Cloning and expression of GPS gene induced by methyl jasmonate in Sanghuangporus baumii

doi:10.12302/j.issn.1000-2006.202108040

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2023, Vol. 47 ›› Issue (4): 123-130.doi: 10.12302/j.issn.1000-2006.202108040

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Cloning and expression of GPS gene induced by methyl jasmonate in Sanghuangporus baumii

LIU Zengcai(), WANG Shuting, TONG Xinyu, ZOU Li()

College of Forestry, Northeast Forestry University, Harbin 150040, China

Received:2021-08-23 Revised:2022-04-29 Online:2023-07-30 Published:2023-07-20

Abstract

Abstract:

【Objective】 To further explore the molecular mechanisms of triterpenoid biosynthesis of Sanghuangporus baumii, in this study, the induced expression of the key enzyme geranyl pyrophosphate synthase (GPS) gene, which is involved in triterpenoid biosynthesis, was cloned and analyzed. 【Method】The full-length cDNA and promoter of the GPS gene were cloned by PCR amplification, and the sequence was analyzed by bioinformatics software. Then, the qRT-PCR technique was used to analyze the effects of different concentrations of MeJA on the GPS gene transcript level of S. baumii, and the change in triterpenoid content of S. baumii induced by different concentrations of MeJA was determined by spectrophotometer. 【Result】The cloned GPS gene and promoter from S. baumii were named SbGPS and SbGPS promoter, respectively. The cDNA sequence of the SbGPS gene was 1 617 bp, encoding 538 amino acids. Signal peptides and transmembrane structures were not detected. The results of SbGPS promoter analysis showed that, in addition to the typical promoter action elements, such as CAAT-box and TATA-box, a methyl jasmonate response element was included. The results of qRT-PCR analysis and triterpenoid content determination showed that the expression of the SbGPS gene and triterpenoid content initially increased and then decreased with an increase in MeJA concentration. The change trend was basically the same, showing a significant positive correlation. 【Conclusions】 The SbGPS gene plays an important role in the biosynthesis of triterpenoids by cloning and expression analysis, which lays a foundation for further study on the regulation mechanisms of the SbGPS gene in the biosynthesis of triterpenoids of S. baumii.

Key words: Sanghuangporus baumii, geranyl pyrophosphate synthase, gene cloning, promoter cloning, methyl jasmonate(MeJA)

CLC Number:

Q718

LIU Zengcai, WANG Shuting, TONG Xinyu, ZOU Li. Cloning and expression of GPS gene induced by methyl jasmonate in Sanghuangporus baumii[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2023, 47(4): 123-130.

Figures/Tables 6

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References 30

[1]	丁云云. 药用真菌桑黄化学成分的研究[D]. 合肥: 安徽医科大学, 2017.
	DING Y Y. Chemical constituents of the medicinal fungus Phellinus igniarius[D]. Hefei: Anhui Medical University, 2017.
[2]	KIM H M, HAN S B, OH G T, et al. Stimulation of humoral and cell mediated immunity by polysaccharide from mushroom Phellinus linteus[J]. Int J Immunopharmacol, 1996, 18(5): 295-303. DOI:10.1016/0192-0561(96)00028-8.
[3]	JEON T I, HWANG S G, LIM B O, et al. Extracts of Phellinus linteus grown on germinated brown rice suppress liver damage induced by carbon tetrachloride in rats[J]. Biotechnol Lett, 2003, 25(24): 2093-2096. DOI:10.1023/b:bile.0000007071.28105.c1.
[4]	张林芳, 孙婷婷, 邹莉. 鲍姆纤孔菌总三萜的提取及其体外抗乳腺癌细胞MCF-7活性[J]. 药物评价研究, 2015, 38(5): 497-502.
	ZHANG L F, SUN T T, ZOU L. Extraction of total triterpenoids from Inonotus baumii and its inhibitory activity on breast cancer cells (MCF-7) in vitro[J]. Drug Eval Res, 2015, 38(5): 497-502. DOI:10.7501/j.issn.1674-6376.2015.05.006.
[5]	SHAO Y, GUO H W, ZHANG J P, et al. The genome of the medicinal macrofungus Sanghuang provides insights into the synthesis of diverse secondary metabolites[J]. Front Microbiol, 2020, 10: 3035. DOI:10.3389/fmicb.2019.03035.
[6]	IKEKAWA T, NAKANISHI M, UEHARA N, et al. Antitumor action of some Basidiomycetes, especially Phellinus linteus[J]. Gann, 1968, 59(2): 155-157. DOI:10.20772/cancersci1959.59.2_155.
[7]	DAI Y C, ZHOU L W, CUI B K, et al. Current advances in Phellinus sensu lato: medicinal species, functions, metabolites and mechanisms[J]. Appl Microbiol Biotechnol, 2010, 87(5):1587-1593.DOI:10.1007/s00253-010-2711-3.
[8]	TAJI S, YAMADA T, WADA S I, et al. Lanostane-type triterpenoids from the sclerotia of Inonotus obliquus possessing anti-tumor promoting activity[J]. Eur J Med Chem, 2008, 43(11): 2373-2379. DOI:10.1016/j.ejmech.2008.01.037.
[9]	冯娜, 吴娜, 杨焱, 等. 鲍姆木层孔菌子实体中化合物的结构鉴定及其抗肿瘤活性[J]. 菌物研究, 2013, 11(2):150.
	FENG N, WU N, YANG Y, et al. Structural identification and antitumor activity of compounds from the fruiting body of Phellinus baumannii[J]. J Fungal Res, 2013, 11(2):150. DOI:10.13341/j.jfr.2013.02.007.
[10]	孙婷婷. 桑黄三萜生物合成途径关键酶基因的挖掘及分析[D]. 哈尔滨: 东北林业大学, 2017.
	SUN T T. Analysis and systematic mining of genes involved in the biosynthetic pathway of triterpenoids in Sanghuangporus baumii[D]. Harbin:Northeast Forestry University, 2017.
[11]	刘增才, 孙婷婷, 王世新, 等. 暴马桑黄MVD基因cDNA全长克隆及表达特性分析[J]. 南京林业大学学报(自然科学版), 2020, 44(4):79-85.
	LIU Z C, SUN T T, WANG S X, et al. The cloning and expression analysis of mevalonate pyrophosphate decarboxylase gene cDNA sequence from Sanghuangporus baumii[J]. J Nanjing For Univ (Nat Sci Ed), 2020, 44(4):79-85. DOI:10.3969/j.issn.1000-2006.201912007.
[12]	WANG S X, LIU Z C, WANG X T, et al. Cloning and characterization of a phosphomevalonate kinase gene from Sanghuangporus baumii[J]. Biotechnol Biotechnol Equip, 2021, 35(1):934-942. DOI:10.1080/13102818.2021.1938678.
[13]	WANG X T, WANG S X, XU X R, et al. Molecular cloning, characterization, and heterologous expression of an acetyl-CoA acetyl transferase gene from Sanghuangporus baumii[J]. Protein Expr Purif, 2020, 170: 105592. DOI:10.1016/j.pep.2020.105592.
[14]	WANG X T, SUN T T, SUN J, et al. Molecular cloning, characterisation, and heterologous expression of farnesyl diphosphate synthase from Sanghuangporus baumii[J]. Mol Biotechnol, 2020, 62(2):132-141. DOI: 10.1007/s12033-019-00231-0.
[15]	LIU Z C, WANG S X, XU X R, et al. Molecular cloning and characterization of a gene encoding HMG-CoA reductase involved in triterpenoids biosynthetic pathway from Sanghuangporus baumii[J]. Biotechnol Biotechnol Equip, 2021, 35(1): 796-804.DOI: 10.1080/13102818.2021.1929482.
[16]	WANG X T, SUNT T, SUN J, et al. Expression analysis of lanosterol synthase gene in dynamic accumulation of triterpenoids in Sanghuangporus baumii[J]. Protein Pept Lett, 2022, 29(1): 37-45.DOI:10.2174/0929866528666210922103059.
[17]	LIU Z C, SUN T T, WANG S X, et al. Cloning, molecular properties and differential expression analysis of the isopentenyl diphosphate isomerase gene in Sanghuangporus baumii[J]. Biotechnol Biotechnol Equip, 2020, 34(1):623-630. DOI:10.1080/13102818.2020.1792342
[18]	刘增才, 孙婷婷, 孙健, 等. 暴马桑黄甲羟戊酸激酶基因的克隆及差异表达分析[J/OL]. 吉林农业大学学报.
	LIU Z C, SUN T T, SUN J, et al. Cloning and differential expression analysis of mevalonate kinase gene from Sanghuangporus baumii[J/OL].[2020-05-20].http://dio.org/10.13327/j.jjlau.2020.5689.J Jilin Univ Med Ed:1-9. DOI:10.13327/j.jjlau.2020.5689.
[19]	COSCIA C J, BOTTA L, GUARNACCIA R. On the mechanism of iridoid and secoiridoid monoterpene biosynthesis[J]. Arch Biochem Biophys, 1970, 136(2): 498-506. DOI: 10.1016/0003-9861(70)90221-3.
[20]	CHEN Q W, FAN D J, WANG G D. Heteromeric geranyl(geranyl) diphosphate synthase is involved in monoterpene biosynthesis in Arabidopsis flowers[J]. Mol Plant, 2015, 8(9): 1434-1437.DOI:10.1016/j.molp.2015.05.001.
[21]	CHEONG J J, DO CHOI Y. Methyl jasmonate as a vital substance in plants[J]. Trends Genet, 2003, 19(7):409-413. DOI:10.1016/S0168-9525(03)00138-0.
[22]	彭亮, 颜永刚, 陈莹, 等. 茉莉酸甲酯诱导下远志幼苗转录组分析及三萜类生物合成途径关键酶基因挖掘[J]. 中草药, 2020, 51(9):2517-2529.
	PENG L, YAN Y G, CHEN Y, et al. Transcriptome analysis of Polygala tenuifolia seedlings induced by methyl jasmonate and key genes mining for triterpenoid biosynthetic pathway[J]. Chin Tradit Herb Drugs, 2020, 51(9):2517-2529. DOI:10.7501/j.issn.0253-2670.2020.09.029.
[23]	任昂. 茉莉酸甲酯对灵芝三萜生物合成的影响及其灵芝应答基因的差异表达研究[D]. 南京: 南京农业大学, 2012.
	REN A. Study on the effect of methyl jasmonate on the ganoderic acid biosynthesis and differently expression of genes in response to methyl jasmonate in Ganoderma lucidum[D]. Nanjing: Nanjing Agricultural University, 2012.
[24]	隋娟娟, 李晓昕, 杨秋燕, 等. 重瓣百合LiSEP3基因克隆与表达分析[J]. 南京林业大学学报(自然科学版), 2017, 41(1):42-48.
	SUI J J, LI X X, YANG Q Y, et al. Cloning and expression analysis of gene LiSEP3 in double lily[J]. J Nanjing For Univ (Nat Sci Ed), 2017, 41(1):42-48. DOI:10.3969/j.issn.1000-2006.2017.01.007.
[25]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods, 2001, 25(4): 402-408. DOI:10.1006/meth.2001.1262.
[26]	张小辉, 祁艳霞. 真核生物启动子TATA-box·GC-box和CAAT-box的分析[J]. 安徽农业科学, 2008, 36(4):1380-1381,1395.
	ZHANG X H, QI Y X. Analysis on TATA-box, GC-box and CAAT-box in eukaryotic promoters[J]. J. Anhui Agric Sci, 2008, 36(4):1380-1381,1395. DOI:10.13989/j.cnki.0517-6611.2008.04.008.
[27]	OHLER U, LIAOG C, NIEMANN H, et al. Computational analysis of core promoters in the Drosophila genome[J]. Genome Biol, 2002, 3(12): RESEARCH0087. DOI:10.1186/gb-2002-3-12-research0087.
[28]	王彩云, 李富生, 李涛, 等. 滇龙胆GrGPPS基因的克隆及其序列分析与原核表达[J]. 中草药, 2014, 45(14): 2060-2068.
	WANG C Y, LI F S, LI T, et al. Cloning, sequence analysis, and prokaryotic expression of GrGGPS gene in Gentiana rigescens[J]. Chin Tradit Herb Drugs, 2014, 45 (14): 2060-2068. DOI:10.7501/j.issn.0253-2670.2014.14.020.
[29]	向蓓蓓, 李晓雪, 王勇, 等. 川西獐牙菜牻牛儿基焦磷酸合成酶基因的克隆及表达分析[J]. 中草药, 2017, 48(5): 962-970.
	XIANG B B, LI X X, WANG Y, et al. Cloning and expression of geranyl pyrophosphate synthase gene in Swertia mussotii[J]. Chin Tradit Herb Drugs, 2017, 48(5): 962-970. DOI: 10.7501/j.issn.0253-2670.2017.05.021.
[30]	张进杰, 徐茂军. NO和茉莉酸甲酯对黄芩悬浮细胞生长及黄芩苷合成的影响[J]. 植物学通报, 2006, 41(4): 374-379.
	ZHANG J J, XU M J. Effects of nitric oxide and methyl jasmonate on the baicalin production and cell growth in suspension cultures of Scutellaria baicalensis[J]. Chin Bull Bot, 2006, 41(4): 374-379. DOI:10.3969/j.issn.1674-3466.2006.04.008.

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引物名称 primer name	引物序列(5'-3') primer sequence	预测片段大小/bp predicted band
GPS-pro-F₁	CCGCAGACGGAGTATTATGGGGAG	1 612
GPS-pro-R₁	AGGCTGAGAAGCGACAGAACGAGT
GPS-F₁	ATAGACTGACTGATAGATAAGAAGA	1 718
GPS-R₁	AGGTAACAATTCTATTCAGCAAAAT
M13F	CGCCAGGGTTTTCCCAGTCACGAC	150
M13R	AGCGGATAACAATTTCACACAGGA
GPS-F₂	CCCCTACTCCTTCTCATCCATCAA	190
GPS-R₂	GCCGCACCATCTATCACATCATCA
α-tubulin-F₂	CCAGCAAGCGTTACCGATT	129
α-tubulin-R₂	TCCACGACGTCCATCGTTC

项目 item	茉莉酸甲酯浓度 MeJA concentration	SbGPS 基因转录水平 SbGPS gene transcript level
茉莉酸甲酯浓度 MeJA concentration	1	-0.201
SbGPS基因转录水平 SbGPS gene transcript level	-0.201	1
暴马桑黄三萜含量 triterpenoids content	0.120	0.835^*

Cloning and expression of GPS gene induced by methyl jasmonate in Sanghuangporus baumii

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