大肠杆菌-枯草芽孢杆菌穿梭质粒的构建及碱性蛋白酶的表达

构建了一个可以在大肠杆菌和枯草芽孢杆菌中复制,并可以在枯草芽孢杆菌中表达外源蛋白的穿梭表达载体pBE2R.该穿梭表达载体是以pBE2为基本骨架,引入来自pWB980质粒的P43强启动子,并在其下游引入嗜碱芽孢杆菌碱性蛋白酶信号肽和前肽构建而成.试验结果表明,该穿梭表达载体pBE2R可以在大肠杆DH5α和枯草芽孢杆菌WB600中稳定存在,并可以使外源蛋白在枯草芽孢杆菌进行分泌表达,同时也为碱性蛋白酶定向改造提供了高通量筛选平台.     

以β-半乳糖苷酶为报告基因的原核启动子检测体系构建

目的:以β-半乳糖苷酶为报告基因构建原核启动子检测体系.方法:以由质粒 pDL 扩增获得的 bgaB 基因为报告基因,将其克隆到大肠杆菌-枯草杆菌穿梭载体 pBE2,构建成一个具有启动子活性检测功能的重组质粒pBEB.将组成型启动子P43和诱导型启动子Pcpac克隆入 pBEB,得到重组表达载体 pBEBP43 和 pBEBPapac,转化至大肠杆菌和枯草芽孢杆菌.结果:两种不同类型的启动子均能在大肠杆菌 BL21 和枯草芽孢杆菌 1A751 中启动 bgaB 基因的表达.结论:成功构建具有较为广泛适用性的原核启动子检测体系.     

以绿色荧光蛋白为报告基因的原核启动子检测体系构建

以质粒pMUTIN-GFP 扩增获得的目的gfp 基因为报告基因,将其克隆到大肠杆菌-枯草芽孢杆菌穿梭载体pBE2,构建成一个具有启动子活性检测功能的重组质粒pBE2-GFP .将组成型启动子P43和诱导型启动子Pspac克隆入pBE2-GFP ,得到重组表达载体pBE-GFP-P43和pBE-GFP-Pspac,转化至大肠杆菌和枯草芽孢杆菌.荧光显微镜检测GFP 蛋白的表达情况.结果 表明,2种不同类型的启动子均能在大肠杆菌BL21和枯草芽孢杆菌1A751中启动gfp 基因的表达.     

细菌麦芽糖淀粉酶在枯草芽孢杆菌中的诱导型异源表达

【目的】实现地衣芽孢杆菌麦芽糖淀粉酶在枯草芽孢杆菌中的高效异源表达,并研究该重组酶的酶学性质。【方法】克隆巨大芽孢杆菌木糖异构酶基因的启动子区域及其调控蛋白,构建一个大肠杆菌/芽孢杆菌穿梭型诱导表达质粒,使用该诱导型启动子介导麦芽糖淀粉酶编码基因,实现其在枯草芽孢杆菌中的功能表达。对重组枯草芽孢杆菌的诱导条件进行优化,提高麦芽糖淀粉酶的产量。【结果】获得了诱导表达麦芽糖淀粉酶基因的重组枯草芽孢杆菌菌株。最适诱导温度为45°C,最适诱导剂添加浓度为1%,最适添加诱导剂时间为接种培养9 h后。重组酶蛋白分子量大小为67 k D,对该酶的酶学性质研究发现,以可溶性淀粉为底物,反应生成麦芽糖和葡萄糖,其中麦芽糖含量为60.42%。重组酶最适作用温度为45°C,最适作用p H为6.5,Ca2+、Co2+、EDTA对该重组麦芽糖淀粉酶具有激活作用。【结论】通过木糖诱导表达系统可以实现麦芽糖淀粉酶在枯草芽孢杆菌中的高效诱导型表达,酶活最高可达296.64 U/m L发酵液,在工业上有着较好的应用前景。     

β-半乳糖苷酶在枯草芽孢杆菌中的分泌表达

为检测本课题组构建的枯草芽孢杆菌分泌表达载体pGPST中启动子和信号肽的活性,将β-半乳糖苷酶基因插入表达载体的多克隆位点,构建含有β-半乳糖苷酶基因的重组质粒pGPST-lacZ,重组质粒转化枯草芽孢杆菌,分别测定液体培养基中上清液和菌体沉淀中β-半乳糖苷酶的活性。培养上清液中的酶活在22h达到峰值,约为26mU/mL,之后开始下降;菌体沉淀中的酶活在14h最高,达6mU/mL,而阴性对照pGPST没有检测到酶活性。结果表明分泌型表达载体中的启动子和信号肽具有较好的活性,能实现异源基因在枯草芽孢杆菌中的分泌表达。本试验为该表达载体的进一步研究和应用提供重要的数据资料,也为外源基因在枯草芽孢杆菌中的表达提供参考资料。该载体将在研究外源基因在枯草芽孢杆菌中表达发挥重要作用。     

利用枯草芽孢杆菌ytkA和ywoF基因启动子与信号肽重组分泌表达mpd基因

用大肠杆菌-枯草芽孢杆菌穿梭载体pNW33N和去除了信号肽编码序列的成熟mpd基因构建了穿梭启动子探针pNW33N-mpd。用该探针从质粒pMPDP3和pMPDP29上克隆来自于枯草芽孢杆菌ytkA和ywoF基因上游的启动子功能片段,构建了穿梭表达载体pNYTM和pNYWM。将表达载体pNYTM和pNYWM转入枯草芽孢杆菌1A751获得表达菌株1A751(pNYTM)和1A751(pNYTM),mpd基因在ytkA和ywoF基因的启动子和信号肽的带动下实现了分泌表达且具有天然活性,结果表明ytkA基因的启动子强度强于ywoF基因的启动子。利用ytkA基因的强启动子和nprB基因的分泌型信号肽编码序列构建了新的穿梭分泌表达载体pYNMK,并使mpd基因在枯草芽孢杆菌WB800中得到了更高水平的分泌表达,表达菌株WB800(pYNMK)在培养到第84h时甲基对硫磷水解酶酶活达到最高值为10.40u/mL,是出发菌株邻单胞菌M6表达量的10.8倍,重组表达产物有91.4%分泌在培养基中。     

枯草芽孢杆菌中一种新型双向启动子的功能鉴定

本研究旨在通过转录组分析预测的方法,由地衣芽孢杆菌中筛选获得一种新型双向启动子,鉴定其启动强度。以已知强组成型启动子pShuttle-09为对照,检测其对克劳氏芽孢杆菌碱性蛋白酶基因的表达活性。成功构建了3种重组碱性蛋白酶表达载体及对应的工程菌株。在新型启动子pLA和其反向启动子pLB调控转录下,克劳氏芽孢杆菌碱性蛋白酶表达活性达到164 U/mL和111 U/mL。结果表明,pLA的启动强度明显高于pShuttle-09和pLB,pLA启动子与pLB启动子均可表达碱性蛋白酶。从而为枯草芽孢杆菌表达系统中异源基因的表达提供一个新的方向,也为原核生物中共同表达两种基因提供了新的思路。     

枯草芽孢杆菌表达和分泌异源蛋白的研究进展

枯草芽孢杆菌是一种广泛应用于基础研究和工业生产的重要模式菌株,具有无致病性、蛋白分泌能力强、遗传背景清晰等多种优势,是生产异源蛋白的理想宿主。目前已有诸多异源蛋白在枯草芽孢杆菌中实现表达和分泌,其中包括淀粉酶、β-半乳糖苷酶和蛋白酶等有价值的工业酶。本文从异源蛋白表达和分泌的关键步骤出发,总结了枯草芽孢杆菌生产异源蛋白的传统策略和最新技术。除此之外,分析了当前研究存在的瓶颈并对如何提高异源蛋白产量提出了新的建议和策略。     

一种高效表达碱性蛋白酶的新型启动子的筛选及研究

碱性蛋白酶广泛的应用价值及市场供不应求的现状,旨在构建碱性蛋白酶高效表达系统。通过蛋白电泳和质谱检测的方法,由地衣芽孢杆菌中筛选获得一种新型启动子p Chi,以两种已知强组成型启动子p Shuttle-09和pyxi E为对照,研究其对克劳氏芽孢杆菌碱性蛋白酶与地衣芽孢杆菌碱性蛋白酶基因的表达活性。成功构建了6种重组表达载体及重组菌,结果表明,p Chi的启动强度是p Shuttle-09的1.25倍,pyxi E的2倍,从而为介导枯草芽孢杆菌表达系统中异源基因的表达奠定基础。     

信号肽优化在枯草芽孢杆菌体系中对脂肪酶LipS分泌表达的影响

枯草芽孢杆菌具有良好的安全性和优异的外分泌能力,是目前常用的脂肪酶异源分泌表达体系。在枯草体系中如何提高脂肪酶分泌量是目前的研究热点。该体系中信号肽与目的基因不匹配和目标蛋白总表达量不高是导致脂肪酶LipS分泌表达效果较差的重要原因。针对这些问题,依据信号肽结构特征的不同(正电荷与疏水氨基酸个数的差异)对枯草芽孢杆菌体系中Sec和Tat这两个主要分泌途径的信号肽进行了筛选,结果显示Tat途径中的phoD信号肽分泌效果明显优于其他信号肽。在此基础上,通过替换使用诱导型表达载体、突变优化phoD信号肽,并考察了表达条件的影响,显著提高了脂肪酶LipS的分泌表达量,发酵液中转酯酶活达到62.07U/L,占总酶活的62.30%,较原始分泌表达体系的分泌量提高了13.7倍。     

嗜盐古菌启动子的研究及应用

嗜盐古菌是古菌域的一个重要代表类群,在遗传与代谢、进化与适应、前沿生物技术及合成生物学领域都显示了其重要的研究价值。嗜盐古菌启动子的认识和利用,可以为嗜盐古菌的基础和应用研究提供必要的条件。本文从古菌启动子的结构与功能出发,就启动子的研究方法、嗜盐古菌启动子的特征及嗜盐古菌启动子的应用3个方面综述了嗜盐古菌启动子的研究现状,并对嗜盐古菌启动子未来研究的重点和方向进行了展望。     

Targeting transgene expression in research,agricultural, and environmental applications: Promoters used in plant transformation

Summary Plant genetic engineering has contributed substantially to the understanding of gene regulation and plant development, in the generation of transgenic organisms for widespread usage in agriculture, and has increased the potential uses of crops for industrial and pharmaceutical purposes. As the application of geneticallly engineered plants has widened, so has the need to develop methods to fine-tune control of transgene expression. The availability of a broad spectrum of promoters that differ in their ability to regulate the temporal and spatial expression patterns of the transgene can dramatically increase the successful application of transgenic technology. Indeed, a variety of promoters in necessary at all levels of genetic engineering in plants, from basic research discoveries, concepts and question to development of economically viable crops and plant commodities, to addressing legitimate concerns raised about the safety and containment of transgenic plants in the environment. This review covers the characterization and usage of a broad range of promoters employed in plant genetic engineering, including the widespread use of plant promoters with viral and plant origin that drive constitutive expression. Also covered are selected tissue-specific promoters from fruit, seed and grain, tubers, flowers, pistils, anther and pollen, roots and root nodules, and leaves and green tissue. Topics also include organellar promoters, and those found in specific cell types, as well as the development and evaluation of inducible (endogenous and exogenous origin) and synthetic plant promoter systems. Discussions on the relevance and potential pitfalls within specific applications are included.     

植物启动子研究进展

植物启动子在转录水平上发挥着重要的调控作用,对其功能进行研究不仅可以反映相应基因的表达模式,还能为利用植物基因工程手段实现基因的高效特异性表达提供有效途径。结合近年来的相关研究,综述了植物启动子的结构特点及其功能研究进展,重点对与生物和非生物胁迫有关的各类诱导型启动子进行了阐述,并展望了植物启动子的未来研究方向。     

植物基因启动子研究进展

综述了植物基因启动子的核心结构与功能、植物基因启动子的种类：异源组成型启动子(CaMV35S、MMV)和从植物自身克隆的组成型启动子(PTSB1和PPHYB),以及植物中通过按物理因素(温、光、旱、热等)、化学因素(离子、有机物、激素等)和生物因素(病菌、组织器官、发育阶段等)诱导表达的一些启动子,发育时期特异的启动子、器官特异的启动子(根、茎、叶等)和植物中应用的人工双向启动子的研究进展和应用前景。     

化学诱导表达系统及其在植物中的应用

化学诱导启动子可以在特定时间和部位激活或抑制目的基因的表达。目前,已经建立了多种化学诱导表达系统,用于基因功能分析、无标记植物转化、特定位点DNA切除、育性恢复和RNA沉默等方面的研究。化学诱导表达系统为基础分子生物学研究和生物技术应用提供了强有力的工具,将大大加快植物转基因技术的应用。     

Spacing requirements for simultaneous recognition of the adenovirus major late promoter TATAAAAG box and initiator element

The distance between the TATAAAAG box and initiator element of the strong adenovirus major late promoter was systematically altered to determine the optimal spacing for simultaneous recognition of both elements. We find that the TATAAAAG element is strongly dominant over the initiator for specification of the start site. The wild type spacing of 23 base pairs between TATAAAAG and +1A is optimal for promoter strength and selective recognition of the A-start. Initiation is constrained to a window spaced 19-26 base pairs downstream of (-31)-TATAAAAG-(-24), and A-starts are favored over alternate starts only when spaced between 21 and 25 base pairs downstream of TATAAAAG. We report an expanded TATAAAAG and initiator promoter consensus for vertebrates and plants. Plant promoters of this class are (A-T)-rich and have an A-rich (non-template strand) core promoter sequence element downstream of +1A.     

植物逆境胁迫耐受性启动子的研究进展

逆境胁迫如干旱、极端温度、损伤等非生物胁迫和病虫害等生物胁迫严重影响植物的生长发育及产量。逆境胁迫耐受性启动子能够接受逆境条件下的诱导信号,激活植物体内胁迫应答基因的表达,使植物感知并适应逆境。本文对逆境胁迫耐受性启动子的克隆及功能研究情况进行综合分析,主要包括抗旱、耐盐、耐高温、抗冻、耐损伤、抗病和抗虫基因启动子。     

Effect of the growth promoter avilamycin on emergence and persistence of antimicrobial resistance in enteric bacteria in the pig

AIM: To assess the effect of the growth promoter avilamycin on emergence and persistence of resistance in enteric bacteria in the pig. METHODS AND RESULTS: Pigs (treated with avilamycin for 3 months and controls) were challenged with multi-resistant Salmonella Typhimurium DT104 and faecal counts were performed for enterococci, Escherichia coli, S. Typhimurium and Campylobacter (before, during and 5 weeks post-treatment). Representative isolates were tested for antibiotic resistance and for the presence of resistance genes. Avilamycin-resistant Enterococci faecalis (speciated by PCR) were isolated from the treated pigs and continued to be detected for the first week after treatment had ceased. The avilamycin-resistance gene was characterized by PCR as the emtA gene and speciation by PCR. MIC profiling confirmed that more than one strain of Ent. faecalis carried this gene. There was no evidence of increased antimicrobial resistance in the E. coli, Salmonella and Campylobacter populations, although there was a higher incidence of tetB positive E. coli in the treated pigs than the controls. CONCLUSION: Although avilamycin selects for resistance in the native enterococci population of the pig, no resistant isolates were detected beyond 1 week post-treatment. This suggests that resistant isolates were unable to persist once selective pressure was removed and were out-competed by the sensitive microflora. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest the risk of resistant isolates becoming carcass contaminants and infecting humans could be minimized by introducing a withdrawal period after using avilamycin and prior to slaughter.