Cloning and Analysis of Chalcone Synthase Gene and Its Promoter from Euphorbia maculata

doi:10.13560/j.cnki.biotech.bull.1985.2022-1130

Abstract

Abstract:

Quercetin is one of the main medicinal ingredients of Euphorbia maculata, and chalcone synthase（CHS）is one of key enzymes in the biosynthesis of quercetin. To explore the mechanism of quercetin biosynthesis in E. maculata, a CHS gene named EmCHS（GenBank access number: ON652865）was cloned from E. maculatabased on transcriptome sequencing results, combined with technologies of 3' RACE and Tail-PCR. The amino acid sequence was aligned, and physicochemical properties, transmembrane region, subcellular localization and phylogenetic relationships of EmCHS protein were analyzed. And the expressions of EmCHS in different tissues during different growth stages were detected with RT-qPCR. The results showed that the ORF of EmCHS gene was 1 194 bp, encoding 397 amino acids. The EmCHS protein was located in the cytoplasm. The theoretical isoelectric point of the protein was 5.96 and the theoretical molecular weight was 43.48 kD, and it did not contain a transmembrane region and was a hydrophilic protein with stable structure. The results of amino acid sequence alignment and phylogenetic relationships analysis revealed that EmCHS had the highest similarity（91.92%）with the amino acid sequence of Manihot esculenta in Euphorbiaceae family, which was consistent with the characteristics of plant taxonomy. The analysis of RT-qPCR confirmed that EmCHS gene was expressed in different tissues during different growth stages, and there were significant differences. The expression of EmCHS gene was the lowest in the leaf at the reproduction stage, while the highest in the root at the reproduction stage. Besides, the promoter（GenBank access number: OP626754）length of EmCHS gene cloned was 971 bp, including TATA-box, CAAT-box, MYB and MYC transcription factor binding sites, multiple light response and hormone response cis-acting elements, and so on. These results will provide a foundation for further research on gene function and expression regulation of EmCHS gene.

Key words: Euphorbia maculata, CHS, promoter, cloning, tail-PCR, bioinformatics, gene expression

GUO San-bao, SONG Mei-ling, LI Ling-xin, YAO Zi-zhao, GUI Ming-ming, HUANG Sheng-he. Cloning and Analysis of Chalcone Synthase Gene and Its Promoter from Euphorbia maculata[J]. Biotechnology Bulletin, 2023, 39(4): 148-156.

Figures/Tables 10

References 26

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引物名称Primer name	引物序列Primer sequence（5'-3'）	用途Purpose
FZL-1	AAGCAGTGGTATCAACGCAGAGTATTTTTTTTTTTTTTTTTTTTTTTTTVN	cDNA合成 cDNA synthesis
EmCHS-XF	TGTTCCTGGATGAGGAGATTCTTAC	3' RACE
EmCHS-3R	AGCAGTGGTATCAACGCAGAG	3' RACE
EmCHS-SR1	CTTTAAGGTCAGTCATATGCTCGCTCTG	热不对称交错PCR Tail-PCR
EmCHS-SR2	ACGATGGACTCCAGTCCGGCCGCTCTGGGTGATCCTGAAGTAGTAGTC
EmCHS-SR3	ATGGCCTGTAACTCGTGTTGTGCAG
ADF1	ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA
ADF2	ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT
ADF3	ACGATGGACTCCAGAGCGGCCGCVVNVNNNCCAA
ADF4	ACGATGGACTCCAGAGCGGCCGCBDNBNNNCGGT
ADF0	ACGATGGACTCCAGAG
EmCHS-CF	CCTTAACTCGCTTGTACCTTTGTG	ORF区克隆 ORF clone
EmCHS-CR	CAGTAGAAAAGTAAAACCACCGATAGG	ORF区克隆 ORF clone
EmCHS-PF	CGGTCCACATGGACAGTAGTG	启动子克隆 Promoter clone
EmCHS-PR	CTGTAACTCGTGTTGTGCAGC	启动子克隆 Promoter clone
EmCHS-qF	CGCCGCTAGGGATTAAGGAC	实时荧光定量PCR RT-qPCR
EmCHS-qR	CCCGTAATCGCTCAACACCT
EmGDI1-qF	TCTAGGTCCCGTTGACGAGT
EmGDI1-qR	TCGAGAGCCTTCCCAGTGAT

引物名称Primer name	引物序列Primer sequence（5'-3'）	用途Purpose
FZL-1	AAGCAGTGGTATCAACGCAGAGTATTTTTTTTTTTTTTTTTTTTTTTTTVN	cDNA合成 cDNA synthesis
EmCHS-XF	TGTTCCTGGATGAGGAGATTCTTAC	3' RACE
EmCHS-3R	AGCAGTGGTATCAACGCAGAG	3' RACE
EmCHS-SR1	CTTTAAGGTCAGTCATATGCTCGCTCTG	热不对称交错PCR Tail-PCR
EmCHS-SR2	ACGATGGACTCCAGTCCGGCCGCTCTGGGTGATCCTGAAGTAGTAGTC
EmCHS-SR3	ATGGCCTGTAACTCGTGTTGTGCAG
ADF1	ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA
ADF2	ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT
ADF3	ACGATGGACTCCAGAGCGGCCGCVVNVNNNCCAA
ADF4	ACGATGGACTCCAGAGCGGCCGCBDNBNNNCGGT
ADF0	ACGATGGACTCCAGAG
EmCHS-CF	CCTTAACTCGCTTGTACCTTTGTG	ORF区克隆 ORF clone
EmCHS-CR	CAGTAGAAAAGTAAAACCACCGATAGG	ORF区克隆 ORF clone
EmCHS-PF	CGGTCCACATGGACAGTAGTG	启动子克隆 Promoter clone
EmCHS-PR	CTGTAACTCGTGTTGTGCAGC	启动子克隆 Promoter clone
EmCHS-qF	CGCCGCTAGGGATTAAGGAC	实时荧光定量PCR RT-qPCR
EmCHS-qR	CCCGTAATCGCTCAACACCT
EmGDI1-qF	TCTAGGTCCCGTTGACGAGT
EmGDI1-qR	TCGAGAGCCTTCCCAGTGAT

元件名称 Element name	序列 Sequence	数量 Amount	功能 Function
TATA-box	TATA	6	-30处基因启动转录核心RNA聚合酶Ⅱ识别位点 Core promoter element around -30 of transcription start
CAAT-box	CAAT/CAAAT	11	启动子和增强区中常见的顺式作用元件Common cis-acting element in promoter and enhancer region
MYB	CAACCA	1	MYB转录因子结合位点MYB transcription factor binding sites
MYC	CATTTG	1	MYC转录因子结合位点MYC transcription factor binding sites
Box-II	CCACGTGGC	1	光反应元件的一部分Part of a light responsive element
Box-IV	ATTAAT	1	参与光反应的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness
G-box	CACGTG	1	参与光反应的顺式作用调节元件Cis-acting regulatory element involved in light responsiveness
L-box	ATCCCACCTAC	1	光反应元件的一部分Part of a light responsive element
ABRE	ACGTG	3	参与脱落酸应答的顺式作用元件Cis-acting element involved in the abscisic acid responsiveness
ERE	ATTTTAAA	1	乙烯反应元件Ethylene responsive element
ARE	AAACCA	1	厌氧诱导必须的顺式作用元件Cis-acting regulatory element essential for the anaerobic induction
STRE	AGGGG	1	应激反应元件Stress responsive element
WUN-motif	AAATTACT	1	伤口反应元件Wound-responsive element
ATCT-motif	AATCTAATCC	1	参与光反应的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness

元件名称 Element name	序列 Sequence	数量 Amount	功能 Function
TATA-box	TATA	6	-30处基因启动转录核心RNA聚合酶Ⅱ识别位点 Core promoter element around -30 of transcription start
CAAT-box	CAAT/CAAAT	11	启动子和增强区中常见的顺式作用元件Common cis-acting element in promoter and enhancer region
MYB	CAACCA	1	MYB转录因子结合位点MYB transcription factor binding sites
MYC	CATTTG	1	MYC转录因子结合位点MYC transcription factor binding sites
Box-II	CCACGTGGC	1	光反应元件的一部分Part of a light responsive element
Box-IV	ATTAAT	1	参与光反应的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness
G-box	CACGTG	1	参与光反应的顺式作用调节元件Cis-acting regulatory element involved in light responsiveness
L-box	ATCCCACCTAC	1	光反应元件的一部分Part of a light responsive element
ABRE	ACGTG	3	参与脱落酸应答的顺式作用元件Cis-acting element involved in the abscisic acid responsiveness
ERE	ATTTTAAA	1	乙烯反应元件Ethylene responsive element
ARE	AAACCA	1	厌氧诱导必须的顺式作用元件Cis-acting regulatory element essential for the anaerobic induction
STRE	AGGGG	1	应激反应元件Stress responsive element
WUN-motif	AAATTACT	1	伤口反应元件Wound-responsive element
ATCT-motif	AATCTAATCC	1	参与光反应的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness