泥蚶乙二醛酶Ⅰ基因的克隆及时空表达特征分析
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浙江万里学院,浙江万里学院,浙江万里学院,浙江万里学院

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国家现代贝类产业技术体系项目(CARS-48);浙江省自然科学基金重点建设专项(LZ12C19001);宁波市科技创新团队项目(2011B82017);浙江省重中之重学科开放基金项目(ZS2012015)


Cloning and spatiotemporal expression analysis of glyoxalase Ⅰ gene in the mud clam(Tegillarca granosa)
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Zhejiang Key Laboratory of Aquatic Germplasm Resources,Zhejiang Wanli University,Zhejiang Key Laboratory of Aquatic Germplasm Resources,Zhejiang Wanli University,Zhejiang Key Laboratory of Aquatic Germplasm Resources,Zhejiang Wanli University,Zhejiang Key Laboratory of Aquatic Germplasm Resources,Zhejiang Wanli University

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    摘要:

    为探索贝类乙二醛酶Ⅰ基因的结构及功能特征,实验利用RACE 技术首次克隆获得泥蚶乙二醛Ⅰ基因(Tg-GLOI)的cDNA全长序列,并对其氨基酸序列特征及时空表达特征进行了研究。结果发现,Tg-GLOI基因的cDNA全长序列为1 807 bp,开放阅读框为540 bp,编码180个氨基酸,在27~169 aa处具有典型的乙二醛酶系保守序列;成熟的乙二醛酶Ⅰ由2个相同的亚基组成,每个亚基由乙二醛Ⅰ基因编码,其二级结构由18个α-螺旋、20个β-折叠片和36个转角组成,每个亚基各含1个Zn2+;氨基酸序列比对发现,Tg-GLOI氨基酸序列与太平洋牡蛎的同源性达到86.1%,与其他脊椎动物的同源性也都在70%以上,表现出高度保守性。qRT-PCR结果显示,Tg-GLOI基因在泥蚶成贝8个组织中均有表达,在内脏团中的表达量最高,极显著地高于其他组织(P<0.01);在各发育时期中,Tg-GLOI表达量随发育进程呈逐渐升高的趋势,在眼点幼虫期达到最高,极显著高于其他时期(P<0.01),而在幼虫变态至稚贝时,表达量又极显著地下降(P<0.01)。研究表明,Tg-GLOI具有类似于高等动物的分子结构,并在泥蚶不同组织、不同发育时期表达差异显著,这为进一步研究该酶在贝类中的功能和作用机制奠定了重要基础。

    Abstract:

    Glyoxalase Ⅰ(GLOI),an enzyme of the glyoxalase system,is involved in the cellular detoxication,and also plays an important role in the cell cycle and cellular growth.In order to explore the molecular structure and biological function of GLOI in molluscs,GLOI gene of Tegillarca granosa(Tg-GLOI)was cloned by SMART RACE techniques,and characteristics of its amino acid sequence and expression in different tissues and developmental stages were analyzed.The full length cDNA of Tg-GLOI was 1 807 bp,and its complete ORF(Open Reading Frame)was 540 bp encoding 180 amino acids.There was a conserved domain of glyoxalase family at the 27-169 aa in amino sequence of Tg-GLOI.Mature glyoxalase Ⅰ of T.granosa contains two identical subunits,and each subunit was encoded by GLOI gene.The second structure of Tg-GLOI contains eighteen α-helices,twenty parallel β-sheets and thirty-six corners,each of the subunits comprising a Zn2+,and the Zn2+ binding sites are located within the gap between subunits.Homologous analysis showed that the amino acid sequence of Tg-GLOI shared 86.1% similarity with Crassostrea gigas,and shared more than 70% similarity with those of vertebrates.The results of tissue-specific expression by real time PCR showed that Tg-GLOI gene was expressed in all tissues,and the expression of digestive gland was significantly higher than other tissues(P<0.01).The results of relative expression in different development stages revealed that the expression of Tg-GLOI gene was gradually increased with the process of development,and showed the highest expression in the eyebot larvae stage,which was significantly higher than other stages(P<0.01).And the expression decreased significantly from eyebot larvae stage to juvenile clams(P<0.01)and then stabilized.The results showed that Tg-GLOI had the similar molecular structure as the higher species,and the relative expression revealed significant differences in different tissues and development stages,which laid the theoretical foundation for further study on function and mechanisms of GLOI in molluscs.

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姚韩韩,董迎辉,姚彬彬,包永波,林志华.泥蚶乙二醛酶Ⅰ基因的克隆及时空表达特征分析[J].水产学报,2014,38(9):1302~1309

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  • 收稿日期:2014-04-16
  • 最后修改日期:2014-06-23
  • 录用日期:2014-09-23
  • 在线发布日期: 2014-09-24
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