光棘球海胆TGFBR2基因的克隆及其对海水酸化的响应  

Cloning and Characterization of TGFBR2 Gene in the Sea Urchin Mesocentrotus nudus and Its Response to Seawater Acidification

作　　者：李莹莹 崔东遥 程操 孙景贤[1] 刘丽[1] 赵谭军 尹文露 常亚青[1] 湛垚垚[1] Li Yingying;Cui Dongyao;Cheng Cao;Sun Jingxian;Liu Li;Zhao Tanjun;Yin Wenlu;Chang Yaqing;Zhan Yaoyao(Key Laboratory of Mariculture&Stock Enhancement in North China's Sea,Ministry of Agriculture and Rural Affairs,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：《基因组学与应用生物学》2020年第12期5507-5519,共13页Genomics and Applied Biology

基　　金：中国水产科学研究院院级基本科研业务费-农业部海洋渔业可持续发展重点实验室开放课题(2019HY-XKQ01);辽宁省自然科学基金(20170540104)共同资助。

摘　　要：为了探究光棘球海胆(Mesocentrotus nudus)转化生长因子-βⅡ型受体(Transforming growth factor-βreceptor typeⅡ,TGFBR2)基因的序列信息及其响应海水酸化的模式和规律。本研究利用同源克隆和cDNA末端快速扩增技术(RACE)首次获得光棘球海胆TGFBR2基因(MnTGFBR2)的cDNA全长序列。结果显示:该基因全长为2260 bp,其中5’非编码区长度为227 bp,3’非编码区长度为254 bp,开放阅读框长度为1776 bp,编码一个含592个氨基酸的多肽。MnTGFBR2蛋白理论分子量为66.11 kD,理论等电点为5.30。同源性及系统进化分析显示,Mn TGFBR2蛋白与紫球海胆(Strongylocentrotus purpuratus)TGFBR2蛋白聚为最近的一支,二者序列一致性高达95.97%。实时荧光定量PCR(qRT-PCR)结果显示,MnTGFBR2在管足、围口膜、体腔液、肠和性腺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>体腔液>肠>性腺。海水酸化处理实验发现,随着酸化时间的延长,与自然海水组相比,当海水pH值下降0.3个单位时,光棘球海胆性腺组织和肠组织中MnTGFBR2的相对表达均呈先极显著降低(p<0.01)后升高的趋势;当海水pH值下降0.4个单位时,光棘球海胆性腺组织中MnTGFBR2的相对表达呈先极显著降低(p<0.01)后升高再降低的趋势,而肠组织中MnTGFBR2的相对表达则一直呈现极显著降低趋势(p<0.01);当海水pH值下降0.5个单位时,光棘球海胆性腺组织中MnTGFBR2的相对表达呈先极显著降低(p<0.01)再升高的趋势,而肠组织中MnTGFBR2的相对表达则呈显著降低(p<0.05)的趋势。This identify and characterize a novel transforming growth factor-β receptor type Ⅱ(TGFBR2)gene and clarify its response to seawater acidification in the sea urchin Mesocentrotus nudus.The full-length cDNA sequence of TGFBR2 gene in the M.nudus(designated as MnTGFBR2)was acquired by homology cloning and rapid amplification of cDNA ends(RACE).The full length of MnTGFBR2 was 2260 bp,including a 5’untranslated region(UTR)of 227 bp and a 3’UTR of 254 bp,with a 1776 bp open reading frame encoding 592 amino acids.The MnTGFBR2 protein had a theoretical molecular weight of 66.11 kD with a theoretical isoelectric point of 5.30.Homology and phylogenetic analysis indicated that MnTGFBR2 protein and the TGFBR2 protein of Strongylocentrotus purpuratus were the closest ones,and their sequence identity was as high as 95.97%.Quantitative real-time PCR(qRT-PCR)data demonstrated that MnTGFBR2 gene was ubiquitously expressed in five examined tissues including tube foot,peristomial membrane,coelomic fluid,intestine and gonad,with the relative expression level from high to low as:tube foot>peristomial membranet>coelomic fluidt>intestine>gonad.Seawater acidification experiment results showed that,as compared to that of natural seawater group,the relative expression of MnTGFBR2 in the gonad and the intestine of M.nudus were firstly significantly decreased(p<0.01)and then increased as incubation time went on when △pHnbs=-0.3.When seawater pHnbs drops by 0.4 units,the relative expression of MnTGFBR2 in the gonad of M.nudus tended to decrease significantly first(p<0.01)then increased and then decreased,while the relative expression of MnTGFBR2 in the intestine extremely decreased(p<0.01).As △pHnbs=-0.5,the relative expression of MnTGFBR2 in the gonad of M.nudus exhibited a tendency of decreasing first(p<0.01)and then increasing,meanwhile,the relative expression of MnTGFBR2 in the intestine of M.nudus exhibited a significantly decreased trend(p<0.05).

关 键 词：光棘球海胆 TGFBR2 基因克隆 表达模式 海水酸化 

分 类 号：S917.4[农业科学—水产科学]