【目的】对‘美人’梅(Prunus mume ‘Meiren’)中谷胱甘肽S-转移酶(glutathione S-transferases,GST)基因家族成员PmMGST81进行克隆和功能研究,旨在解析梅花GST基因在花青素苷积累中的作用,为深入揭示梅花呈色机制奠定理论基础。【方法】以同源序列法克隆PmMGST81基因,对其序列进行生物信息学分析,通过荧光定量PCR(qPCR)、亚细胞定位、烟草的稳定遗传转化验证PmMGST81参与花青素苷积累的功能。【结果】①克隆得到‘美人梅’PmMGST81编码序列(coding sequence,CDS)全长642 bp,上游启动子预测所含顺式作用元件多为光响应元件和激素响应元件,表明其在蔷薇科中与李属物种同源蛋白亲缘关系最近。②qPCR结果表明PmMGST81在‘美人’梅叶片不同发育阶段表达量与花青素苷含量呈显著正相关。③亚细胞定位显示PmMGST81蛋白分布位置是细胞核与细胞质。④PmMGST81过表达烟草株系中花冠着色变深,上游花青素苷合成通路相关结构基因上调表达,花青素苷含量显著增加。【结论】PmMGST81在花青素苷积累过程中起正向调控作用,促进花青素苷的积累从而影响‘美人’梅紫色表型的形成。

【Objective】Prunus mume, a renowned traditional ornamental flower in China, is extensively utilized in landscape gardening due to its high aesthetic value. Developing P. mume cultivars with colorful foliage represents a pivotal objective in current genetic improvement programs. Among them, the ‘Meiren’ cultivar group, distinguished by its unique purple-leaf phenotype, serves as an ideal model for investigating the genetic regulation of anthocyanin biosynthesis. Anthocyanin accumulation is the primary determinant of the purple-leaf trait. Although glutathione S-transferases (GSTs) play a crucial role in the anthocyanin transport process, research on GSTs in P. mume remains scarce. This study aimed to clone and functionally characterize PmMGST81, a member of the GST gene family from P. mume ‘Meiren’, to elucidate the role of GST genes in anthocyanin accumulation and lay a theoretical foundation for understanding the coloration mechanism of P. mume.【Method】The PmMGST81 gene was cloned using the homology-based cloning method. Bioinformatics analyses, including prediction of signal peptides and transmembrane domains, gene structure analysis, and prediction of cis-acting elements in the 2 000 bp upstream promoter region, were conducted. Amino acid sequences of homologous proteins in Rosaceae were retrieved for multiple sequence alignment. RNA was extracted from leaves at different developmental stages (S1. apical leaves; S2. middle leaves; S3. basal leaves) of the same annual shoots of P. mume ‘Meiren’and reverse-transcribed into cDNA. The expression patterns of PmMGST81 and structural genes in the anthocyanin synthesis pathway were analyzed by quantitative real time PCR (qPCR) to explore the correlation between the expression of PmMGST81 and anthocyanin content phenotypes. Agrobacterium tumefaciens strains harboring the empty vector plasmid pSuper1300-GFP and the recombinant plasmid PmMGST81-pSuper1300-GFP were injected into Nicotiana benthamiana leaves. Fluorescent signals were observed and photographed using a laser confocal microscope to confirm the subcellular localization of PmMGST81 protein based on the position of GFP green fluorescence.The flower color phenotypes of transgenic tobacco(N. tabacum) plants overexpressing PmMGST81 were observed. Anthocyanins were extracted from tobacco petals, and their contents were determined by spectrophotometry. Five structural genes related to the anthocyanin synthesis pathway in tobacco, namely NtF3'H, NtFLS, NtANS, NtDFR, and NtUFGT, were selected. qPCR was employed to detect their expression levels in the over expressed PmMGST81 lines, verifying the function of PmMGST81 in anthocyanin accumulation through stable genetic transformation of tobacco.【Result】(1) The full-length coding sequence (CDS) of PmMGST81, which was 642 bp and encoded 213 amino acids, was successfully cloned. It was classified as a stable protein, and subcellular localization prediction indicated that it was located in both the cytoplasm and nucleus. The PmMGST81 protein lacked signal peptides and obvious transmembrane structures but contained the typical GST-C and GST-N conserved domains of GST family proteins. The predicted cis-acting elements in the upstream promoter were predominantly light-responsive and hormone-responsive elements, and numerous binding sites for MYB genes were present in the promoter sequence. Sequence alignment of the PmMGST81 protein with homologous proteins from Rosaceae plants revealed that it had the closest genetic relationship with those of Prunus species, indicating its conservation during evolution. (2)qPCR results showed that the expression patterns of structural genes in the anthocyanin synthesis pathway, such as PmMF3'H, generally exhibited a decreasing trend from S1 to S3 in P. mume ‘Meiren’ leaves. The leaf color gradually changed from purple to green during the S1—S3 stages. Similarly, the expression levels of PmMGST81 differed significantly among the three stages, and a significant positive correlation was observed between the expression of PmMGST81 and anthocyanin content. (3)Subcellular localization demonstrated that the PmMGST81 protein was distributed in both the nucleus and cytoplasm. (4)In the transgenic tobacco lines overexpressing PmMGST81, the expression level of PmMGST81 was significantly higher than that in the control group. The corolla color deepened, and the anthocyanin content was significantly increased. Expression analysis of anthocyanin synthesis pathway genes showed that, except for NtFLS, whose expression level did not differ significantly from that of the wild-type, the expression levels of NtF3'H, NtANS, NtDFR, and NtUFGT were upregulated to varying degrees. This indicated that the deepening of tobacco flower color was due to increased anthocyanin accumulation. 【Conclusion】In this study, the GST family gene PmMGST81 (with a CDS length of 642 bp) was cloned from P. mume ‘Meiren’, and its protein was found to be distributed in both the nucleus and cytoplasm. The expression of PmMGST81 at different developmental stages of purple leaves was significantly positively correlated with anthocyanin content. Overexpression of PmMGST81 in tobacco promoted anthocyanin accumulation in the corolla and upregulated the expression of structural genes in the anthocyanin biosynthesis pathway, suggesting that PmMGST81 regulates the purple leaf phenotype of P. mume ‘Meiren’ by positively modulating anthocyanin metabolism.