暴马桑黄GPS基因克隆及响应茉莉酸甲酯诱导表达研究

doi:10.12302/j.issn.1000-2006.202108040

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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

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()

东北林业大学林学院,黑龙江哈尔滨 150040

收稿日期:2021-08-23 修回日期:2022-04-29 出版日期:2023-07-30 发布日期:2023-07-20
通讯作者: * 邹莉(shyj@nefu.edu.cn),教授。
作者简介:刘增才(1758458181@qq.com),博士生。
基金资助:
国家自然科学基金项目(32171792);中央高校基本科研业务费专项资金项目(2572018AA34)

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

摘要/Abstract

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。

关键词: 暴马桑黄, 牻牛儿基焦磷酸合酶, 基因克隆, 启动子克隆, 茉莉酸甲酯

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)

中图分类号:

Q718

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.

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 (Natural Science Edition), 2023, 47(4): 123-130.DOI: 10.12302/j.issn.1000-2006.202108040.

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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^*

暴马桑黄GPS基因克隆及响应茉莉酸甲酯诱导表达研究

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

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