Cloning\Expression and Bioactivity of the Chitinase Gene <i>ChiA</i> from the Endophytes of <i>Periplaneta americana</i>

doi:10.13523/j.cb.20190104

China Biotechnology

2019, Vol. 39

Issue (1): 31-37 DOI: 10.13523/j.cb.20190104

Cloning\Expression and Bioactivity of the Chitinase Gene ChiA from the Endophytes of Periplaneta americana

Min-hua XU,Jing-jing ZHANG,Xiao-bao JIN,Xia-bo LI,Yan WANG,Yan MA(

)

Guangdong Pharmaceutical University, Guangdong Key Laboratory of Pharmaceutical Bioactive Substances, Guangzhou 510006, China

Download:

HTML

PDF(993KB) HTML
Export: BibTeX | EndNote (RIS)

Abstract

Objective: To clone the chitinase gene ChiA from the endophytes of Periplaneta americana soluble expression of the protein and to identify its function.Methods:The chitinase gene ChiA was amplified by PCR from the DNA of Serratia marcescens,which was separated from the gut of Periplaneta americana and obtained by subcloning. The expression plasmid ChiA/pET21b was constructed and analyzed by bioinformatics. The plasmid was transformed into E. coli BL21(DE3) and the postive strains were induced by IPTG at 20℃ for 20h. The bioactivity of the protein was determined by small punch test.Results:The cloned sequence was associated with Serratia marcescens ChiA gene of GenBank and their homology was 99%. The sequence encoded a protein containing of 571 amino acids and expressed stably in prokaryotic system.SDS-PAGE/Western blot show that the soluble target protein was obtained. The small punch test suggested that the target protein had the activity of decomposing chitin and was stronger than that of the Serratia marcescens.Conclusion:The chitinase gene ChiA of the Serratia marcescens from the gut of Periplaneta americana was cloned successfully. The soluble chitinase that shows marked bioactivity was attained by prokaryotic expression system, which has provided theoretical basis for its further application.

Key words： Periplaneta americana Serratia marcescens Chitinase ChiA Soluble expression Bioactivity

Received: 25 July 2018 Published: 28 February 2019

ZTFLH:

Q78

Corresponding Authors: Yan MA E-mail: mayan820901@126.com

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	Min-hua XU
	Jing-jing ZHANG
	Xiao-bao JIN
	Xia-bo LI
	Yan WANG
	Yan MA

Cite this article:

Min-hua XU,Jing-jing ZHANG,Xiao-bao JIN,Xia-bo LI,Yan WANG,Yan MA. Cloning\Expression and Bioactivity of the Chitinase Gene ChiA from the Endophytes of Periplaneta americana. China Biotechnology, 2019, 39(1): 31-37.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190104 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I1/31

Fig.1 PCR amplification and sucloning of ChiA gene M:DL2000 DNA marker;1: PCR product of ChiA gene;M1: DL10000 DNA marker; 2: PCR product of ChiA/pMD18-T; 3: ChiA/pMD18-T digested with Xho I; 4: ChiA/pMD18-T digested with Nde I/Xho I

Fig.2 Screening of recombinant plasmids ChiA/pET21b M: DL2000 DNA mMarker;1: PCR product of ChiA gene from positive colonies;2,3: PCR product of negative colonies; M1: DL10000 DNA marker;4: ChiA/pET21b digested with Xho I; 5: ChiA/pET21b digested with Nde I/Xho I

Fig.3 Amino acid sequence

Fig.4 Hydrophilicity or Hydrophobicity prediction result

Fig.5 The result of the SDS-PAGE and Western blot M: Protein molecular weight marker; 1: Total proteins of ChiA/pET21b/BL21 by uninduced; 2: Total proteins of ChiA/pET21b/BL21 by induced; 3: Total proteins of pET21b/BL21 by uninduced; 4: Total proteins of pET21b/BL21; 5: Total proteins of ChiA/pET21b/BL21 by induced; 6: Total proteins of liquid supernatant; 7:Total proteins of sediment

Fig.6 Bioactivity test 1: ChiA/pET21b/BL21;2: WA12-1-18; 3: Liquid supernatant of ChiA/pET21b/BL21;4: Buffer solution

Table 1 The result of bioactivity test of chitinase


[1]	Yuli P E, Suhartono M T, Rukayadi Y , et al. Characteristics of thermostable chitinase enzymes from the indonesian Bacillus sp.13.26. Enzyme and Microbial Technology, 2004,35(2/3):147-153. doi: 10.1016/j.enzmictec.2004.03.017

[2]	Dutta J, Tripathi S, Dutta P K , et al. Progress in antimicrobial activities of chitin, chitosan and its oligosaccharides: a system-atic study needs for food applications. Food Science and Technology International, 2012,18(1):3-34. doi: 10.1177/1082013211399195 pmid: 6821198

[3]	Horn S J, Sikorski P, Cederkvist J B , et al. Costs and benefits of processivity in enzymatic degradation of recalcitrant polysaccharides. P Natl Acad Sci USA, 2006,103(48):18089-18094. doi: 10.1073/pnas.0608909103

[4]	李海浪 . 壳聚糖衍生物的制备及其在药物载体中的应用研究. 上海: 中国科学院大学, 2014.

[4]	Li H L . Study on the preparation of chitosan derivatives and their applications in drug delivery. Shanghai: University of Chinese Academy of Sciences, 2014.

[5]	Benecke U . Bacillus chitinovorous einen chitin zersetzenden Spaltpilz. Bot Ztg, 1905,63(8):227-242.

[6]	Merzendorfer H . The cellular basis of chitin synjournal in fungi and insect: common principles and differaices. Eur[J] Cell Biol. 2011,90(9):759-769.

[7]	糜艳霞, 任慧, 张常 , 等. 几丁质酶的研究进展. 生命科学研究, 2015,19(5):437-442. doi: 10.3969/j.issn.1004-311X.2001.05.012

[7]	Mi Y X, Ren H, Zhang C , et al. Advances in study and application on chitinase. Life Science Research, 2015,19(5):437-442. doi: 10.3969/j.issn.1004-311X.2001.05.012

[8]	白腾飞, 刘月芹 . 沙雷氏菌抗生性次级代谢产物合成机制. 微生物学杂志, 2017,37(4):115-119. doi: 10.3969/j.issn.1005-7021.2017.04.017

[8]	Bai T F, Liu Y Q . A Survey of synthesis mechanism of antibiotic secondary metabolites by Serratia spp. Journal of Microbiology, 2017,37(4):115-119. doi: 10.3969/j.issn.1005-7021.2017.04.017

[9]	方霞, 沈娟, 王影姣 , 等. 药用昆虫美洲大蠊内生放线菌的分离和鉴定. 中国病原生物学杂志, 2016,11(6):550-565.

[9]	Fang X, Shen J, Wang Y J , et al. Isolation and identification of endophytic actinomycetes from medical insects (Periplaneta Americana). Journal of Pathogen Biology, 2016,11(6):550-565.

[10]	Van Arnam E B, Currie C R, Clardy J . Defense contracts: molecular protection in insect-microbe symbioses. Chemical Society Reviews, 2018,47(57):1638-1651. doi: 10.1039/C7CS00340D

[11]	贺淹才, 刘爱花, 柴思捷 , 等. 改进RBB染色法初步筛选几丁质酶基因chiA的表达. 华侨大学学报(自然科学版), 2008,29(4):563-566. doi: 10.11830/issn.1000-5013.2008.04.0563

[11]	He Y C, Liu A H, Chai S J , et al. Improved RBB dyeing method screen bacteria of expression of chintinase chiA. Journal of Huaqiao University (Natural Science), 2008,29(4):563-566. doi: 10.11830/issn.1000-5013.2008.04.0563

[12]	Chung W C, Chen L L, Lo W S , et al. Complete genome sequence of Serratia marcescens WW4. Genome Announc, 2013,4(2):12-13.

[13]	Francesco M, Sushmita M . Natural computing methods in bioin-formatics: A survey. Information Fusion, 2009,10(3):211-216. doi: 10.1016/j.inffus.2008.12.005

[14]	Balzer S, Kucharova V, Megerle J , et al. A comparative analysis of the properties of regulated promoter systemscommonly used for recombinant gene expression in Escherichia coli. Microb Cell Fact, 2013,12(1):1-14. doi: 10.1186/1475-2859-12-1 pmid: 23282100

[15]	刘丹, 孟凡欢, 李爽 , 等. 一株产几丁质酶渤海丝状真菌(Alternaria tenuissima)的筛选及鉴定. 中国酿造, 2012,31(2):45-28. doi: 10.3969/j.issn.0254-5071.2012.02.012

[15]	Liu D, Meng F H, Li S , et al. Screening and identification of chitinase-producing fungus (Alternaria tenuissima) from Bohai Bay. China Brewing, 2012,31(2):45-28. doi: 10.3969/j.issn.0254-5071.2012.02.012

[16]	李晶, 于丽萍, 刘宇帅 , 等. 黏质沙雷氏菌几丁质酶基因的研究进展. 基因组学与应用生物学, 2017,36(9):3837-3841.

[16]	Li J, Yu L P, Liu Y S , et al. Research progress of chitinase gene from Serratia marcescens. Genomics and Applied Biology, 2017,36(9):3837-3841.

[17]	孟利强, 沙长青, 张先成 , 等. 黏质沙雷氏菌几丁质酶基因ChiA克隆及生物信息学分析. 微生物学杂志, 2016,36(1):62-68. doi: 10.3969/j.issn.1005-7021.2016.01.011

[17]	Meng L Q, Sha C Q, Zhang X C , et al. Cloning and bioinformatics analysis of chitinase gene ChiA from Serratia marcescens. Journal of Microbiology, 2016,36(1):62-68. doi: 10.3969/j.issn.1005-7021.2016.01.011

[18]	Shapira R., Ordentlich A., Chet I , et al. Control of plant diseases by chitinase expressed from cloned DNA in Escherichia coli. Phytopathology, 1989,79(11):1246-1249. doi: 10.1094/Phyto-79-1246

[1]	Jian-xue TANG,Yong-le XIAO,Jun-jie PENG,Shi-ji ZHAO,Xiao-ping WAN,Rong GAO. Expression of Fusion Antibacterial Peptide in Recombinant Pichia pastoris and Its Bioactivity In Vitro[J]. China Biotechnology, 2018, 38(6): 9-16.

[2]	ZHANG He-ming, CAI Chu-fan, LIU Yang, GAN Long-zhan, JIAO Xue-miao, TIAN Yong-qiang. Soluble Expression of Human Leukemia Inhibitory Factor in Prokaryotic Cells and Its Purification[J]. China Biotechnology, 2017, 37(9): 7-14.

[3]	JI Jun, ZHU Chen-chen, XU Xin, LIU Xiau, LENG Chao-liang, SHI Hong-fei, YAO Lun-guang, KAN Yun-chao. *Soluble Fusion-expression and Antitumor Activity Analysis of Apoptin* of Chicken Anemia Virus**[J]. China Biotechnology, 2017, 37(2): 26-32.

[4]	ZHANG Yu-meng, TONG Mei, LU Xiao-dong, MI Yue, XU Chen, YAO Wen-bing. Advances in Promoting Soluble Expression of Recombinant Protein in Escherichia coli[J]. China Biotechnology, 2016, 36(5): 118-124.

[5]	LIU Pan-rao, ZHOU Xue-chen, ZHU Xue-jiao, BAI Juan, WANG Xian-wei, JIANG Ping. *The Soluble Expression of Porcine Circovirus Type 2 Cap Gene in Escherichia coli* and Its Immunogenicity in Mice**[J]. China Biotechnology, 2016, 36(4): 50-56.

[6]	WANG Lan, XU Gang-ling, GAO Kai, WANG Jun-zhi. Progress in Research and Development of Bioactivity Determination of Antibody-based Therapeutics[J]. China Biotechnology, 2015, 35(6): 101-108.

[7]	LU Qing-shan, QIAO Yuan-yuan, LI Jin-feng, WANG Yun-liang, WANG Shan-shan, SHI Cheng-he, YANG Xiao-peng, ZHANG Da-jin. Soluble Expression of Human HPPCn Recombinant Protein and Detection of Its Proliferation Activity[J]. China Biotechnology, 2015, 35(12): 15-20.

[8]	ZHANG Chao, GONG Wei, GUO Ying-ying, SUN Wei-guo, YAO Min, YU Ai-ping. The Prokaryotic Expression of an Anti-coagulant Protein of EH[J]. China Biotechnology, 2014, 34(12): 69-77.

[9]	SUN Jing, WANG Bin, DUAN Zhi-qing, HU Ning-zhu, LI Jian-fang, LI Yan-han, HU Yun-zhang. Expression, Purification and Bioactivity Identification of Recombinant Human Leukemia Inhibitory Factor (hLIF) Fusion Protein[J]. China Biotechnology, 2013, 33(5): 50-55.

[10]	LU Qing-peng, XU Zhen-hong, SI Jin-song, DOU Wen-fang. *The Disruption of STE13* Gene in Pichia pastoris Improves the Expression and Bioactivity of GGH**[J]. China Biotechnology, 2013, 33(4): 28-33.

[11]	FENG Cui, ZHAO Da-wei, ZHANG Chun, WANG Jian, QIN Pei-yong, LIU Yong-dong, SU Zhi-guo. Purification and Characterization of a New Recombinant Cilinary Neuronotrophic Factor Mutant Expressed in Soluble Form by E.coli[J]. China Biotechnology, 2013, 33(10): 21-27.

[12]	TAO Feng-yun, YIN Xue-wei, LI Dan, JIANG Dan, ZHAO Wei. Prokaryotic Soluble Expression of a Novel Ribonuclease Gene Rdrlec from Rana dybowskii[J]. China Biotechnology, 2013, 33(1): 27-32.

[13]	LUO Li, HE Yong-zhi, ZHANG Yong-xia, WANG Ming-rong. Advances in the Study of non-classical Inclusion Bodies[J]. China Biotechnology, 2013, 33(1): 114-121.

[14]	HAN Cui-xiao, LI Feng, YAN Xiao-jin, FENG Duo, LI Qian-qian, GAO Hong-bo, GAO Wei. *Optimization of Conditions for Soluble Expression and Purification of the Cytosolic Region of Arabidopsis thaliana* PDV1**[J]. China Biotechnology, 2012, 32(6): 35-42.

[15]	LI Qian-qian, LI Zhong-yuan, FENG Duo, HUANG Huo-qing, HAN Cui-xiao, YANG Pei-long, YAO Bin, GAO Wei. Optimizing Soluble Expression and Inclusion Body Renature Research of β-Propeller Phytase of Bacillus sp. HJB17 in E.coli[J]. China Biotechnology, 2012, 32(08): 49-55.

Viewed

Full text

Abstract

Cited

Shared

Discussed