Gene Cloning and Functional Analysis of the Anthocyanin-related VcGSTF19 Gene in Blueberry （Vaccinium corymbosum L.）

doi:10.13560/j.cnki.biotech.bull.1985.2025-0190

Abstract

Abstract:

Objective Glutathione S-transferase Phi (F) subfamily members (GSTFs) play key roles in anthocyanin accumulation and transportation in many plants. To study the function of blueberry (Vaccinium corymbosum L.) GSTF, the blueberry anthocyanin-related GSTF19 gene was cloned and its function was studied. Method Reverse transcription PCR was used to clone the coding sequence (CDS) of VcGSTF19 related toanthocyanin from blueberry and construct overexpressing vector. Based on the blueberry fruit transient overexpression and Arabidopsis tt19 complementary transgenic transformation assays, the functions of VcGSTF19 were further studied. Result Transient overexpression of VcGSTF19 significantly promoted the accumulation of anthocyanins in blueberry peel. The transient overexpression of VcGSTF19 improved significantly the anthocyanin accumulation by 6.61-fold in blueberry fruit peel, compared to the empty vector control. Moreover, its transient overexpression significantly upregulated the expressions of anthocyanin biosynthesis related structural genes, including VcCHS, VcCHI, VcF3H, VcDFR, VcANS and VcUFGT. Heterologous transformation of Arabidopsis tt19 mutant with VcGSTF19 restored the anthocyanin accumulation in the mutant, with anthocyanin contents in rosette leaves accounting for approximately 6.21-fold of the tt19 mutant. Conclusion Blueberry VcGSTF19 gene plays an important role in anthocyanin accumulation and transport.

Key words: anthocyanin, blueberry, glutathione S-transferase F subfamily member (GSTF), gene cloning, functional analysis

ZHANG Yong-yan, GUO Si-jian, LI Jing, HAO Si-yi, LI Rui-de, LIU Jia-peng, CHENG Chun-zhen. Gene Cloning and Functional Analysis of the Anthocyanin-related VcGSTF19 Gene in Blueberry （Vaccinium corymbosum L.）[J]. Biotechnology Bulletin, 2025, 41(9): 139-146.

Figures/Tables 5

Table 1 Information for the primers used in this study

引物名称	引物序列	退火温度	用途
Primer name	Primer sequence （5′-3′）	Annealing temperature （℃）	Applications
VcGSTF19F	ATGGTGGTCAAAGTTTATGGTCCAATTAGAG	63	基因克隆 Gene cloning
VcGSTF19R	CTAATCCATAAGCTTCATTATTTTCTTCCAAGCAG	63	基因克隆 Gene cloning
VcGSTF19-SF	CTCTACAAATCTATCTCTGGATCCATGGTGGTCAAAGTTTATGGTCCAATTAGAG	68	载体构建
VcGST19F-SR	GATTTTTGCGGACTCTAGGAGCTCCTAATCCATAAGCTTCATTATTTTCTTCCAAGCAG	68	Vector construction
VcGSTF19-qF	GGAGAGAGCCCTTGTTGACC	60	实时荧光定量
VcGSTF19-qR	GCTCTTCGACAACCTCTGCT	60	RT-qPCR
VcCHS-qF	CGCATGTGTGACAAATCCCA	60
VcCHS-qR	ACCATATCCTGCCTAGCGTC	60
VcCHI-qF	TCTTTCCTCCGTCGGTCAAA	60
VcCHI-qR	ACTCTACGGCGCTAACTTGT	60
VcF3H-qF	GTGGACGGAGCTTTTGTTGT	60
VcF3H-qR	GTGATTGGCTCGTCGAGAAC	60
VcDFR-qF	GCTTCTTGAACGGGGCTATG	60
VcDFR-qR	CTTCAATGGCCTCGTCGAAG	60
VcANS-qF	ACCTGAGAGCCCTAACAACC	60
VcANS-qR	CTGTGATCCATTTGCCCTCG	60
VcUFGT-qF	ATTGGTGTGAGAGTGGAGGG	60
VcUFGT-qR	GTCCAACAGCCTTCAAAGCA	60
VcGAPDH-qF	ACTACCATCCACTCTATCACCG	60
VcGAPDH-qR	AACACCTTACCAACAGCCTTG	60

Fig. 1 Gene cloning and sequence analysis of blueberry VcGSTF19 geneA: Electrophoresis detection results of amplified VcGSTF19. M: DL2000 marker; 1-3 indicate three replications. B: Nucleotide acid sequences alignment results of amplified VcGSTF19 and reference VcGSTF19. C: Protein sequences alignment result of protein sequences encoded by amplified VcGSTF19 with reference VcGSTF19. Ref: Reference sequences

Fig. 2 Predicted interacting proteins of VcGSTF19 based on the Arabidopsis protein databaseTT8: Transparent testa 8. DFRA: Dihydroflavonol 4-reductase. LDOX: Leucoanthocyanidin dioxygenase. A3G2XYLT: Anthocyanidin 3-O-glucoside 2'″- O-xylosyltransferase. CYP75B1: Flavonoid 3'-monooxygenase. AHA10: Arabidopsis H (+)-ATPase proton pump 10. DTX41: detoxification 41. UGT75C1: UDP-glycosyltransferase 75C1

Fig. 3 Influences of VcGSTF19 transient overexpression on the anthocyanin metabolism in blueberry fruit peelsA: Fruit phenotype and extracted anthocyanin solution; B: Influences of VcGSTF19 transient overexpression on the blueberry fruit color parameters. C: Influences of VcGSTF19 transient overexpression on anthocyanin contents in blueberry fruit peels. D-J: Influences of VcGSTF19 overexpression on the relative expressions of VcGSTF19, VcCHS, VcCHI, VcF3H, VcDFR, VcANS and VcUFGT in blueberry fruit peels. Different letters above columns indicate significant differences between samples at P<0.05 level. EV: Empty vector. FW: Fresh weight. GSTF: F subfamily glutathione S- transferase. CHS: Chalcone synthase. CHI: Chalcone isomerase. F3H: Flavonoid 3-hydroxylase. DFR: dihydroflavonol 4-reductase. ANS: anthocyanin synthase. UFGT: UDP Glucose. flavonoid 3-glycosyltransferase

Fig. 4 Influences of VcGSTF19 overexpression on the rosette leaves of Arabidopsistt19 mutantsA: Arabidopsistt19 mutant. B: Transgenicplants overexpressing VcGSTF19. C-E: Influences of VcGSTF19 overexpression on the contents of flavonoids, anthocyanin and procyanidins in Arabidopsis rosette leaves, respectively. MT: Transgenic tt19 plants overexpressing VcGSTF19

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