马氏珠母贝(Pinctada fucata martensii) FBP基因的克隆及其对温度胁迫的响应

cDNA Cloning of FBP Gene in Pinctada fucata martensii and Its Response to Temperature Stress

中文关键词: 马氏珠母贝  果糖-1,6-二磷酸酶基因  温度胁迫  表达分析  基因克隆

英文关键词: Pinctada fucata martensii  FBP  Temperature stress  Expression analysis  Gene cloning

中文摘要:

      果糖-1,6-二磷酸酶(FBP)是催化糖异生过程中的限速酶，当动植物处于温度胁迫等不良环境条件时，FBP通过参与糖异生途径以维持机体的糖平衡，在动植物抗逆过程中起着重要作用。本研究通过RACE技术获得了马氏珠母贝(Pinctada fucata martensii) FBP(Pm-FBP)基因cDNA全长，并使用实时荧光定量PCR技术检测了该基因在马氏珠母贝不同组织中的表达量，以及在17℃(低温组)、22℃(对照组)、32℃(高温组)条件下鳃中的时序表达模式。序列分析显示，Pm-FBP全长为1381 bp，具有54 bp的5¢ UTR和62 bp的3¢ UTR，开放阅读框(ORF)为1020 bp，编码339个氨基酸，预测分子量为37.13 kDa，等电点为6.02。Pm-FBP具有一个Pfam FBPase保守结构域，6个潜在的O-连接糖基化位点(Ser36、Ser56、Ser57、Ser76、Ser80和Thr115)，1个潜在的N-糖基化位点，1个金属结合位点(Asp-Pro-Ile/Leu-Asp-Gly/Ser-Thr/Ser)和46个磷酸化位点。多序列比对结果显示，Pm-FBP与长牡蛎(Crassostrea gigas)FBP的相似性最高，为83%；系统进化树显示，Pm-FBP与长牡蛎等贝类聚为一支，然后再与其他软体动物聚为一大支，节肢动物和脊椎动物分别聚类，进化树总体聚为三大支。实时荧光定量结果显示，Pm-FBP在所检测的闭壳肌、鳃、性腺、肝胰腺、足和外套膜等组织中均有表达，在性腺表达量最高，肝胰腺和鳃中有较高表达；对Pm-FBP时序表达的分析发现，Pm-FBP在低温组和高温组实验时间范围内均出现先上升后下降的趋势，且在72 h时达到最大值，表明Pm-FBP参与了马氏珠母贝对温度胁迫的响应；在120 h时，高温组和低温组的Pm-FBP表达量均显著下降，表明Pm-FBP可能主要在短期的温度胁迫中发挥作用。本研究结果为进一步探索马氏珠母贝对温度胁迫的适应性提供了参考资料。

英文摘要:

      Fructose 1,6-bisphosphatase (FBP) is the rate-limiting enzyme in the gluconeogenesis process. When animals and plants are exposed to adverse environmental conditions, such as temperature stress, FBP participates in the gluconeogenesis pathway to maintain the body’s sugar balance, playing an important role in animal and plant stress resistance. In this study, we obtained the FBP gene of the pearl oyster Pinctada fucata martensii (Pm-FBP) using the RACE technique and detected the expression level of this gene in six tissues of P. fucata martensii as well as its temporal expression pattern in the gill at 17℃ (low-temperature group), 22℃ (control group), and 32℃ (high-temperature group). According to the results of sequence analysis, the full-length Pm-FBP sequence was 1381 bp long, including a 54 bp 5¢ UTR and a 62 bp 3¢ UTR. The open reading frame was 1020 bp long, encoding a peptide of 339 amino acids with a predicted molecular mass of 37.13 kDa. The isoelectric point of Pm-FBP was 6.02. Pm-FBP had a Pfam FBPase conserved domain, 6 potential O-linked glycosylation sites (Ser36, Ser56, Ser57, Ser76, Ser80, and Thr115), a potential N-glycosylation site, a metal-binding site (Asp-Pro-Ile/Leu-Asp- Gly/Ser-Thr/Ser), and 46 phosphorylation sites. Multiple sequence alignment showed that Pm-FBP had the highest homology with the corresponding gene in Crassostrea gigas, with a similarity value of 83%. The phylogenetic tree indicated that Pm-FBP was clustered with shellfish such as C. gigas, and then clustered with other mollusks, and arthropods and vertebrates were clustered together, and the evolutionary tree was clustered into three branches. Real-time fluorescence quantitative results showed that Pm-FBP was expressed in the adductor muscle, gill, gonads, hepatopancreas, foot, mantle, and other tissues. The gonads had the highest expression of Pm-FBP, followed by the hepatopancreas and gill. Time-series analysis showed that Pm-FBP expression first increased and then decreased, reaching the maximum at 72 h, which indicates that it may be involved in the response of P. fucata martensii to temperature stress. The Pm-FBP expression levels in the high-temperature and low-temperature groups decreased significantly at 120 h, suggesting that Pm-FBP may play an important role in short-term temperature stress. The results of this study provide reference data for further exploration of the adaptability of P. fucata martensii under temperature stress.