重组枯草芽胞杆菌不对称还原产d-伪麻黄碱

为了实现羰基还原酶基因mldh在枯草芽胞杆菌Bacillus subtilis中的表达并通过细胞内的葡萄糖脱氢酶完成辅酶的再生,以枯草芽胞杆菌rpsD基因的启动子PrpsD和终止子TrpsD为表达元件,将羰基还原酶基因mldh连接至构建好的质粒(pHY300plk-PrpsD-TrpsD上,得到质粒pHY300plk-PrpsD-mldh-TrpsD;进一步将重组质粒转化入B. subtilis Wb600中获得重组菌B. subtilis Wb600 (pHY300plk-PrpsD-mldh-Trps     

外源木聚糖酶在枯草芽胞杆菌中信号肽筛选

[目的]本试验旨在筛选引导表达外源木聚糖酶基因高效分泌的信号肽,为枯草芽胞杆菌木聚糖酶高效分泌表达系统提供元件.[方法]构建信号肽筛选载体,载体是以含壮观霉素抗性基因的大肠-枯草穿梭载体为基本骨架,目标蛋白为耐碱性木聚糖酶,可在麦芽糖启动子Pglv诱导下表达.从枯草芽胞杆菌A1747基因组中扩增获得24个Sec途径信号肽,并将其全部链接到至筛选载体上,并在枯草芽胞杆菌WB700中实现表达分泌.重组菌在3%麦芽糖诱导下培养24h后用DNS法测定上清酶活.[结果]成功构建信号肽筛选载体pGPSX及24个表达载体,实现木聚糖酶表达分泌.且不同信号肽对于引导外源木聚糖酶分泌能力不同,其中YnfF信号肽引导分泌目标蛋白效率最高,上清酶活为37.2IU/mL.[结论]试验证明在枯草杆菌中对外源蛋白进行信号肽筛选是提高其分泌的有效途径,并获得了针对木聚糖酶高效分泌信号肽YnfF.     

苏云金芽胞杆菌营养期杀虫蛋白基因vip3A在枯草芽胞杆菌中的表达

枯草芽胞杆菌Bacillus subtilis常被用于表达杀虫和抗菌蛋白.为了探讨苏云金芽胞杆菌B. thuringiensis营养期杀虫蛋白基因（vip3A）在枯草芽胞杆菌中的表达情况,促进杀虫防病工程菌构建,将枯草芽胞杆菌168菌株核糖体小亚基S4蛋白基因的启动子与苏云金芽胞杆菌WB7菌株vip3A基因的编码序列连接,插入大肠杆菌Escherichia coli与枯草芽胞杆菌穿梭载体pAD123,得到重组原核表达质粒pADpvip,将重组质粒转化枯草芽胞杆菌标准菌株168和分离自辣椒体内的生防内生枯草芽胞杆菌BS-2菌株中,获得工程菌株.SDS-PAGE分析表明在枯草芽胞杆菌168菌株的部分工程菌株中有约88 kDa大小的VIP条带,而BS-2的工程菌株中未见相应的条带,表明Vip3A蛋白仅在168菌株中表达.生物测定表明有5株168的工程菌株（168vip1-4,6）表现较高的杀虫活性,工程菌株发酵稀释液（约10⁷CFU/mL）处理的小白菜叶片饲喂斜纹夜蛾2龄幼虫72 h的杀虫效果可达87.64%~92.13%,但vip3A基因转入内生枯草芽胞杆菌BS-2中不表现杀虫作用.毒力测定表明168vip2菌株对斜纹夜蛾2龄幼虫72 h的LC₅₀为0.0194 mL/mL.这些结果为进一步研究基因在枯草芽胞杆菌中的表达构建杀虫防病工程菌打下了基础.     

过量表达DegQ有利于地衣芽胞杆菌表达高温α-淀粉酶

研究了过量表达DegQ对地衣芽胞杆菌表达高温α-淀粉酶的影响。通过PCR从地衣芽胞杆菌基因组中扩增得到degQ基因,将其克隆到pHY—P43载体中,得到重组质粒pHY—P43-degQ。将其分别转化至地衣芽胞杆菌ATCC14580和B0204中,得到重组菌ATCC14580（pHY—P43-degQ）和B0204（pHY—P43-degQ）。通过发酵试验,证实degQ基因的表达能够显著提高高温α-淀粉酶的表达水平。     

极细链格孢菌peaT1基因在毕赤酵母中的表达与功能 分析

建立了在毕赤酵母(Pichiapastoris)中分泌表达PeaT1蛋白的技术。将来源于极细链格孢菌(Alternaria tenuissima)的基因peaT1亚克隆至酵母分泌型表达载体pPIC9K,构建了重组表达载体pPIC9K-peaT1,分别用SalI或BglII酶切线性化后电击转入毕赤酵母GS115菌株中,经MD平板筛选、PCR鉴定获得整合有外源基因的重组菌株。在α-Factor及AOX1基因启动子和终止信号的调控下,PeaT1在酵母中大量表达并分泌到胞外,SDS-PAGE检测表明表达蛋白的表观分子量约为35kD。表达蛋白上清稀释液能诱导烟草产生对TMV的抗性,其枯斑数抑制率可达到30.37%。每升表达上清液经超滤浓缩和离子交换层析可纯化目的蛋白16.13mg,该纯化蛋白能显著地促进小麦幼苗的生长。     

以CotX为分子载体在枯草芽胞杆菌芽胞表面展示绿色荧光蛋白

芽胞衣壳蛋白CotB、CotC、CotG等可作为芽胞表面展示外源蛋白的分子载体,制备口服重组疫苗或具有催化活性的重组酶。CotX为枯草芽胞杆菌Bacillussubtilis芽胞衣壳中的另一种结构蛋白。为证明CotX能否作为分子载体将外源蛋白展示在芽胞表面,本研究将cotX基因与绿色荧光蛋白基因gfp的编码序列进行基因重组,构建融合表达CotX-GFP的整合型重组质粒,将该质粒转化枯草芽胞杆菌,筛选重组菌株并诱导产生芽胞,观察到重组芽胞表面具有GFP绿色荧光。结果表明枯草芽胞杆菌的芽胞衣壳蛋白CotX位于芽胞衣壳外层,可作为芽胞表面展示外源蛋白的载体分子。     

细极链格孢菌peaT2基因在毕赤酵母中的表达及蛋白功能确定

从细极链格孢菌表达文库获得阳性克隆子,序列分析表明,克隆的DNA片段中含有完整的开放阅读框架,将该基因命名为peaT2(GenBank登录号为EF212880)。用PCR法扩增peaT2基因的编码序列并亚克隆到毕赤酵母表达系统的表达载体pPIC9K上,得到重组质粒pPIC9K/peaT2。重组质粒经SacⅠ线性化后用电穿孔法导入到毕赤酵母(Pichia pastoris)GS115中,采用MD、G418-YPD平板和PCR法筛选Mut 表型,获得了分泌表达的重组毕赤酵母。随机挑取一菌株作为表达菌,用甲醇诱导PeaT2蛋白表达。SDS-PAGE及Western blot检测结果均表明PeaT2在毕赤酵母中成功地分泌表达。用peaT2基因的表达蛋白处理小麦种子,生物测定表明,表达蛋白能明显促进小麦的生长,具有蛋白激发子作用。     

细极链格孢菌peaT2基因在毕赤酵母中的表达及蛋白功能确定

从细极链格孢菌表达文库获得阳性克隆子,序列分析表明,克隆的DNA片段中含有完整的开放阅读框架,将该基因命名为peaT2(GenBank登录号为EF212880)。用PCR法扩增peaT2基因的编码序列并亚克隆到毕赤酵母表达系统的表达载体pPIC9K上,得到重组质粒pPIC9K/peaT2。重组质粒经SacⅠ线性化后用电穿孔法导入到毕赤酵母(Pichia pastoris)GS115中,采用MD、G418-YPD平板和PCR法筛选Mut+表型,获得了分泌表达的重组毕赤酵母。随机挑取一菌株作为表达菌,用甲醇诱导PeaT2蛋白表达。SDS-PAGE及Western blot检测结果均表明PeaT2在毕赤酵母中成功地分泌表达。用peaT2基因的表达蛋白处理小麦种子,生物测定表明,表达蛋白能明显促进小麦的生长,具有蛋白激发子作用。     

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。     

表达淀粉酶、糖化酶葡糖酸醋杆菌工程菌的构建

采用基因融合技术,将葡糖酸醋杆菌Gluconacetobacter hansenii ATCC23769分泌蛋白CMCax的信号肽序列分别与来源于枯草芽胞杆菌的淀粉酶基因、黑曲霉的糖化酶基因融合构建融合蛋白,连入能在G.hansenii ATCC23769自主复制的载体pbs-H1S中,电击转入G.hansenii ATCC23769,构建能内源表达淀粉酶、糖化酶,以及淀粉酶-糖化酶的葡糖酸醋杆菌。淀粉平板透明圈检测结果和DNS测酶活结果显示,构建的3种工程菌能成功表达并分泌淀粉酶和糖化酶。     

Purification and expression of a protein elicitor from Alternaria tenuissima and elicitor-mediated defence responses in tobacco

A new protein elicitor, PeaT1, was purified from the mycelium of Alternaria tenuissima by column chromatography. PeaT1 was identified as a heat-stable and acidic protein. It induced systemic acquired resistance to tobacco mosaic virus (TMV) in tobacco plants but did not cause hypersensitive response. The elicitor-encoding gene was cloned by rapid amplification of cDNA ends method. Sequence analysis revealed that the cDNA is 624 bp in length and the open reading frame encodes for a polypeptide of 207 amino acids with a nascent polypeptide-associated complex domain. The peaT1 gene was cloned into the expression vector pET-28a and transformed into Escherichia coli BL21 (DE3). The recombinant elicitor also triggered defence responses in intact tobacco plants. The availability of the pure protein offers the possibility to isolate the corresponding receptor and links it to the downstream signalling pathway.     

地衣芽孢杆菌YP1A耐有机溶剂蛋白酶基因的克隆与功能表达

根据B．licheniformis YP1A来源的碱性蛋白酶具有的高强度耐有机溶剂性能及相关数据库分析,采用PCR克隆B．licheniformis YP1A耐有机溶剂碱性蛋白酶基因,序列分析显示该基因（1264bp）包含启动子与编码380个氨基酸的开放阅读框（ORF）,ORF包括信号肽、前肽及编码254个氨基酸的成熟肽序列。相关基因分析表明,YP1A耐有机溶剂碱性蛋白酶基因与地衣芽孢杆菌ATCC14580的碱性蛋白酶基因仅有6个氨基酸残基差异：构建2种含YP1A碱性蛋白酶CDS的组成型穿梭表达载体pHY／aprYP与pHY／aprP43,前者采用YP1A蛋白酶自带的启动子,后者则采用来自于质粒pP43NMK的P43强启动子。利用这2种表达载体在枯草芽孢杆菌WB800中成功进行蛋白酶的功能表达．其中P43强启动子的表达能力明显优于碱性蛋白酶自带的启动子,表达的蛋白酶比酶活为395U／ml。重组菌表达的碱性蛋白酶在体积分数50％的亲水及疏水有机溶剂中表现出了很好的耐受性,验证了克隆基因为地衣芽孢杆菌YP1A的高强度耐有机溶剂碱性蛋白酶基因.     

Cloning and expression of a bpr gene encoding Bacillopeptidase F from Bacillus amyloliquefaciens CH86-1

A gene encoding bacillopeptidase F, bpr86-1, was cloned from B. amyloliquefaciens CH86-1 isolated from cheonggukjang. This gene could encode a preproenzyme of 1,431 amino acids. When bpr86-1 was introduced into B. subtilis WB600 via pHY300PLK, an E. coli-Bacillus shuttle vector, the transformant showed fibrinolytic activity. During growth on LB, the fibrinolytic activity of cells increased sharply when they entered the stationary phase. The highest activity (761.4 mU/mg protein) was observed at 96 h of cultivation.     

Cloning and expression of subtilisin amylosacchariticus gene

The gene encoding subtilisin Amylosacchariticus from Bacillus subtilis var. amylosacchariticus was isolated and the entire nucleotide sequence of the coding sequence was determined. The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprising 275 residues. There were discrepancies in 10 amino acids between the sequence elucidated from the nucleotide sequence and the published protein sequence (Kurihara et al. (1972) J. Biol. Chem. 247, 5619-5631). The nucleotide sequence was highly homologous to that of subtilisin E gene from B. subtilis 168, with discrepancies at 12 nucleotides out of 1,426 nucleotides we sequenced. Ten of them were found in mature subtilisin coding sequence, which resulted in two amino acid changes and another one was in the putative promoter region between two genes. The productivity of subtilisin in culture broth of B. subtilis var. amylosacchariticus was much higher than that of B. subtilis 168. The enzyme gene was inserted in a shuttle vector pHY300PLK, with which B. subtilis ISW1214 was transformed. The proteolytic activity found in the culture broth of the transformed bacterium was 20- and 4-fold higher than those of the host strain and B. subtilis var. amylosacchariticus, respectively. Subtilisin Amylosacchariticus was easily purified to a crystalline form from culture filtrate of cloned B. subtilis, after a single step of chromatography on CM-cellulose.     

Nucleotide sequence of the gene for an alkaline endoglucanase from an alkalophilic Bacillus and its expression in Escherichia coli and Bacillus subtilis.

The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp. KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-1 and ORF-2. The protein deduced from ORF-1 was composed of 244 amino acids with an M(r) of 27,865. Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an M(r) of 91,040). Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like sequence (TGTAAGCGGTTAACC). The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E. coli and B. subtilis. One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expression in both host organisms.     

利用短短小芽孢杆菌启动子和信号肽编码序列构建穿梭分泌表达载体

以短短小芽孢杆菌B15的总DNA为模板,利用PCR技术克隆到其细胞壁蛋白基因串联启动子和信号肽编码序列,测序分析后提交GenBank,登录号为AY956423。重新设计引物扩增该片段并在PCR产物两侧引入BamHⅠ和PstⅠ酶切位点,将PCR产物双酶切后克隆至穿梭载体pP43NMK的相应位点构建分泌表达载体pP15MK,插入片段置于该载体中mpd基因的上游,并使信号肽编码序列与去除了自身信号肽编码序列的mpd基因阅读框恰好融合。将pP15MK导入枯草杆菌构建表达菌株1A751(pP15MK),在短短小芽孢杆菌启动子和信号肽元件的带动下,mpd基因能够在表达菌株的对数生长期和稳定期持续性高效分泌表达,表达产物结合在细胞膜上;发酵液在48h酶活达到最高值7.79U/mL,是出发菌株邻单胞菌M6表达量的8.1倍。     

通过信号肽筛选优化耐高温α-淀粉酶在枯草芽孢杆菌中的分泌

以大肠-枯草穿梭载体p MA5质粒为基本骨架,以来源于嗜热脂肪地芽孢杆菌Geobacillus stearothermophilus NUB3621的耐高温α-淀粉酶基因为目标基因,利用POE-PCR法,成功构建针对淀粉酶的信号肽筛选载体。从枯草芽孢杆菌168基因组中扩增得到46个信号肽,利用POE-PCR法,使46个信号肽分别与线性化的筛选载体形成对应的multimer产物,直接转化枯草芽孢杆菌1A751,得到含不同信号肽的重组菌株。发酵结果显示,除了5个与淀粉酶适配性很低的信号肽,其它信号肽均有不同的引导淀粉酶细胞外分泌的能力,其中bgls引导淀粉酶细胞外分泌的能力最强,上清酶活的峰值达1 393.3 U/m L。     

A DREPP protein interacted with PeaT1 from <Emphasis Type="Italic">Alternaria tenuissima</Emphasis> and is involved in elicitor-induced disease resistance in <Emphasis Type="Italic">Nicotiana</Emphasis> plants

PeaT1 is a proteinaceous elicitor from fungal pathogen Alternaria tenuissima. Our previous research revealed that this elicitor could induce defense response and enhance disease resistance in various plants including Nicotiana plants. However, immune activation mechanisms whereby PeaT1 elicits defense response remain unclear. In this study, the association between elicitor protein PeaT1 and the plasma membrane was assessed using the FITC (Fluorescein isothiocyanate) labeling method. A PeaT1-interacting protein was isolated via ¹²⁵I-PeaT1 cross-linking and Far Western blot analyses, and designated PtBP1 (PeaT1 Binding Protein 1). From the data of Mass spectrometry (MS) and bioinformatics analysis, the 22 kDa plasma membrane protein PtBP1 was inferred to be a member of DREPP (developmentally regulated plasma membrane polypeptide) family that is induced in plants under stress conditions and might get involved in downstream signaling. For further verification of this association, Far Western blot, co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) analyses were performed, showing PtBP1 could bind with PeaT1 in vitro and in vivo. Virus-induced gene silencing (VIGS) analysis exhibited that PtBP1 silencing in Nicotiana benthamiana attenuated tobacco mosaic virus (TMV) resistance compared to the tobacco rattle virus (TRV) control after PeaT1 treatment.