蓝莓花青素相关VcGSTF19基因的克隆及功能研究

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

摘要/Abstract

摘要：

目的 Phi亚家族谷胱甘肽转移酶（GSTF）在多种植物花青素积累和转运中发挥关键作用。为研究蓝莓GSTF的功能，对蓝莓花青素相关GSTF19基因进行克隆和功能研究。方法利用反转录PCR克隆获得蓝莓花青素相关GSTF19的编码序列并构建过表达载体，基于蓝莓果实瞬时转化和拟南芥tt19突变体互补实验研究其功能。结果 VcGSTF19的瞬时过表达可以显著促进蓝莓果皮中花青素的积累。过表达VcGSTF19的蓝莓果皮花青素含量达到空载对照的6.61倍。其瞬时过表达显著提高了蓝莓果皮中VcCHS、VcCHI、VcF3H、VcDFR、VcANS 和 VcUFGT等花青素合成结构基因的表达水平。此外，VcGSTF19异源转化拟南芥tt19突变体恢复了突变体花青素积累，转基因莲座叶中花青素含量约为突变体的6.21倍。结论蓝莓VcGSTF19基因在花青素积累和转运中发挥着重要作用。

关键词: 花青素, 蓝莓, Phi亚家族谷胱甘肽转移酶, 基因克隆, 功能研究

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

张永艳, 郭思健, 李晶, 郝思怡, 李瑞得, 刘嘉鹏, 程春振. 蓝莓花青素相关VcGSTF19基因的克隆及功能研究[J]. 生物技术通报, 2025, 41(9): 139-146.

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.

图/表 5

表1 本研究所用引物信息

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

图1 蓝莓VcGSTF19基因的克隆和序列分析A：VcGSTF19全长CDS扩增产物电泳检测结果。M：DL2000 Marker；1-3代表3个重复。B：VcGSTF19与参考核苷酸序列比对结果。C：VcGSTF19与参考序列蛋白序列比对结果。Ref：参考序列

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

图2 基于拟南芥蛋白数据库的VcGSTF19互作蛋白预测TT8：透明种皮8；DFRA：二氢黄酮醇4-还原酶；LDOX：无色花青素双加氧酶； A3G2XYLT：花青素3-O-葡萄糖苷2'″-O-木糖基转移酶；CYP75B1：类黄酮3'-单加氧酶；5MTA：丙二酰辅酶A：花青素5-O-葡萄糖苷-6''-O-丙二酰转移酶；AHA10：拟南芥H（+）-ATP酶质子泵10；DTX41：解毒蛋白41；UGT75C1：UDP-糖基转移酶75C1

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

图3 VcGSTF19瞬时过表达对蓝莓果皮花青素代谢的影响A：果实表型及花青素提取溶液；B：瞬时过表达VcGSTF19对蓝莓果皮色差指标的影响；C：瞬时过表达VcGSTF19对蓝莓果皮花青素含量的影响。D-J：分别为瞬时过表达VcGSTF19的蓝莓果皮中VcGSTF19、VcCHS、VcCHI、VcF3H、VcDFR、VcANS和VcUFGT基因表达分析结果。柱上不同小写字母表示差异显著（P<0.05）。EV：空载对照；FW：鲜重；GSTF：F亚家族谷胱甘肽转移酶；CHS：查尔酮合酶；CHI：查尔酮异构酶；F3H：黄酮3-羟化酶；DFR：二氢黄酮醇4-还原酶；ANS：花青素合酶；UFGT：UDP葡萄糖：类黄酮3-糖基转移酶

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

图4 VcGSTF19过表达对tt19突变体莲座叶的影响A：拟南芥tt19突变体；B：过表达VcGSTF19的转基因植株；C-E：VcGSTF19过表达对拟南芥莲座叶类黄酮、花青素和原花青素含量的影响；MT：过表达VcGSTF19的tt19转基因植株

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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