Effects of hypoxia stress on glycolysis pathway of Onchidium reevesii

【Objective】 To explore the effects of hypoxia stress on the glycolysis pathway of Onchidium reevesii, clarify the conversion mechanism of its energy metabolism pathway, which could study the adaptation strategy of intertidal species to hypoxia environments. 【Method】 The glucose transporter 1 gene（OrGLUT-1） of Onchidium reevesii was cloned and subjected to bioinformatics analysis. Real-time fluorescence quantitative PCR was used to detect the expression of OrGLUT-1 gene in different tissues of Onchidium reevesii, as well as the expressions of glycolysis-related genes（OrGLUT-1）, hexokinase gene（HK） and pyruvate kinase gene（PK） in ganglion and hepatopancreas tissues under hypoxia stress at 0,1,3,6,12 h. The activities of glycolytic enzymes（HK, PK） and lactate dehydrogenase（LDH） in the ganglion tissue of Onchidium reevesii under different hypoxia stress times were determined. 【Result】 The full-length cDNA sequence of OrGLUT-1 gene was 2386 bp, with an open reading frame（ORF） of 1656 bp, encoding 551 amino acid residues. The molecular formula of the OrGLUT-1 protein was C₂₇₀₆H₄₂₈₂N₇₀₂O₇₈₆S₂₄, with a molecular weight of 59.995 kD and a theoretical isoelectric point（pI） of 5.79. The secondary structure of the OrGLUT-1 protein was mainly composed of α-helices and random coils. The phylogenetic tree constructed based on the amino acid sequence similarity of GLUT-1 showed that Onchidium reevesii had a close genetic relationship with California sea hare（Aplysia californica）. OrGLUT-1 was expressed in different tissues but with obvious tissue specificity, and its relative expression levels were higher in the hepatopancreas, ganglion and gonad tissues. After different periods of hypoxia stress, the relative expression levels of OrGLUT-1, HK and PK genes in the ganglion and hepatopancreas tissues were all up-regulated compared with those before hypoxia stress, and the response of the ganglion tissue was more rapid than that of the hepatopancreas tissue. After different hypoxia stress times, the activities of HK, PK and LDH in the ganglion tissue all increased compared with those before hypoxia stress, indicating that Onchidium reevesii could obtain energy through the glycolytic pathway under hypoxia stress. 【Conclusion】 Under hypoxia stress, the glycolysis-related genes of Onchidium reevesii are greatly upregulated, and the activities of glycolytic enzymes are greatly increased, indicating a substantial enhancement of glycolytic flux. The strategy of Onchidium reevesii to adapt to the hypoxic environment in the intertidal zone may be to switch the energy metabolism pathway from aerobic metabolism to anaerobic metabolism, thereby activating the glycolysis pathway to obtain energy.