枯草芽孢杆菌基因的安全改良研究

为了构建能稳定表达外源基因且无抗性标记基因的枯草芽孢杆菌(Bacillus subtilis)工程菌。采用聚合酶链式反应(PCR)技术,分两段扩增定位于B.subtilis BJ3-2染色体上的谷氨酰胺酶基因(glsA)上下游基因片段作为同源臂。将同源臂及卡那霉素基因(Kan)克隆入温敏型载体pKSV7,构建了双交换载体pKUKD。并利用pKUKD质粒将kan基因定点整合入BJ3-2染色体上,通过Kan抗性筛选获得glsA敲除菌株BJ-kan。经检测BJ3-2菌株谷氨酰胺酶活力比重组菌株高3.2倍。结果显示:BJ3-2染色体上的基因可通过同源重组方式进行敲除与置换,为今后BJ3-2菌株以及野生型B.subtilis基因敲除与置换提供了更加便捷的方法。     

生物合成γ-氨基丁酸酿酒酵母谷氨酸脱羧酶基因的克隆与表达

为了提高酵母菌的γ-氨基丁酸产量,本研究以十六烷基三甲基溴化铵法(hexadecyl trimethy ammonium bromide,CTAB)提取的酿酒酵母28基因组DNA为模板,扩增得到序列长度为1 758 bp的GAD1基因,经比对与S.cerevisiae S288c的一致性达到98.58%,并设计引物扩增包括GAD1基因上游启动子及调控序列,下游终止子在内的全长基因序列,长度为3 490 bp。将全长基因序列克隆至高拷贝质粒p UG6,构建重组质粒p28。以菌株28作为出发菌株进行转化,经G418抗性筛选得到转化子,进一步经过聚合酶链式反应(polymerase chain reaction,PCR)实验验证,质粒提取及酶切验证,确证得到重组子DL28。经重组质粒p28的特性研究得知,重组质粒p28具有良好的遗传稳定性,无抗性传代培养10次重组质粒不丢失。转化后进行酶活力测定,重组子DL28的谷氨酸脱羧酶(glutamic acid decarboxylase,GAD)酶活力比菌株28提高73.8%。通过以上实验结果得出结论,GAD基因高表达菌株DL28具有更高的G418抗性和GAD酶活性。     

采用同源重组技术构建金黄色葡萄球菌sae基因缺失突变株

针对金黄色葡萄球菌sae基因前后两段序列设计两对引物,PCR扩增出sae基因上下游同源臂序列,克隆到穿梭载体pBT2中;两段序列之间用来自质粒p646的Em抗性基因片段连接,作为筛选标记,从而构建同源重组穿梭质粒pBT2△sae;将pBT2△sae电转化到金黄色葡萄球菌菌株RN4220中,40℃经过七轮培养,进行抗性培养基筛选和PCR验证,以及RT-PCR观察基因表达水平.获得一株金黄色葡萄球菌sae基因缺失突变株RN△sae,为进一步研究sae基因的调控机制等提供有用的实验材料.     

葡萄糖氧化酶基因在黑曲霉中的分泌表达

通过PCR扩增从一株葡萄糖氧化酶生产菌黑曲霉中克隆得到1818bp葡萄糖氧化酶基因GOD,将GOD基因与糖化酶基因的5’同源臂、3’同源臂进行重叠延伸,构建葡萄糖氧化酶基因的表达框。将此表达框插入载体pSZH中,构建黑曲霉同源重组表达载体pSZH-GOD。将载体pSZH-GOD通过冻融法转化农杆菌AGL1,进而通过农杆菌介导法转化高产糖化酶的黑曲霉。经潮霉素筛选和PCR鉴定获得8株重组菌株。对8株重组菌株的发酵液上清液进行酶活测定,结果表明葡萄糖氧化酶基因在高产糖化酶的黑曲霉中得到了分泌表达,静置培养6d后其分泌表达的葡萄糖氧化酶酶活最高可达1.166U/mL,而在出发菌株中检测不到葡萄糖氧化酶的分泌表达。同时,重组菌株胞内表达的葡萄糖氧化酶最高酶活可达11.45U/mL,是出发菌株的266倍。此研究结果为简化葡萄糖氧化酶发酵生产工艺、提高酶产量提供了新的途径。     

门多萨假单胞菌Pseudomonas mendocina P10脂肪酶基因的克隆及其在酵母中的表达

采用同源克隆法获得了门多萨假单胞菌Pseudomonas mendocina P10脂肪酶基因的全长序列,DNA测序结果表明,该基因的DNA全长序列为801bp,编码267个氨基酸,推测蛋白相对分子量为29.95ku.将该脂肪酶基因连接到pPICZαA载体上得到重组表达质粒,转化Pichia pastoris X-33.脂肪酶基因在Pichia pasttoris X-33中实现了分泌性表达,摇瓶发酵液上清酶活最大可达8U/mL.     

同源片段长度对副干酪乳杆菌同源重组频率的影响

目的:探索同源片段大小与副干酪乳杆菌同源重组率之间的关系。方法:分别构建以氨苄青霉素和四环素抗性标记基因为遗传转化筛选标记的,用于敲除prc K,prc R基因的4个同源重组质粒p2NIL-KA(625 bp,650bp),pUC18-KT(1 350 bp,1 350 bp),p2NIL-RT(433 bp,474 bp),pUC18-RT(1 350 bp,1 350 bp),并分别电转化于副干酪乳杆菌HD1.7感受态细胞中,检测筛选同源重组转化子,计算发生同源重组的频率。结果:成功构建同源重组质粒,p2NIL-KA(625 bp,650 bp),pUC18-KT(1 350 bp,1 350 bp),p2NIL-RT(433 bp,474 bp),pUC18-RT(1 350 bp,1 350 bp)同源重组频率分别为0,1.85%,0,1.78%。结论:当同源片段长度为1 350 bp或更长时,可与副干酪乳杆菌染色体进行同源重组。     

四川泡菜中植物乳杆菌亚硝酸盐还原酶基因克隆与表达

目的:对植物乳杆菌中亚硝酸盐还原酶基因进行克隆并表达,并测定酶的活力。方法:根据Gen Bank中亚硝酸盐还原酶(NIR)基因序列,设计引物。提取植物乳杆菌的DNA,采用PCR技术扩增nir基因,TA克隆构建获得重组质粒p MD19-nir,分析其核苷酸序列同源性。通过双酶切连接到表达载体p ET-32a(+)中,获得重组表达载体p ET-32a-nir,构建成功后转化到E.coli BL(DE3)中进行表达研究。经IPTG诱导表达,得重组蛋白,超声波破碎,提取粗酶液,测定酶活力。结果:克隆的目的基因序列长度为1638 bp。获得重组蛋白分子量为80 ku,酶活力为4.2 U/mg。结论:成功克隆了植物乳杆菌中的亚硝酸盐还原酶基因,成功的导入到E.coli BL(DE3)中,表达产物有酶活。     

乳酸菌胞外蛋白水解酶基因的克隆及在大肠杆菌中的表达

目的：构建连接乳酸菌胞外蛋白水解酶基因的非抗性重组质粒。方法：以瑞士乳杆菌基因组DNA 为模板,克隆胞外蛋白水解酶基因,连接到以thyA 为选择压力的非抗性穿梭表达载体pW425et 上,构建重组质粒pW425et-R,转化入thyA 基因缺陷型E.coli X13 感受态细胞,SDS-PAGE 电泳检测显示该水解酶得到了表达。结果：成功构建了重组质粒pW425et-R,胞外蛋白水解酶基因在E.coli X13 中得到正确表达。结论：成功地表达了乳酸菌胞外蛋白水解酶基因,可为进一步制备具降血压功能基因工程乳酸菌提供参考。     

Bar基因双元载体的快速构建及其在蛹虫草中的功能验证

为获得草铵膦完全抑制蛹虫草生长的最小浓度和一种快速构建Bar基因双元载体的方法,本文首先采用浓度梯度法研究草铵膦对蛹虫草的抑制效果,其次采用双接头PCR(DJ-PCR)构建Bar基因表达盒,然后分别采用T4 DNA连接酶法和同源重组法把Bar基因表达盒插入双元载体pAg1-H3的T-DNA区域,以构建Bar基因双元载体pAg-Bar,并比较T4DNA连接酶法和同源重组法的连接效果,最后通过农杆菌介导转化法来验证载体pAg-Bar的有效性及Bar基因在蛹虫草中的功能。结果表明:草铵膦完全抑制蛹虫草分生孢子生长的最小质量浓度为400μg/mL;采用DJ-PCR方法可快速构建Bar基因表达盒;采用T4 DNA连接酶法和同源重组法均可实现双元载体pAg-Bar的构建,而同源重组法更快速、高效;采用农杆菌介导转化法可把双元载体pAg-Bar中的Bar基因表达盒整合入蛹虫草的基因组,使蛹虫草转化子具有草铵膦抗性。本研究建立的载体构建方法具有快速、高效的特点,适用于抗性基因载体、敲除载体、超表达载体等载体的构建,为蛹虫草基因功能的鉴定提供技术支撑。     

A 2-microm DNA-based marker recycling system for multiple gene disruption in the yeast Saccharomyces cerevisiae

A molecular FRT (Flp recombinase recognition target)-based cassette system for multiple gene disruption in the yeast Saccharomyces cerevisiae was developed. FRT DNA sequences were designed with different core mutations and subsequently cloned in direct orientation upstream and downstream of a marker gene to serve as template for the amplification of a set of different gene disruption cassettes. After each disruption, the marker can be easily eliminated from its integration site by in vivo site-specific recombination between the two identical, mutated FRT sequences flanking the marker, leaving behind one FRT sequence with a particular point mutation. Since recombination between two FRTs with a different core mutation is extremely rare, the possibility of chromosome rearrangements, due to site-specific recombination between residual FRTs, is very low. In strains containing 2-microm ([cir+]) the site-specific reaction is catalysed by the endogenous Flp gene product, whereas in strains without 2-microm ([cir0]), the FLP gene is carried on the cassette, together with the marker gene. This system can be applied for haploid and diploid [cir+] and [cir0] strains.     

Disruption of six Saccharomyces cerevisiae ORFs on chromosome XII results in three lethal disruptants.

Six ORFs of unknown function from the left arm of chromosome XII of Saccharomyces cerevisiae were chosen for a reverse genetic approach to provide materials to assist in assignment of function. A two-step PCR using long-flanking homology was employed to amplify disruption cassettes consisting of a kanMX gene as selectable marker flanked by 250-350 bp long regions homologous to the target gene. The diploid strains FY1679 and CEN.PK2 were transformed with the replacement cassettes and transformants were selected for geneticin (G418) resistance. Correct targeting of the replacement cassettes at the genomic locus was verified by Southern blot analysis with the kanMX gene as a probe. Disruption cassettes were cloned in pUG7 plasmid for systematic gene inactivation in other yeast strains and the cognate genes were cloned in pRS416 plasmid for gene complementation studies. Sporulation and tetrad analysis of heterozygous disruptants showed that three of the six ORFs [YLR141w (RRN5), YLR145w and YLR147c (SMD3)] were essential genes that were complemented by their cognate genes. ylr146c Delta (spe4) homozygous diploids showed enhanced sporulation efficiency, whereas ylr147c Delta heterozygous diploids failed to sporulate in the FY1679 but not in the CEN.PK2 genetic background. The other two disruptants [ylr143w and ylr144c (acf2)] gave no phenotype.     

Disruption and phenotypic analysis of six novel genes from chromosome IV of Saccharomyces cerevisiae reveal YDL060w as an essential gene for vegetative growth

The disruption of six novel genes (YDL059c, YDL060w, YDL063c, YDL065c, YDL070w and YDL110c), localized on the left arm of chromosome IV in Saccharomyces cerevisiae, is reported. A PCR-based strategy was used to construct disruption cassettes in which the kanMX4 dominant marker was introduced between two long flanking homology regions, homologous to the promoter and terminator sequences of the target gene (Wach et al., 1994). The disruption cassettes were used to generate homologous recombinants in two diploid strains with different genetic backgrounds (FY1679 and CEN. PK2), selecting for geneticin (G418) resistance conferred by the presence of the dominant marker kanMX4. The correctness of the cassette integration was tested by PCR. After sporulation and tetrad analysis of the heterozygous deletant diploids, geneticin-resistant haploids carrying the disrupted allele were isolated. YDL060w was shown to be an essential gene for vegetative growth. A more detailed phenotypic analysis of the non-lethal haploid deletant strains was performed, looking at cell and colony morphology, growth capability on different media at different temperatures, and ability to conjugate. Homozygous deletant diploids were also constructed and tested for sporulation. Only minor differences between parental and mutant strains were found for some deletant haploids.     

甘蓝型油菜BnADC基因的克隆及原核表达

利用同源克隆和RACE（Rapid amplification of cDNA ends）技术从甘蓝型油菜中克隆到精氨酸脱羧酶（Arginine decarboxylase,ADC）基因cDNA全长序列,命名为BnADC。BnADC全长为2 649bp,包含344bp的5＇非翻译区（5＇Untranslated region,5＇-UTR）、226bp的3＇非翻译区（3＇Untranslated region,3＇-UTR）和2 079bp的完整开放阅读框（open reading frame,ORF）,编码75.1kD的蛋白质。5＇-UTR中含有12bp的uORF（Upstream open readingframe）,编码MIRE 4个氨基酸。氨基酸同源性比对表明,BnADC蛋白与其他植物ADC蛋白具有很高的相似性,与芥菜的同源性高达93%;系统进化树分析表明,BnADC与芥菜、拟南芥的ADC亲缘关系较近。在获得全长cDNA的基础上,构建融合表达载体pET30a（＋）-BnADC,转化大肠杆菌（Escherichia coli）表达菌株BL21（DE3）,SDS-PAGE检测到一个约81.1kD的融合蛋白被E.coil表达,且融合蛋白主要存在于菌体沉淀中。     

基于PCR方法敲除黄酒酵母精氨酸酶基因的工程菌构建

用一种高效快速的免克隆方法-长侧翼同源PCR(LFH-PCR),构建基因敲除组件,然后通过化学转化方法把敲除组件转入黄酒酵母细胞中,利用同源重组机制精确敲除精氨酸酶基因(CAR1),构建了黄酒酵母工程菌株。结果表明,敲除CAR1基因的工程菌与出发菌株相比,酒液中尿素的含量降低了72%,氨基甲酸乙酯含量降低了38%,并且该菌株的遗传稳定性好,发酵性能与出发菌株基本一致。     

基于尿嘧啶磷酸核糖转移酶为负向筛选标记的生酮古龙酸杆菌基因组无痕修饰系统的构建

通过构建尿嘧啶磷酸核糖转移酶基因(upp)敲除打靶质粒,采用同源重组的方法获得了生酮古龙酸杆菌(Ketogulonigenium vulgare)的upp基因缺失突变株K.vulgareΔupp,可作为基因组无痕修饰系统底盘细胞的upp基因表达载体,转化突变株K.vulgareΔupp,获得upp基因回补菌株PBB-upp,并验证了upp作为负向筛选标记的可行性。实验结果表明:突变株Δupp在1 mg/m L的5-氟尿嘧啶培养基上可生长,野生型和回补菌株PBB-upp不能生长,3株菌对5-氟尿嘧啶的抗性存在显著差异,说明可以将upp基因作为负向筛选标记,与正向筛选标记相结合,在upp基因缺失的底盘细胞基础上通过两次同源重组,实现基因组的无痕修饰与改造。     

A two-step cloning-free PCR-based method for the deletion of genes in the opportunistic pathogenic yeast Candida lusitaniae

We describe a new cloning-free strategy to delete genes in the opportunistic pathogenic yeast Candida lusitaniae. We first constructed two ura3 Δ strains in C. lusitaniae for their use in transformation experiments. One was deleted for the entire URA3 coding sequence; the other possessed a partial deletion within the coding region, which was used to determine the minimum amount of homology required for efficient homologous recombination by double crossing-over of a linear DNA fragment restoring URA3 expression. This amount was estimated to 200 bp on each side of the DNA fragment. These data constituted the basis of the development of a strategy to construct DNA cassettes for gene deletion by a cloning-free overlapping PCR method. Two cassettes were necessary in two successive transformation steps for the complete removal of a gene of interest. As an example, we report here the deletion of the LEU2 gene. The first cassette was constituted by the URA3 gene flanked by two large fragments (500 bp) homologous to the 5' and 3' non-coding regions of LEU2. After transformation of an ura3 Δ recipient strain and integration of the cassette at the LEU2 locus, the URA3 gene was removed by a second transformation round with a DNA cassette made by the fusion between the 5' and 3' non-coding regions of the LEU2 gene. The overall procedure takes less than 2 weeks and allows the creation of a clean null mutant that retains no foreign DNA sequence integrated in its genome.     

Puromycin‐ and methotrexate‐resistance cassettes and optimized Cre‐recombinase expression plasmids for use in yeast

Here we expand the set of tools for genetically manipulating Saccharomyces cerevisiae. We show that puromycin‐resistance can be achieved in yeast through expression of a bacterial puromycin‐resistance gene optimized to the yeast codon bias, which in turn serves as an easy‐to‐use dominant genetic marker suitable for gene disruption. We have constructed a similar DNA cassette expressing yeast codon‐optimized mutant human dihydrofolate reductase (DHFR), which confers resistance to methotrexate and can also be used as a dominant selectable marker. Both of these drug‐resistant marker cassettes are flanked by loxP sites, allowing for their excision from the genome following expression of Cre‐recombinase. Finally, we have created a series of plasmids for low‐level constitutive expression of Cre‐recombinase in yeast that allows for efficient excision of loxP‐flanked markers. Copyright © 2015 John Wiley & Sons, Ltd.     

肠炎沙门氏菌rpoH 基因缺陷株的构建及其热激响应特性

构建肠炎沙门氏菌Salmonella enteritidis IFO3313 株的rpoH 基因缺陷株IFO3313- ΔrpoH,比较不同温度下缺陷株与野生株的热激响应特性。利用λ-Red 重组系统对Salmonella enteritidis IFO3313 的rpoH 基因进行缺失突变,并使用PCR方法对其进行验证,在此基础上使用不同培养基对缺陷株与野生株进行不同温度下的热激应答结果和亚致死热损伤修复能力的考察：野生株比缺陷株有更强的热激耐受能力,在DHL 平板上对亚致死热损伤的肠炎沙门氏菌的分离检测可能具有假阴性,野生株比缺陷株在TSYA 平板上有更强的修复能力。利用λ-Red 重组系统敲除了肠炎沙门氏菌rpoH 基因,有助于了解肠炎沙门氏菌的热激亚致死及其修复机制,对亚致死状态食源性病原微生物的检测做出初步研究。     

Heterologous URA3MX cassettes for gene replacement in Saccharomyces cerevisiae

Heterologous gene replacement cassettes are powerful tools for dissecting gene function in Saccharomyces cerevisiae. Their primary advantages over homologous gene replacement cassettes include reduced gene conversion (leading to efficient site-specific integration of the cassette) and greater independence of strain background. Perhaps the most widely used cassettes are the MX cassettes containing the dominant selectable kanamycin resistance gene (kanr), which confers resistance to G418 (Wach et al., 1994). One limitation of the kanMX cassettes is that they are not counterselectable and therefore not readily recyclable, which is important when constructing strains with more than one gene deletion. To address this limitation, and to expand the choices of heterologous markers, we have created two new MX cassettes by replacing the kanr ORF from plasmids pFA6-kanMX3 and pFA6-kanMX4 with the Candida albicans URA3 ORF. These plasmids, pAG60 (CaURA3MX4) and pAG61 (CaURA3MX3) are identical to the kanMX cassettes in all other respects but have the added advantage of being counterselectable and therefore readily recyclable in S. cerevisiae.