Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (16): 3234-3249.doi: 10.3864/j.issn.0578-1752.2024.16.011

• HORTICULTURE • Previous Articles     Next Articles

Genome-Wide Identification of GST Gene Family and Functional Analysis of the BcGSTF6 Gene Related to Anthocyanin in Pak Choi

YIN YuQin(), XU HuanHuan, TANG LiPing, WANG XinYa, HU ChunMei, HOU XiLin, LI Ying()   

  1. College of Horticulture, Nanjing Agricultural University/National Key Laboratory of Crop Genetics and Germplasm Innovation and Utilization/Engineering Research Center for Horticultural Crop Germplasm Innovation and Utilization of the Ministry of Education, Nanjing 210000
  • Received:2024-01-25 Accepted:2024-04-15 Online:2024-08-16 Published:2024-08-27
  • Contact: LI Ying

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Abstract:

【Background】Anthocyanin is one of the important nutritional components in purple pak choi (Brassica campestris ssp. chinensis), and the glutathione S-transferase (GST) plays an important role in anthocyanin transport. 【Objective】This study aimed to identify the genes encoding GST in pak choi and to elucidate the role of BcGSTF6 in anthocyanin transport through transcriptome and molecular biology methods, which would help to elucidate the molecular basis of anthocyanin accumulation in pak choi. 【Method】Using bioinformatics methods to identify members of the GST gene family and to analyze their positions and motifs on chromosomes. The transcriptome analysis was performed in purple and green pak choi to screen genes involving in anthocyanin transport. To further validate the functions of BcGSTF6 and BcTT19, the expression pattern analysis, subcellular localization, and heterologous expression of BcGSTF6 and BcTT19 in the Arabidopsis tt19 mutant were conducted. By comparing the amino acid sequences of BcGSTF6 and BcTT19 with anthocyanin transporters in other species, the differences between BcGSTF6 and others were analyzed. In vitro binding experiments and gene silencing experiments of the BcGSTF6 protein with cyanidin (Cya) demonstrated whether BcGSTF6 played a crucial role in anthocyanin transport and accumulation in pak choi.【Result】In total, 83 members of the GST gene family were identified. They were divided into 8 subfamilies, and they were distributed on all 10 chromosomes of the pak choi. The transcriptome analysis results showed that BcGSTF6 and BcTT19 may be important genes involved in anthocyanin transport in pak choi. Furthermore, qRT-PCR analysis revealed that both BcGSTF6 and BcTT19 were highly expressed during the anthocyanin accumulation; subcellular localization results indicated that both BcGSTF6 and BcTT19 were located in the endoplasmic reticulum (ER) and tonoplast; multiple comparisons of amino acid sequences revealed high homology between BcTT19 and other anthocyanin transport proteins in other species, while BcGSTF6 showed significant differences in the conserved domain; the overexpression of BcGSTF6 and BcTT19 in the Arabidopsis tt19 mutant rescued the phenotype that seedlings could accumulate anthocyanin, but the brown color of the seed coats was not rescued. These results suggested that both BcGSTF6 and BcTT19 were involved in anthocyanin transport but could not transport proanthocyanidins (PAs); In vitro binding assay showed that BcGSTF6 protein could increase the solubility of Cya at room temperature; gene silencing of BcGSTF6 in pak choi not only resulted a significant decrease in anthocyanin content in the leaves, but also led to a decrease in the expression of genes related to anthocyanin synthesis and regulation. These results demonstrated that BcGSTF6 was involved in anthocyanin transport and accumulation in pak choi, and played an important role in the coloring process of pak choi leaves. 【Conclusion】 This study identified the GST gene family and further elucidated the functions of BcTT19 and BcGSTF6 in anthocyanin transport in pak choi. This experiment elucidated the partial mechanism of BcGSTs regulation of anthocyanin transport and accumulation in pak choi.

Key words: pak choi (Brassica campestris ssp. chinensis), anthocyanins, glutathione S-transferase, BcGSTF6, transport

Fig. 1

Member identification and classification of BcGST in pak choi A: The phylogenetic tree of GST genes in pak choi; B: Chromosomal mapping analysis of GST genes in pak choi; C: Analysis of GST protein motifs in pak choi"

Fig. 2

The expression patterns of BcGSTF6 and BcTT19 A：Differential expression heatmap of BcGST in ‘HG072’ and ‘HP072’; B: The total anthocyanin content of ‘HG072’and ‘HP072’, and the expression levels of BraC10g001530, BraC10g024210 and BraC08g036270 in ‘HG072’ and ‘HP072’; C: S1-S8 during the development process of ‘G125’ leaves, total anthocyanin content in leaves, and expression levels of BcGSTF6 and BcTT19. The scale is 2 cm. Different lowercase letters indicate significant difference (P<0.05). The same as below"

Fig. 3

Protein sequence alignment and subcellular localization analysis of BcGSTF6 and BcTT19 A: Protein sequence alignment of BcGSTF6 and BcTT19 with other species; B: Subcellular localization of BcGSTF6 and BcTT19"

Fig. 4

Transformation of Arabidopsis mutant tt19 by BcGSTF6 and BcTT19 A:7-day-old Arabidopsis seedlings were grown on MS medium containing 5% sucrose, and the scale is 2 mm; B: Images of Arabidopsis seeds, and the scale is 1 mm; C: Relative expression levels of BcGSTF6 and BcTT19 in transgenic Arabidopsis"

Fig. 5

In vitro binding of BcGSTF6 protein to Cya A: Denatured protein gel electrophoresis; B: UPLC detection of the absorbance of Cya in BcGSTF6 protein and empty protein solution, * indicate the absorbance after being placed at room temperature for 2.5 minutes"

Fig. 6

BcGSTF6 silencing inhibits anthocyanin accumulation in pak choi A: Phenotypes of ‘G125’ after silencing BcGSTF6, and PTY-Empty was used as a control; B: The total anthocyanin content of silenced plants and control plants; C: The content of Cya in both silenced and control plants were measured by UPLC; D: The gene expression levels of BcGSTF6 in both silenced and control plants; E: Relative expression levels of anthocyanin synthesis pathway genes in both silenced and control plants. LODX: Leucoanthocyanidin dioxygenase; UF3GT: UDP-glucose flavonoid-3-O-glucosyltransferase; F3H: Flavanone 3-hydroxylase; CHI: Chalcone isomerase; F3′H: Flavonoid 3'-hydroxylase; DFR: Dihydroflavonol-4-reductase"

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