The cloning and prokaryotic expression of the alkaline protease gene TvALP from <i>Trichoderma viride</i>

XU Yangyu; LIU Fuxiang; HONG Yanbin; CHEN Xiaoping; LI Haifeng; WEN Shijie; LI Xingyu; LI Ling; LIANG Xuanqiang

doi:10.15886/j.cnki.rdswxb.20240077

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Volume 15 Issue 6

Nov. 2024

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Article Navigation > Journal of Tropical Biology > 2024 > 15(6): 683-690

XU Yangyu, LIU Fuxiang, HONG Yanbin, CHEN Xiaoping, LI Haifeng, WEN Shijie, LI Xingyu, LI Ling, LIANG Xuanqiang. The cloning and prokaryotic expression of the alkaline protease gene TvALP from Trichoderma viride[J]. Journal of Tropical Biology, 2024, 15(6): 683-690. doi: 10.15886/j.cnki.rdswxb.20240077

Citation:

XU Yangyu, LIU Fuxiang, HONG Yanbin, CHEN Xiaoping, LI Haifeng, WEN Shijie, LI Xingyu, LI Ling, LIANG Xuanqiang. The cloning and prokaryotic expression of the alkaline protease gene TvALP from Trichoderma viride[J]. Journal of Tropical Biology, 2024, 15(6): 683-690. doi: 10.15886/j.cnki.rdswxb.20240077

The cloning and prokaryotic expression of the alkaline protease gene TvALP from Trichoderma viride

doi: 10.15886/j.cnki.rdswxb.20240077

1. Crops Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, Guangdong 510640;
2. Guangdong Agribusiness Tropical Agriculture Research Co., Ltd., Guangzhou, Guangdong 511365;
3. Guangdong Zhongshan Experimental Middle School, Zhongshan, Guangdong 528404;
4. School of Life Science, South China Normal University, Guangzhou, Guangdong 510631, China

Received Date: 2024-05-05
Rev Recd Date: 2024-06-15

Abstract

A peptide sequence was identified from antimicrobial proteins in Trichoderma viride based on the denovo mass spectrometry determination. Through BLASTp database searching, it was found that this peptide sequence is encoded by a alkaline proteinase gene, and the target gene fragment was amplified using homologybased cloning. Bioinformatics analysis reveals that the TvALP gene（GenBank number KJ659907） has a complete open reading frame of 1 230 bp and encodes a protein composed of 409 amino acids. This protein has a predicted molecular weight of about 43 kD, and an isoelectric point of 7.96, and contains a signal peptide composed of 20amino acids. According to classification, the protein belongs to the peptidase inhibitor＿I9 superfamily and is a member of the subtilisin serine protease S8 family. The sequenced plasmid was ligated into the prokaryotic expression vector pET30a using double digestion, and the expression was induced in the host bacterium BL21（DE3）pLysS. The result showed a specific protein band at 45 kD, which was consistent with the predicted size of the target protein, indicating that the expression plasmid pET30a-ΔTvALP（“Δ” Signaling peptide excision） was successfully expressed in the host bacterium BL21（DE3）pLysS. After sonication, the protein formed inclusion bodies. However, after in vitro renaturation, no active protein was obtained for antibacterial mechanism research.
- Trichoderma viride,
- alkaline proteinase gene,
- prokaryotic expression,
- inclusion bodies

References

[1]	曾立,程万里,余豪,等.多粘类芽孢杆菌KM2501-1发酵液对番茄根结线虫的防治效果[J].应用与环境生物学报, 2020, 26(5):1046-1050.
[2]	范亚磊,赵敏,邓晟,等.侵染江苏猕猴桃的北方根结线虫(Meloidogyne hapla)形态学描述和分子特征分析[J].江苏农业学报, 2021, 37(1):75-82.
[3]	李通,毛维兴,薛应钰,等.绿色木霉B3菌株发酵液杀线活性分析及其稳定性测定[J].西北农业学报, 2019,28(9):1535-1542.
[4]	PENG K C, LIN C C, LIAO C F, et al. Expression of Lamino acid oxidase of Trichoderma harzianum in tobacco confers resistance to Sclerotinia sclerotiorum and Botrytis cinerea[J]. Plant Science:an International Journal of Experimental Plant Biology, 2021, 303:110772.
[5]	SWAIN H, ADAK T, MUKHERJEE A K, et al. Novel Trichoderma strains isolated from tree barks as potential biocontrol agents and biofertilizers for direct seeded rice[J].Microbiological Research, 2018, 214:83-90.
[6]	YOUSSEF S A, TARTOURA K A, ABDELRAOUF G A.Evaluation of Trichoderma harzianum and Serratia proteamaculans effect on disease suppression, stimulation of ROS-scavenging enzymes and improving tomato growth infected by Rhizoctonia solani[J]. Biological Control,2016, 100:79-86.
[7]	刘畅,张欣玥,蔡汶妤,等.绿色木霉与哈茨木霉对黄瓜幼苗促生作用机理的研究[J].江苏农业科学, 2020,48(16):156-160.
[8]	王禹佳,武佶,李享,等.发根农杆菌与绿色木霉对玉米幼苗根系生长的影响[J].江苏农业科学, 2020, 48(15):112-117.
[9]	RODRIGUEZ-KABANA R, KELLEY W D, CURL E A.Proteolytic activity of Trichoderma viride in mixed culture with Sclerotium rolfsii in soil[J]. Canadian Journal of Microbiology, 1978, 24(4):487-490.
[10]	叶思帆,赵媛.微生物源碱性蛋白酶的生产及其应用[J].青海科技, 2018, 25(2):73-76.
[11]	王兴吉,佟新伟,王克芬,等.芽孢杆菌产碱性蛋白酶的研究进展[J].食品与发酵科技, 2019, 55(1):62-65.
[12]	KUMAR C G, TAKAGI H. Microbial alkaline proteases:from a bioindustrial viewpoint[J]. Biotechnology Advances,1999, 17(7):561-594.
[13]	RAJAN S, MURUGESAN A. Optimization and Production of Alkaline Protease enzyme from Bacillus subtilis168 isolated from food industry waste[J]. International Journal of Current Microbiology and Applied Sciences,2014, 3(6):36-44.
[14]	BARZKAR N. Marine microbial alkaline protease:an efficient and essential tool for various industrial applications[J]. International Journal of Biological Macromolecules, 2020, 161:1216-1229.
[15]	SHARMA K M, KUMAR R, PANWAR S, et al. Microbial alkaline proteases:Optimization of production parameters and their properties[J]. Journal, Genetic Engineering&Biotechnology, 2017, 15(1):115-126.
[16]	BHATT H B, SINGH S P. Cloning, expression, and structural elucidation of a biotechnologically potential alkaline serine protease from a newly isolated haloalkaliphilic Bacillus lehensis JO-26[J]. Frontiers in Microbiology,2020, 11:941.
[17]	SZEKERES A, KREDICS L, ANTAL Z, et al. Isolation and characterization of protease overproducing mutants of Trichoderma harzianum[J]. FEMS Microbiology Letters, 2004, 233(2):215-222.
[18]	SUÁREZ M B, VIZCAÍNO J A, LLOBELL A, et al. Characterization of genes encoding novel peptidases in the biocontrol fungus Trichoderma harzianum CECT 2413using the TrichoEST functional genomics approach[J].Current Genetics, 2007, 51(5):331-342.
[19]	纪明山,李博强,陈捷,等.绿色木霉TR-8菌株对尖镰孢的拮抗机制[J].中国生物防治, 2005, 21(2):104-108.
[20]	迟玉杰,杨谦,宋颖琦.对哈茨木霉转化子菌体形态观察及抗药性测定[J].哈尔滨工业大学学报, 2002,34(6):784-788.
[21]	POZO M J, BAEK J M, GARCıA J M, et al. Functional analysis of tvsp1, a serine protease-encoding gene in the biocontrol agent Trichoderma virens[J]. Fungal Genetics and Biology, 2004, 41(3):336-348.
[22]	KAPAT A, ZIMAND G, ELAD Y. Effect of two isolates of Trichoderma harzianumon the activity of hydrolytic enzymes produced by Botrytis cinerea[J]. Physiological and Molecular Plant Pathology, 1998, 52(2):127-137.
[23]	徐杨玉,刘付香,洪彦彬,等.绿色木霉对花生黄曲霉毒素污染的防治[J].热带生物学报,2019,10(4):367-371. DOI: 10.15886/j.cnki.rdswxb.2019.04.010.
[24]	康彬,童哲.一种利于蛋白质回收的快速SDS-聚丙烯酰胺凝胶电泳染色-脱色方法[J].生物化学与生物物理进展, 2000, 27(2):210-211.
[25]	BRADFORD M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry, 1976, 72:248-254.
[26]	RAWLINGS N D, BARRETT A J. Proteolytic enzymes:serine and cysteine peptidases[J]. Methods in Enzymology, 1994, 244(26):19-61.
[27]	黄萱,徐子勤,陈立余,等.小麦NBS-LRR类抗病基因同源序列的分离与鉴定[J].分子细胞生物学报, 2006,39(2):91-96.
[28]	YAHIAOUI N, SRICHUMPA P, DUDLER R, et al.Genome analysis at different ploidy levels allows cloning of the powdery mildew resistance gene Pm3b from hexaploid wheat[J]. The Plant Journal:for Cell and Molecular Biology, 2004, 37(4):528-538.
[29]	姜海霞,刘永生,杨孝朴,等.人朊蛋白基因的克隆与原核表达[J].甘肃农业大学学报, 2006, 41(3):11-15.
[30]	王晓东,牟玉莲,张莉,等.近交五指山小型猪SLADQA基因的克隆及在大肠杆菌的原核表达[J].甘肃农业大学学报, 2006, 41(4):23-26.

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通讯作者: 陈斌, bchen63@163.com

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沈阳化工大学材料科学与工程学院沈阳 110142

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The cloning and prokaryotic expression of the alkaline protease gene TvALP from Trichoderma viride

1. Crops Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, Guangdong 510640;

2. Guangdong Agribusiness Tropical Agriculture Research Co., Ltd., Guangzhou, Guangdong 511365;

3. Guangdong Zhongshan Experimental Middle School, Zhongshan, Guangdong 528404;

4. School of Life Science, South China Normal University, Guangzhou, Guangdong 510631, China

Received Date: 2024-05-05

Rev Recd Date: 2024-06-15

Key words:

Abstract: A peptide sequence was identified from antimicrobial proteins in Trichoderma viride based on the denovo mass spectrometry determination. Through BLASTp database searching, it was found that this peptide sequence is encoded by a alkaline proteinase gene, and the target gene fragment was amplified using homologybased cloning. Bioinformatics analysis reveals that the TvALP gene（GenBank number KJ659907） has a complete open reading frame of 1 230 bp and encodes a protein composed of 409 amino acids. This protein has a predicted molecular weight of about 43 kD, and an isoelectric point of 7.96, and contains a signal peptide composed of 20amino acids. According to classification, the protein belongs to the peptidase inhibitor＿I9 superfamily and is a member of the subtilisin serine protease S8 family. The sequenced plasmid was ligated into the prokaryotic expression vector pET30a using double digestion, and the expression was induced in the host bacterium BL21（DE3）pLysS. The result showed a specific protein band at 45 kD, which was consistent with the predicted size of the target protein, indicating that the expression plasmid pET30a-ΔTvALP（“Δ” Signaling peptide excision） was successfully expressed in the host bacterium BL21（DE3）pLysS. After sonication, the protein formed inclusion bodies. However, after in vitro renaturation, no active protein was obtained for antibacterial mechanism research.

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Reference (30)

[1]	曾立,程万里,余豪,等.多粘类芽孢杆菌KM2501-1发酵液对番茄根结线虫的防治效果[J].应用与环境生物学报, 2020, 26(5):1046-1050.
[2]	范亚磊,赵敏,邓晟,等.侵染江苏猕猴桃的北方根结线虫(Meloidogyne hapla)形态学描述和分子特征分析[J].江苏农业学报, 2021, 37(1):75-82.
[3]	李通,毛维兴,薛应钰,等.绿色木霉B3菌株发酵液杀线活性分析及其稳定性测定[J].西北农业学报, 2019,28(9):1535-1542.
[4]	PENG K C, LIN C C, LIAO C F, et al. Expression of Lamino acid oxidase of Trichoderma harzianum in tobacco confers resistance to Sclerotinia sclerotiorum and Botrytis cinerea[J]. Plant Science:an International Journal of Experimental Plant Biology, 2021, 303:110772.
[5]	SWAIN H, ADAK T, MUKHERJEE A K, et al. Novel Trichoderma strains isolated from tree barks as potential biocontrol agents and biofertilizers for direct seeded rice[J].Microbiological Research, 2018, 214:83-90.
[6]	YOUSSEF S A, TARTOURA K A, ABDELRAOUF G A.Evaluation of Trichoderma harzianum and Serratia proteamaculans effect on disease suppression, stimulation of ROS-scavenging enzymes and improving tomato growth infected by Rhizoctonia solani[J]. Biological Control,2016, 100:79-86.
[7]	刘畅,张欣玥,蔡汶妤,等.绿色木霉与哈茨木霉对黄瓜幼苗促生作用机理的研究[J].江苏农业科学, 2020,48(16):156-160.
[8]	王禹佳,武佶,李享,等.发根农杆菌与绿色木霉对玉米幼苗根系生长的影响[J].江苏农业科学, 2020, 48(15):112-117.
[9]	RODRIGUEZ-KABANA R, KELLEY W D, CURL E A.Proteolytic activity of Trichoderma viride in mixed culture with Sclerotium rolfsii in soil[J]. Canadian Journal of Microbiology, 1978, 24(4):487-490.
[10]	叶思帆,赵媛.微生物源碱性蛋白酶的生产及其应用[J].青海科技, 2018, 25(2):73-76.
[11]	王兴吉,佟新伟,王克芬,等.芽孢杆菌产碱性蛋白酶的研究进展[J].食品与发酵科技, 2019, 55(1):62-65.
[12]	KUMAR C G, TAKAGI H. Microbial alkaline proteases:from a bioindustrial viewpoint[J]. Biotechnology Advances,1999, 17(7):561-594.
[13]	RAJAN S, MURUGESAN A. Optimization and Production of Alkaline Protease enzyme from Bacillus subtilis168 isolated from food industry waste[J]. International Journal of Current Microbiology and Applied Sciences,2014, 3(6):36-44.
[14]	BARZKAR N. Marine microbial alkaline protease:an efficient and essential tool for various industrial applications[J]. International Journal of Biological Macromolecules, 2020, 161:1216-1229.
[15]	SHARMA K M, KUMAR R, PANWAR S, et al. Microbial alkaline proteases:Optimization of production parameters and their properties[J]. Journal, Genetic Engineering&Biotechnology, 2017, 15(1):115-126.
[16]	BHATT H B, SINGH S P. Cloning, expression, and structural elucidation of a biotechnologically potential alkaline serine protease from a newly isolated haloalkaliphilic Bacillus lehensis JO-26[J]. Frontiers in Microbiology,2020, 11:941.
[17]	SZEKERES A, KREDICS L, ANTAL Z, et al. Isolation and characterization of protease overproducing mutants of Trichoderma harzianum[J]. FEMS Microbiology Letters, 2004, 233(2):215-222.
[18]	SUÁREZ M B, VIZCAÍNO J A, LLOBELL A, et al. Characterization of genes encoding novel peptidases in the biocontrol fungus Trichoderma harzianum CECT 2413using the TrichoEST functional genomics approach[J].Current Genetics, 2007, 51(5):331-342.
[19]	纪明山,李博强,陈捷,等.绿色木霉TR-8菌株对尖镰孢的拮抗机制[J].中国生物防治, 2005, 21(2):104-108.
[20]	迟玉杰,杨谦,宋颖琦.对哈茨木霉转化子菌体形态观察及抗药性测定[J].哈尔滨工业大学学报, 2002,34(6):784-788.
[21]	POZO M J, BAEK J M, GARCıA J M, et al. Functional analysis of tvsp1, a serine protease-encoding gene in the biocontrol agent Trichoderma virens[J]. Fungal Genetics and Biology, 2004, 41(3):336-348.
[22]	KAPAT A, ZIMAND G, ELAD Y. Effect of two isolates of Trichoderma harzianumon the activity of hydrolytic enzymes produced by Botrytis cinerea[J]. Physiological and Molecular Plant Pathology, 1998, 52(2):127-137.
[23]	徐杨玉,刘付香,洪彦彬,等.绿色木霉对花生黄曲霉毒素污染的防治[J].热带生物学报,2019,10(4):367-371.	DOI:10.15886/j.cnki.rdswxb.2019.04.010.
[24]	康彬,童哲.一种利于蛋白质回收的快速SDS-聚丙烯酰胺凝胶电泳染色-脱色方法[J].生物化学与生物物理进展, 2000, 27(2):210-211.
[25]	BRADFORD M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry, 1976, 72:248-254.
[26]	RAWLINGS N D, BARRETT A J. Proteolytic enzymes:serine and cysteine peptidases[J]. Methods in Enzymology, 1994, 244(26):19-61.
[27]	黄萱,徐子勤,陈立余,等.小麦NBS-LRR类抗病基因同源序列的分离与鉴定[J].分子细胞生物学报, 2006,39(2):91-96.
[28]	YAHIAOUI N, SRICHUMPA P, DUDLER R, et al.Genome analysis at different ploidy levels allows cloning of the powdery mildew resistance gene Pm3b from hexaploid wheat[J]. The Plant Journal:for Cell and Molecular Biology, 2004, 37(4):528-538.
[29]	姜海霞,刘永生,杨孝朴,等.人朊蛋白基因的克隆与原核表达[J].甘肃农业大学学报, 2006, 41(3):11-15.
[30]	王晓东,牟玉莲,张莉,等.近交五指山小型猪SLADQA基因的克隆及在大肠杆菌的原核表达[J].甘肃农业大学学报, 2006, 41(4):23-26.

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The cloning and prokaryotic expression of the alkaline protease gene TvALP from Trichoderma viride

doi: 10.15886/j.cnki.rdswxb.20240077

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通讯作者: 陈斌, bchen63@163.com

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The cloning and prokaryotic expression of the alkaline protease gene TvALP from Trichoderma viride

doi: 10.15886/j.cnki.rdswxb.20240077

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通讯作者: 陈斌, bchen63@163.com

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