Ata4CL基因在紫菀黄酮合成与抗旱性功能研究

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

摘要/Abstract

摘要：

目的紫菀（Aster tataricus L.）作为止咳润肺药材，黄酮类化合物是其主要活性成分，4-香豆酸辅酶A连接酶（4CL）为黄酮合成的关键限速酶，解析Ata4CL分子特征及在黄酮合成与抗旱中的双重功能，为紫菀药用成分优化及抗逆育种提供理论依据。方法硝酸铝比色法测定紫菀不同组织和生长阶段的总黄酮含量，克隆Ata4CL基因全长，结合生物信息学工具分析其结构特征，利用农杆菌介导转化烟草进行亚细胞定位，构建35S∶∶Ata4CL-6HA过表达载体并转化拟南芥，经干旱胁迫处理验证其功能。结果紫菀根部总黄酮呈双峰积累，为黄酮积累的主要部位，进入开花期的植株叶片和根茎中含量最高。克隆获得Ata4CL基因全长1 623 bp，编码540个氨基酸，分子量为59.04 kD，编码蛋白以无规则卷曲为主，含AAE保守结构域，定位于叶绿体；其氨基酸序列在菊科植物中高度保守，与小蓬草同源性高达96.48%；该基因在根部和叶片的表达量均呈“上升-下降-上升”的趋势，在开花植株中根部表达最强，盛花期的花中表达量最高。过表达Ata4CL的拟南芥总黄酮含量较野生型提高1.47-1.76倍，基因表达量上调5.41-12.23倍，同时在黄酮通路上下游合成基因表达量均上调；干旱胁迫下，转基因拟南芥比野生型存活率提高76.67%-86.67%，株高、根长及莲座直径均显著增加，SOD和POD活性增强，MDA含量降低。结论 Ata4CL基因通过协同调控黄酮生物合成从而提高植物抗旱性，为紫菀药用成分优化及抗逆品种选育提供了关键分子靶点。

关键词: 紫菀, Ata4CL基因, 黄酮合成, 抗旱性

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

王婷, 孟义江, 王晗, 贾凯旋, 乔晓宇, 韩冰冰, 刘晓清, 葛淑俊. Ata4CL基因在紫菀黄酮合成与抗旱性功能研究[J]. 生物技术通报, 2026, 42(1): 230-240.

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.

图/表 13

表1 试验所用引物信息

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

图1 紫菀不同发育阶段总黄酮含量大写字母和小写字母分别表示根部和叶片间差异显著。误差线为3次重复的平均数±标准差，Duncan检验差异显著水平小于0.05，下同

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

图2 开花期紫菀的总黄酮含量

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

图3 紫菀Ata4CL基因克隆M：DNA marker DL2000；1，2：目的条带

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

图4 紫菀Ata4CL 编码蛋白的生物信息学特性分析

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

图5 Ata4CL编码氨基酸的多序列比对分析

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

图6 4CL同源进化分析

Fig. 6 Homologous evolutionary analysis of 4CL

图7 Ata4CL编码蛋白的亚细胞定位A：过表达35S：：Ata4CL-6HA载体构建；B：过表达拟南芥除草剂筛选；C：PCR验证过表达株系（M：2 000 bp标记；水：空白对照；WT：野生型；编号1-8：过表达植株）

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)

图8 不同发育阶段和不同组织中Ata4CL的表达水平A：不同月份Ata4CL的表达量；B：花期开花植株不同组织Ata4CL的表达量；C：不同花时期Ata4CL的表达量

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

图10 野生型和过表达Ata4CL拟南芥中总黄酮含量

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

图11 过表达Ata4CL拟南芥中Ata4CL、AtPAL和AtCHI的相对表达量误差线表示3次重复平均值±标准差，采用t检验进行统计学分析（*P<0.05，**P<0.01）

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)

图12 Ata4CL过表达拟南芥的耐旱性分析A：成株期拟南芥干旱胁迫12 d前后生长表型及根系形态；B：干旱胁迫12 d前后WT和Ata4CL相对含水量；C-F：干旱12 d后WT和Ata4CL株高、存活率、根长及莲座直径

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

图13 干旱胁迫前后成株期拟南芥POD/SOD活性及MDA含量变化

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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