当归转录因子AsMYB44的克隆与功能研究

doi:10.3969/j.issn.1004-1524.2023.06.03

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (6): 1253-1264.DOI: 10.3969/j.issn.1004-1524.2023.06.03

当归转录因子AsMYB44的克隆与功能研究

刘光瑞¹^,²(), 宗渊², 李云², 曹东², 刘宝龙², 包雪梅¹^,²^,³, 李建民¹^,^*()

1.青海师范大学生命科学学院,青海西宁 810008
2.中国科学院西北高原生物研究所,青海省作物分子育种重点实验室,青海西宁 810008
3.中国科学院藏药研究重点实验室,青海西宁 810008

收稿日期:2022-07-02 出版日期:2023-06-25 发布日期:2023-07-04
通讯作者: *李建民,E-mail:beyond_3862740@163.com
作者简介:刘光瑞(1998—),男,贵州毕节人,硕士研究生,主要从事分子植物育种研究。E-mail:liugr1208@163.com
基金资助:
青海省创新平台建设专项(2022-ZJ-Y04)

Cloning and functional research of MYB transcription factor AsMYB44 from Angelica sinensis

LIU Guangrui¹^,²(), ZONG Yuan², LI Yun², CAO Dong², LIU Baolong², BAO Xuemei¹^,²^,³, LI Jianmin¹^,^*()

1. School of Life Sciences, Qinghai Normal University, Xining 810008, China
2. Qinghai Provincial Key Laboratory of Crop Molecular Breeding, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, China
3. CAS Key Laboratory of Tibetan Medicine Research, Xining 810008, China

Received:2022-07-02 Online:2023-06-25 Published:2023-07-04

摘要/Abstract

摘要：

为明确AsMYB44调控次生代谢产物生物合成的功能,从当归(Angelica sinensis)中分离克隆得到MYB转录因子AsMYB44,对AsMYB44进行生物信息学分析,利用根癌农杆菌介导的遗传转化体系在Samsun烟草中过表达,并对转基因烟草进行多组学联合分析。结果表明,AsMYB44基因CDS (coding sequence)全长903 bp,编码300个氨基酸,具有完整的SANT、MYB domains结构域。系统发育树结果表明,AsMYB44与拟南芥AtMYB11(AF062863.1)、AtMYB12(AF062864.1)和丹参SmMYB97(KF059561.1)同源性较高。AsMYB44转基因烟草中差异表达基因主要富集在苯丙烷合成代谢途径,其中苯丙氨酸解氨酶基因(PAL)、肉桂酸-4-羟化酶基因(C4H)、4-香豆酸:辅酶A连接酶基因(4CL)、咖啡酸3-O-甲基转移酶基因(COMT)、肉桂醇脱氢酶基因(CAD)等表达量上调。广泛靶向代谢组学结果显示,转基因烟草中酚酸、脂质、黄酮类化合物、生物碱等代谢产物含量变化显著。AsMYB44转录因子可正向调控酚酸的代谢合成,这可以为当归酚酸次生代谢物的生物合成机制提供理论基础。

关键词: 当归, AsMYB44转录因子, 基因克隆, 转基因烟草, 多组学分析, 酚酸

Abstract:

In order to study the mechanism of biosynthetic regulation of Angelica sinensis phenolic acids, a MYB transcription factor AsMYB44 gene was cloned from Angelica sinensis and the relevant bioinformatics analysis of the sequence was carried out using biological software. The Agrobacterium tumefaciens-mediated genetic transformation system was overexpressed in Samsun tobacco, and a multi-omics combined analysis of the transgenic tobacco was performed. The CDS (coding sequence) of the AsMYB44 was 903 bp, encoding 300 amino acids, and with the complete SANT and MYB domains. Phylogenetic analysis showed that AsMYB44 had higher homology with Arabidopsis thaliana AtMYB11 (AF062863.1), AtMYB12 (AF062864.1) and Salvia miltiorrhiza SmMYB97 (KF059561.1). The results of transcriptome sequencing showed that the differentially expressed genes were mostly enriched in the phenylpropane synthesis and metabolism pathway in AsMYB44 transgenic tobacco. Expression levels of phenylalanine ammonia lyase gene (PAL), cinnamic acid-4-hydroxyl (C4H), 4-coumaric acid: coenzyme A ligase gene (4CL), caffeic acid 3-O-methyltransferase gene (COMT), cinnamyl alcohol dehydrogenase gene (CAD) and other structural genes were upregulated. Metabolomic analysis showed that the contents of metabolites such as phenolic acids, lipids, alkaloids, flavonoids and other compounds in transgenic tobacco changed significantly. These results indicated that the AsMYB44 transcription factor could positively regulate the metabolic synthesis of phenolic acids, which provided a theoretical basis for the biosynthesis mechanism of phenolic acid secondary metabolites in Angelica sinensis.

Key words: Angelica sinensis, AsMYB44 transcription factor, gene cloning, transgenic tobacco, multi-omics analysis, phenolic acid

中图分类号:

S567.239

刘光瑞, 宗渊, 李云, 曹东, 刘宝龙, 包雪梅, 李建民. 当归转录因子AsMYB44的克隆与功能研究[J]. 浙江农业学报, 2023, 35(6): 1253-1264.

LIU Guangrui, ZONG Yuan, LI Yun, CAO Dong, LIU Baolong, BAO Xuemei, LI Jianmin. Cloning and functional research of MYB transcription factor AsMYB44 from Angelica sinensis[J]. Acta Agriculturae Zhejiangensis, 2023, 35(6): 1253-1264.

图/表 6

表1 引物序列

Table 1 Primer sequences

用途 Purpose	基因名称 Gene name	正向引物 Forward primer(5’→3’)	反向引物 Reverse primer(5’→3’)
实时荧光定量PCR	NtActin	AATGATCGGAATGGAAGCTG	TGGTACCACCACTGAGGACA
qRT-PCR	NtPAL	ATAGACTTGAGGCATTTGG	GCATCAGTGGGTAGTTAGC
	NtC4H	GAGGCTGAACAGAAGGGAG	TGTGGGACTAAAAGAGGGA
	Nt4CL	GTTCTCGTCCCTGTTTGAT	GAGGCTTTGGCTTGTTTTA
	NtF5H	TCCTTCTCTCCAAATCTCG	AACTTATGGCTACGGTCGC
	NtCOMT	GATGCTTAGGCTACTTGCT	TCATTGACATAGTGGAGTG
	NtCAD	CAGCGAAAACAGTATGCGT	CAGCGAAAACAGTATGCGT
	NtCCR	CAGCATCTCCATTTCCCTA	ATCCCTTTCTCCTTCACAA
基因克隆Gene cloning	AsMYB44	acgcGAGCTCATGTCCAGGAGAGATATGGATCG	ctagTCTAGACTAATCGATTTTACTAATCCCCATTC

图1 AsMYB44基因克隆、结构域分析和系统进化分析 A,AsMYB44基因克隆;M,DL5000 marker;1~3,当归叶片cDNA为模板的PCR产物。B,结构域分析。C,AsMYB44系统进化分析。

Fig.1 Cloning, domain analysis and phylogenetic analysis of AsMYB44 gene A, AsMYB44 gene clone; M, DL5000 marker; 1-3, PCR product of Angelica sinensis leaf cDNA. B, Domain analysis. C, Phylogenetic analysis of AsMYB44.

图2 大肠埃希菌菌落、农杆菌菌液与转基因烟草PCR检测 A,1~10为大肠埃希菌菌落PCR,11为以AsMYB44质粒为模板的PCR,12为阴性对照;B,1~9为农杆菌菌落PCR,10为以AsMYB44质粒为模板的PCR,11为阴性对照;C,1~12为以转基因烟草DNA为模板的PCR,13为以AsMYB44质粒为模板的PCR,14为阴性对照;M,DL5000 marker。

Fig.2 PCR detection of Escherichia coli colony, Agrobacterium liquid and transgenic tobacco A, 1-10 were PCR products of E. coli colonies, 11 was PCR product with AsMYB44 plasmid as template, 12 was negative control; B, 1-9 were PCR products of Agrobacterium colonies, 10 was PCR product with AsMYB44 plasmid as template, 11 was negative control; C, 1-12 were PCR products with transgenic tobacco DNA as templates, 13 was PCR product with AsMYB44 plasmid as template, 14 was negative control; M, DL5000 marker.

图3 差异表达基因统计图、火山图、KEGG富集因子图 A,差异表达基因统计图;B,差异表达基因火山图;C,差异表达基因KEGG富集因子图。

Fig.3 Statistical graph, volcano graph and KEGG enrichment factor graph of differentially expressed genes A, Statistical map of differentially expressed genes; B, Volcano map of differentially expressed genes; C, Map of KEGG enrichment factors of differentially expressed genes.

图4 差异代谢物类别统计、KEGG通路富集和苯丙烷代谢途径多组学分析 A,差异代谢物类别统计;B,差异代谢物KEGG通路富集;C,苯丙烷代谢途径多组学分析。

Fig.4 Statistics of differential metabolite categories, KEGG pathway enrichment and multi-omics analysis of phenylpropane metabolic pathways A, Statistics of differential metabolite categories; B, KEGG pathway enrichment of differential metabolites; C, Multi-omics analysis of phenylpropane metabolic pathways.

图5 AsMYB44转基因烟草苯丙烷代谢途径部分关键基因的相对表达量 *代表P ≤0.05,**代表P ≤0. 01,***代表P ≤0.001,****代表P ≤0.000 1。

Fig.5 Relative expression levels of some key genes in the phenylpropane metabolism pathway of AsMYB44 transgenic tobacco *represented P ≤0.05, **represented P ≤0.01, ***represented P ≤0.001, ****represented P ≤0.000 1.

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当归转录因子AsMYB44的克隆与功能研究

Cloning and functional research of MYB transcription factor AsMYB44 from Angelica sinensis

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