Cloning and Functional Analysis of ZhGSTF Related to Anthocyanin Transport Zantedeschia hybrida

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

Abstract

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

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.

Figures/Tables 7

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

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

Fig. 2 PCR amplified products of Z. hybridaZhGSTF

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

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

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

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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