CAJ | 학술논문

【目的】克隆猴头菌 CB1锰过氧化物酶( MnP)基因，用于分析 He-mnp1基因及蛋白序列、基因的结构与功能，为研究猴头菌 MnPs基因功能、转录调控和构建优良工程菌株提供参考。【方法】根据 GenBank 中已报道的白腐菌MnPs基因cDNA序列的保守区域设计引物，采用简并PCR、逆转录RT-PCR、cDNA末端的快速扩增( RACE)等方法扩增出全长 cDNA基因序列，命名为 He-mnp1( GenBank 登录号为 HM116841.3)，并对其猴头菌 He-mnp1基因进行生物信息学的分析。通过 NCBI 数据库进行 BLAST 同源搜索； ORF Finder 查找该基因的完整开放阅读框( ORF)；利用Expasy数据库和BioEdit软件预测He-mnp1蛋白质的理化特性及氨基酸的组成；同时进行亲/疏水性及跨膜区的分析；采用 SignalP 4.1软件进行蛋白信号肽的预测；并利用 Clustal W 和 MEGA 5.1软件对 He-mnp1蛋白序列同源性比对和构建白腐菌MnPs系统发育树；利用数据库Conserved Domain Database( CDD)蛋白保守结构域的预测，查看 He-mnp1血红素、基质及锰、钙等结合位点；采用 PredictProtein 软件和 SWISS-MODEL 软件进行He-mnp1蛋白二级结构的预测和同源三维建模。【结果】该基因 He-mnp1的 cDNA 全长1279 bp，完整开放阅读框1080 bp，起始密码子 ATG，终止密码子 TAA，5'端非翻译区有68个核苷酸，3'端非翻译区有131个核苷酸，编码蛋白359 aa；生物信息学分析表明，猴头菌 He-mnp1氨基酸组成中丙氨酸含量最高，无酪氨酸，分子量为38.18 kDa，等电点为4.35，He-mnp1的蛋白有明显的亲水区，存在81－105、121－141间有2处疏水区域，此蛋白为亲水蛋白。He-mnp1蛋白质多肽前体包含1个18 aa的信号肽及1个5 aa的中间前导短肽。【结论】蛋白系统进化分析表明He-mnp1分布在第2组群，与平菇侧耳、冬生多孔菌、变色栓菌的 MnPs亲缘关系最为接近。He-mnp1基因存在1个保守结构域，分析显示为 Class Ⅱ类的真菌血红素过氧化物酶蛋白家族。He-mnp1蛋白二级结构预测α－螺旋占30.99%，β－折叠占3.38%，无规则卷曲占65.63%，属于稳定蛋白。He-mnp1蛋白三维建模，结果得到1个 Fe 血红素，2个 Ca2+、1个 Mn2+的结合位点及组氨酸残基等。【目的】剋隆猴頭菌 CB1錳過氧化物酶( MnP)基因，用于分析 He-mnp1基因及蛋白序列、基因的結構與功能，為研究猴頭菌 MnPs基因功能、轉錄調控和構建優良工程菌株提供參攷。【方法】根據 GenBank 中已報道的白腐菌MnPs基因cDNA序列的保守區域設計引物，採用簡併PCR、逆轉錄RT-PCR、cDNA末耑的快速擴增( RACE)等方法擴增齣全長 cDNA基因序列，命名為 He-mnp1( GenBank 登錄號為 HM116841.3)，併對其猴頭菌 He-mnp1基因進行生物信息學的分析。通過 NCBI 數據庫進行 BLAST 同源搜索； ORF Finder 查找該基因的完整開放閱讀框( ORF)；利用Expasy數據庫和BioEdit軟件預測He-mnp1蛋白質的理化特性及氨基痠的組成；同時進行親/疏水性及跨膜區的分析；採用 SignalP 4.1軟件進行蛋白信號肽的預測；併利用 Clustal W 和 MEGA 5.1軟件對 He-mnp1蛋白序列同源性比對和構建白腐菌MnPs繫統髮育樹；利用數據庫Conserved Domain Database( CDD)蛋白保守結構域的預測，查看 He-mnp1血紅素、基質及錳、鈣等結閤位點；採用 PredictProtein 軟件和 SWISS-MODEL 軟件進行He-mnp1蛋白二級結構的預測和同源三維建模。【結果】該基因 He-mnp1的 cDNA 全長1279 bp，完整開放閱讀框1080 bp，起始密碼子 ATG，終止密碼子 TAA，5'耑非翻譯區有68箇覈苷痠，3'耑非翻譯區有131箇覈苷痠，編碼蛋白359 aa；生物信息學分析錶明，猴頭菌 He-mnp1氨基痠組成中丙氨痠含量最高，無酪氨痠，分子量為38.18 kDa，等電點為4.35，He-mnp1的蛋白有明顯的親水區，存在81－105、121－141間有2處疏水區域，此蛋白為親水蛋白。He-mnp1蛋白質多肽前體包含1箇18 aa的信號肽及1箇5 aa的中間前導短肽。【結論】蛋白繫統進化分析錶明He-mnp1分佈在第2組群，與平菇側耳、鼕生多孔菌、變色栓菌的 MnPs親緣關繫最為接近。He-mnp1基因存在1箇保守結構域，分析顯示為 Class Ⅱ類的真菌血紅素過氧化物酶蛋白傢族。He-mnp1蛋白二級結構預測α－螺鏇佔30.99%，β－摺疊佔3.38%，無規則捲麯佔65.63%，屬于穩定蛋白。He-mnp1蛋白三維建模，結果得到1箇 Fe 血紅素，2箇 Ca2+、1箇 Mn2+的結閤位點及組氨痠殘基等。
【목적】극륭후두균 CB1맹과양화물매( MnP)기인，용우분석 He-mnp1기인급단백서렬、기인적결구여공능，위연구후두균 MnPs기인공능、전록조공화구건우량공정균주제공삼고。【방법】근거 GenBank 중이보도적백부균MnPs기인cDNA서렬적보수구역설계인물，채용간병PCR、역전록RT-PCR、cDNA말단적쾌속확증( RACE)등방법확증출전장 cDNA기인서렬，명명위 He-mnp1( GenBank 등록호위 HM116841.3)，병대기후두균 He-mnp1기인진행생물신식학적분석。통과 NCBI 수거고진행 BLAST 동원수색； ORF Finder 사조해기인적완정개방열독광( ORF)；이용Expasy수거고화BioEdit연건예측He-mnp1단백질적이화특성급안기산적조성；동시진행친/소수성급과막구적분석；채용 SignalP 4.1연건진행단백신호태적예측；병이용 Clustal W 화 MEGA 5.1연건대 He-mnp1단백서렬동원성비대화구건백부균MnPs계통발육수；이용수거고Conserved Domain Database( CDD)단백보수결구역적예측，사간 He-mnp1혈홍소、기질급맹、개등결합위점；채용 PredictProtein 연건화 SWISS-MODEL 연건진행He-mnp1단백이급결구적예측화동원삼유건모。【결과】해기인 He-mnp1적 cDNA 전장1279 bp，완정개방열독광1080 bp，기시밀마자 ATG，종지밀마자 TAA，5'단비번역구유68개핵감산，3'단비번역구유131개핵감산，편마단백359 aa；생물신식학분석표명，후두균 He-mnp1안기산조성중병안산함량최고，무락안산，분자량위38.18 kDa，등전점위4.35，He-mnp1적단백유명현적친수구，존재81－105、121－141간유2처소수구역，차단백위친수단백。He-mnp1단백질다태전체포함1개18 aa적신호태급1개5 aa적중간전도단태。【결론】단백계통진화분석표명He-mnp1분포재제2조군，여평고측이、동생다공균、변색전균적 MnPs친연관계최위접근。He-mnp1기인존재1개보수결구역，분석현시위 Class Ⅱ류적진균혈홍소과양화물매단백가족。He-mnp1단백이급결구예측α－라선점30.99%，β－절첩점3.38%，무규칙권곡점65.63%，속우은정단백。He-mnp1단백삼유건모，결과득도1개 Fe 혈홍소，2개 Ca2+、1개 Mn2+적결합위점급조안산잔기등。
Objective]The cDNA gene sequences of Manganese peroxidase ( MnP) were isolated from H. erinaceum CB1,and used for analyzing structure and function of the He-mnp1.[Method]Degenerate primers were designed according to conservative domain of white-rot fungi MnPs gene cDNA sequences reported in GenBank,the full-length cDNA gene sequence was obtained by using the methods of PCR,Reverse transcription-PCR and Rapid Amplification of cDNA Ends (RACE) and named as He-mnp1(GenBank No. HM116841. 3),and the bioinformatics of He-mnp1 gene were analyzed. BLAST homology search was conducted through the NCBI database; ORF Finder was used to look up the complete open reading frame of the gene; The Expasy database and BioEdit software were used to predict physicochemical properties and amino acid composition of He-mnp1 protein,and analyze the hydrophilicity/hydrophobicity and transmembrane region;The SignalP 4. 1 software was used to predict protein signal peptide; The clustal W with MEGA 5. 1 software was adopted to complete the He-mnp1 protein sequence homology alignment and to construct the phylogenetic trees of white rot fungi MnPs,respectively. The CDD database was used to predict protein conserved domains,and check the He-mnp1 heme,substrate and manganese,calcium binding site etc. The PredictProtein software and SWISS-MODEL software were used to complete the He-mnp1 protein secondary structure prediction and to construct homologous 3D modeling,respectively.[Result]The full-length cDNA of He-mnp1 was 1 279 bp,the ORF of 1 080 bp with starting codon of ATG and stopping codon of TAA,including 5'UTR of 68 bps and 3'UTR of 131 bps and encoded 359 amino acids. Bioinformatics analysis showed that the He-mnp1 protein has the highest content of Ala,without Tyr,and the Mw 38. 18 is kDa,with the pI of 4. 35 . The He-nmp1 protein has an obvious hydrophilic region and two hydrophobic regions in the area of 81 -105 and 121 -141,and belongs to hydrophilic protein. He-mnp1 protein precursor polypeptides consists of a 18 aa signal peptide and a 5 aa the intermediate leader peptide.[Conclusion]Protein phylogenetic analysis revealed that He-mnp1 is distributed in Group II,and has closely evolutionary relationship to MnPs of Pleurotus ostreatus,Polyporus brumalis,and Trametes versicolor. He-mnp1 has a conserved domain,and belongs to Class II fungal heme-dependent peroxidase superfamily, predicting that the protein secondary structure accounts for α-helix of 30 . 99%,β-sheet of 3 . 38% and random coil of 65. 63%,and it is a stable protein. He-mnp1 protein 3D modeling showed that there are 1 Fe heme,2 Ca2 +,1 Mn2 +binding sites and the histidine residues.