紫杉烯合酶基因遗传转化金针菇的研究

利用紫杉烯合酶基因(ts)和潮霉素抗性基因(hph),采用PEG转化法共转化金针菇的原生质体.研究结果表明,18个拟转化子中有8个转化子同时整合了ts基因和hph基因,两个基因的共转化率为44.44％;RT-PCR鉴定结果表明.7个金针菇转化子实现了外源ts基因的转录.金针菇转化子在不含潮霉素(HmB)的PDA平板上经5次继代培养后仍检测到有HmB抗性,说明外源基因在金针菇转化子的无性繁殖(即有丝分裂)过程中是稳定的.本研究为通过基因工程手段定向、快速改良金针菇品种以及利用金针菇作为生物反应器生产一些具有重大经济价值的外源基因产物奠定了基础.     

木糖还原酶基因整合表达载体构建

木糖还原酶是酵母代谢木糖发酵的关键酶之一。根据已报道的木糖还原酶基因的全序列设计引物,从休哈塔假丝酵母中克隆得到1 110 bp大小的片段。将木糖还原酶基因亚克隆到酿酒酵母的整合表达载体p406ADH1中,醋酸锂转化法将线性化重组质粒p YX-AK-xyl1转化到酿酒酵母W5,对阳性转化子进行酶活测定,结果表明,以辅酶DANH和NADPH为底物诱导,转化子均表现出活性,酶活分别为6.513 U/mg和7.080 U/mg,是受体菌W5酶活的1.4倍(NADH)和1.3倍(NADPH),其酶活比为0.919。     

As(Ⅲ)和As(Ⅴ)在香菇栽培基质和子实体中的迁移规律

在两个生长年份里向香菇培养料中添加不同含量的亚砷酸盐（As(III)）和砷酸盐（As(V)）标准溶液,利用高效液相色谱联用电感耦合等离子体质谱法检测香菇及其培养料中5 种形态砷含量,研究香菇子实体对培养料中高毒无机砷的迁移规律。结果表明,香菇子实体对添加0.3～10 mg/kg As(III)和As(V)培养料中As(III)和As(V)的富集系数范围分别为0.36～0.84和0.82～7.53;添加0.33～0.38 mg/kg As(III)和As(V)培养料中As(III)和As(V)的迁移规律方程分别为y＝－1.138 9x2＋0.921 6x－0.004和y＝－9.024 3x2－4.388 1x＋0.838 7（x、y分别为培养料和香菇子实体中的As(III)或As(V)含量）。培养料中As(III)和As(V)含量的增加会导致香菇子实体的减产甚至绝收,其临界值为0.33 mg/kg。培养料中的As(III)含量随着栽培时间的延长逐渐减少,而As(V)含量则略有增加,香菇子实体中的有机砷含量也会随着As(III)和As(V)添加量的增加而提高,这可能和As(III)在香菇生长时转化为As(V)或有机砷有关。     

黑曲霉高效敲除体系构建及tpsA基因的敲除

为完善高效的黑曲霉工程菌10142基因敲除体系,提高其基因敲除效率,构建含有致死基因单纯疱疹病毒胸苷激酶(herpes simplex virus thymidine kinase,HSVtk)的基因敲除质粒,通过农杆菌介导的黑曲霉转化(Agrobacterium tumefaciens-mediated transformation,ATMT)法,使致死基因随机插入黑曲霉基因组,其阳性转化子在添加10μmol/L的5-氟脱氧尿苷的培养基上无法生长,从而得到致死基因随机插入转化子的"反向筛选"方法。利用该方法对黑曲霉海藻糖-6-磷酸合成酶进行基因敲除,结果表明阳性转子占总转化子的比例为63%。成功构建高效黑曲霉基因敲除体系。     

热带假丝酵母磷酸甘油酸激酶基因启动子功能鉴定

热带假丝酵母是重要的工业生产菌株,但表达系统还不完善导致代谢工程育种手段受到限制。探索新的表达元件对热带假丝酵母的基因工程育种十分重要。本文通过多重序列比对从热带假丝酵母ATCC 20336基因组中扩增得到一个具有较高活性的磷酸甘油酸激酶基因(PGK1)启动子,以酵母增强型绿色荧光蛋白(yeGFP3)作为报告基因,整合到热带假丝酵母基因组上并对其表达活性进行研究。通过分离300、550、750 bp和846 bp4个长度的启动子片段分别表达yeGFP3,转化XZX宿主菌获得P-1、P-2、P-3和P-4共4个转化子。荧光显微镜结果表明,随着启动子长度的截短,荧光逐渐减弱,前3个转化子的相对荧光强度分别为P-4的4.98%、9.84%和48.4%。同时分析了4个转化子的yeGFP3转录水平,转录水平分别为P-4的5.6%、13.8%和60%。初步判断PGK1启动子至少存在2个上游激活序列(UAS)。     

羟酸还原异构酶基因多拷贝重组酵母的构建

利用PCR技术扩增出啤酒工业酵母QY的羟酸还原异构酶基因ILV5 ,在酵母YS5 8中表达时 ,羟酸还原异构酶活力提高 2～ 3倍。将ILV5基因与铜抗性基因插入到YEp3 5 2载体中得到重组质粒 pLZ -5C ,转化QY ,通过铜抗性筛选转化子 ,所得转化子的羟酸还原异构酶的活力明显高于对照菌株QY ,在发酵测试中 ,转化子产生双乙酰的量比原始菌株降低了 60 %。     

日本曲霉β-呋喃果糖苷酶基因克隆及在酿酒酵母中的表达

通过反转录PCR法成功地扩增出日本曲霉β-呋喃果糖苷酶基因，基因片断长度为1917bp，编码638个氨基酸。将所得片断定向克隆到pYX212载体上，并转化至酿酒酵母中，通过酵母菌落PCR检验后，确定该基因在酿酒酵母中获得表达，转化子平均酶活为26．4U／mg。     

酱油工业生产菌沪酿3042基因工程转化体系的构建

以工业酿造常用菌株米曲霉沪酿3042为出发菌株,采用紫外诱变和氯酸钾压迫相结合的方法,得到硝酸盐营养缺陷型(niaD-)宿主菌,将含有niaD基因的载体转化到该宿主中,转化率为0.7个转化子/μg DNA,相对比较低,但转化子传代数次后遗传仍很稳定,该转化体系可用于工业生产菌沪酿3042的遗传改造.     

利用RNAi抑制cre1基因转录提高里氏木霉表达纤维素酶

本研究采用RNA干扰技术(RNAi)对里氏木霉主要的转录抑制因子cre1基因的表达进行抑制,以期提高里氏木霉生产纤维素酶的能力。通过PCR,从里氏木霉的基因组中扩增得到cbh1启动子、反向的cre1基因片段(568~963 bp)、正向的cre1基因片段(655~961 bp)和cbh2终止子,利用DNA assembler方法将这些基因连接到pRS424质粒上,构建成p Cre1-i质粒。再将RNAi盒分作两个片段扩增,同时转化里氏木霉并通过PCR鉴定阳性转化子。摇瓶发酵显示其中一株转化子在纤维素诱导培养基中培养4.5 d后,其纤维素酶的滤纸酶活、内切葡聚糖酶活和CBHI酶活为0.67、3.70和0.46 U/m L,较出发菌株分别提高了1.3、1.8和5.6倍。通过实时荧光RT-PCR检测,发现该转化子中cre1基因的转录水平比出发菌株降低了43%。     

植物乳杆菌melA基因的克隆及其作为食品级筛选标记的初步研究

以L. plantarum1.557的基因组DNA为模板,通过PCR技术成功克隆α-半乳糖苷酶基因(melA基因),与报道的melA基因序列同源性达到99%以上。再以大肠杆菌-乳酸菌穿梭表达载体pMG36e为基本骨架,将melA基因插入该载体,构建melA基因标记的穿梭表达载体pMG36e-melA。重组表达质粒pMG36e-melA经Sac I和Sph I双酶切和PCR鉴定与预期片段大小相符。结果表明已初步构建了以melA基因为食品级筛选标记的重组载体。     

嗜碱耐盐芽孢杆菌乙醛脱氢酶基因aldC的克隆及组氨酸融合酶蛋白的表达

应用PCR扩增嗜碱耐盐芽孢杆菌乙醛脱氢酶基因aldC,重组至原核表达载体pET30b（＋）,转化大肠杆菌BL21（DE3）,诱导表达的重组乙醛脱氢酶在碳端含有一个组氨酸标签。SDS-PAGE考察蛋白的表达量和亚基分子量,并对Km值进行了考察。成功构建了pET30b-aldC原核表达质粒;诱导表达了亚基分子量约为56 kDa的组氨酸融合蛋白;乙醛脱氢酶活性比对照菌增加了约3.5倍;Km值为2.874 mmol/L。成功克隆并诱导表达了含组氨酸标签的嗜碱耐盐芽孢杆菌乙醛脱氢酶基因aldC。     

海洋放线菌（Salinispora arenicola）基因转移系统的建立

该实验旨在建立海洋放线菌Salinispora arenicola基因转移系统,以便基因敲除和外源基因表达等遗传操作。以整合型质粒pSET152和pIB139为出发质粒,通过接合转移构建了海洋放线菌Salinispora arenicola的基因转移系统。结果表明,25 μg/mL阿泊拉霉素可有效筛选接合子,大肠杆菌与孢子的比例为8∶1时可获得较多的转化子。经聚合酶链式反应（PCR）验证,质粒成功整合到菌株海洋放线菌S. arenicola基因组中,接合子经多次传代后,导入的质粒pSET152和pIB139仍稳定整合于接合子基因组上。成功构建了海洋放线菌S. arenicola接合转移系统。     

高效表达淀粉酶的工业酿酒酵母菌株的构建

将PCR扩增的扣囊腹膜孢酵母菌淀粉酶基因 ,与磷酸甘油酸激酶基因 1启动子和α 分泌序列一起插入含 2 μm的酵母穿梭质粒YEp3 5 2 ,构建酵母菌重组表达质粒并命名为pLA8α。用醋酸锂转化法转化工业酿酒酵母Sc 1 6,转化子培养上清液的淀粉酶活性为 6 8U/mL ,46%的淀粉被降解 ,SDS PAGE检测到约 5 5ku的蛋白条带。转化子在非选择条件下的遗传稳定性为82 %。     

菊粉酶基因克隆表达与油脂的组分分析

为了实现解脂亚罗威亚酵母（Yarrowia lipolytica）直接利用菊粉进行油脂生产,将外切菊粉酶基因INU1与表达质粒pINA1317连接,在解脂亚罗威亚酵母（Y. lipolytica）ACA-DC尿嘧啶缺陷突变菌株中表达。以尿嘧啶缺陷型筛选作为筛选标记获得转化子C37,经过培养菊粉酶酶活达到37.15 U/mL。在2 L发酵罐中,转化子C37以菊粉为底物进行发酵,油脂产量和细胞干质量分别为49%和14 g/L。脂肪酸分析结果显示棕榈酸、硬脂酸和油酸总和占总脂肪酸的92%以上,其中油酸含量高达59%,表明通过菊粉酶基因在解脂亚罗威亚酵母中的表达,实现了以菊粉为底物一步发酵产单细胞油脂。     

Use of the gfp gene in monitoring bacteriocin-producing Lactobacillus plantarum N014, a potential starter culture in nham fermentation

Lactobacillus plantarum N014 is a bacteriocin-producing lactic acid bacteria originally isolated from nham, a traditional Thai fermented sausage, and in the process of development to be used as a starter culture for nham fermentation. During the fermentation process, there is a need to identify the starter culture among several naturally occurring bacteria. In this study, a new plasmid carrying the gfp (green fluorescent protein) gene was constructed based on pGKV210, an Escherichia coli/ Lactococcus shuttle vector containing an erythromycin resistance marker. The gfp gene derived from pGFPuv was placed under the control of an L-lactate dehydrogenase promoter and then inserted at the EcoRI site of pGKV210, leading to pN014-GFP. The novel plasmid was used to transform L. plantarum N014, which is a bacteriocin-producing lactic acid bacteria isolated from nham. The resulting transformant, L. plantarum N014-GFP+, was brightly fluorescent and harbored the expected plasmid. A plasmid stability test revealed that pN014-GFP was stable after 100 generations of growth under nonselective pressure. L. plantarum N014-GFP+ and its parent strain were shown to be very similar in growth rate, bacteriocin production, and lactate production. L. plantarum N014-GFP+ was able to survive in a nham model. The survival clones were still fluorescent and harbored pN014-GFP.     

乙醇脱氢酶I基因敲除的酿酒酵母重组菌构建的初步研究

本实验根据酿酒酵母乙醇代谢途径,构建一株低乙醇产量的酿酒酵母基因工程菌株,以满足人们对低醇啤酒的需要。利用抗性基因筛选基因敲除突变体的方法,通过引物L1和L2扩增潮霉素B基因(两翼与酿酒酵母同源),按常规醋酸锂法转化酵母细胞后,筛选标记与酵母adhI基因发生同源重组,得到一株ADHI酶活性降低的工程菌株。发酵实验结果表明,转化菌株乙醇含量平均值为1.8%(V/V),较原始菌株低了65%。说明转化菌株体内乙醇生成途径受到干扰。     

PROPERTIES OF A GENETICALLY-ENGINEERED DEXTRIN-FERMENTING STRAIN OF BREWERS' YEAST

A dextrin-fermenting strain of brewer's yeast was obtained by transformation with a recombinant plasmid (pLHCD6) containing a DEX gene. The transformant produced five-fold more extracellular amyloglucosidase (AMG) than the strain of Saccharomyces diastaticus from which DEX was cloned. The growth rate, however, was adversely affected and, consequently, the transformant fermented wort more slowly than the parental (untransformed) strain. A mixed culture was therefore used at pilot-scale, to maintain a rate of fermentation similar to that of the parental strain. The transformant by itself was capable of superattenuating wort (original gravity 1.040) to a specific gravity of 1.0023 (compared with 1.0046 for the parental strain). This represents the expected degree of dextrin breakdown by an AMG with no “debranching” activity which can only hydrolyse alpha-1,4 linkages. A high copy number for the plasmid pLHCD6 was maintained throughout fermentation. The beers produced using the Dex+ transformants were judged to be of sound quality.     

植物乳杆菌亚硝酸盐还原酶基因在大肠杆菌中表达

构建乳杆菌亚硝酸盐还原酶基因的重组质粒并表达。以植物乳杆菌基因组DNA为模板,PCR扩增亚硝酸盐还原酶基因,连接到表达载体pET-32a(+)上,构建的重组载体转入大肠杆菌BL21(DE3)中,经IPTG诱导表达后,菌体总蛋白经SDS-PAGE鉴定显示重组质粒诱导表达产物有特异性蛋白条带。该表达产物经细菌亚硝酸盐还原酶试剂盒测定显示有明显的酶活性。实验结果为深入研究植物乳杆菌亚硝酸盐还原酶提供有益的参考。     

Overexpression and purification of Aspergillus aculeatus beta-mannosidase and analysis of the integrated gene in Aspergillus oryzae

An expression plasmid for the manB gene encoding Aspergillus aculeatus beta-d-mannosidase (MANB) was constructed by using an expression vector carrying an improved promoter. After transformation of A. oryzae by the plasmid, several transformants formed colonies emitting fluorescence on a plate containing 4-methylumbelliferyl beta-d-mannopyranoside (MU-Man) under UV-irradiation. The transformant that displayed the strongest fluorescence, named A. oryzae BMN1, produced about 270 mg MANB/l in liquid culture. Recombinant MANB overproduced in BMN1 was purified by two steps of column chromatography to a single protein band on SDS-polyacrylamide gel electrophoresis and had a molecular weight of 130,000. Analyses by Southern blotting and genomic PCR demonstrated that a single copy of the plasmid was integrated into the chromosome by recombination at the niaD locus.