虫草棒束孢类枯草杆菌蛋白酶基因克隆及分析
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摘要
采用逆转录PCR方法克隆获得1个虫草棒束孢类枯草杆菌蛋白酶基因的开放阅读框(ORF)序列,并对其进行生物信息学分析。该基因的开放阅读框全长1 599bp,编码的蛋白质由532个氨基酸组成(包括18个氨基酸的信号肽)。预测蛋白质的等电点和分子量大小分别为6.256、56.936 ku。进行序列分析和进化树构建,发现该蛋白酶与已知的几个种类枯草杆菌蛋白酶具有很高的同源性,且与虎甲寄生菌的粉拟青霉位于同一进化分支。实验首次鉴定了虫草棒束孢类枯草杆菌蛋白酶基因的编码区序列,为进一步研究虫草棒束孢致死蝙蝠蛾幼虫的作用机理提供理论依据。
In the present study,the coding cDNA sequence of Isaria farinosa subtilisin-like protease(IsSUB)was cloned by reverse transcription PCR and analyzed by bioinformatical tools in detail. The subtilisin contains 1 599 nucleotides with a putative open reading frame( ORF) encoding 532 amino acid residues( with a signal peptide of 18 amino acid residues). The isoelectric point( pI) and predicted molecular weight of IsSUB were 6.256 and56.936 kDa,respectively. Amino acid sequence and phylogenetic tree analysis among I. farinosa and other insects represented that these fungal subtilisin had a high homology,and IsSUB existed in one evolutionary branching with Cicindelidae sp. Paecilomyces farinosus. It was concluded that the ORF of subtilisin-like protease from I. farinosa provided theoretical basis for damaging on Hepialus larvae body wall.
In the present study,the coding cDNA sequence of Isaria farinosa subtilisin-like protease(IsSUB)was cloned by reverse transcription PCR and analyzed by bioinformatical tools in detail. The subtilisin contains 1 599 nucleotides with a putative open reading frame( ORF) encoding 532 amino acid residues( with a signal peptide of 18 amino acid residues). The isoelectric point( pI) and predicted molecular weight of IsSUB were 6.256 and56.936 kDa,respectively. Amino acid sequence and phylogenetic tree analysis among I. farinosa and other insects represented that these fungal subtilisin had a high homology,and IsSUB existed in one evolutionary branching with Cicindelidae sp. Paecilomyces farinosus. It was concluded that the ORF of subtilisin-like protease from I. farinosa provided theoretical basis for damaging on Hepialus larvae body wall.
引文
[1]重庆市科委.重庆市虫草抚育技术研究取得可喜进展[EB/OL].(http://www.most.gov.cn/dfkj/cq/zxdt/201406/t20140625_113939.htm,2014-06-26.
[2]刘飞,伍晓丽,刘莹,等.冬虫夏草人工培殖过程中寄主昆虫粉拟青霉病原菌的分子生物学研究[J].中国中药杂志,2016,41(3):403-409.
[3]鲁增辉,伍晓丽,刘飞.虫草棒束孢(Isariafarinosa)活性成分及药效研究进展[J].中国药学杂志,2013,48(11):841-844.
[4]鲁增辉,石萍,陈仕江.侵染昆虫前后冬虫夏草菌类枯草杆菌蛋白酶基因表达研究[J].药学学报,2013,48(7):1164-1168.
[5]李娟.真菌枯草杆菌素丝氨酸蛋白酶超家族分子进化研究[D].昆明:云南大学,2016.
[6]Letunic I,Doerks T,Bork P.SMART 6:recent updates and new developments[J].Nucleic Acids Research,2008,37(1):229-232.
[7]Siezen R J,Leunissen J A M.Subtilases:the superfamily of subtilisin-like serine proteases[J].Protein Science A Publication of the Protein Society,1997,6(3):642-658.
[8]李河,周国英,刘君昂.双孢蘑菇褐腐病病原菌的分离及分子鉴定[J].食用菌学报,2009,16(2):74-76.
[9]Uehara R,Angkawidjaja C,Koga Y,et al.Formation of the high-affinity calcium binding site in pro-subtilisin E with the inser tion s e q u e n c e I S1 o f P r o-T k-s u b t i l i si n[J].Biochemistry,2013,52(50):9080-9088.
[10]Guntelberg A V,Ottesen M.Purification of the proteolytic enzyme from Bacillus subtilis[J].Comptes Rendus Des Travaux Du Laboratoire Carlsberg Serie Chimique,1954,29(3-4):36-48.
[11]Dang X,Pan G,Li T,et al.Characterization of a subtilisin-like protease with apical localization from microsporidian Nosema bombycis[J].Journal of Invertebrate Pathology,2013,112(2):166-174.
[12]Shang Y,Xiao G,Zheng P,et al.Divergent and convergent evolution of fungal pathogenicity[J].Genome Biology&Evolution,2016,8(5):1374-1387.
[2]刘飞,伍晓丽,刘莹,等.冬虫夏草人工培殖过程中寄主昆虫粉拟青霉病原菌的分子生物学研究[J].中国中药杂志,2016,41(3):403-409.
[3]鲁增辉,伍晓丽,刘飞.虫草棒束孢(Isariafarinosa)活性成分及药效研究进展[J].中国药学杂志,2013,48(11):841-844.
[4]鲁增辉,石萍,陈仕江.侵染昆虫前后冬虫夏草菌类枯草杆菌蛋白酶基因表达研究[J].药学学报,2013,48(7):1164-1168.
[5]李娟.真菌枯草杆菌素丝氨酸蛋白酶超家族分子进化研究[D].昆明:云南大学,2016.
[6]Letunic I,Doerks T,Bork P.SMART 6:recent updates and new developments[J].Nucleic Acids Research,2008,37(1):229-232.
[7]Siezen R J,Leunissen J A M.Subtilases:the superfamily of subtilisin-like serine proteases[J].Protein Science A Publication of the Protein Society,1997,6(3):642-658.
[8]李河,周国英,刘君昂.双孢蘑菇褐腐病病原菌的分离及分子鉴定[J].食用菌学报,2009,16(2):74-76.
[9]Uehara R,Angkawidjaja C,Koga Y,et al.Formation of the high-affinity calcium binding site in pro-subtilisin E with the inser tion s e q u e n c e I S1 o f P r o-T k-s u b t i l i si n[J].Biochemistry,2013,52(50):9080-9088.
[10]Guntelberg A V,Ottesen M.Purification of the proteolytic enzyme from Bacillus subtilis[J].Comptes Rendus Des Travaux Du Laboratoire Carlsberg Serie Chimique,1954,29(3-4):36-48.
[11]Dang X,Pan G,Li T,et al.Characterization of a subtilisin-like protease with apical localization from microsporidian Nosema bombycis[J].Journal of Invertebrate Pathology,2013,112(2):166-174.
[12]Shang Y,Xiao G,Zheng P,et al.Divergent and convergent evolution of fungal pathogenicity[J].Genome Biology&Evolution,2016,8(5):1374-1387.