酒类酒球菌mleP基因的克隆及其在酿酒酵母中的表达

苹果酸通透酶具有协助苹果酸 乳酸发酵 (MLF)的重要功能。以酒类酒球菌 (Oenococcusoeni)优良菌系Oenococcus Lee SD 2a的总DNA为模板 ,用PCR方法克隆到苹果酸通透酶基因mleP ,构建了重组质粒pBMmleP。序列分析表明克隆到的基因序列与已报道的序列同源性为 99%。为使目的基因在酿酒酵母中表达 ,以大肠杆菌 酿酒酵母穿梭质粒YEp35 2为载体 ,以PGK1强启动子和ADH1终止子为调控元件 ,构建了重组表达质粒YEpmleP ,并转化酿酒酵母 (Saccharomycescerevisiae)YS5 8。酵母转化子用含有亮氨酸、组氨酸和色氨酸的YNB平板筛选鉴定。获得的转化子在添加了L 苹果酸 (5g L)的培养基中培养 4d ;取培养液上清用HPLC检测 ,结果显示重组转化子YSP的培养液中L 苹果酸剩余含量均低于空载体转化子YS35 2 ,因此所得酵母重组转化子对苹果酸的转运能力有所提高     

核盘菌属一新种——人参核盘菌

本文报道了采自人参(Panax ginseng C. A．Mey (保留名nom. Coiiscry))的核盘菌属一新种——人参核盘菌(Sclerotinla．Ginseng Wang C. R.,C. F. Chen et J. Chen)。该种在形态学以及可溶性蛋白、果胶(甲)酯酶和多聚半乳糖醛酸酶谱带等方面,均不同于已知种核盘菌(S. sclerotiorum (Lib．)de Bary),小核盘菌(S. minor Jagger)车轴草核盘菌(S．Trifoliorum Erikss．)和细辛核盘菌(S．Asari Wu et C．R. Wang)。模式标本(800719)保存于沈阳农业大学植物免疫研究室。     

核盘菌角质酶的克隆、表达及活性分析

角质酶可降解脂肪族或芳香族聚酯,对聚对苯二甲酸乙二醇酯也具有较好的降解作用,但目前市面上角质酶产品非常稀缺,因此寻求一种高效表达的角质酶用于工程酶的开发非常有必要.本研究从核盘菌中克隆得到8个角质酶基因,利用PCR结合RT-PCR技术筛选出主效基因SsCut-52,利用大肠杆菌Escherichia coli BL21...     

mel基因在酿酒酵母菌中的克隆和表达*

构建了含mel基因的重组质粒pAJM,并转化到酿酒酵母菌中,获得了在平板上产生黑色表型的转化重组子,为酵母菌的遗传操作提供了一种新的选择标记。由于mel基因经酪氨酸产生的黑色素无毒、无害,安全稳定,无需另外加显色化合物或使用特殊的仪器设备,直接在平板上观察结果。因此,是一种便捷、价廉、无污染的新型报告基因。此外,实验中还发现含mel基因的酵母菌生长迅速,这不仅作为报告基因更能显示其优势,而且也为进一步研究其在酵母菌中的功能提出了新的内容。     

酿酒酵母海藻糖合成酶基因的克隆和在大肠村菌中的表达

用PCR方法克隆了1．5kb的酿酒母Sacchromyces cerevisiae海藻糖合成酶基因TPSI,将该片段连接到pUC19载体,通过转化分别引入海藻糖合成酶基因缺失和缺陷的大肠杆菌Escherichia coli FF4169 和FF4050,对转化株的质粒DNA酶切分析表明均含有1．5kb PCR克隆片段,生长曲线实验证明,带有克隆片段的转化株在含0．5mol/L NaCl的高渗透压基础培养基中生长良好;用高效液相色谱（HPLC）结合蒸发散射（ELSD）技术测定细胞内海藻糖实验证明转化株能够合成海藻糖。     

AtWRKY63基因过表达拟南芥对核盘菌抗性的研究

为研究草酸在核盘菌致病过程中可能的作用,以模式植物拟南芥为材料,采用30mmol/L草酸喷施3周龄拟南芥,发现草酸显著诱导拟南芥AtWRKY63的表达。通过构建AtWRKY63过表达载体转化拟南芥,获得过表达AtWRKY63的纯系转基因植株,再用核盘菌活体接种拟南芥,结果表明过表达AtWRKY63植株对核盘菌的抗性显著增强。组织化学染色结果表明,AtWRKY63是通过诱导植物的氧爆发,抑制核盘菌菌丝的生长来抵御核盘菌的侵染;qRT-PCR对拟南芥转录水平分析表明,AtWRKY63可能激活了过表达植株的水杨酸与茉莉酸依赖的抗病信号途径,从而增强对核盘菌的抗性。     

酿酒酵母GAL基因的表达调控

酵母菌一直是人们进行遗传分析的理想材料,对许多酵母基因的表达调节途径人们也已有了较深人的了解。酵母中研究最多的基因表达调节途径是GAL基因表达调节过程,它不仅为比较研究原核和真核生物的协同表达调节机制提供了可能性,而且由于酵母GAL基因表达可以被生长条件所控制,所以GAL基因区域对外源基因在酵母中的克隆表达也具有潜在的应用价值I‘,‘］。现在就酵母GAL基因的表达调节做一概述。IGAL基因组成及转录调节模型半乳糖是通过转化为6一磷酸葡萄糖后进人糖解途径而被酵母利用的。其中至少有五种基因产物参与从半乳糖的运…     

苹果酸降解相关基因在酿酒酵母中的表达

微生物降酸是现代葡萄酒酿造重要工艺。将裂殖酵母苹果酸通透酶基因(mae1)和苹果酸酶基因(mae2)克隆到酿酒酵母中,构建了苹果酸酒精酵母;将mae1基因和乳酸乳球菌的苹果酸乳酸酶基因(mleS)克隆到酿酒酵母中,构建了苹果酸乳酸酵母。构建的酵母重组子能够有效地分解发酵基质中的苹果酸。     

花生油酸脱氢酶基因在酿酒酵母中的高效表达

将克隆的油酸脱氢酶基因(AF900663)亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES6/CT,从大肠杆菌中筛选到含有目的基因的重组质粒pYES/HO-A,用醋酸锂方法转化到酿酒酵母缺陷型菌株INVScI中,经半乳糖诱导后,收集菌体,用气相色谱质谱(GC-MS)仪分析转化酵母的脂肪酸色谱的结果表明,HO-A所编码的酶具有油酸脱氢酶活性,能将酵母内源性油酸转化为亚油酸,油酸脱氢酶的表达量为15.6%,高于已有的报道。     

aspS encoding an unusual aspartyl protease from Sclerotinia sclerotiorum is expressed during phytopathogenesis

The gene aspS encoding an aspartyl protease has been cloned from Sclerotinia sclerotiorum by screening a genomic library with a PCR-amplified fragment of the gene. The open reading frame of 1368 bp interrupted by one intron would encode a preproprotein of 435 amino acids. The catalytic aspartyl residues characteristic of aspartyl proteases are conserved; however, the active-site motif (DSG) in the N-terminal lobe is unusual in that Ser replaced Thr used in the active-site motif (DTG) of the C-terminal lobe and in all other fungal aspartyl proteases. RT-PCR revealed that aspS expression in axenic culture is not subjected to catabolite repression and demonstrated that aspS is expressed from the beginning of infection of sunflower cotyledons.     

酿酒酵母INVSC1对油菜菌核病菌抑制作用的研究

以油菜菌核病菌为供试病原菌,在摇瓶发酵备件下,研究了酿酒酵母菌INVSC1在各种培养条件下对油菜菌核病菌的抑菌效果,确定了酿酒酵母的最佳培养条件：最佳培养温度为28℃,培养时间为20h,接种量为5％。酿酒酵母能抑制油菜菌核病可能与它发酵产乙醇有关。酿酒酵母抑制油菜菌核病的发现使生物防治又多了一类新的可供选择的生防菌。     

PGA基因在酿酒酵母中的表达研究

本文根据GenBank 中巨大芽孢杆菌（Bacillus megaterium）的PGA基因序列设计了上下游引物,通过PCR扩增出巨大芽孢杆菌1.1741中的PGA基因。将该基因连接到T7lac启动子控制下的表达载体pYES2(amp+,ura+)上,构建了重组质粒pYES2-PGA。用LiAc/SSDNA/PEG方法将其转化进酿酒酵母(Saccharomyces cerevisiae)H158中表达,在不需要苯乙酸诱导的重组菌株发酵液中检测到了青霉素酰化酶活性,最高酶活达到0.75 U/ml。将该PGA基因测序结果与GenBank中巨大芽孢杆菌L04471.1、U07682.1和Z37542三株的PGA基因序列比对,表现出很高的同源性,分别达到97.1%、99.8% 和99.8%。     

絮凝基因的克隆和在工业啤酒酵母菌株中表达

The partial genomic library of Saccharomyce cerevisiae FL189 possessing strong flocculation ability was constructed using Yeast-E.coli shuttle plasmid YCp50 as vector.Recombinant plasmid containing flocculation gene was obtained by screening the growth of transformants on the selective medium and measurement flocculation,designated as pCF1.pCF1 was introduced into industrial yeast strain PJ208-5-15.Six transformants PJ208-5-15-1(pCF1)～PJ208-5-15-6(pCF1)possessing strong flocculation ability were obtained.Th…     

酿酒酵母乙醇脱氢酶2(ADH2)基因的克隆表达及活性验证

为了从酿酒酵母Saccharomyces cerevisiae中克隆出乙醇脱氢酶2(Alcoholdehy drogenase2,ADH2)基因并使之在大肠杆菌中高效表达。以酿酒酵母细胞中提取的总RNA为模板,通过反转录获得酿酒酵母乙醇脱氢酶2基因,连接到表达载体pTAT上,得到重组表达质粒pTAT-ADH2,将此重组质粒转化到大肠杆菌BL21中,重组工程菌株经IPTG诱导表达得到ADH2蛋白。将该蛋白纯化后,在体外进行活性检测和小鼠体内进行毒理试验,检测ADH2的酶活性。测序结果表明克隆的基因与GenBank中所报道的adh2基因序列有90%的同源性,经SDS-PAGE电泳分析,目的蛋白得到了有效表达,蛋白条带扫描分析表明,表达量占总蛋白的50%左右,纯化得到的蛋白在小鼠体内进行毒理试验,显示出一定的活性。酿酒酵母adh2基因的克隆正确,不仅在大肠杆菌中进行了高效表达而且表现出了较好的酶活性。     

Polycistronic gene expression in yeast versus cryptic promoter elements

Saccharomyces cerevisiae is a much preferred host for biotechnological applications. However, the expression of entire heterologous pathways, required for some potential products, is technically challenging in yeast. A possible tool would be polycistronic gene expression. Recent studies demonstrated that short 5' untranslated regions (5'UTRs) found upstream of certain genes support cap-independent translation in vitro. In this study 5'UTRs were used as linkers between genes in polycistronic constructs. Expression levels of genes located in the first, second and third position after a promoter were studied by replacing the respective gene by a promoterless green fluorescence protein (GFP) gene. S. cerevisiae transformed with these constructs was grown on different carbon sources and GFP expression was assayed. Our results demonstrate that (i) ribosomal read-through does not suffice for polycistronic gene expression in vivo, (ii) 5'TFIID and 5'HAP4 but not 5'L-A significantly improve the expression of a reporter gene located second in a bicistron, (iii) 5'TFIID, 5'HAP4 and 5'YAP1 but not 5'L-A can drive expression of a promoterless reporter gene, and (iv) expression driven from 5'TFIID, 5'HAP4 and 5'YAP1 is induced in the presence of raffinose or galactose but not in the presence of glucose. This implies that these elements unlike typical internal ribosome entry site-like structures contain small, potentially useful promoters which support carbon source-regulated expression.