【作者】 杨明明；

【导师】 陈玉林；

【作者基本信息】 西北农林科技大学 ， 动物营养与饲料科学， 2013， 博士

【摘要】 枯草芽孢杆菌广泛存在于土壤和动物胃肠道。因其易于分离培养，具有较清晰的遗传背景和良好的分泌性，又无致病性等特点，已成为生物技术基础研究和应用研究中使用较多的重要菌种。而且，枯草芽孢杆菌作为益生菌和饲用酶产生菌越来越多的被应用于畜禽生产中。为此，需要针对枯草芽孢杆菌载体、表达元件以及遗传操作方法等关键环节，建立较为完善的枯草芽孢杆菌遗传操作系统。本研究围绕构建新的枯草芽孢杆菌质粒载体，构建以麦芽糖利用操纵元启动子为调控元件的表达载体，并基于枯草芽孢杆菌的电转化整合和定点突变，进一步优化表达系统。同时，期望克隆筛选获得强启动子元件作为枯草芽孢杆菌的调控元件。取得的主要研究结果如下：1.把具有完全复制功能的700bp大肠杆菌ColE1复制子与2500bp枯草芽孢杆菌质粒复制子连接，成功构建了E. coli-B. subtilis穿梭载体pGJ103。利用B. subtilis麦芽糖操纵元启动子和穿梭载体pGJ103，成功构建了B. subtilis表达载体pLJ，利用其在枯草杆菌中高水平表达了β-半乳糖苷酶、BioA和VHB等蛋白。另外，证明葡萄糖严重抑制pLJ的表达。2.通过降低盐离子浓度,提高细胞活力,在枯草芽孢杆菌中建立了优化的电转化整合方法。并据此实现了在B. subtilis基因组DNA中的环形DNA单交叉整合和线性DNA双交叉整合，敲除了B. subtilis早期生孢基因，获得了枯草芽孢杆菌生孢基因缺失株B. subtilis GJ09。3.以β-半乳糖苷酶编码基因为报告基因，对枯草芽孢杆菌麦芽糖操纵元启动子进行了定点突变。正突变子pJRINM1在B. subtilis1A747中实现了β-半乳糖苷酶高效表达，其表达量占总可溶性蛋白的18%，5%的麦芽糖诱导24h酶活达到14U/mL。同时，基于同源重组方法，用枯草芽孢杆菌组成型启动子P43替换了野生型菌株B. subtilis1A747的麦芽糖操纵元上游调控序列，获得了优化重组表达宿主菌B. subtilis BCYL。将pJRINM1转入B. subtilis BCYL后，β-半乳糖苷酶活性有大幅度提高，经5%麦芽糖诱导24h后，β-半乳糖苷酶活达到21U/mL，葡萄糖的抑制作用明显减弱。分别PCR扩增了大肠杆菌和枯草芽孢杆菌维生素B12合成前期途径的谷氨酰-tRNA合成酶编码基因hemA，构建了其表达载体，并将其转化到B. subtilis BCYL中，实现了诱导表达。因此，通过对启动子和表达宿主的优化，获得了枯草芽孢杆菌高效表达系统，为枯草芽孢杆菌基因工程研究提供了有效实用的工具。4.利用bgaB作为报告基因，构建了启动子检测载体pShuttlF，并据此筛选获得了200个可能含有地衣芽孢杆菌启动子的重组克隆，从中获得了β-半乳糖苷酶活性可在大肠杆菌和枯草芽孢杆菌中分别达14016Mill U和16417Mill U的重组克隆pShuttle-09，表明在pShuttle-09中克隆获得了一个强启动子PluxS。通过对PluxS进行重构，得到了优化的启动子P_LapS，其活性达到原来的1.6倍。同时，利用P_LapS成功构建了表达载体pLus-Hyb和pLus-His，并实现了bgaB和bioI基因的高效表达与纯化。综上所述，本研究主要构建了大肠杆菌-枯草芽孢杆菌穿梭载体、枯草芽孢杆菌麦芽糖诱导表达载体和枯草芽孢杆菌启动子筛选克隆载体。同时，利用麦芽糖诱导表达载体实现了外源基因的高效表达，并利用建立的枯草芽孢杆菌电转化整合方法优化提高了麦芽糖诱导表达系统的表达效果。利用枯草芽孢杆菌启动子筛选克隆载体系统筛选启动子元件获得了强启动子元件。建立和完善了枯草芽孢杆菌遗传操作体系，为枯草芽孢杆菌的基因工程研究提供了有效方法和工具。更多还原

【Abstract】 Bacillus subtilis is a non-pathogenic gram-positive bacterium and has been an attractivehost for production of recombinant proteins from both prokaryotic and eukaryotic origins. Thesuitability and popularity of B. subtilis as one of main hosts for recombinant proteinproduction is due to several reasons: generally recognized as safe as a result of its lack ofpathogenicity and the absence of endotoxins; capable of secreting functional proteins directlyinto culture media, easy for genetic manipulation and handling, and capable of large-scalefermentation. Bacillus subtilis has been applied in animal production, which acts as probioticsand feed enzyme source. It is attractive to employed genetic engineering of Bacillus subtilisfor alleviate the feed resource and enhance feed efficiency. However, the genetic manipulationof Bacillus subtilis involved in the vector, regulation element and methods limited itsbiotechnological application. To easy the manipulation, we develop and improve the geneticmanipulation of Bacillus subtilis in this study through constructing the E. coli-B. subtilisshuttle vector, expression system and promoter trapping system. The results were shown asfollows:1. The E. coli-B. subtilis shuttle vector pGJ103was developed in this study by ligation ofColE1replicon (about700bp) and pGDV1replicon (about2500bp). A maltose-inducibleexpression vector in Bacillus subtilis based on pGJ103has been developed and characterized.The vector permitted β-galactosidase expression at a high level. Using this vector, wesuccessfully expressed the other two genes, bioA and vgb. However, the system constructed inthis study was repressed badly by the glucose.2. Here, we present the electroporation as a feasible and efficient method for introducingcircularized and linearized DNA into Bacillus subtilis chromosome. Two integrationexperiments were carried out and demonstrated the feasibility and efficiency ofelectroporation to introduce the target DNA into the B. subtilis chromosome. To furtherdemonstrate the application of electroporation in genetic research, the early sporulation genespo0A of B. subtilis was knocked out (named B. subtilis GJ09) and, consequently, the null ofsporulation and logged growth was observed in this study.3. Site-directed mutagenesis was facilitated to enhance the expression strength of Pglv. The production of β-Gal driven by the mutant promoter in B. subtilis1A747reached14U/mLat24h when induced by5%maltose. To further improve the promoter system, the B. subtilisexpression host was reconstructed, in which B. subtilis well-characterized constitutivepromoter P43replaced the promoter of the glv operon in B. subtilis chromosome through adouble crossover event, yelding B. subtilis BCYL. The β-galactosidase production from theimproved system (21U/mL) increased compared to that from origin system. Meanwhile, therepression caused by glucose was further alleviated.4. To screen and isolate strong promoters, a promoter trap vector pShuttleF was used inthis study. Using the vector, approximately200colonies containing likely promoters fromBacillus licheniformis genomic DNA were obtained. Amongst them, pShuttle-09exhibited thehighest β-Gal activities in both Escherichia coli and B. subtilis (14016Mill U and16417MillU, respectively). A sequencing analysis showed that pShuttle-09contained PluxSand truncatedluxS in-frame fused with the reporter gene as well as another fragment upstream of PluxScontaining a putative promoter. Reconstructing the hybrid promoter from pShuttle-09to PlapSfurther improved the β-Gal production by1.6folds. By using of the PlapS, β-Gal and BioI weresuccessfully expressed in B. subtilis.In conclusion, a facilitated E.coli-B. subtilis shuttle vecoter, inducible expression systemand promoter trap system were developed in this study. By using of these expression elements,the target genes were high-level expressed in B. subtilis. Thus, we provided a potential tooland manipulation approach for B. subtilis genetic engendering.更多还原