Cloning and response of pyruvate kinase(PK) gene to seawater acidification in sea urchin Strongylocentrotus intermedius

doi:10.16535/j.cnki.dlhyxb.2019-175

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Journal of Dalian Fisheries University ›› 2020, Vol. 35 ›› Issue (3) : 360-367. DOI: 10.16535/j.cnki.dlhyxb.2019-175

Cloning and response of pyruvate kinase(PK) gene to seawater acidification in sea urchin Strongylocentrotus intermedius

YIN Wenlu, CUI Dongyao, LI Yingying, SUN Jingxian, LI Wenqi, CHANG Yaqing, ZHAN Yaoyao*

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Abstract

Sea urchin Strongylocentrotus intermedius with body weight of (55.27±3.56)g were exposure to natural seawater group (control, pH=8.10±0.03), seawater with pH=7.82±0.03 (group SA1), seawater with pH=7.68±0.03 (group SA2), and seawater with pH=7.55±0.04 (group SA3) prepared by pure gas for 60 days and the full length cDNA of the pyruvate kinase (PK) gene (SiPK) in the sea urchin was cloned by homologous cloning and rapid amplification of cDNA ends (RACE) to clarify the sequence characteristics, tissue expression pattern and the responses of the sea urchin to seawater acidification. Results showed that the full length of the SiPK gene was 3616 bp, including a 1614 bp open reading frame encoding 537 amino acids and two non-coding regions (a 91 bp 5′ non-coding region and a 1911 bp 3′ non-coding region). Phylogenetic analysis revealed that SiPK protein and the PK proteins from sea urchin Strongylocentrotus purpuratus, sea cucumber Apostichopus japonicus and star fish Acanthaster planci were clustered together, with the closest evolution distance between SiPK protein and PK protein from S.purpuratus. Real-time quantitative PCR (qRT-PCR) detection showed that SiPK gene was expressed in the tube foot, intestine, gonad and coelomic fluid of adult S.intermedius. The relative expression levels from high to low were described as: gonad>tube foot> coelomic fluid> intestine. Enzyme activity tests showed that total PK activitieswere detected in all examined tissues of S.intermedius with levels from high to low as: tube foot>gonad>coelomic fluid>intestine. Seawater acidification stress test showed that the relative expression level of PK gene in sea urchin intestines was decreased very significantly first and then very significantly increased as pH decline (P<0.01), with very significant increase in total PK enzyme activity compared that in the control (P<0.01). The relative expression level of SiPK in gonad was shown to be decreased very significantly as seawater pH decreased (P<0.01), while total PK activity in gonad showed a increased significantly first and then decreased trend (P<0.05). The alterations of SiPK mRNA expression and total PK enzyme activities were consistent in the tube foot and coelomic fluid, both showing a decreased trend as seawater pH decline (P<0.05). The findings provide more sequence and phylogengy information on PK and tissue specific response patterns coping with sea water acidification in sea urchin.

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Strongylocentrotus intermedius / pyruvate kinase / gene cloning / enzyme activity / seawater acidification

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YIN Wenlu, CUI Dongyao, LI Yingying, SUN Jingxian, LI Wenqi, CHANG Yaqing, ZHAN Yaoyao. Cloning and response of pyruvate kinase(PK) gene to seawater acidification in sea urchin Strongylocentrotus intermedius[J]. Journal of Dalian Fisheries University, 2020, 35(3): 360-367 https://doi.org/10.16535/j.cnki.dlhyxb.2019-175