为阐明菲律宾蛤仔(Ruditapes philippinarum)谷胱甘肽转移酶(glutathione S-transferase, GST)基因(RpGST)家族特征及其在应对急性盐度变化中所起的作用, 本研究通过生物信息学方法对 RpGST 基因家族进行了全基因组水平的鉴定、染色体定位、结构特征分析、进化分析和表达特征分析, 共鉴定出 7 个 RpGST 基因家族成员, 分别命名为 RpGSTA1、RpGSTA2、RpGSTA3、RpGSTA4、RpGST_C_3a、RpGST_N_M1、RpGST_N_M2。RpGSTs 不均匀地定位在 5 条染色体上, 其编码蛋白的理化性质呈现出不同程度的亲水性。RpGST 蛋白均定位于细胞质中, 该基因家族成员都具有谷胱甘肽转移酶结构域(PF00043、PF02798), 表明 RpGSTs 蛋白参与抗氧化和解毒过程; 系统发育分析显示, RpGSTs 分为 3 个亚家族且在进化上保守。实时荧光定量分析显示, 急性盐度胁迫菲律宾蛤仔 0 h、12 h 和 24 h 后, 急性高盐(40)胁迫下肝胰腺中 RpGSTA 和 RpGST_N_M 亚家族的基因整体表达水平随时间逐步升高, RpGST_C_3 亚家族的基因表达水平随时间先降低后升高; 急性低盐(15)胁迫下肝胰腺中 RpGSTA、RpGST_C_3a 和 RpGST_N_M 亚家族的基因整体表达水平随时间逐步升高; 急性低盐胁迫下谷胱甘肽转移酶表达量显著高于急性高盐胁迫, 说明急性低盐胁迫对菲律宾蛤仔的影响大于急性高盐胁迫。以上研究结果明确了 RpGST 基因家族的序列结构特征、系统进化以及对急性高盐和急性低盐胁迫的响应机制, 证明 RpGST 基因家族在菲律宾蛤仔抗氧化和解毒过程中发挥了重要作用, 为菲律宾蛤仔抗逆分子选择育种研究提供了理论依据。

Glutathione S-transferase (GSTs, EC 2.5.1.18), a class of multifunctional detoxification enzymes widely distributed in organisms, is involved in the metabolism of many toxic substances from both internal and external sources, and plays an important role in protecting cells against biological and abiotic stresses. This study investigated the characteristics of the Ruditapes philippinarum gene family (RpGST) and its function in response to acute hypersalinity and acute hyposalinity stress. Bioinformatics methods were used to identify RpGST gene family members and analyze their structural characteristics, chromosome localization, phylogeny, and expression characteristics in the hepatopancreas after acute salt stress. A total of seven RpGST genes were identified, namely GSTA1, GSTA2, GSTA3, GSTA4, GST_C_3a, GST_N_M1, and GST_N_M2. Chromosome localization results revealed that these seven GST genes were located on five chromosomes. The hydrophobicity analysis showed that all protein members had different degrees of hydrophilicity. The average aliphatic amino acid index was 82.08; subcellular localization found that RpGST proteins were all located in the cytoplasm; and all members of this gene family possess the glutathione transferase domain (PF00043, PF02798), which is associated with antioxidant and detoxification functions. Phylogenetic analysis revealed that RpGSTs were divided into three subfamilies and were evolutionarily conserved. The seven RpGSTs were divided into three distinct classes, with the GSTA class being the largest, consisting of four members within the Ruditapes philippinarum family, whereas the GSTM class consisted of two members and the GST_C_3 class of one member. All GSTs in the Ruditapes philippinarum family evolved into separate clusters, such as RpGSTA and RpGSTM, related to the wide distribution and strong adaptability of the family. These results can provide a reference for the subsequent breeding of Ruditapes philippinarum with high and low salinity tolerance. After acute salinity stress in Ruditapes philippinarum for 0 h, 12 h and 24 h, the hepatopancreatic expression levels of RpGST members in acute hypersaline (40) and hyposaline (15) stress were detected by qRT-PCR. The results evidenced that the relative expression levels of RpGSTA1, RpGSTA2, RpGST_N_M1, and RpGST_N_M2 in the hepatopancreas of Ruditapes philippinarum increased with time, and the expression levels of RpGSTA1 and RpGSTM2 were significantly different from those of the control group (P＜0.01), whereas the relative expression levels of RpGSTA3, RpGSTA4 and Rp GST_C_3a first decreased and subsequently increased. The relative hepatopancreatic expression levels of RpGSTA1, RpGSTA4, RpGST_C_3a, RpGST_N_M1, and RpGST_N_M2 in Ruditapes philippinarum increased over time under acute hyposaline stress. Specifically, those of RpGSTA1 and RpGSTA4 significantly changed during acute hyposaline stress, gradually increasing with time, and this difference was significant compared with the control group (P＜0.05). In contrast, the relative expression levels of RpGSTA2 initially decreased and subsequently increased over time, while the relative expression of RpGSTA3 first increased and then decreased. The expression of glutathione transferase under acute low-salt stress was significantly higher than that under acute high-salt stress, indicating that acute low-salt stress had a more pronounced effect on Philippine clams than acute high-salt stress. This study clarified the gene characteristics, phylogeny and response mechanism of the RpGST gene family in the context of acute hypersaline and hyposaline stress. It enriches the research data on the RpGST gene family in bivalve shellfish and provides a reference for further study on the role of this gene family in regulating salinity stress.

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国家自然科学基金面上项目(32273107)； 崂山实验室科技创新项目(LSKJ202203803)； 中国水产科学研究院黄海水产研究所基本科研业务费项目(20603022022001)； 青岛市市南区科技计划项目(2022-2-026-ZH)； 莆田市科技计划项目(2021NJJ002)