不结球白菜GST基因家族的全基因组鉴定及花青素相关基因BcGSTF6的功能分析

doi:10.3864/j.issn.0578-1752.2024.16.011

摘要/Abstract

摘要：

【背景】花青素是紫色不结球白菜中重要的营养成分之一，谷胱甘肽转移酶（glutathione S-transferase，GST）在花青素转运中发挥着重要功能。【目的】鉴定不结球白菜中编码GST的基因，并通过转录组和分子生物学手段揭示BcGSTF6在不结球白菜花青素转运中的作用，解析不结球白菜花青素积累的分子机制。【方法】通过生物信息学方法，鉴定GST基因家族成员并分析其在染色体上的位置和基序；在紫色和绿色的不结球白菜中进行转录组分析，筛选不结球白菜中参与花青素转运的基因，并通过表达模式分析、亚细胞定位、拟南芥突变体tt19的异源回补试验进一步验证BcGSTF6和BcTT19的功能；通过将BcGSTF6和BcTT19与其他物种中参与花青素转运基因的氨基酸序列进行比较，分析BcGSTF6存在的差异；并通过BcGSTF6蛋白与矢车菊素（Cya）的体外结合和基因沉默试验验证BcGSTF6是否为参与不结球白菜花青素转运和积累的重要基因。【结果】从不结球白菜中鉴定得到83个GST基因家族成员，分为8个亚家族，分布在10条染色体上。转录组分析结果表明BcGSTF6和BcTT19可能是参与不结球白菜花青素转运的基因，进一步利用qRT-PCR发现BcGSTF6与BcTT19都在不结球白菜花青素积累阶段高度表达；亚细胞定位结果表明BcGSTF6和BcTT19都定位于内质网和液泡膜中。氨基酸序列多重比较发现BcTT19与其他物种中参与花青素转运的蛋白存在高度同源性，而BcGSTF6则在保守结构域中存在较大差异。在拟南芥突变体tt19中对BcGSTF6和BcTT19进行过表达均可恢复拟南芥幼苗tt19积累花青素的表型，但不能恢复其种皮棕色的表型，表明BcGSTF6和BcTT19都参与了花青素的转运但不能转运原花青素（PAs）。体外结合试验发现BcGSTF6蛋白在室温下可增加Cya的可溶性；对BcGSTF6在不结球白菜中进行基因沉默发现叶片中花青素含量显著下降，同时也导致参与花青素合成与调控相关基因表达量降低。证明BcGSTF6参与了花青素的转运和积累，在不结球白菜叶片着色过程中发挥重要功能。【结论】本研究鉴定了不结球白菜GST基因家族，并进一步解析了BcTT19和BcGSTF6在不结球白菜花青素转运和积累中的功能，阐明了BcGSTs在不结球白菜中花青素转运调控的部分机制。

关键词: 不结球白菜, 花青素, 谷胱甘肽S-转移酶, BcGSTF6, 转运

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

尹雨钦, 徐欢欢, 唐丽萍, 王欣雅, 胡春梅, 侯喜林, 李英. 不结球白菜GST基因家族的全基因组鉴定及花青素相关基因BcGSTF6的功能分析[J]. 中国农业科学, 2024, 57(16): 3234-3249.

YIN YuQin, XU HuanHuan, TANG LiPing, WANG XinYa, HU ChunMei, HOU XiLin, LI Ying. Genome-Wide Identification of GST Gene Family and Functional Analysis of the BcGSTF6 Gene Related to Anthocyanin in Pak Choi[J]. Scientia Agricultura Sinica, 2024, 57(16): 3234-3249.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2024.16.011

https://www.chinaagrisci.com/CN/Y2024/V57/I16/3234

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