链霉菌MY0504纤溶酶YG4基因的克隆及生物信息学分析
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摘要
本研究获得了海洋链霉菌MY0504纤溶酶YG4基因,并对其进行生物信息学分析。利用依据同源序列设计的兼并引物,对提取的链霉菌MY0504的基因组DNA进行PCR扩增,将得到的DNA片段连接到pMD18-T载体后转化感受态Trans10细胞,然后对阳性克隆进行测序,并对序列进行生物信息学分析。最终克隆了1083bp的海洋链霉菌MY0504纤溶酶YG4基因。将获得的YG4基因输入Gen Bank网站,进行检索比对,结果显示与丝氨酸蛋白酶基因碱基序列同源性100%。对16s rDNA序列分析结果表明,该菌株与达格斯地链霉菌、氢化链霉菌、嫩白黄链霉菌、浅紫链霉菌的亲缘关系较近;通过对YG4基因编码蛋白的一级结构、二级结构、亚细胞定位和三级结构的预测和分析,结果表明:该基因编码360个氨基酸,其编码产物为稳定的亲水蛋白;二级结构以α螺旋和β折叠为主,无信号肽和跨膜结构域,有40个磷酸化位点;高级结构以无规则卷曲为主。本研究结果为研究海洋链霉菌MY0504纤溶酶YG4基因的表达机制及目的蛋白表达水平的提高提供了重要信息。
In this study, the fibrinolytic enzyme YG4 gene from marine Streptomyces sp.MY0504 was obtained and analyzed by bioinformatics methods. The genomic DNA of the extracted Streptomyces MY0504 was amplified by PCR using the degenerate primers designed based on the homologous sequences. The obtained DNA fragments were ligated to the p MD18-T vectors and transformed into competent Trans10 cells. The positive clones were then sequenced, and the obtained sequences were analyzed bioinformatically. Finally, the fibrinolytic enzyme YG4 gene from marine Streptomyces strain MY0504 sized 1083 bp was cloned. The obtained YG4 gene was introduced into the Gen Bank website, and then an alignment search and comparison was performed. The result showed that the YG4 gene exhibited 100% homology with the base sequence of the serine protease gene. The results of the 16s rDNA sequence analysis showed that the strain was closely related to Streptomyces daghestanicus, Streptomyces hydrogenans, Streptomyces albidoflavus and Streptomyces violascens. The primary structure, secondary structure, subcellular localization and tertiary structure of the protein encoded by YG4 gene were predicted and analyzed.The analysis results showed that the gene encoded 360 amino acids, and the encoded product was a stable hydrophilic protein. The secondary structure was mainly composed of alpha helix and beta sheet without signal peptides and transmembrane domains but with 40 phosphorylation sites. The high-order structure was dominated by random coils. The results of this study provided important information for investigating the expression mechanism of the fibrinolytic enzyme YG4 gene from marine Streptomyces MY0504 and for improving the expression level of a target protein.
In this study, the fibrinolytic enzyme YG4 gene from marine Streptomyces sp.MY0504 was obtained and analyzed by bioinformatics methods. The genomic DNA of the extracted Streptomyces MY0504 was amplified by PCR using the degenerate primers designed based on the homologous sequences. The obtained DNA fragments were ligated to the p MD18-T vectors and transformed into competent Trans10 cells. The positive clones were then sequenced, and the obtained sequences were analyzed bioinformatically. Finally, the fibrinolytic enzyme YG4 gene from marine Streptomyces strain MY0504 sized 1083 bp was cloned. The obtained YG4 gene was introduced into the Gen Bank website, and then an alignment search and comparison was performed. The result showed that the YG4 gene exhibited 100% homology with the base sequence of the serine protease gene. The results of the 16s rDNA sequence analysis showed that the strain was closely related to Streptomyces daghestanicus, Streptomyces hydrogenans, Streptomyces albidoflavus and Streptomyces violascens. The primary structure, secondary structure, subcellular localization and tertiary structure of the protein encoded by YG4 gene were predicted and analyzed.The analysis results showed that the gene encoded 360 amino acids, and the encoded product was a stable hydrophilic protein. The secondary structure was mainly composed of alpha helix and beta sheet without signal peptides and transmembrane domains but with 40 phosphorylation sites. The high-order structure was dominated by random coils. The results of this study provided important information for investigating the expression mechanism of the fibrinolytic enzyme YG4 gene from marine Streptomyces MY0504 and for improving the expression level of a target protein.
引文
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[18]张树波,赖剑煌.分子系统发育分析的生物信息学方法[J].计算机科学,2010,37(8):47-51ZHANG Shu-bo,LAI Jian-huang.Molecular system growth analysis biological information study method[J].Computer Science,2010,37(8):47-51
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[20]Li J B,Luan Y S.Molecular cloning and characterization of a pathogen-induced WRKY transcription factor gene from late blight resistant tomato varieties Solanum pimpinellifolium L3708[J].Physiological&Molecular Plant Pathology,2014,87:25-31
[21]赵慧,郑文岭,马文丽.信号肽对外源蛋白分泌效率的影响[J].生物学杂志,2003,20(5):177-179ZHAO Hui,ZHENG Wen-ling,MA Wen-li.Signal peptide to extraneous source protein secretion efficiency influence[J].Biology Magazine,2003,20(5):177-179
[22]马怡茗,柯欣,李晓霞,等.角蛋白酶基因gm2886在密旋链霉菌ACT12中的表达及鉴定[J].生物工程学报,2017,12:1968-1978MA Yi-ming,KE Xin,LI Xiao-xia,et al.Because the keratin substrat gm2886 is turning on lathe in streptomycete ACT12densely the expression and appraises[J].Bio-engineering Journal,2017,12:1968-1978
[2]Narasimhan M K,Chandrasekaran M,Rajesh M.Fibrinolytic enzyme production by newly isolated Bacillus cereus SRM-001 with enhanced in vitro blood clot lysis potential[J].J Gen Appl Microbiol,2015,61(5):157-164
[3]Banerjee A,Chisti Y,Banerjee U C.Streptokinase-a clinically useful thrombolytic agent[J].Biotechnol Adv,2004,22(4):287-307
[4]郝树站,王素英.海洋微生物生物活性物质的研究进展[J].生命科学研究,2005,S1:35-38HAO Shu-zhan,WANG Su-ying.The sea microorganism biological activity material research progresses[J].Life Sciences Research,2005,S1:35-38
[5]田新朋,张偲,李文均.海洋放线菌研究进展[J].微生物学报,2011,51(2):161-169TIAN Xin-peng,ZHANG Si,LI Wen-jun.The sea ray fungi research progresses[J].Microorganism Journal,2011,51(2):161-169
[6]SV,SSS,OSA,et al.Antimicrobial potential of Actinomycetes species isolated from marine environment[J].Asian Pacific Journal of Tropical Biomedicine,2012,6:469-473
[7]Elich E,Schreinemakers P,Vullings M.Partha N Ultrasound induced production of thrombinase by marine actinomycetes:kinetic and optimization studies[J].Biochemical Engineering Journal,2012,61(8):34-42
[8]Zotchev S B.Marine actinomycetes as an emerging resource for the drug development pipelines[J].J Biotechnol,2012,158(4):168-175
[9]Ju X,Cao X,Yong S,et al.Purification and characterization of a fibrinolytic enzyme from Streptomyces sp.XZNUM00004[J].World J Microbiol Biotechnol,2012,28(7):2479-2486
[10]侯正欣,董超,马萱,等.海洋来源链霉菌MY0504产纤溶酶的发酵条件优化[J].微生物学通报,2017,5:1009-1016HOU Zheng-xin,DONG Chao,MA Xuan,et al.Sea origin streptomycete MY0504 produces the plasmin the fermentation condition to optimize[J].Microbiology Notification,2017,5:1009-1016
[11]米阳,董超,侯正欣,等.海洋链霉菌发酵纤溶酶的分离纯化和酶学性质研究[J].中国海洋药物,2016,35(3):43-48MI Yang,DONG Chao,HOU Zheng-xin,et al.The sea streptomycete fermentation plasmin separation purification and the zymology nature study[J].Chinese Sea Medicine,2016,35(3):43-48
[12]董超,米阳,原晋波,等.产纤溶酶海洋放线菌的筛选及初步鉴定[J].中国酿造,2015,7:59-64DONG Chao,MI Yang,YUAN Jin-bo,et al.Produces the plasmin sea ray fungi screening and appraises[J].China Brews,2015,7:59-64
[13]Xiang L,Moore BS.Characterization of benzoyl coenzyme a biosynthesis genes in the enterocin-producing bacterium“Streptomyces maritimus”[J].Journal of Bacteriology,2003,185(2):399-404
[14]敬俊锋,陈斌,李莹,等.纳豆激酶基因的克隆及其在毕赤酵母中的表达[J].生物学杂志,2011,5:55-57JIN Jun-feng,CHEN Bin,LI Ying,et al.Accepts the bean activating enzyme gene the clone and in finishes in the red yeast expression[J].Biology Magazine,2011,5:55-57
[15]王志坤,常健敏,李丹丹,等.大豆Gm WRI1a基因克隆及生物信息学分析[J].东北农业大学学报,2013,7:11-16WANG Zhi-kun,CHANG Jian-min,Li Dan-dan,et al.Soybean Gm WRI1a gene clone and biological information study analysis[J].Northeast Agricultural College Journal,2013,7:11-16
[16]Petersen T N,Brunak S,von Heijne G,etal.Signal P 4.0:discriminating signal peptides from transmembrane regions[J].Nat Methods,2011,8(10):785-786
[17]Gallego Albiach V,Martínez Pastor F,Mazzeo I,et al.Intracellular changes in Ca2+,K+and pH after sperm motility activation in the European eel(Anguilla):Preliminary results[J].Aquaculture,2014,418(1):155-158
[18]张树波,赖剑煌.分子系统发育分析的生物信息学方法[J].计算机科学,2010,37(8):47-51ZHANG Shu-bo,LAI Jian-huang.Molecular system growth analysis biological information study method[J].Computer Science,2010,37(8):47-51
[19]Jensen L J,Gupta R,Staerfeldt H H,et al.Prediction of human protein function according to gene ontology categories[J].Bioinformatics,2003,19(5):635-642
[20]Li J B,Luan Y S.Molecular cloning and characterization of a pathogen-induced WRKY transcription factor gene from late blight resistant tomato varieties Solanum pimpinellifolium L3708[J].Physiological&Molecular Plant Pathology,2014,87:25-31
[21]赵慧,郑文岭,马文丽.信号肽对外源蛋白分泌效率的影响[J].生物学杂志,2003,20(5):177-179ZHAO Hui,ZHENG Wen-ling,MA Wen-li.Signal peptide to extraneous source protein secretion efficiency influence[J].Biology Magazine,2003,20(5):177-179
[22]马怡茗,柯欣,李晓霞,等.角蛋白酶基因gm2886在密旋链霉菌ACT12中的表达及鉴定[J].生物工程学报,2017,12:1968-1978MA Yi-ming,KE Xin,LI Xiao-xia,et al.Because the keratin substrat gm2886 is turning on lathe in streptomycete ACT12densely the expression and appraises[J].Bio-engineering Journal,2017,12:1968-1978