光棘球海胆(Mesocentrotus nudus)TGF-β基因克隆及其对海水酸化的响应  

Molecular Cloning and Characterization of TGF-β and Its Response to Seawater Acidification in the Sea Urchin Meconocrotus nudus

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作  者:李莹莹 崔东遥 常亚青[1] 柳林 张宝警 姜惠婷 刘印 湛垚垚[1] LI Ying-ying;CUI Dong-yao;CHANG Ya-qing;LIU Lin;ZHANG Bao-jing;JIANG Hui-ting;LIU Yin;ZHAN Yao-yao(Key Laboratory of Mariculture & Stock Enhancement in North China's Sea,Ministry of Agriculture and Rural Affairs,Dalian Ocean University,Dalian 116023)

机构地区:[1]大连海洋大学农业农村部北方海水增养殖重点实验室,大连116023

出  处:《生物技术通报》2019年第2期116-128,共13页Biotechnology Bulletin

基  金:国家自然科学基金项目(31672652);辽宁省自然基金项目(20170540104);农业部农业科研人才及其创新团队项目.

摘  要:旨在明确光棘球海胆(Mesocentrotus nudus)转化生长因子-β(transforming growth factor β,TGF-β)基因的序列及结构信息,探明该基因在海水酸化胁迫下的表达模式。利用同源克隆和cDNA末端快速扩增(RACE)技术首次获得光棘球海胆TGF-β基因(命名为MnTGF-β)的全长cDNA序列。结果显示:(1)光棘球海胆TGF-β基因的cDNA全长为2299bp,其中5'非编码区长度为745bp,3'非编码区长度为261bp;开放阅读框(ORF)长度为1290bp,编码430个氨基酸,相对分子量为48.31kD,理论等电点为5.34。(2)同源性及系统进化分析显示,光棘球海胆MnTGF-β蛋白序列与紫球海胆(Strongylocentrotus purpuratus)SpTGF-β2蛋白序列高度保守(同源性97.69%)。(3)实时荧光定量PCR(qRT-PCR)检测发现,MnTGF-β基因在光棘球海胆性腺、体腔液、肠、围口膜、管足5种组织中均有表达,其相对表达量从高到低依次为:管足 >围口膜 >肠 >体腔液 >性腺。(4)与自然海水组(pH8.06±0.01)相比,当海水△pH为-0.3时,随酸化时间延长,MnTGF-β基因在光棘球海胆性腺中的相对表达量呈先升高而后极显著降低(P<0.01)再极显著升高(P<0.01)趋势,在管足中的相对表达量呈现极显著上调趋势(P<0.01),而在肠组织中的相对表达量则先极显著降低(P<0.01)而后显著升高(P<0.05);当海水△pH为-0.4和-0.5时,随酸化时间的延长,MnTGF-β基因在光棘球海胆性腺和管足中的相对表达量呈现先降低后升高的趋势,而在肠组织中的相对表达量则呈现极显著降低趋势(P<0.01)。This work aims to clarify the sequences and structure of transforming growth factor-β gene(TGF-β)and explore its expression mode under seawater acidification.We used homology-based cloning and rapid amplification of cDNA ends(RACE)to acquire the full-length cDNA sequence of novel gene TGF-β from Meconocrotus nudus(here designated MnTGF-β).Results are as such:(1)The full-length cDNA of MnTGF-β were 2299 bp,the length of 5'noncoding region was 745 bp,the length of 3'noncoding region was 261 bp,the length of ORF was 1 290 bp,and it encoded 430 amino acid with the relative molecular weight of 48.31 kD,and the theoretical pI of 5.34.(2)Homology and phylogenetic analysis showed that MnTGF-β protein sequence was highly conserved with SpTGF-β2 protein sequence in Strongylocentrotus purpuratus(homology was 97.69%).(3)Quantitative real-time PCR analysis demonstrated that MnTGF-β was ubiquitously expressed in gonad,coelomic fluid,intestines,peristomial membrane and tube foot of healthy adult M.nudus,and the order of its expression was as tube foot >peristomial membrane >intestines >coelomic fluid >gonad.(4)Compared with the natural seawater group(pH 8.06 ±0.01),when △pH was -0.3,the relative expression of gene MnTGF-β in the gonad of sea urchin tended to increase first,then decrease significantly(P<0.01),and then increased significantly(P<0.01)with the increase of acidification time;the relative expression level in tube foot was significantly up-regulated(P<0.01),but that in intestines significantly decreased(P<0.01)and then increased(P<0.05).When the △pH in seawater were -0.4 and -0.5,the relative expression of gene MnTGF-β in the gonads and tube foot of sea urchins showed a tendency of first decreasing and then increasing with the prolongation of acidification time,while that in the intestines decreased significantly(P<0.01).

关 键 词:光棘球海胆 转化生长因子-Β 基因克隆 海水酸化 表达模式 

分 类 号:Q257[生物学—细胞生物学]

 

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