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

doi:10.3969/j.issn.1004-1524.2023.06.03

Acta Agriculturae Zhejiangensis ›› 2023, Vol. 35 ›› Issue (6): 1253-1264.DOI: 10.3969/j.issn.1004-1524.2023.06.03

• Crop Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S567.239

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.

Figures/Tables 6

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

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.

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.

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.

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.

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

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[8]	WANG Weike, SONG Jiling, LU Na, YUAN Weidong, YAN Jing, CHEN Guanping. Cloning and expression of the PpFBD1 involved in primordium formation of Pleurotus pulmonarius [J]. , 2020, 32(1): 93-97.
[9]	GONG Shasha, MENG Qingling, QIAO Jun, ZHONG Wenqiang, HUANG Yunfu, ZHANG Guowu, CHEN Ying, CAI Xuepeng. Gene cloning and bioactivity analysis of chitinase gene AO-483 from Arthrobotrys oligospora XJ-A1 [J]. , 2019, 31(2): 222-228.
[10]	XU Qingsong, ZHAO Jia, WEI Yunmin, HAN Rongrong, LIU Lusheng, JIANG Caode, YU Yong-xiong. Cloning and expression analysis of oxalate oxidase gene MsOXO from Medicago sativa [J]. , 2019, 31(1): 11-19.
[11]	YANG Zhou, LYU Ke, LYU Shan, WANG Junjie, ZHANG Di. Cloning and sequence analysis of two ARF genes and two Aux/IAA genes in Agapanthus praecox ssp. orientalis [J]. , 2019, 31(1): 86-97.
[12]	TANG Xiao, DENG Mengsheng, ZOU Xue, Li Liqin, ZHU Yuanzhi, WANG Xiyao. Cloning and expression analysis of StDWF1 in Solanum tuberosum [J]. , 2018, 30(6): 909-917.
[13]	XUE Shengling, JIANG Min, CHANG Jiaqi, LIU Yang, WEI Lin, ZHOU Jiankun, ZHANG Fen, SUN Bo. Cloning and prokaryotic expression of 1-deoxy-D-xylulose-5-phosphate synthase encoding gene (BaDXS1) in Brassica alboglabra [J]. , 2018, 30(5): 771-777.
[14]	LIANG Minhua, YANG Zhenfeng, SU Xinguo, SONG Chunbo. Cloning and expression analysis of phytoene synthase gene from peach fruit [J]. , 2018, 30(3): 399-405.
[15]	CAO Xing, ZHANG Xiusheng, HOU Dong, SUI Juanjuan, MU Hongmei, GAO Xiangbin, LYU Futang, GUO Shangjing, WANG Guiqing. Cloning and characterization of LlMBF1a in lily induced by gray mold pathogen [J]. , 2018, 30(12): 2024-2030.