里氏木霉绿色荧光蛋白表达载体的构建及功能鉴定
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摘要
为了后续研究里氏木霉(Trichoderma reesei)纤维素酶基因的表达与调控,利用overlap PCR及分子克隆技术构建了含有Col E1原核复制起始位点、氨苄青霉素抗性、里氏木霉的丙酮酸脱羧酶启动子、丙酮酸脱羧酶终止子、潮霉素B抗性的筛选标记并能表达增强型绿色荧光蛋白(Zs Green)的表达载体p LXT-Zs Green。将该载体转化里氏木霉QM9414原生质细胞,使用潮霉素B筛选平板得到阳性转化子,随后使用荧光显微镜在488 nm激发光下观察菌丝,并随机挑取4个转化菌株进行Western-blot验证。结果显示,里氏木霉菌丝体可发出明亮的绿色荧光,而且Western-blot验证了该载体能够在里氏木霉中有效地表达增强型绿色荧光蛋白。上述研究表明,载体p LXT-Zs Green在里氏木霉中能够稳定高效地表达外源基因,为研究里氏木霉的基因表达调控奠定了实验基础。
In order to study the expression and regulation of cellulase gene in Trichoderma reesei, overlap PCR amplification and molecular cloning techniques were employed to construct a vector p LXT-Zs Green expressing bright green fluorescence protein in T. reesei. The vector contains prokaryotic replication origin Col E1, the ampicillin resistance gene, promoter of pyruvate decarboxylase(PDC), terminator of PDC, Zs Green gene, and the hygromycin B resistance gene. The vectors were transformed into the protoplasts of T. reesei QM9414. The hyphae of the transformants were observed using a fluorescence microscope with 488 nm exci-tation light, and four colonies were randomly selected for Western-blot. The results showed that the trans-formants were able to express bright green fluorescence under fluorescence microscope. The Western-blot analysis further verified that the constructed plasmid could express Zs Green protein effectively in T. reesei.The vector p LXT-Zs Green is capable of stable and efficient expression of exogenous genes in T. reesei, and this lays a foundation for the study of gene expression regulation of T. reesei.
In order to study the expression and regulation of cellulase gene in Trichoderma reesei, overlap PCR amplification and molecular cloning techniques were employed to construct a vector p LXT-Zs Green expressing bright green fluorescence protein in T. reesei. The vector contains prokaryotic replication origin Col E1, the ampicillin resistance gene, promoter of pyruvate decarboxylase(PDC), terminator of PDC, Zs Green gene, and the hygromycin B resistance gene. The vectors were transformed into the protoplasts of T. reesei QM9414. The hyphae of the transformants were observed using a fluorescence microscope with 488 nm exci-tation light, and four colonies were randomly selected for Western-blot. The results showed that the trans-formants were able to express bright green fluorescence under fluorescence microscope. The Western-blot analysis further verified that the constructed plasmid could express Zs Green protein effectively in T. reesei.The vector p LXT-Zs Green is capable of stable and efficient expression of exogenous genes in T. reesei, and this lays a foundation for the study of gene expression regulation of T. reesei.
引文
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[17]冯飞,王绍文,王娟,等.里氏木霉GH61家族糖苷酶的高效表达及酶学特性研究[J].微生物学通报(Feng Fei,Wang Shaowen,Wang Juan,et al.High-level expression and characterization of GH61 glycoside hydrolase from Trichoderma reesei[J].Microbiology China),2014,41(7):1261-1269.
[18]曾敏,邓丽瑜,汤新,等.CBHⅡ和EGⅣ的重构基因在里氏木霉中的组成型表达[J].食品研究与开发(Zeng Min,Deng Li-yu,Tang Xin,et al.Construction of CBH II and EG IV for homologous protein expression with constitutive promoter in Trichoderma reesei[J].Food Research and Development),2015,36(13):114-117.
[19]Penttil覿M,Nevalainen H,R覿tt觟M.A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei[J].Gene,1987,61(2):155-164.
[20]Kelly J M,Lockington R A.The WD40-repeat protein Cre C interacts with and stabilizes the deubiquitinating enzyme Cre B in vivo in Aspergillus nidulans[J].Molecular Microbiology,2002,43(5):1173-1182.
[21]Margeot A,Portnoy T,Linke R,et al.The CRE1 carbon catabolite repressor of the fungus Trichoderma reesei:a master regulator of carbon assimilation[J].Bio Med Central Genomics,2011,12:269.
[2]Martinez D,Berka R M,Henrissat B,et al.Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei(syn.Hypocrea jecorina)[J].Nature Biotechnology,2008,26(5):553-560.
[3]Nevalainen H,Suominen P,Taimisto K.On the safety of Trichoderma reesei[J].Journal of Biotechnology,1994,37(3):193-200.
[4]Wang B,Xia L.High efficient expression of cellobiase gene from Aspergillus niger in the cells of Trichoderma reesei[J].Bioresource Technology,2011,102(6):4568-4572.
[5]Schuster A,Schmoll M.Biology and biotechnology of Trichoderma[J].Applied Microbiology and Biotechnology,2010,87(3):787-799.
[6]Meyer V.Genetic engineering of filamentous fungi-progress,obstacles and future trends[J].Biotechnology Advances,2008,26(2):177-185.
[7]Gen Z,Shi S,Jiang Y,et al.Construction of a cellulase hyperexpression system in Trichoderma reesei by promoter and enzyme engineering[J].Microbial Cell Factories,2012,11:21.
[8]唐偲洋,魏东芝,杨忠,等.启动子cbh1的改造与里氏木霉通用质粒载体的构建[J].工业微生物(Tang Cai-yang,Wei Dong-zhi,Yang Zhong,et al.Improvement of promoter cbh1 and construction of heterologous gene expression multi-purpose vector of Trichoderma reesei[J].Industrial Microbiology),2014,44(4):33-38.
[9]林涛,黄建忠.丝状真菌启动子研究进展[J].安徽农业科学(Lin Tao,Huang Jian-zhong.Research advance on promoters for heterologous gene expression in filamentous fungi[J].Journal of Anhui Agricultural Sciences),2013,41(7):2862-2863.
[10]Xu J,Nogawa M,Okada H,et al.Regulation of xyn3 gene expression in Trichoderma reesei PC-3-7[J].Applied Microbiology and Biotechnology,2000,54(3):370-375.
[11]Mach R L,Zeilinger S.Regulation of gene expression in industrial fungi:Trichoderma[J].Applied Microbiology and Biotechnology,2003,60(5):515-522.
[12]Seiboth B,Gamauf C,Pail M,et al.The D-xylose reductase of Hypocrea jecorina is the major aldose reductase in pentose and D-galactose catabolism and necessary forβ-galactosidase and cellulase induction by lactose[J].Molecular Microbiology,2007,66(4):890-900.
[13]Kurzatkowski W,Torronen A,Filipek J,et al.Glucose-induced secretion of Trichoderma reesei xylanases[J].Applied and Environmental Microbiology,1996,62(8):2859-2865.
[14]Eriksson T,Stals I,Collen A,et al.Heterogeneity of homologously expressed Hypocrea jecorina(Trichoderma reesei)Cel7B catalytic module[J].European Journal of Biochemistry,2004,271(7):1266-1276.
[15]Wang S W,Liu G,Wang J,et al.Enhancing cellulase production in Trichoderma reesei RUT C30 through combined manipulation of activating and repressing genes[J].Journal of Industrial Microbiology and Biotechnology,2013,40(6):633-641.
[16]梁智伟,王娟,田生礼,等.里氏木霉中小分子RNA对纤维素酶表达调控的作用[J].生物技术通讯(Liang Zhi-wei,Wang Juan,Tian Sheng-li,et al.Study on the regulation fuction of small RNAs on cellulase expression in Trichoderma reesei[J].Letters in Biontonology),2015,26(4):535-540.
[17]冯飞,王绍文,王娟,等.里氏木霉GH61家族糖苷酶的高效表达及酶学特性研究[J].微生物学通报(Feng Fei,Wang Shaowen,Wang Juan,et al.High-level expression and characterization of GH61 glycoside hydrolase from Trichoderma reesei[J].Microbiology China),2014,41(7):1261-1269.
[18]曾敏,邓丽瑜,汤新,等.CBHⅡ和EGⅣ的重构基因在里氏木霉中的组成型表达[J].食品研究与开发(Zeng Min,Deng Li-yu,Tang Xin,et al.Construction of CBH II and EG IV for homologous protein expression with constitutive promoter in Trichoderma reesei[J].Food Research and Development),2015,36(13):114-117.
[19]Penttil覿M,Nevalainen H,R覿tt觟M.A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei[J].Gene,1987,61(2):155-164.
[20]Kelly J M,Lockington R A.The WD40-repeat protein Cre C interacts with and stabilizes the deubiquitinating enzyme Cre B in vivo in Aspergillus nidulans[J].Molecular Microbiology,2002,43(5):1173-1182.
[21]Margeot A,Portnoy T,Linke R,et al.The CRE1 carbon catabolite repressor of the fungus Trichoderma reesei:a master regulator of carbon assimilation[J].Bio Med Central Genomics,2011,12:269.