黑木耳内切葡聚糖酶基因克隆与原核表达
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摘要
克隆了黑木耳(Auricularia auricular-judae)内切葡聚糖酶(endoglucanase)基因并进行序列分析,此外,在大肠杆菌BL21(DE3)中成功表达。研究基于前期测得的黑木耳转录组数据,同时结合基因组数据(GenBank登录号为NEKD00000000. 1),筛选到黑木耳内切葡聚糖酶基因序列并设计特异引物,以黑木耳菌丝总RNA为模板,采用RT-PCR技术克隆黑木耳内切葡聚糖酶基因c DNA全长,命名为Aa-eg,构建了重组原核表达载体p ET32a-Aa-eg并成功在大肠杆菌中表达。序列分析表明,c DNA全长为1 242 bp,编码413个氨基酸,预测该蛋白分子量为43. 59 ku,理论等电点为4. 42,存在信号肽。由于p ET-32a载体包含20. 0 ku的标签,SDS-PAGE分析在45. 0 ku和66. 2 ku之间出现目的蛋白条带,与理论预期值大小一致。通过对黑木耳内切葡聚糖酶基因的克隆及分析,为进一步揭示黑木耳内切葡聚糖酶基因功能奠定基础。
The endoglucanase gene was cloned and analyzed from Auricularia auricular-judae,and the recombinant protein was obtained by prokaryotic expression. Combined with the genomic data of Auricularia auricular-judae in NCBI( The GenBank login number is NEKD00000000. 1) and the transcriptome data obtained in the early stage,the sequence of endoglucanase gene was screened out and specific primers were designed. The full length of the cDNA of the endoglucanase gene was cloned by RT-PCR and named as Aa-eg. The recombinant prokaryotic expression vector pET32 aAa-eg was constructed and successfully expressed in E. coli BL21. Sequence analysis showed that the total length of cDNA was 1 242 bp,encoding 413 amino acid polypeptides. The molecular weight of the protein was predicted to be 43. 59 ku,the theoretical isoelectric point was 4. 42,and there was signal peptide in the endoglucanase. Due to the fact that a tag protein with the molecular weight of 20 ku was involved in the vector,the SDS-PAGE analysis showed that the target protein band was located between 45. 0 ku and 66. 2 ku,which was consistent with the theoretical expectation. The cloning and analysis of the endoglucanase gene laid a foundation for further revealing the function of the endoglucanase gene in A.auricular-judae.
The endoglucanase gene was cloned and analyzed from Auricularia auricular-judae,and the recombinant protein was obtained by prokaryotic expression. Combined with the genomic data of Auricularia auricular-judae in NCBI( The GenBank login number is NEKD00000000. 1) and the transcriptome data obtained in the early stage,the sequence of endoglucanase gene was screened out and specific primers were designed. The full length of the cDNA of the endoglucanase gene was cloned by RT-PCR and named as Aa-eg. The recombinant prokaryotic expression vector pET32 aAa-eg was constructed and successfully expressed in E. coli BL21. Sequence analysis showed that the total length of cDNA was 1 242 bp,encoding 413 amino acid polypeptides. The molecular weight of the protein was predicted to be 43. 59 ku,the theoretical isoelectric point was 4. 42,and there was signal peptide in the endoglucanase. Due to the fact that a tag protein with the molecular weight of 20 ku was involved in the vector,the SDS-PAGE analysis showed that the target protein band was located between 45. 0 ku and 66. 2 ku,which was consistent with the theoretical expectation. The cloning and analysis of the endoglucanase gene laid a foundation for further revealing the function of the endoglucanase gene in A.auricular-judae.
引文
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[15] Manavalan T,Manavalan A,Thangavelu K P,et al. Characterization of a novel endoglucanase from Ganoderma lucidum[J].Journal of Basic Microbiology,2015,55(6):761-771.
[16]卢敏,王帅豪,狄元冉,等.纤维素酶基因克隆与表达[J].动物营养学报,2012,24(6):1013-1018.
[17]任大明,毕霏,陈红漫,等.绿色木霉纤维素酶CBHI基因的克隆与原核表达[J].沈阳农业大学学报,2012,43(3):366-369.
[18]张宇婷,曲柏宏,宁云山,等.桦褐孔菌纤维素酶基因真核表达载体构建[J].延边大学农学学报,2017,39(3):1-6,18
[19] Claydon N,Allan M,Wood D A. Fruit body biomass regulated production of extracellular endocellulase during periodic fruiting by Agaricus bisporus[J].Transactions of the British Mycological Society,1988,90(1):85-90.
[20]李杰,高博,江连洲,等.内切葡聚糖酶基因在黑曲霉中的同源表达[J].东北农业大学学报,2014,45(9):56-61.
[2]朱雪琼.黑木耳多糖的提取、功能及单糖组成的研究[D].南宁:广西大学,2014:1-5.
[3]白海娜,王振宇,张华,等.多酚类化合物与黑木耳多糖协同抗氧化作用研究[J].食品工业科技,2013,34(22):124-127,134.
[4]周国华.黑木耳多糖抗衰老及降血脂生物功效的研究[D].哈尔滨:东北农业大学,2005.
[5]尹红力,赵鑫,佟丽丽,等.黑木耳多糖体外和体内降血糖功能[J].食品科学,2015,36(21):221-226.
[6]黄滨南,张秀娟,邹翔,等.黑木耳多糖抗肿瘤作用的研究[J].哈尔滨商业大学学报(自然科学版),2004,20(6):648-651.
[7]丁少军,宋美静,池杏微,等.不同碳源条件下草菇内切型纤维素酶基因(eg1)转录表达的分析[J].应用与环境生物学报,2005,11(4):419-422.
[8] Baldrian P,ValáskováV.Degradation of cellulose by basidiomycetous fungi[J]. Fems Microbiology Reviews,2010,32(3):501-521.
[9]余兴莲,王丽,徐伟民.纤维素酶降解纤维素机理的研究进展[J].宁波大学学报(理工版),2007,20(1):78-82.
[10] Tomme P,Warren R A,Gilkes N R. Cellulose hydrolysi by bacteria and fungi[J]. Advances in Microbial Physiology,1995(37):1-81.
[11] Murphy L,Cruysbagger N,Damgaard H D,et al. Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose[J].Journal of Biological Chemistry,2012,287(2):1252
[12] Ling Z,Ma T,Li J,et al. Functional expression of trypsin from Streptomyces griseus by Pichia pastoris[J]. Journal of Industrial Microbiology&Biotechnology,2012,39(11):1651-1662.
[13]李青青,黄晓梅,范金霞,等.木霉内切葡聚糖酶的研究现状及应用[J].基因组学与应用生物学,2015,34(10):2277-2286.
[14]靳振江.纤维素酶降解纤维素的研究进展[J].广西农业科学,2007,38(2):127-130.
[15] Manavalan T,Manavalan A,Thangavelu K P,et al. Characterization of a novel endoglucanase from Ganoderma lucidum[J].Journal of Basic Microbiology,2015,55(6):761-771.
[16]卢敏,王帅豪,狄元冉,等.纤维素酶基因克隆与表达[J].动物营养学报,2012,24(6):1013-1018.
[17]任大明,毕霏,陈红漫,等.绿色木霉纤维素酶CBHI基因的克隆与原核表达[J].沈阳农业大学学报,2012,43(3):366-369.
[18]张宇婷,曲柏宏,宁云山,等.桦褐孔菌纤维素酶基因真核表达载体构建[J].延边大学农学学报,2017,39(3):1-6,18
[19] Claydon N,Allan M,Wood D A. Fruit body biomass regulated production of extracellular endocellulase during periodic fruiting by Agaricus bisporus[J].Transactions of the British Mycological Society,1988,90(1):85-90.
[20]李杰,高博,江连洲,等.内切葡聚糖酶基因在黑曲霉中的同源表达[J].东北农业大学学报,2014,45(9):56-61.
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