斑地锦查尔酮合酶基因及启动子的克隆与分析

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

摘要/Abstract

摘要：

黄酮类化合物槲皮素是斑地锦主要药效成分之一，而查尔酮合酶（CHS）是槲皮素生物合成过程中的关键酶。为阐明斑地锦中槲皮素生物合成机制，本研究以斑地锦为材料，根据转录组测序数据结合3' RACE、Tail-PCR技术，克隆了CHS基因及其启动子，将该基因命名为EmCHS（GenBank登录号为ON652865）。对EmCHS蛋白进行氨基酸序列比对、理化性质、跨膜结构域、亚细胞定位和系统进化树等分析，并采用实时荧光定量PCR技术检测EmCHS在不同生长期不同组织中的表达情况。结果显示，EmCHS开放阅读框（ORF）全长为1 194 bp，编码397个氨基酸，EmCHS蛋白定位于细胞质，理论等电点为5.96，相对分子质量为43.48 kD，不含跨膜区域，为结构稳定的亲水性蛋白。氨基酸序列比对和系统进化树分析结果显示，EmCHS与同为大戟科的木薯氨基酸序列相似性最高（91.92%），符合植物分类学的特点。RT-qPCR实验表明，EmCHS基因在斑地锦不同生长期不同组织中均有表达，且存在明显差异，在生殖生长期叶内表达水平最低，生殖生长期根内表达水平最高。克隆到的EmCHS启动子（GenBank登录号为OP626754），长度为971 bp，生物信息学分析结果显示，其既含TATA-box、CAAT-box等基本序列，也含有MYB、MYC等转录因子结合位点、多个光反应元件（G-box等）和激素反应元件（ABRE等）等顺式作用元件。实验结果为进一步研究斑地锦CHS基因功能及表达调控奠定基础。

关键词: 斑地锦, CHS, 启动子, 克隆, 热不对称交错PCR, 生物信息学, 基因表达

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

郭三保, 宋美玲, 李灵心, 尧子钊, 桂明明, 黄胜和. 斑地锦查尔酮合酶基因及启动子的克隆与分析[J]. 生物技术通报, 2023, 39(4): 148-156.

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.

图/表 10

参考文献 26

[1]	中国科学院中国植物志编辑委员会. 中国植物志(第三分册)[M]. 北京: 科学出版社, 1997.
	Delectis Florae Reipublicae Popularis Sinicae Agendae Academiae Sinicae Edita. Flora Reipublicae Popularis Sinicae(Volume 3)[M]. Beijing: Science Press, 1997.
[2]	国家药典委员会. 中华人民共和国药典(一部)[M]. 北京: 中国医药科技出版社, 2020.
	Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China(Volume I)[M]. Beijing: China Medical Science Press, 2020.
[3]	曹旭, 王文春, 周泐, 等. 3种地锦草总黄酮和鞣质质量分数及体外抗菌活性比较[J]. 西北农业学报, 2012, 21(9): 184-188.
	Cao X, Wang WC, Zhou L, et al. Comparison on mass fraction of total flavonoids and tannin of three kinds of herba euphorbiae humifusae and their antibacaterial activity in vitro[J]. Acta Agric Boreali Occidentalis Sin, 2012, 21(9): 184-188.
[4]	安惠霞, 李治建, 古丽娜·达吾提, 等. 地锦草的研究进展[J]. 时珍国医国药, 2008, 19(12): 2866-2868.
	An HX, Li ZJ, Gulina D, et al. Research advance of the uighur medicine Dijincao[J]. Lishizhen Med Mater Med Res, 2008, 19(12): 2866-2868.
[5]	王叶情, 王宇红, 邹蔓姝, 等. 槲皮素及其糖苷衍生物抗抑郁作用及其机制的研究进展[J]. 中草药, 2022, 53(5): 1548-1557.
	Wang YQ, Wang YH, Zou MS, et al. Research progress on antidepressant effect and mechanism of quercetin and its glycoside derivatives[J]. Chin Tradit Herb Drugs, 2022, 53(5): 1548-1557.
[6]	张声祥, 施圆圆, 王晨凯, 等. 异叶天南星查尔酮合成酶和异构酶基因的克隆及蛋白的结构性质分析[J]. 中国中药杂志, 2019, 44(9): 1799-1807.
	Zhang SX, Shi YY, Wang CK, et al. Cloning and characterization of chalcone synthase and chalcone isomerase genes in Arisaema heterophyllum[J]. China J Chin Mater Med, 2019, 44(9): 1799-1807.
[7]	包颖, 郭昌锋, 陈少华, 等. 植物查尔酮合成酶超基因家族的分子进化[J]. 植物学报, 2015, 50(1): 55-71. doi: 10.3724/SP.J.1259.2015.00055
	Bao Y, Guo CF, Chen SH, et al. Molecular evolution of chalcone synthase gene superfamily in plants[J]. Chin Bull Bot, 2015, 50(1): 55-71.
[8]	白洁, 杨杞, 郭琳, 等. 中间锦鸡儿查尔酮合成酶基因的克隆和表达分析[J]. 基因组学与应用生物学, 2016, 35(1): 87-93.
	Bai J, Yang Q, Guo L, et al. Cloning and expression analysis of CiCHS from Caragana intermedia[J]. Genom Appl Biol, 2016, 35(1): 87-93.
[9]	安利清, 杨凯, 张克, 等. 百合查尔酮合成酶基因的克隆与分析[J]. 西北植物学报, 2011, 31(3): 492-498.
	An LQ, Yang K, Zhang K, et al. Cloning and analysis of chalcone synthase gene of lily[J]. Acta Bot Boreali Occidentalia Sin, 2011, 31(3): 492-498.
[10]	尚洁, 陈阿丽, 潘伟志. 宁夏枸杞查耳酮合成酶基因cDNA片段的克隆及生物信息学分析[J]. 中草药, 2013, 44(20): 2898-2903.
	Shang J, Chen AL, Pan WZ. Cloning and bioinformation analysis of chalcone synthase gene from Lycium barbarum in Ningxia[J]. Chin Tradit Herb Drugs, 2013, 44(20): 2898-2903.
[11]	岳春华, 张初梅, 林爽, 等. 布渣叶查耳酮合酶基因全长cDNA克隆及其表达模式分析[J]. 中草药, 2018, 49(17): 4118-4124.
	Yue CH, Zhang CM, Lin S, et al. Cloning and expression pattern analysis of chalcone synthase gene in Microcos paniculata[J]. Chin Tradit Herb Drugs, 2018, 49(17): 4118-4124.
[12]	Guo DD, Xue YR, Li DQ, et al. Overexpression of CtCHS1 increases accumulation of quinochalcone in safflower[J]. Front Plant Sci, 2017, 8: 1409. doi: 10.3389/fpls.2017.01409 URL
[13]	张蕾, 朱立新, 徐川, 等. 查尔酮合酶基因对桃果实花色苷代谢的影响[J]. 园艺学报, 2015, 42(1): 31-37. doi: 10.16420/j.issn.0513-353x.2014-0554
	Zhang L, Zhu LX, Xu C, et al. The effect of silencing chalcone synthase on anthocyanin metabolism in peach[J]. Acta Hortic Sin, 2015, 42(1): 31-37. doi: 10.16420/j.issn.0513-353x.2014-0554
[14]	夏玉凤, 耿晓娜, 王万双, 等. 转查尔酮合酶基因对烟草花色及花器官的影响[J]. 生物技术通报, 2010(1): 93-98.
	Xia YF, Geng XN, Wang WS, et al. The influence of CHS gene expression on flower colour and flower morphology of transgenic tobaccos[J]. Biotechnol Bull, 2010(1): 93-98.
[15]	徐秀荣, 马燕, 臧德奎. 桂花查耳酮合酶chs基因的克隆及序列分析[J]. 中国农学通报, 2016, 32(1): 118-124. doi: 10.11924/j.issn.1000-6850.casb15070012
	Xu XR, Ma Y, Zang DK. Cloning and sequencing analysis of Osmanthus fragrans chalcone synthase gene[J]. Chin Agric Sci Bull, 2016, 32(1): 118-124.
[16]	Wani TA, Pandith SA, Gupta AP, et al. Molecular and functional characterization of two isoforms of Chalcone synthase and their expression analysis in relation to flavonoid constituents in Grewia asiatica L[J]. PLoS One, 2017, 12(6): e0179155. doi: 10.1371/journal.pone.0179155 URL
[17]	黄琼林, 曾湘达, 卓一南, 等. 青天葵查尔酮合成酶基因的克隆及序列分析[J]. 中药新药与临床药理, 2018, 29(2): 205-210.
	Huang QL, Zeng XD, Zhuo YN, et al. Cloning and sequence analysis of CHS gene from endangered Nervilia fordii[J]. Tradit Chin Drug Res ＆ Clin Pharmacol, 2018, 29(2): 205-210.
[18]	Liu YG, Chen YL. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J]. BioTechniques, 2007, 43(5): 649-650, 652, 654 passim. doi: 10.2144/000112601 URL
[19]	郭三保, 黄胜和, 邹嘉轩, 等. 斑地锦肉桂酸4-羟基化酶基因及启动子的克隆与分析[J]. 分子植物育种, 2023, 21(3): 819-825.
	Guo SB, Huang SH, Zou JX, et al. Cloning and analysis of cinnamate acid 4-hydroxylase gene and its promoter from Euphorbia maculata L.[J]. Mol Plant Breed, 2023, 21(3): 819-825.
[20]	镐青青, 姚圣, 刘佳禾, 等. 马尾松NAC转录因子基因PmNAC8的克隆及表达分析[J]. 生物技术通报, 2022, 38(4): 202-216. doi: 10.13560/j.cnki.biotech.bull.1985.2021-0830
	Hao QQ, Yao S, Liu JH, et al. Cloning and expression analysis of NAC transcription factor PmNAC8 in Pinus massoniana[J]. Biotechnol Bull, 2022, 38(4): 202-216.
[21]	卢婷, 杨莉, 胡威, 等. 柑橘抗逆基因WRKY75的克隆与表达分析[J]. 江西农业大学学报, 2021, 43(1): 82-93.
	Lu T, Yang L, Hu W, et al. Cloning and expression analysis of Citrus WRKY75 genes in response to abiotic stresses[J]. Acta Agric Univ Jiangxiensis, 2021, 43(1): 82-93.
[22]	李文静, 孙艳香, 付亚娟, 等. 菊芋查尔酮合成酶基因的克隆与表达分析[J]. 西北农业学报, 2020, 29(4): 603-612.
	Li WJ, Sun YX, Fu YJ, et al. Cloning and expression analysis of chalcone synthase gene from Helianthus tuberosus[J]. Acta Agric Boreali Occidentalis Sin, 2020, 29(4): 603-612.
[23]	Yang HL, Liu J, Huang SM, et al. Selection and evaluation of novel reference genes for quantitative reverse transcription PCR(RT-qPCR)based on genome and transcriptome data in Brassica napus L[J]. Gene, 2014, 538(1): 113-122. doi: 10.1016/j.gene.2013.12.057 URL
[24]	姜翠翠, 王玉珍, 叶新福. 油㮈果实中查尔酮合成酶基因PsCHS的克隆表达分析及其启动子的分离[J]. 福建农业学报, 2016, 31(1): 16-21.
	Jiang CC, Wang YZ, Ye XF. Cloning, expression analysis and promoter isolation of chalcone-synthase gene from fruits of nane, Prunus salicina Lindl. var. cordata[J]. Fujian J Agric Sci, 2016, 31(1): 16-21.
[25]	李琳玲, 程华, 程水源, 等. 银杏查尔酮合成酶基因启动子(GbCHSP)调控元件及功能分析[J]. 园艺学报, 2010, 37(12): 1919-1928.
	Li LL, Cheng H, Cheng SY, et al. Regulatory elements and functional analysis of Chalcone synthase gene promoter from Ginkgo biloba L[J]. Acta Hortic Sin, 2010, 37(12): 1919-1928.
[26]	曹天骏, 戴好富, 李辉亮, 等. 白木香查尔酮合成酶基因(AsCHS1)启动子克隆及激素应答元件功能的初步鉴定[J]. 热带作物学报, 2014, 35(10): 1950-1956.
	Cao TJ, Dai HF, Li HL, et al. Cloning and function analysis of the chalcone synthase gene(AsCHS1)promoter in Aquilaria sinensis[J]. Chin J Trop Crops, 2014, 35(10): 1950-1956.

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