载体pBMB801。将其转化苏云金芽胞杆菌无晶体突变体,再导入含解离酶基因的辅助质粒pBMB1200。在解离酶作用下,两个解离位点间发生重组,消除基在操作中的抗性标记基因等非必需基因片段,获得仅保留有完整cry1Ac10基因和来 自苏云金芽胞杆菌质粒复制起始区,在无抗生素选择压力下能稳定遗传的重组质粒pBMB801B 。     

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。     

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。      

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.     

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.     

苏云金芽胞杆菌质粒pBMB2062的克隆及遗传稳定载体的构建

从苏云金芽胞杆菌ＹＢＴ－１５２０菌株中克隆到１个小质粒ｐＢＭＢ２０６２,序列分析表明该质粒由２ ０６２个核苷酸组成。该质粒含２个编码框（ｏｒｆ１和ｏｒｆ２）,可分别编码由２８９个氨基酸和８０个氨基酸组成的蛋白质。这２个潜在的蛋白质分别与质粒的复制启始蛋白和复制蛋白同源。ｐＢＭＢ２０６２与２个已知同源质粒之间有２３个核苷酸的差异,这些差异引起了ｏｒｆ１编码框的变化。ｃＤＮＡ合成和ＰＣＲ检测显示与ｐＢ