彩色马蹄莲花青素苷转运相关ZhGSTF的克隆及功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0747

摘要/Abstract

摘要：

目的 GSTF转运蛋白参与植物花青素苷的转运过程，研究其对彩色马蹄莲（Zantedeschia hybrida）佛焰苞着色和花青素苷积累的作用及其分子机制，为彩色马蹄莲花青素苷转运机制解析提供理论依据，为新品种选育提供有效基因资源。方法基于课题组前期对彩色马蹄莲转录组数据的分析，筛选并克隆彩色马蹄莲佛焰苞花青素苷转运相关基因ZhGSTF，分析其进化关系和序列特征，并测定在不同品种佛焰苞中的基因表达量和花青素苷含量。同时利用病毒诱导的基因沉默（virus induced gene silencing, VIGS）技术构建ZhGSTF沉默体系，比较沉默后佛焰苞表型、基因表达和3个类黄酮合成途径下游结构基因表达量的变化。结果 ZhGSTF的CDS长度为666 bp，编码221个氨基酸。系统进化树分析表明，ZhGSTF与其他物种花青素苷转运相关的GST聚为一类；氨基酸序列比对显示，ZhGSTF与紫苏（Perilla frutescens）PfGST1相似性最高。ZhGSTF在玫红色‘佳人’和橙色‘奥迪安’中表达量均显著高于黄色‘金城’，在白色‘范图拉’中显著低表达，ZhGSTF在4个品种中的表达量与其花青素苷积累趋势一致。经VIGS沉默后，ZhGSTF表达量显著下调，佛焰苞表型发生明显褪色，而ZhGSTF的沉默对ZhANS、ZhANR和ZhDFR表达量无显著影响。结论彩色马蹄莲ZhGSTF在佛焰苞着色过程中促进花青素苷积累并影响其呈色。

关键词: 彩色马蹄莲, 花青素苷, 佛焰苞, ZhGSTF, 转运蛋白, 生物信息学分析, VIGS, 表型

Abstract:

Objective GSTF transporter is involved in the transport process of anthocyanins in plants. Studying its role and molecular mechanism in the coloration of spathe coloration and anthocyanin accumulation of Zantedeschia hybrida may provide theoretical basis for the analysis of anthocyanin transport mechanism of Z. hybrida and provide effective gene resources for breeding new varieties. Method Based on the analysis of transcriptome data of Z. hybrida, the gene ZhGSTF related to anthocyanin transport in the spatula of Z. hybrida was screened and cloned, its evolutionary relationship and sequence characteristics were analyzed, and the gene expressions and anthocyanin contents in different varieties of spatula were determined. Concurrently, virus induced gene silencing technology was used to construct ZhGSTF silencing system, and the variations of phenotype, gene expression and gene expression of three flavonoid synthesis pathways downstream after silencing were compared. Result The CDS of ZhGSTF gene was 666 bp long, encoding 221 amino acids. Phylogenetic tree analysis showed that ZhGSTF was clustered with GST related to anthocyanin transport in other species. The amino acid sequence comparison showed that ZhGSTF had the highest similarity with Perilla frutescens PFGST1. The expressions of ZhGSTF in rose ‘Lover’ and orange ‘Odean’ was significantly higher than that in yellow ‘Jincheng’, and it was significantly lower in white ‘Ventura’. The expressions of ZhGSTF in the four varieties were consistent with the accumulation trend of anthocyanins. After VIGS silencing, the expressions of ZhGSTF significantly decreased, and the phenotype of spathe faded obviously, but ZhGSTF silencing had no significant effect on the expression of ZhANS, ZhANR and ZhDFR. Conclusion The Z. hybridaZhGSTF promotes the accumulation of anthocyanins and affects its coloration in the process of spathe coloration.

Key words: Zantedeschia hybrida, anthocyanins, spatula, ZhGSTF, transport protein, bioinformatics analysis, VIGS, phenotype

吕呈聪, 衡蒙, 陈思琪, 金雪花. 彩色马蹄莲花青素苷转运相关ZhGSTF的克隆及功能分析[J]. 生物技术通报, 2026, 42(1): 161-169.

LYU Cheng-cong, HENG Meng, CHEN Si-qi, JIN Xue-hua. Cloning and Functional Analysis of ZhGSTF Related to Anthocyanin Transport Zantedeschia hybrida[J]. Biotechnology Bulletin, 2026, 42(1): 161-169.

图/表 7

图1 四个品种彩色马蹄莲佛焰苞（A）和玫红色品种佛焰苞的4个着色阶段（B）S1：佛焰苞未着色；S2：佛焰苞微着色；S3：佛焰苞部分着色；S4：佛焰苞完全着色。下同

Fig. 1 Four varieties of Z. hybrida spatula (A) and four coloring stages of rose spatula (B)S1: The spatula is uncolored; S2: micro-coloring the spatula; S3: partially coloring the spathe; S4: completely coloring the spatula. The same below

表1 实验所用引物

Table 1 Primers used in the study

引物 Primer	引物序列 Primer sequence （5′‒3′）	引物用途 Primer usage
ZhGSTF-F	TATGCCGTTACGTGTGCGAC	基因克隆
ZhGSTF-R	GGTTGTAGGACTGCCCCTCT	Gene clone
ZhGSTF-F-q	AGATCCGTATCAATCCCT	基因表达 Gene expression
ZhGSTF-R-q	TAAGAGTATGAAAGTGCC
ZhANS-F	GATCCCGTGGTTGACGATGT
ZhANS-R	ATCGTGGACTTCACGTCTGG
ZhANR-F	TGGAAGACATAGTCGCAGCC
ZhANR-R	GACGAAGCTCGCTCACCTTA
ZhDFR-F	CGACGAGATGAAAGGACCCA
ZhDFR-R	TGTATCGAAGACGTTGGCGG
ZhActin-F	GAAGGAGAAACTTGCGTATGTAGC	内参基因
ZhActin-R	GTCCATCTGGCAGCTCATAACT	Reference gene
ZhGSTF-Sac I-F	CTCTAGAAGGCCTCCATGGGGATCCGCAGAGGGGCAGTCCTACAA	载体构建
ZhGSTF-BamH I-R	GCCTCGAGACGCGTGAGCTCGGAGCCTTCTGCTTCTGCA	Vectors construction

图2 彩色马蹄莲ZhGSTF的PCR扩增产物

Fig. 2 PCR amplified products of Z. hybridaZhGSTF

图3 彩色马蹄莲ZhGSTF与其他物种GST系统进化树（A）及氨基酸序列比对（B）

Fig. 3 Phylogenetic tree (A) and amino acid sequence alignment of Z. hybrida ZhGSTF with GST of other species (B)

图4 ZhGSTF在4个彩色马蹄莲品种中的表达特性分析A：ZhGSTF在4个彩色马蹄莲品种的4个着色阶段的表达水平；B：4个不同品种彩色马蹄莲在S4阶段的佛焰苞花青素苷含量；不同小写字母代表不同处理间差异显著（P<0.05），下同

Fig. 4 Expressions of ZhGSTF in four coloring stages of four Z. hybrida varietiesA: The expressions of ZhGSTF in four coloring stages of four colored Z. hybrida varieties. B: Anthocyanin content in spatula of four different varieties of Z. hybrida at S4 stage. Different lowercase letters indicate significant differences among different treatments (P<0.05), the same below

图5 TRV2-ZhGSTF菌落PCR检测M：DL 2000 marker；1‒12：ZhGSTF菌落PCR扩增产物

Fig. 5 PCR detection of TRV2-ZhGSTF colonyM: DL 2000 marker; 1‒12: PCR-amplified product of ZhGSTF colony

图6 ZhGSTF沉默后佛焰苞的表型及相关基因表达分析A：经VIGS沉默ZhGSTF后佛焰苞的表型；B：经VIGS沉默后ZhGSTF在佛焰苞中的相对表达水平；C：类黄酮合成途径下游3个关键基因在ZhGSTF沉默后佛焰苞中的表达水平；D0：处理第0天；D7：处理第7天；D14：处理第14天

Fig. 6 Phenotype and related gene expression analysis of spathe after ZhGSTF silencingA: Phenotype of spathe after VIGS silencing ZhGSTF. B: Relative expression of ZhGSTF in spathe after VIGS silencing. C: Expressions of three key genes downstream of flavonoid synthesis pathway in spathe after ZhGSTF silencing. D0: 0 d of treatment; D7: 7th day of treatment; D4: 14th day of treatment

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