In Silico Cloning,Expression and Bioinformatic Analysis of LaeA from Ganoderma lucidum

doi:10.7668/hbnxb.20194614

Abstract

Abstract:

Obtaining the LaeA gene sequence of Ganoderma lucidum through electronic cloning,analyzing its gene sequence information and preliminarily exploring its regulatory function.This study adopts the method of electronic cloning,using the known LaeA protein sequence of Penicillium citrinum as a template,perform sequence similarity search and alignment(Blast)in the EST database of Ganoderma lucidum and obtain the cDNA sequence of the LaeA gene of Ganoderma lucidum through electronic cloning methods such as sequence splicing,sequence validation and sequence extension.Some characters of amino acids encoded by LaeA gene,including the physical and chemical properties,hydrophobicity/hydrophilicity,subcellular localization,secondary and tertiary structure of protein,and phylogenetic relationship were analyzed by bioinformatics tools.The length of LaeA gene from G.lucidum was 1 134 bp,encoded 378 amino acids.The protein encoded molecular weight of 42.895 3 ku.The protein was an instability protein that was present in the cytoplasm and not secreted to the extracellular.Furthermore,the structure of LaeA protein was mainly composed by 47.88% random coil,33.33%α-helix and 18.78% extension strand,contained SAM binding site,belonged to AdoMet_MTases superfamily proteins.Phylogenetic analysis showed that LaeA was closely related to white rot basidiomycetes such as Trametes versicolor and Dichomitus squalens;the Real-time Fluorescence Quantitative PCR results showed that the expression level of LaeA in Ganoderma lucidum cells during liquid static culture was significantly higher than in oscillatory culture.It is speculated that the LaeA protein as a methyltransferase protein that participates in histone methylation modification,thereby affecting the expression level of gene clusters.

Key words: Ganoderma lucidum, LaeA, Silico cloning, Bioinformatics

HE Wangxing, LI Wenjin, JIANG Yongmei, TONG Zhongfei, CHEN Hualing, LI Yansheng, XIE Xiaoqun, LI Chen. In Silico Cloning,Expression and Bioinformatic Analysis of LaeA from Ganoderma lucidum[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(2): 47-54. doi: 10.7668/hbnxb.20194614.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20194614

http://www.hbnxb.net/EN/Y2024/V39/I2/47

Figures/Tables 13

Tab.1 Bioinformatics analysis software and website

检索内容 Iterns	分析工具 Softwares
基因序列 Gene sequence	http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi
氨基酸组成Amino acid composition	http://www.bioinformatics.org/sms/
理化性质Physical and chemical properties	http://web.expasy.org/protparam/
亲/疏水性 Pro/hydrophobic	http://web.expasy.org/protscale/
亚细胞定位Subcellular localization	http://www.psort.org/
信号肽 Signal peptide	http://www.cbs.dtu.dk/services/SignalP/
二级结构Secondary structure	http://npsa-pbil.ibcp.fr/cgi-bin/secpred_sopma.pl
三级结构 Tertiary structure	http://swissmodel.expasy.org/repository/
同源进化树Homologous evolutionary tree	MEGA 5.0

Fig.1 Gene sequence alignment results

Fig.2 PCR electrophoretogram of LaeA M.DNA Marker;1.LaeA。

Fig.3 The LaeA cDNA sequence of G.luncidum

Fig.4 ORF and conserved domain analysis of the LaeA sequence in G.lucidum

Fig.5 Predicted hydrophobicity/hydrophilicity of the LaeA protein

Fig.6 Signal peptide prediction for LaeA protein from G.lucidum

Fig.7 Secondary structure composition of LaeA protein from G.lucidum

Fig.8 The conformation simulated maps of LaeA protein and template protein A.The conformation simulated maps of LaeA protein;B.The template protein simulated maps of Methanosarcina Mazei.

Fig.9 SAM binding site

Fig.10 The result of multiple sequence alignment between LaeA protein from G.lucidum and those from Basidiomycete species

Fig.11 Phylogenetic tree of LaeA based on the protein sequence

Fig.12 Expression levels of LaeA gene in G.lucidum under different fermentation methods

References 31

[7]	黄泰康. 现代本草纲目[M]. 北京: 中国医药科技出版社, 2001.
	Huang T K. Compendium of modern materia medica[M]. Beijing: China Medical Science and Technology Press, 2001.
[8]	冯道俊. 灵芝的化学成分、功效及药理作用[J]. 特种经济动植物, 2006, 9(8):39-40.doi:10.3969/j.issn.1001-4713.2006.08.031.
	Feng D J. Chemical composition,efficacy and pharmacological action of Ganoderma lucidum[J]. Special Economic Animals and Plants, 2006, 9(8):39-40.
[9]	姜芳燕, 马军, 陈永敢, 陈光宙, 李珍, 王沛正, 王辉芳. 灵芝活性成分的研究进展[J]. 黑龙江农业科学, 2014(8):137-142.doi:10.3969/j.issn.1002-2767.2014.08.039.
	Jiang F Y, Ma J, Chen Y G, Chen G Z, Li Z, Wang P Z, Wang H F. Research progress on active ingredients of Ganoderma lucidum[J]. Heilongjiang Agricultural Sciences, 2014(8):137-142.
[10]	Ahmad M F. Ganoderma lucidum: a rational pharmacological approach to surmount cancer[J]. Journal of Ethnopharmacology, 2020,260:113047.doi:10.1016/j.jep.2020.113047.
[11]	Ryu D H, Cho J Y, Bin Sadiq N, Kim J C, Lee B, Hamayun M, Lee T S, Kim H S, Park S H, Nho C W, Kim H Y. Optimization of antioxidant,anti-diabetic,and anti-inflammatory activities and ganoderic acid content of differentially dried Ganoderma lucidum using response surface methodology[J]. Food Chemistry, 2021,335:127645.doi:10.1016/j.foodchem.2020.127645.
[12]	Zhang W X, Zhong J J. Effect of oxygen concentration in gas phase on sporulation and individual ganoderic acids accumulation in liquid static culture of Ganoderma lucidum[J]. Journal of Bioscience and Bioengineering, 2010, 109(1):37-40.doi:10.1016/j.jbiosc.2009.06.024. URL
[13]	Shi L, Ren A, Mu D S, Zhao M W. Current progress in the study on biosynthesis and regulation of ganoderic acids[J]. Applied Microbiology and Biotechnology, 2010, 88(6):1243-1251.doi:10.1007/s00253-010-2871-1. pmid: 20859739
[14]	Choi D, Ward B L, Bostock R M. Differential induction and suppression of potato 3-hydroxy-3-methylglutaryl coenzyme A reductase genes in response to Phytophthora infestans and to its elicitor arachidonic acid[J]. The Plant Cell, 1992, 4(10):1333-1344.doi:10.1105/tpc.4.10.1333.
[15]	Zhao M W, Zhong J Y, Liang W Q, Wang N, Chen M J, Zhang D B, Pan Y J, Jong S C. Analysis of squalene synthase expression during the development of Ganoderma lucidum[J]. Journal of Microbiology and Biotechnology, 2004, 14(1):116-120.doi:10.1016/S0378-4290(97)00055-5.
[16]	Shang C H, Zhu F, Li N, Ouyang X, Shi L, Zhao M W, Li Y X. Cloning and characterization of a gene encoding HMG-CoA reductase from Ganoderma lucidum and its functional identification in yeast[J]. Bioscience,Biotechnology,and Biochemistry, 2008, 72(5):1333-1339.doi:10.1271/bbb.80011.
[17]	Zhao M W, Liang W Q, Zhang D B, Wang N, Wang C G, Pan Y J. Cloning and characterization of squalene synthase(SQS)gene from Ganoderma lucidum[J]. Journal of Microbiology and Biotechnology, 2007, 17(7):1106-1112.doi:10.1007/s10295-007-0211-y.
[18]	Shang C H, Shi L, Ren A, Qin L, Zhao M W. Molecular cloning,characterization,and differential expression of a lanosterol synthase gene from Ganoderma lucidum[J].Bioscience,Biotechnology,and Biochemistry, 2010, 74(5):974-978.doi:10.1271/bbb.90833.
[19]	周锐, 廖国建, 胡昌华. 丝状真菌次级代谢产物生物合成的表观遗传调控[J]. 生物工程学报, 2011, 27(8):1142-1148.doi:10.13345/j.cjb.2011.08.008. pmid: 22097802
	Zhou R, Liao G J, Hu C H. Epigenetic regulation of secondary metabolite biosynthesis in filamentous fungi:a review[J]. Chinese Journal of Biotechnology, 2011, 27(8):1142-1148. pmid: 22097802
[20]	何懿菡, 孙坤. 生物信息学研究进展[J]. 青海师范大学学报(自然科学版), 2011, 27(3):69-72.doi:10.16229/j.cnki.issn1001-7542.2011.03.028.
	He Y H, Sun K. Bioinformatics research progress and application[J]. Journal of Qinghai Normal University (Natural Science Edition), 2011, 27(3):69-72.
[21]	张瑶瑶, 曹广力, 薛仁宇, 贡成良. 家蚕同源异型结构域蛋白基因Bmvis的克隆及组织表达和亚细胞定位[J]. 蚕业科学, 2011, 37(4):606-613.doi:10.13441/j.cnki.cykx.2011.04.015.
	Zhang Y Y, Cao G L, Xue R Y, Gong C L. Cloning, expression profile and subcellular localization of Bombyx mori homeodomain protein gene Bmvis[J]. Science of Sericulture, 2011, 37(4):606-613.
[22]	Xu J W, Xu Y N, Zhong J J. Production of individual ganoderic acids and expression of biosynthetic genes in liquid static and shaking cultures of Ganoderma lucidum[J]. Applied Microbiology and Biotechnology, 2010, 85(4):941-948.doi:10.1007/s00253-009-2106-5. URL
[23]	董晨, 胡会刚, 贾利强, 决登伟, 赵秋芳, 陈宏良. 香蕉α-1,4-葡聚糖淀粉磷酸化酶基因家族全基因组鉴定与进化分析[J]. 湖北农业科学, 2016, 55(12):3200-3204.doi:10.14088/j.cnki.issn0439-8114.2016.12.051.
	Dong C, Hu H G, Jia L Q, Jue D W, Zhao Q F, Chen H L. Genome-wide identification and phylogenetic analysis of α-1,4-glucan starch phosphorylase gene family of banana[J]. Hubei Agricultural Sciences, 2016, 55(12):3200-3204.
[24]	Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins M R, Appel R D, Bairoch A. Protein identification and analysis tools on the ExPASy server[M]//The Proteomics Protocols Handbook. Totowa, NJ: Humana Press,2005:571-607.doi:10.1385/1-59259-890-0:571.
[25]	王丽芳, 杜希华, 于涌鲲, 赵福宽, 孙清鹏. 番茄WRKY转录因子基因片段的克隆及序列分析[J]. 中国农学通报, 2009, 25(23):70-73.
	Wang L F, Du X H, Yu Y K, Zhao F K, Sun Q P. Cloning and sequence analysis of WRKY transcription factor gene fragment in tomato[J]. Chinese Agricultural Science Bulletin, 2009, 25(23):70-73. doi: 10.11924/j.issn.1000-6850.2009-1332
[26]	翟中和. 细胞生物学[M]. 北京: 高等教育出版社, 2000.
	Zhai Z H. Cell biology[M]. Beijing: Higher Education Press, 2000.
[27]	Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL workspace:a web-based environment for protein structure homology modelling[J]. Bioinformatics, 2006, 22(2):195-201.doi:10.1093/bioinformatics/bti770. URL
[28]	Schwede T, Kopp J, Guex N, Peitsch M C. SWISS-MODEL:an automated protein homology-modeling server[J]. Nucleic Acids Research, 2003, 31(13):3381-3385.doi:10.1093/nar/gkg520. URL
[29]	Guex N, Peitsch M C. SWISS-MODEL and the Swiss-PdbViewer:an environment for comparative protein modeling[J]. Electrophoresis, 1997, 18(15):2714-2723.doi:10.1002/elps.1150181505. pmid: 9504803
[30]	高静, 程汉, 蔡海滨, 安泽伟, 黄华孙. 巴西橡胶树HbRD22基因的电子克隆与生物信息学分析[J]. 热带作物学报, 2010, 31(9):1439-1445.doi:10.3969/j.issn.1000-2561.2010.09.002.
	Gao J, Cheng H, Cai H B, An Z W, Huang H S. In silico cloning and bioinformatics analysis of HbRD22 gene from Hevea brasiliensis[J]. Chinese Journal of Tropical Crops, 2010, 31(9):1439-1445.
[1]	Bok J W, Keller N P. LaeA,a regulator of secondary metabolism in Aspergillus spp[J]. Eukaryotic Cell, 2004, 3(2):527-535.doi:10.1128/EC.3.2.527-535.2004.
[2]	Bok J W, Balajee S A, Marr K A, Andes D, Nielsen K F, Frisvad J C, Keller N P. LaeA,a regulator of morphogenetic fungal virulence factors[J]. Eukaryotic Cell, 2005, 4(9):1574-1582.doi:10.1128/EC.4.9.1574-1582.2005.
[3]	Bok J W, Noordermeer D, Kale S P, Keller N P. Secondary metabolic gene cluster silencing in Aspergillus nidulans[J]. Molecular Microbiology, 2006, 61(6):1636-1645.doi:10.1111/j.1365-2958.2006.05330.x.
[4]	Kosalková K, García-Estrada C, Ullán R V, Godio R P, Feltrer R, Teijeira F, Mauriz E, Martín J F. The global regulator LaeA controls penicillin biosynthesis,pigmentation and sporulation,but not roquefortine C synthesis in Penicillium chrysogenum[J]. Biochimie, 2009, 91(2):214-225.doi:10.1016/j.biochi.2008.09.004. pmid: 18952140
[5]	Perrin R M, Fedorova N D, Bok J W, Cramer R A, Wortman J R, Kim H S, Nierman W C, Keller N P. Transcriptional regulation of chemical diversity in Aspergillus fumigatus by LaeA[J]. PLoS Pathogens, 2007, 3(4):e50.doi:10.1371/journal.ppat.0030050. pmid: 17432932
[6]	Hu Y R, Ahmed S, Li J W, Luo B B, Gao Z Y, Zhang Q Y, Li X H, Hu X B. Improved ganoderic acids production in Ganoderma lucidum by wood decaying components[J]. Scientific Reports, 2017,7:46623.doi:10.1038/srep46623.
[31]	郑建立, 高峰, 田永生, 高建杰, 帅建军, 彭日荷, 熊爱生, 姚泉洪. 葡萄中一类AP2/EREBP-B1亚族转录因子的电子克隆及生物信息学分析[J]. 上海农业学报, 2011, 27(3):9-15.doi:10.3969/j.issn.1000-3924.2011.03.003.
	Zheng J L, Gao F, Tian Y S, Gao J J, Shuai J J, Peng R H, Xiong A S, Yao Q H. In silico cloning and characterization of AP2/EREBP-B1 subfamily transcription factors from Vitis vinifera by using bioinformatics tools[J]. Acta Agriculturae Shanghai, 2011, 27(3):9-15.

Please choose a citation manager

Content to export