产甘油假丝酵母TRP1基因的克隆与功能分析

产甘油假丝酵母(Candida glycerinogenes) WL2002-5 是我国发酵甘油生产菌种, 具有高产甘油和耐高渗透压的优良性能。本文采用遗传互补的方法从产甘油假丝酵母基因文库中克隆了TRP1基因(CgTRP1)。序列分析显示, 该基因编码区全长735 bp, 编码的磷酸核糖氨基苯甲酸同分异构酶(CgPRAI)氨基酸序列与其他酵母来源的PRAI蛋白同源性在32.9%~49.2%之间。功能互补实验显示, CgTRP1基因在高拷贝情况下可以互补酿酒酵母trp1基因功能但在低拷贝情况下只能部分互补酿酒酵母trp1基因功能, 是一条功能明确、结构完整的酵母新基因。在CgTRP1 基因下游发现另一蛋白编码基因, 编码的氨基酸序列与酵母无机焦磷酸酶有很高的相似性。     

产甘油假丝酵母HOG1 MAPK同源基因CgHOG1的克隆及特征分析

摘要:【目的】获得产甘油假丝酵母(Candida glycerinogenes)耐高渗和过量合成甘油的关键调控基因—丝裂原活化蛋白激酶基因(CgHOG1),并考察其渗透压调节功能。【方法】运用简并PCR 结合Self-Formed Adaptor PCR技术从产甘油假丝酵母基因组中克隆CgHOG1基因并进行生物信息学相关分析,将CgHOG1基因在酿酒酵母(Saccharomyces cerevisiae W303-1A)hog1Δ缺失突变株中互补表达,考察菌株耐渗透压能力变化。【结果】所获得CgHOG1基因全长1164 bp,编码387个氨基酸序列(GenBank No. KC480066);氨基酸序列与来源于Ogataea parapolymorpha的Hog1p同源性最高,为86%;该基因在酿酒酵母hog1Δ缺失突变株中异源表达能够显著提高菌株的抗盐耐高渗和甘油合成能力。【结论】本文所获得的基因CgHOG1是一个具有耐高渗和过量合成甘油调控功能的新基因,研究结果为产甘油假丝酵母超高渗应答机制的研究及抗盐耐旱作物改造提供了新的基因。     

产甘油假丝酵母甘油代谢关键酶的研究

本文对产甘油假丝酵母的甘油代谢关键酶进行了研究,发现产甘油假丝酵母同化甘油能力极弱,少量葡萄糖明显改善其同化甘油的能力;线粒体3磷酸甘油脱氢酶受3磷酸甘油的强烈诱导,受葡萄糖代谢的阻遏。在甘油发酵过程中,产甘油假丝酵母胞浆3磷酸甘油脱氢酶酶活处于较高水平并在36h和60h时出现两次酶活高峰,其中第一次酶活峰值水平决定产甘油假丝酵母的甘油合成和积累水平,成为甘油高速积累期(18～48h)甘油合成的关键性的限速酶。在甘油发酵18～48h内,3磷酸甘油酯酶的酶活处于高水平,并在36h时出现酶活峰值;处于缓慢甘油积累阶段的48～72h间,3磷酸甘油酯酶已处于低水平表达,此时,3磷酸甘油酯酶则成为甘油合成的限速酶。产甘油假丝酵母稳定并高表达其胞浆3磷酸甘油脱氢酶基因并且其所表达的3磷酸甘油酯酶酶活远高于胞浆3磷酸甘油脱氢酶这一特征是其高产甘油根本所在。     

产甘油假丝酵母胞浆3-磷酸甘油脱氢酶基因（CgGPD）功能分析

【目的】从高产甘油生产菌株产甘油假丝酵母（Candida glycerinogenes）基因组中克隆了NAD+依赖3-磷酸甘油脱氢酶编码基因（CgGPD）,但是该基因及其上游调控序列具体的功能还是未知的。本文研究了CgGPD基因及其上游调控序列的功能。【方法】本文以酿酒酵母（Saccharomyces cerevisiae）及其渗透压敏感型突变株为宿主,构建3种不同的酵母表达载体导入酵母细胞,研究了不同酵母转化子在渗透压胁迫条件下CgGPD基因表达对细胞的耐高渗透压胁迫应答及其细胞的甘油合成能力的影响。【结果】实验结果表明无论是以来源于S. cerevisiae 的TPI启动子还是来源于CgGPD基因的启动子,过量表达CgGPD基因的转化子均能够显著加速葡萄糖消耗速度和提高甘油合成能力,在gpd1/gpd2突变株中表达CgGPD基因能够消除细胞对外界高渗透压的敏感性,同时转化子胞内甘油大量积累。【结论】CgGPD基因在野生型酵母S. cerevisiae W303-1A表达显著提高细胞的甘油合成能力,在gpd/1gpd2突变株中能够互补GPD1基因的功能,CgGPD基因表达受渗透压诱导 调控。     

少根根霉Δ6-脂肪酸脱氢酶基因的克隆和表达

根据真菌Δ6 脂肪酸脱氢酶保守的氨基酸序列设计简并引物进行RT PCR ,获得一个 5 93bp的cDNA片段 ,再根据获得的部分序列设计基因特异性引物 ,通过cDNA末端扩增技术 (RACE)获得该cDNA的 3′和 5′序列 ,从而得到全长为 14 82bp的cDNA序列。序列分析结果表明 ,该序列具有一个长度为 1377bp、编码 4 5 8个氨基酸的开放阅读框 ,所编码蛋白质的大小为 5 2kD。与报道的Δ6 脂肪酸脱氢酶一样 ,推测的氨基酸序列具有膜整合脂肪酸脱氢酶特异性的 3个组氨酸保守区和疏水结构 ,在其氨基酸序列的N 末端具有类似于细胞色素b5的血红素结合区。该序列为一个新的编码Δ6 脂肪酸脱氢酶的基因 ,为了验证其功能 ,把开放阅读框序列RAD6亚克隆到表达载体 pYES2 0 ,构建重组表达载体pYRAD6 ,并转化到酿酒酵母的缺陷型菌株INVScl进行表达。通过气相色谱(GC)和气相色谱 /质谱 (GC MS)分析表明 ,该序列在酿酒酵母中获得表达。所编码的酶具有Δ6 脂肪酸脱氢酶活性 ,能将外源性的底物亚油酸转化为γ 亚麻酸 ,γ 亚麻酸的含量占酵母总脂肪酸的 3 85 %。     

新一代高产甘油重组菌的构建及其初步发酵

产甘油假丝酵母(Candida glycerinogenes WL2002-5)是一株发酵生产甘油的工业化菌株。为进一步提高其产甘油能力,本研究利用前期研究中成功克隆的产甘油假丝酵母中甘油合成关键酶3-磷酸甘油脱氢酶基因CgGPD1,构建根癌农杆菌双元载体pCAM3300-zeocin-CgGPD1后,电击转化根癌农杆菌LBA4404,通过根癌农杆菌介导法(ATMT)转化产甘油假丝酵母,构建了产甘油假丝酵母重组菌。并从中筛选出一株酶活力和产甘油性能较好的产甘油假丝酵母重组菌株C.g-G8。以葡萄糖为底物摇瓶发酵96h后,重组菌C.g-G8的甘油产量比野生型菌株Candida glycerinogene提高18.06%,平均耗糖速率提高12.97%,平均酶活力提高27.55%。本研究成功利用ATMT法转化产甘油假丝酵母构建新一代高产甘油菌株。     

烟草二脂酰甘油酰基转移酶基因(NtDGAT2)的克隆与功能分析

二脂酰甘油酰基转移酶(diacylglycerol acyltransferase,DGAT2)是植物储存油脂生物合成过程中的关键酶,对种子储存油脂累积具有重要的生理作用。本文采用电子克隆与实验相结合的方法,从烟草种子cDNA中克隆到DGAT2基因的开放阅读框序列,命名为NtDGAT2(GenBank登录号JX843807),其序列长999bp,编码332个氨基酸。多序列比对和进化分析表明该基因编码蛋白与其他植物DGAT2具有较高相似性和典型的DGAT2结构域。利用Real-time PCR定量表达分析显示Nt-DGAT2在烟草种子、花、茎、叶和根里面都有表达,且在发育中的种子和花的发育过程大量表达。酵母互补实验证实该基因编码蛋白具有DGAT酶活性。     

少根根霉△^6-脂肪酸脱氢酶基因的克隆和表达

根据真菌△^6-脂肪酸脱氢酶保守的氨基酸序列设计简并引物进行RT-PCR,获得一个593 bp的cDNA片段,再根据获得的部分序列设计基因特异性引物,通过cDNA末端扩增技术(RACE)获得该cDNA的3’和5’序列,从而得到全长为1482bp的cDNA序列。序列分析结果表明,该序列具有一个长度为1377bp、编码458个氨基酸的开放阅读框,所编码蛋白质的大小为52kD。与报道的△^6-脂肪酸脱氢酶一样,推测的氨基酸序列具有膜整合脂肪酸脱氢酶特异性的3个组氨酸保守区和疏水结构,在其氨基酸序列的N-末端具有类似于细胞色素b5的血红素结合区。该序列为一个新的编码△^6-脂肪酸脱氢酶的基因,为了验证其功能,把开放阅读框序列RAD6亚克隆到表达载体pYES2．0,构建重组表达载体pYRAD6,并转化到酿酒酵母的缺陷型菌株INVScl进行表达。通过气相色谱(GC)和气相色谱／质谱(GC-MS)分析表明,该序列在酿酒酵母中获得表达。所编码的酶具有△^6-脂肪酸脱氢酶活性,能将外源性的底物亚油酸转化为γ-亚麻酸,γ-亚麻酸的含量占酵母总脂肪酸的3．85％。     

雅致枝霉Δ6-脂肪酸脱氢酶基因的克隆及在酿酒酵母中的表达

根据真菌Δ6-脂肪酸脱氢酶基因保守的组氨酸Ⅱ区和Ⅲ区附近保守序列设计兼并引物进行RT-PCR,得到雅致枝霉(Thamnidium elegans)As3.2806Δ6-脂肪酸脱氢酶基因459bp部分cDNA序列,然后通过快速扩增cDNA末端技术(RACE)向两端延伸得到1504bp的Δ6-脂肪酸脱氢酶基因全长cDNA序列。序列分析表明有一个1377bp、编码459个氨基酸的开放阅读框TED6。推测的氨基酸序列与已知其他真菌的Δ6-脂肪酸脱氢酶基因的氨基酸序列比对,具有3个组氨酸保守区、2个疏水区及N末端细胞色素b5融合区。将此编码区序列亚克隆到酿酒酵母缺陷型菌株INVSc1的表达载体pYES2.0中,构建表达载体pYTED6,并在酿酒酵母INVSc1中异源表达。通过气相色谱(GC)和气相色谱/质谱(GC-MS)分析表明,该序列在酿酒酵母中获得表达,产生γ-亚麻酸(GLA)的含量占酵母总脂肪酸的7.5%。证明此序列编码的蛋白能将外加的亚油酸转化为γ-亚麻酸,是一个新的有功能的Δ6-脂肪酸脱氢酶基因(GenBank,AY941161)。     

利用荧光蛋白研究产甘油假丝酵母胞浆3-磷酸甘油脱氢酶基因CgGPD启动子

[目的]克隆产甘油假丝酵母(Candida glycerinogenes)胞浆3-磷酸甘油脱氢酶基因CgGPD的启动子(PCggpd),并通过报告基因gfp的差异表达来研究葡萄糖浓度对PCggpd在酿酒酵母(Saccharomyces cerevisiae)中的诱导特性.[方法]采用PCR扩增的方法分别从产甘油假丝酵母基因组和pCAMBIA1302载体中克隆出CgGPD的启动序列PCggpd和绿色荧光蛋白基因gfp.将两个基因同时构建到酿酒酵母表达载体pYX212-zeocin中,构建时将绿色荧光蛋白基因gfp置于CgGPD的启动序列下游,获得重组质粒pYX212-zeocin-PCggpd-gfp.通过电击转化酿酒酵母W303-lA.将重组酿酒酵母S.cerevisiae W303-1A-GFP置于不同葡萄糖浓度培养基中进行培养,利用荧光显微技术对其进行荧光检测.[结果]重组酿酒酵母能产生稳定的荧光,当葡萄糖浓度为2%时,重组酿酒酵母在YEPD培养基中产生较弱的荧光,随着葡萄糖浓度的升高,荧光强度有明显的增强.[结论]PCggpd属于环境胁迫诱导型启动子,高浓度的葡萄糖能诱导PCggpd启动绿色荧光蛋白的高水平表达,这对完善产甘油假丝酵母的遗传背景研究,阐明其高产甘油的机理具有重要意义.     

Cloning and characterization of a NAD+-dependent glycerol-3-phosphate dehydrogenase gene from Candida glycerinogenes, an industrial glycerol producer.

The osmotolerant yeast Candida glycerinogenes produces glycerol as a major metabolite on an industrial scale, but the underlying molecular mechanisms are poorly understood. We cloned and characterized a 4900-bp genomic fragment containing the CgGPD gene encoding a glycerol-3-phosphate dehydrogenase homologous to GPD genes in other yeasts using degenerate primers in conjunction with inverse PCR. Sequence analysis revealed a 1167-bp open reading frame encoding a putative peptide of 388 deduced amino acids with a molecular mass of 42 695 Da. The CgGPD gene consisted of an N-terminal NAD(+)-binding domain and a central catalytic domain, whereas seven stress response elements were found in the upstream region. Functional analysis revealed that Saccharomyces cerevisiae gpd1Delta and gpd1Delta/gpd2Delta osmosensitive mutants transformed with CgGPD were restored to the wild-type phenotype when cultured in high osmolarity media, suggesting that it is a functional GPD protein. Transformants also accumulated glycerol intracellularly and GPD-specific activity increased significantly when stressed with NaCl, whereas the S. cerevisiae mutants transformed with the empty plasmid showed only slight increases. The full-length CgGPD gene sequence including upstream and downstream regions has been deposited in GenBank under accession no. EU186536.     

Cloning and characterization of a NAD+-dependent glycerol-3-phosphate dehydrogenase gene from Candida glycerinogenes, an industrial glycerol producer

The osmotolerant yeast Candida glycerinogenes produces glycerol as a major metabolite on an industrial scale, but the underlying molecular mechanisms are poorly understood. We cloned and characterized a 4900-bp genomic fragment containing the CgGPD gene encoding a glycerol-3-phosphate dehydrogenase homologous to GPD genes in other yeasts using degenerate primers in conjunction with inverse PCR. Sequence analysis revealed a 1167-bp open reading frame encoding a putative peptide of 388 deduced amino acids with a molecular mass of 42 695 Da. The CgGPD gene consisted of an N-terminal NAD⁺-binding domain and a central catalytic domain, whereas seven stress response elements were found in the upstream region. Functional analysis revealed that Saccharomyces cerevisiae gpd1 Δ and gpd1 Δ/ gpd2 Δ osmosensitive mutants transformed with CgGPD were restored to the wild-type phenotype when cultured in high osmolarity media, suggesting that it is a functional GPD protein. Transformants also accumulated glycerol intracellularly and GPD-specific activity increased significantly when stressed with NaCl, whereas the S. cerevisiae mutants transformed with the empty plasmid showed only slight increases. The full-length CgGPD gene sequence including upstream and downstream regions has been deposited in GenBank under accession no. EU186536 .     

Identification of the gene encoding DNA topoisomerase I from Candida albicans

Abstract A gene encoding a type I topoisomerase (TOP1) was isolated from Candida albicans , sequenced, and expressed in Saccharomyces cerevisiae . The TOP1 gene was identified from a C. albicans genomic library by hybridization with the product of a polymerase chain reaction with degenerate primer sets encoding regions conserved in other TOP1 genes. A clone containing an open reading frame of 2463 bp and predicted to encode a protein of 778 amino acids with sequence similarity to eukaryotic type I topoisomerases was identified. The C. albicans TOP1 gene restored camptothecin sensitivity and increased the topoisomerase activity in S. cerevisiae , indicating that the DNA fragment encodes a functional C. albicans topoisomerase I.     

The Effect of NADP+ on Electrophoretic Behavior of Glucose 6-Phosphate Dehydrogenase from Sweet Potato

The gene from Bacillus brevis TT02–8 encoding arginase was cloned into Escherichia coli, and its nucleotide sequence was identified. The nucleotide sequence contained an open reading frame that encoded a polypeptide of 298 amino acid residues with a predicted molecular weight of 31,891, which was consistent with that previously calculated for arginase purified from this bacterium. Comparison of the deduced amino acid sequence of the B. brevis TT02–8 arginase with that of the prokaryotic and eukaryotic arginases of Bacillus caldovelox, Bacillus subtilis, Agrobacterium Ti plasmid C58, Saccharomyces cerevisiae, Coccidioides immitis, Xenopus laevis, Rana catesbeiana, rat liver, and human liver, showed 33–66% of the sequences to be similar; there were several highly conserved regions. Arginase activity was detected in Escherichia coli cells transformed with an expression plasmid of the cloned arginase gene.     

Cloning and characterization of the DAS gene encoding the major methanol assimilatory enzyme from the methylotrophic yeast Hansenula polymorpha.

A gene library from the methanol utilizing yeast Hansenula polymorpha, constructed in a lambda Charon4A vector, was used to clone the gene encoding a key methanol assimilating enzyme, dihydroxyacetone synthase (DHAS) by differential plaque hybridization. The nucleotide sequence of the 2106 bp structural gene and the 5' and 3' non-coding regions was determined. The deduced amino acid sequence of the protein is in agreement with the apparent molecular weight and amino acid composition of the purified protein. The codon bias is not so pronounced as in some Saccharomyces cerevisiae genes.