苍白杆菌SY286菌株纤维素酶基因的克隆与表达
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摘要
由卵菌引起的植物病害大多难以控制,常因再侵染次数多,暴发流行而导致严重损失。卵菌细胞壁的主要成分为纤维素,因此具有产生纤维素酶能力的生防菌能够更好的用于防治卵菌门真菌引起的植物病害。SY286菌株对葡萄霜霉病菌具有较强抑制活性。纤维素酶活性检测试验结果显示,SY286菌株代谢产物中含纤维素酶,菌落周围透明带宽度达5.9mm。根据已报道的纤维素酶基因序列设计引物,经聚合酶链式反应(PCR)扩增得到了561bp纤维素酶基因相关片段,经Blast比对,该与内切β~(-1),4-葡聚糖酶基因序列相似性达99%,即为SY286基因的核心序列。根据此片段的序列设计特异性引物,通过交错式热不对称PCR(TailPCR)得到其上下游序列,经拼接获得完整的纤维素酶基因的开放阅读框(ORF)序列(命名为SY286基因)。该基因序列ORF长度为1494 bp,编码1个含497个氨基酸的蛋白质,预测其蛋白分子量为55.04k Da,理论等电点PI为8.12。经Blast比对,该序列与内切β~(-1),4-葡聚糖酶编码序列相似性达99%,确定SY286基因为内切β~(-1),4-葡聚糖酶基因。并利用SDS-PAGE电泳对该蛋白在E.coli BL21(DE3)中的表达结果进行了检测。经0.5mmol·L~(-1)的IPTG诱导下,重组菌在15℃、22℃、30℃和37℃条件下的表达产物均可在PAGE凝胶上出现特异性条带。经检测重组菌具有纤维素酶活性。试验结果为研究纤维素酶在植物病害生物防治中的应用,以及SY286菌株抑菌分子机理研究和菌株的遗传改良奠定了基础。
The plant disease s caused by oomycetes mostly were hard to be controlled. The pathogens could infect plant repeatedly, and cause diseases outbreak leading to serious losses. Cellulose is the main component of oomycetes' cell wall.Biocontrol agents which could develop the capacity to produce celluase would be better used to control oomycete disease. SY286 strain had strong inhibitory activity against Plasmopara viticola. The result of cellulase activity detection showed that there were celluases in the metabolite of strain SY286, with the breadth of transparent tape of 5.9 mm. The primers were designed according to the known cellulase gene. The related gene segment of cellulose was amplified by polymerase chain reaction(PCR).The specific primers were designed according this related gene segment. The sequence showed 99% identity as endo-β-1, 4-glucanase gene through blast aligning in Gen Bank. The upstream and downstream sequence were amplified by thermal asymmetric interlaced PCR, and the open reading frame of cellulase gene was obtained from jointing upstream sequence,downstream sequence and the related gene segment. The cellulase gene was named SY286 gene. The open reading frame of SY286 gene was 1494 bp. The protein which was coded by SY286 gene contained 497 amino acids, and the molecular weight of protein was predicted as 55.04 k Da, with the theoretical isoelectric point of 8.12. The amino acid sequence showed 99% identity to endo-β-1, 4-glucanasee, and SY286 gene was identified as endo-β-1, 4-glucanasee gene. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used for detecting whether the recombinant plasmid was expressed in E.coli BL21(DE3).The result showed that recombinant plasmid was expressed successfully in the condition of 15 ℃, 22 ℃, 30 ℃, 37 ℃ under ITPG concentration of 0.5 mmol·L~(-1), and recombination strains had cellulase activity. The result would lay the foundation for cellulase applying to biocontrol of plant disease, and researching on inhibitory molecular mechanisms and genetic modification.
The plant disease s caused by oomycetes mostly were hard to be controlled. The pathogens could infect plant repeatedly, and cause diseases outbreak leading to serious losses. Cellulose is the main component of oomycetes' cell wall.Biocontrol agents which could develop the capacity to produce celluase would be better used to control oomycete disease. SY286 strain had strong inhibitory activity against Plasmopara viticola. The result of cellulase activity detection showed that there were celluases in the metabolite of strain SY286, with the breadth of transparent tape of 5.9 mm. The primers were designed according to the known cellulase gene. The related gene segment of cellulose was amplified by polymerase chain reaction(PCR).The specific primers were designed according this related gene segment. The sequence showed 99% identity as endo-β-1, 4-glucanase gene through blast aligning in Gen Bank. The upstream and downstream sequence were amplified by thermal asymmetric interlaced PCR, and the open reading frame of cellulase gene was obtained from jointing upstream sequence,downstream sequence and the related gene segment. The cellulase gene was named SY286 gene. The open reading frame of SY286 gene was 1494 bp. The protein which was coded by SY286 gene contained 497 amino acids, and the molecular weight of protein was predicted as 55.04 k Da, with the theoretical isoelectric point of 8.12. The amino acid sequence showed 99% identity to endo-β-1, 4-glucanasee, and SY286 gene was identified as endo-β-1, 4-glucanasee gene. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used for detecting whether the recombinant plasmid was expressed in E.coli BL21(DE3).The result showed that recombinant plasmid was expressed successfully in the condition of 15 ℃, 22 ℃, 30 ℃, 37 ℃ under ITPG concentration of 0.5 mmol·L~(-1), and recombination strains had cellulase activity. The result would lay the foundation for cellulase applying to biocontrol of plant disease, and researching on inhibitory molecular mechanisms and genetic modification.
引文
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[4]康兴娇,申红妙,贾招闪,等.葡萄霜霉病生防菌甲基营养型芽胞杆菌T3的鉴定及其防治效果[J].中国生物防治学报,2016,32(6):775-782.
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[8]BAAKZA A,VALA A K,DAVE B P,et al.A comparative study of siderophore production by fungi from marine and terrestrial habitats[J].Journal of Experimental Marine Biology and Ecology,2004,311(1):1-9.
[9]WHITTLE D J,KILBURN D G,WARREN R A J,et al.Molecular cloning of a Cellulomonas fimi cellulase gene in Escherichia coli:Recombinant DNA;plasmid p BR322;immunoassay[J].Gene,1982,17(2):139-145.
[10]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.
[11]HONG J,TAMAKI H,AKIBA S,et al.Cloning of a gene encoding a highly stable endo-beta-1,4-glucanase from Aspergillus niger and its expression in yeast[J].Journal of Bioscience&Bioengineering,2001,92(5):434-441.
[12]LI Y H,DING M,WANG J,et al.A novel thermoacidophilic endoglucanase,Ba-EGA,from a new cellulose-degrading bacterium,Bacillus sp.AC-1[J].Applied Microbiology and Biotechnology,2006,70(4):430-436.
[13]柴国华,付先虎,白泽涛,等.植物内切β-1,4-葡聚糖酶基因研究进展[J].中国农业科技导报,2009,11(5):17-22.
[14]M覫LH覫J M,PAGANT S,H魻FTE H.Towards understanding the role of membrane-bound endo-β-1,4-glucanases in cellulose biosynthesis[J].Plant and Cell Physiol,2002,43(12):1399-1406.
[15]BUSE E L,LATIES G G.Ethylene-mediated posttranscriptional regulation in ripening avocado(Persea americana)mesocarp discs[J].Plant Physiology,1993,102(2):417-423.
[16]TAKASHI A,HANAE K,NAOTO S.Purification and partial characterization of anendo-β-1,3-1,4-glucanase from rice,Oryzasatival[J].Biosci Biotech Biochem,1996,60(12):2078-2080.
[17]范晓静,杨瑞先,邱思鑫,等.内生芽孢杆菌BS2的β-1,4-内切葡聚糖酶基因与定殖相关性[J].中国农业科学,2014,47(2):262-272.
[2]叶正和,王文相,张爱芳,等.我国卵菌病害化学防治概况[J].安徽农业科学,2000,28(4):530-533.
[3]臧超群,赵奎华,刘长远,等.生防细菌SY286的筛选及其对葡萄霜霉病的防治效果研究[J].中国生物防治学报,2014,30(3):402-407.
[4]康兴娇,申红妙,贾招闪,等.葡萄霜霉病生防菌甲基营养型芽胞杆菌T3的鉴定及其防治效果[J].中国生物防治学报,2016,32(6):775-782.
[5]GHOSE TK.Measurement of cellulase activities[J].Pure and Applied Chemistry,2009,59(2):257-268.
[6]LIU Y G,WHITTER R F.Thermal asymmetric interlaced PCR:automatable amplification and sequencing of insert end fragment from P1 and YAC clones for chromosome walking[J].Genomics,1995,25(3):674-681.
[7]张铎.棉花黄萎病拮抗内生细菌的筛选及抗菌物质研究[D].石家庄:河北师范大学,2008.
[8]BAAKZA A,VALA A K,DAVE B P,et al.A comparative study of siderophore production by fungi from marine and terrestrial habitats[J].Journal of Experimental Marine Biology and Ecology,2004,311(1):1-9.
[9]WHITTLE D J,KILBURN D G,WARREN R A J,et al.Molecular cloning of a Cellulomonas fimi cellulase gene in Escherichia coli:Recombinant DNA;plasmid p BR322;immunoassay[J].Gene,1982,17(2):139-145.
[10]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.
[11]HONG J,TAMAKI H,AKIBA S,et al.Cloning of a gene encoding a highly stable endo-beta-1,4-glucanase from Aspergillus niger and its expression in yeast[J].Journal of Bioscience&Bioengineering,2001,92(5):434-441.
[12]LI Y H,DING M,WANG J,et al.A novel thermoacidophilic endoglucanase,Ba-EGA,from a new cellulose-degrading bacterium,Bacillus sp.AC-1[J].Applied Microbiology and Biotechnology,2006,70(4):430-436.
[13]柴国华,付先虎,白泽涛,等.植物内切β-1,4-葡聚糖酶基因研究进展[J].中国农业科技导报,2009,11(5):17-22.
[14]M覫LH覫J M,PAGANT S,H魻FTE H.Towards understanding the role of membrane-bound endo-β-1,4-glucanases in cellulose biosynthesis[J].Plant and Cell Physiol,2002,43(12):1399-1406.
[15]BUSE E L,LATIES G G.Ethylene-mediated posttranscriptional regulation in ripening avocado(Persea americana)mesocarp discs[J].Plant Physiology,1993,102(2):417-423.
[16]TAKASHI A,HANAE K,NAOTO S.Purification and partial characterization of anendo-β-1,3-1,4-glucanase from rice,Oryzasatival[J].Biosci Biotech Biochem,1996,60(12):2078-2080.
[17]范晓静,杨瑞先,邱思鑫,等.内生芽孢杆菌BS2的β-1,4-内切葡聚糖酶基因与定殖相关性[J].中国农业科学,2014,47(2):262-272.