Study on the Function of Ata4CL Gene in Flavonoid Synthesis and Drought Resistance in Aster tataricus

doi:10.13560/j.cnki.biotech.bull.1985.2025-0546

Abstract

Abstract:

Objective As a cough-suppressing and lung-moistening medicinal herb, Aster tataricus L. contains flavonoid compounds as its main active components. 4-coumarate: CoA ligase (4CL) is the key rate-limiting enzyme for flavonoid synthesis. This study is aimed to explore the molecular characteristics of Ata4CL and its dual role in flavonoid synthesis and drought resistance, offering a theoretical basis for enhancing medicinal components of A. tataricus and breeding drought-resistant varieties. Method Total flavonoid content in different A. tataricus tissues and growth stages was measured by the aluminum nitrate colorimetric method. The full-length Ata4CL gene was cloned and its structural features analyzed using bioinformatics tools. Agrobacterium-mediated transformation was used for subcellular localization. A 35S::Ata4CL-6HA overexpression vector was constructed and transformed into Arabidopsis thaliana. Drought-stress experiments were carried out to verify its function. Result The total flavonoids in the roots of A. tataricus show a bimodal accumulation pattern, with the root being the main site for flavonoid accumulation. The highest flavonoid contents in the leaves and rhizomes are observed in plants entering the flowering stage. The full-length cDNA of the Ata4CL gene was cloned, which is 1 623 bp in length and encodes a protein of 540 amino acids with a molecular weight of 59.04 kD. The encoded protein is mainly composed of random coils, contains a conserved AAE domain, and is localized in chloroplasts. Its amino acid sequence is highly conserved among A. tataricus plants, with a homology of up to 96.48% with Conyza canadensis. The expressions of this gene in the roots and leaves both show an "increase-decrease-increase" trend, with the strongest expression in the roots of flowering plants and the highest expression in the flowers at the full flowering stage. The total flavonoid content in A. tataricus overexpressing Ata4CL is 1.47-1.76 times higher than that in wild-type plants, and the gene expression is up-regulated by 5.41-12.23 times. Meanwhile, the expressions of both upstream and downstream synthesis genes in the flavonoid pathway are up-regulated. Under drought stress, the survival rate of transgenic Arabidopsis is 76.67%-86.67% higher than that of wild-type plants, with significantly increased plant height, root length, and rosette diameter, enhanced SOD and POD activities, and decreased MDA content. Conclusion The Ata4CL gene enhances resistance in plants to drought by coordinately regulating flavonoid biosynthesis, thereby providing a key molecular target for the optimization of medicinal components in A. tataricus and the breeding of stress-resistant varieties.

Key words: Aster tataricus, Ata4CL gene, flavonoid biosynthesis, resistance to drought

WANG Ting, MENG Yi-jiang, WANG Han, JIA Kai-xuan, QIAO Xiao-yu, HAN Bing-bing, LIU Xiao-qing, GE Shu-jun. Study on the Function of Ata4CL Gene in Flavonoid Synthesis and Drought Resistance in Aster tataricus[J]. Biotechnology Bulletin, 2026, 42(1): 230-240.

Figures/Tables 13

Table 1 Information on the primers used in the experiment

名称 Name	上游引物 Forward primer sequence （5′‒3′）	下游引物 Reverse primer sequence（5′‒3′ ）	引物用途Application
Ata4CL	ATGGAATCACAAAAGGAAATCATTTTC	ATTTGGAACACCGGCTGCAA	基因克隆
Ata4CL-35s	GCCCAAGCTACGCGTC ATGGAATCACAAAAGGAAATCATTTTC	ATCGTATGGGTAACTAGAAC ATTTGGAACACCGGCTGCAA	构建pGreen-Ata4CL-6HA载体
Ata4CL-GFP	CGACTCTAGAGGATCC ATGGAATCACAAAAGGAAATCATTTTC	CTCACCATGGGCCCGGTACC ATTTGGAACACCGGCTGCAA	构建pCameE-Ata4CL-GFP载体
35spro/Pgp2	ACGAGGAGCATCGTGGAAAA	GCTCCGAGAAGTGCAAGCAG	植株转基因阳性鉴定
Ata4CL	GAAACCAACGAGGCGAGATT	TCGGAAATGTCGGGATGAGT	RT-qPCR
AtaActin	ACATCGCTCTTGACTATGAACAGG	ATGGCTGGAACAACACCTCTG
Atactin2	TCCATGAAACAACTTACAACTCCA	CGTACTCACTCTTTGAAATCCACA
AtPAL1	ACACTGTCTCTCAAGTGGCG	ACGTTGCGCTACAAGGATCA
AtPAL2	AGTCGTGAATCTTGGCGGAG	TCACACCGGCTCTTGAAGTC
AtPAL4	TACTTAGTCGCGCTTTGCCA	CTTGACGGATGTAGCTCCCC
AtCHI	TCTTCGCTCTCTCCCCTACC	AGGTGACACACCGTTCTTCC

Fig. 1 Total flavonoid contents at different developmental stages of Aster tataricusCapital letters and lowercase letters respectively indicate significant differences between roots and leaves. Error bars indicate the average values of 3 repetitions ± standard deviation. Duncan test indicates a significant difference level less than 0.05, the same below

Fig. 2 Total flavonoid contents of Aster tataricus during the flowering period

Fig. 3 Cloning and characterization of the Ata4CL gene in A. tataricusM: DNA marker DL2000; 1, 2: Target bands

Fig. 4 Bioinformatics characterization of the Ata4CL-encoded protein in A. tataricus

Fig. 5 Multiple sequence alignment analysis of the amino acid sequences encoded by Ata4CL

Fig. 6 Homologous evolutionary analysis of 4CL

Fig. 7 Subcellular localization of protein encoded by Ata4CLA: Constructing vector for overexpressing 35S::Ata4CL-6HA. B: Screening of transgenic Arabidopsis herbicides. C: PCR was used to validate transgenic lines (M: 2 000 bp label. Water: Blank control; WT: Wild type; Number 1-8: overexpressed plants)

Fig. 8 Ata4CL expression patterns across tissues and developmental stagesA: The expressions of Ata4CL in different months; B: Expressions of Ata4CL in different tissues of bolting plants during flowering; C: Expressions of Ata4CL in different flower stages

Fig. 10 Total contents in wild type and overexpressed Ata4CL A. thaliana

Fig. 11 Relative expressions of Ata4CL, AtPAL and AtCHI in Ata4CL overexpressing A. thalianaThe error line indicates the mean ± standard deviation of three repetitions, and statistical analysis was performed using the t-test (*P<0.05, **P<0.01)

Fig. 12 Drought tolerance of Ata4CL-overexpressing A. thalianaA: Growth phenotype and root morphology of adult A. thaliana after 12 d of drought stress. B: Relative water content of WT and Ata4CL before and after 12 d of drought stress. C-F: Plant length, root length, rosette diameter and survival rate of WT and Ata4CL after 12 d of drought stress

Fig. 13 Changes in POD/SOD activities and MDA contents of A. thaliana at the mature plant stage before and after drought stress

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