暴马桑黄GPS基因克隆及响应茉莉酸甲酯诱导表达研究

刘增才; 王淑婷; 佟鑫宇; 邹莉

doi:10.12302/j.issn.1000-2006.202108040

暴马桑黄GPS基因克隆及响应茉莉酸甲酯诱导表达研究

刘增才, 王淑婷, 佟鑫宇, 邹莉

南京林业大学学报（自然科学版） ›› 2023, Vol. 47 ›› Issue (4) : 123-130.

PDF(2643 KB)

国家林草科技领军期刊
中国精品科技期刊
中国高校百佳科技期刊
江苏省新闻出版政府奖期刊奖
RCCSE林学权威期刊（A+）
CSCD核心期刊
Scopus数据库收录期刊
中文核心期刊
SCD核心期刊

作者加群：102861116

微信公众号：南京林业大学学报

高级检索

PDF(2643 KB)

南京林业大学学报（自然科学版） ›› 2023, Vol. 47 ›› Issue (4) : 123-130. DOI: 10.12302/j.issn.1000-2006.202108040

研究论文

暴马桑黄GPS基因克隆及响应茉莉酸甲酯诱导表达研究

作者信息 +

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

Author information +

文章历史 +

摘要

【目的】对参与暴马桑黄(Sanghuangporus baumii)三萜合成途径的关键酶牻牛儿基焦磷酸合酶(GPS)基因进行克隆及诱导表达分析,以期深入探究暴马桑黄三萜合成分子机理。【方法】采用PCR扩增技术克隆暴马桑黄GPS基因cDNA全长及启动子,利用生物信息学软件对序列进行分析;采用qRT-PCR技术分析不同浓度茉莉酸甲酯(MeJA)对暴马桑黄GPS基因转录水平的影响,并用分光光度计测定不同浓度MeJA诱导下暴马桑黄三萜含量的变化。【结果】将克隆得到的暴马桑黄GPS基因和启动子分别命名为SbGPS和SbGPS启动子。基因分析发现SbGPS基因cDNA序列全长1 617 bp,共编码538个氨基酸,没有信号肽和跨膜结构。SbGPS启动子分析结果显示,除了含有CAAT-box、TATA-box等典型的启动子作用元件,还含有茉莉酸甲酯响应元件。荧光定量分析及三萜含量测定结果表明,SbGPS基因表达和三萜含量均随着MeJA浓度增加而呈现出先上升后下降的趋势,并且二者变化趋势基本一致,呈显著正相关。【结论】通过对SbGPS基因的克隆及诱导表达分析发现,该基因在暴马桑黄三萜生物合成途径中具有重要作用,为深入研究SbGPS基因在暴马桑黄三萜合成过程中的调控机理奠定基础。

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.

导出引用

刘增才, 王淑婷, 佟鑫宇, 等. 暴马桑黄GPS基因克隆及响应茉莉酸甲酯诱导表达研究[J]. 南京林业大学学报（自然科学版）. 2023, 47(4): 123-130 https://doi.org/10.12302/j.issn.1000-2006.202108040

LIU Zengcai, WANG Shuting, TONG Xinyu, et al. Cloning and expression of GPS gene induced by methyl jasmonate in Sanghuangporus baumii[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY. 2023, 47(4): 123-130 https://doi.org/10.12302/j.issn.1000-2006.202108040

中图分类号： Q718

参考文献

列表( 原文顺序 | 文献年度倒序 | 文中引用次数倒序 ) 可视化分析

[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. 本文引用 [1]

[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. 本文引用 [1]

[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. 本文引用 [1]

[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

. 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.

本文引用 [1]

[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. 本文引用 [1]

[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. 本文引用 [1]

[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. 本文引用 [1]

[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. 本文引用 [1]

[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. 本文引用 [1]

[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. 本文引用 [4]

[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.

本文引用 [2]

[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. 本文引用 [1]

[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. 本文引用 [2]

[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. 本文引用 [1]

[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. 本文引用 [1]

[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. 本文引用 [1]

[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 本文引用 [2]

[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

, 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.

本文引用 [1]

[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. 本文引用 [1]

[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. 本文引用 [1]

[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. 本文引用 [1]

[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

, YAN

Y G

, CHEN

, 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.

本文引用 [2]

[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. 本文引用 [2]

[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. 本文引用 [1]

[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. 本文引用 [1]

[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. 本文引用 [1]

[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. 本文引用 [1]

[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

, 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.

本文引用 [2]

[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. 本文引用 [1]

[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.

本文引用 [1]