紫丁香So4CL的克隆及功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0557

摘要/Abstract

摘要：

目的 4-香豆酸∶辅酶A连接酶（4-coumarate: CoA lig-ase, 4CL）是苯丙烷代谢途径中的核心酶，克隆并探究4CL基因在紫丁香（Syringa oblata）花青苷合成中的功能。方法基于紫丁香基因组及转录组数据筛选并克隆出So4CL基因，对其编码的蛋白进行生物信息学分析和亚细胞定位；利用RT-qPCR研究So4CL在不同花期及不同组织的相对表达量；构建So4CL过表达和沉默载体，利用农杆菌侵染法分别转化紫丁香和烟草（Nicotiana benthamiana），观察其表型变化并测定花青苷含量；通过RT-qPCR检测花青苷生物合成途径中So4CL上下游基因的表达水平。结果 So4CL基因CDS区全长为1 659 bp，编码552个氨基酸，具有BOX I和BOX Ⅱ保守结构域。系统进化分析显示，紫丁香So4CL与水曲柳（Fraxinus mandshurica）的4CL蛋白相似性最高，为97%。RT-qPCR分析结果显示，随着花发育，紫丁香花瓣褪色，So4CL的表达整体呈降低的趋势；So4CL在盛花期中的根、茎、叶、花中均有表达。亚细胞定位分析显示，So4CL主要存在于细胞质上。过表达So4CL的紫丁香花瓣着色明显，花青苷含量显著升高，烟草叶片出现砖红色变化；沉默So4CL后花瓣显著褪色，花青苷含量显著降低；RT-qPCR分析表明，瞬时转化So4CL会影响花青苷生物合成途径中SoPAL、SoCHS、SoDFR和SoUFGT的表达。结论 So4CL在紫丁香花瓣花青苷合成中发挥着重要作用，是合成关键基因。

关键词: 紫丁香, 4-香豆酸：辅酶A连接酶, 花青苷, 基因克隆, 功能验证, 基因表达

Abstract:

Objective 4-coumarate∶CoA ligase (4CL) is a core enzyme of the phenylpropane metabolic pathway, and the function and expression characteristics of 4CL gene in Syringa oblata were investigated. Method Based on the genomic and transcriptomic data of S. oblata, the So4CL gene was screened and cloned. Bioinformatics analysis and subcellular localization were performed on its encoded protein. The relative expressions of So4CL in different flowering stages and tissues were investigated using RT-qPCR. The overexpressing and silencing vectors of So4CL were constructed and transformed into S. oblata and Nicotiana benthamian via Agrobacterium-mediated infection, followed by observation of phenotypic changes and measurement of anthocyanin content; and the expressions of the structural genes upstream and downstream of the anthocyanin biosynthesis pathway were determined by RT-qPCR. Result The CDS region of the So4CL gene was 1 659 bp in full length, encoding 552 amino acids with conserved structural domains BOXI and BOXII. Systematic evolutionary analysis revealed that So4CL had the highest 97% similarity to the 4CL protein of Fraxinus mandshurica. RT-qPCR analysis showed that the expression of the So4CL tended to decrease and then increase as the petals of lilac fading with flower development. So4CL was expressed in the roots, stems, leaves, and flowers in flowering stage. Subcellular localization analysis showed that So4CL was mainly present in the cytoplasm. The petals color of S. oblata got obviously darker after overexpressing So4CL, and the anthocyanin contents were significantly elevated. The N. benthamian leaves also showed brick-red changes. While petals were significantly discolored and anthocyanin contents significantly reduced after silencing So4CL. RT-qPCR analysis showed that transient transformation of So4CL affected the expressions of SoPAL, SoCHS, SoDFR, and SoUFGT in the anthocyanin biosynthesis pathway. Conclusion So4CL plays an important role in anthocyanin synthesis in S. oblata petals and is a key gene in this process.

Key words: Syringa oblata, 4-coumarate: CoA ligase, anthocyanin, gene cloning, function verification, gene expression

倪莹, 李雷, 汪进萱, 马波, 孟昕, 冷平生, 吴静, 胡增辉. 紫丁香So4CL的克隆及功能分析[J]. 生物技术通报, 2026, 42(1): 139-149.

NI Ying, LI Lei, WANG Jin-xuan, MA Bo, MENG Xin, LENG Ping-sheng, WU Jing, HU Zeng-hui. Cloning and Functional Analysis of So4CL Gene in Syringa oblata[J]. Biotechnology Bulletin, 2026, 42(1): 139-149.

图/表 11

图1 紫丁香不同花发育阶段（A）和盛花期不同组织部位（B）

Fig. 1 Different flowering stages (A) and tissues (B) at full flower opening stage of S. oblata

表1 引物序列

Table 1 Primer sequences

引物用途

Primer function

引物名称

Primer name

引物序列

Primer sequence （5′‒3′）

克隆基因

Gene cloning

So4CL-F

So4CL-R

ATGGAGACTAAGACGACGCAAGAAGA

TCAATTGGGAACACCTGCAGCTAATC

实时荧光定量PCR

RT-qPCR

qSoActin-F

qSoActin-R

qSo4CL-F

qSo4CL-R

qSoPAL-F

qSoPAL-R

qSoCHS-F

qSoCHS-R

qSoDFR-F

qSoDFR-R

qSoUFGT-F

qSoUFGT-R

TGGAATGTGCTGAGAGATGC

TGCTGACCGTATGAGCAAAG

CTTGCGGTCTGTCTTGTCTG

TTCCGTCAAACCATAGCCCT

TGGACTATGGCTTCAAGGGGR

CTCGCCGAAATCAAACCCAA

TCTCGTAGTGTGCTCGGAAA

GTCAGAACCCACAATCACGG

GCATTGGAAGCGGCTAAAGA

CCAGTGATAGGCGAAAGTGC

TACCACCAGAATAGAGT

AGAAGTATTGCCAGAAG

基因过表达

Gene overexpression

PRI101-So4CL-F

PRI101-So4CL-R

ATGCCCGTCGACCCCGGGGGCGGAACTCAACCAGC

CTCACCATGGATCCGGTACCTAGTTTAGATACGGCAAGCTTTATTAGATCCTTCC

基因沉默

Gene silencing

TRV2-So4CL-F

TRV2-So4CL-R

GTCCAGTCCTGGCCTCGTCGGCCATGGCGGAACTCAACCAGC

GACCACAAGTGGCCAGACTGGCCAAGCTGAGCTGATGCCCG

So4CL-SubN-F

So4CL-SubN-R

AGTGGTCTCTGTCCAGTCCTATGGAGACTAAGACGACGCAAGAAGA

GGTCTCAGCAGACCACAAGTATTGGGAACACCTGCAGCTAATC

表2 So4CL蛋白质理化性质分析

Table 2 Analysis of physical and chemical properties of So4CL protein

蛋白名称

Protein name

分子式

Formula

相对分子质量

Molecular weight （Da）

总原子数

Total number of atoms

酸碱性氨基酸

Acid-base amino acid

理论等电点

Theoretical pI

脂溶指数

Aliphatic index

Asp + Glu

Arg + Lys

So4CL

C₂₇₁₆H₄₃₈₃N₇₀₉O₈₀₄S₁₈

图2 So4CL蛋白的二级结构（A）及三级结构（B）预测

Fig. 2 Secondary structure (A) and tertiary structure (B) prediction of So4CL protein

图3 So4CL与其他物种4CL蛋白序列比对分析黑色、红色、蓝色部分分别表示同源性=100%、≥75%、≥50%；红色框为SSGTTGLPKGV保守基序，绿色框为GEXXIXG保守基序

Fig. 3 Alignment and analysis of So4CL with 4CL protein sequences of other speciesThe black, red, and blue parts indicate homology =100%, ≥75%, ≥50% respectively. Red boxes are SSGTTGLPKGV conserved motifs, and green boxes are GEXXIXG conserved motifs

图4 紫丁香So4CL与其他植物同源蛋白的系统发育树

Fig. 4 Phylogenetic tree of So4CL from S. oblata and homologous proteins of other plant species

图5 So4CL在不同器官（A）和不同花发育阶段（B）的表达模式不同小写字母表示在P<0.05水平上差异显著

Fig. 5 Expression patterns of So4CL at different organs (A) and in different flower developmental stages (B)Different lowercase letters indicate significant differences at the P<0.05 level

图6 So4CL的亚细胞定位

Fig. 6 Subcellular localization of So4CL

图7 紫丁香过表达So4CL植株的基因表达水平（A）、花青苷含量（B）、花色（C）和烟草过表达So4CL叶色变化（D）*P<0.05， **P<0.01， ****P<0.000 1. The same below

Fig. 7 Expressions of So4CL (A), anthocyanin contents (B), and petal color (C) in So4CL-overexpressed S. oblata and leaf color in So4CL-overexpressing N. benthamian (D)

图8 紫丁香沉默So4CL 植株的基因表达水平（A）、花青苷含量（B）和花色（C）的变化

Fig. 8 Expressions of So4CL (A), anthocyanin contents(B), and petal color (C) in So4CL-silenced S. oblata

图9 瞬时过表达（A）和沉默（B）So4CL对上下游基因表达的影响

Fig. 9 Effects of transient overexpression (A) and silencing (B) of So4CL on the expressions of upstream and downstream genes

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