刺参多功能表皮生长因子6(megf6)cDNA克隆及在肠再生过程中的表达分析  

cDNA cloning and expression of multiple epidermal growth factor 6(megf6)gene in sea cucumber Apostichopus japonicus during intestine regeneration

作　　者：陈雷[1,2] 姚锋 唐爽[1] 郭良勇 王珊 方蕾 秦玉雪 尹增强 侯林 CHEN Lei;YAO Feng;TANG Shuang;GUO Liangyong;WANG Shan;FANG Lei;QIN Yuxue;YIN Zengqiang;HOU Lin(School of Life Science,Liaoning Normal University,Dalian 116081,China;School of Marine Science and Environment,Dalian Ocean University,Dalian 116023,China;Liaoning Fishing Port and Aquatic Seedling Center,Dalian 116000,China)

机构地区：[1]辽宁师范大学生命科学学院,辽宁大连116081 [2]大连海洋大学海洋科技与环境学院,辽宁大连116023 [3]辽宁省渔港与水产种苗中心,辽宁大连116000

出　　处：《大连海洋大学学报》2020年第2期239-246,共8页Journal of Dalian Ocean University

基　　金：辽宁省教育厅产业技术研究院项目(DL201904);辽宁省教育厅科学研究项目(QL201706)。

摘　　要：为了探究刺参Apostichopus japonicus多功能表皮生长因子6(multiple epidermal growth factor 6)的生物学信息和功能,利用RACE技术克隆了体质量为(92.0±4.0)g的成体刺参megf6基因(Aj-megf6)cDNA全长序列,并分析了该基因的信息学特征及其在肠再生过程中表达水平的变化。结果表明:刺参megf6基因的cDNA序列全长为5665 bp,共编码了1604个氨基酸,具有37个EGF样结构域;该基因编码蛋白的相对分子质量为172500,理论等电点为4.74,该蛋白具有信号肽,为分泌蛋白,属于亲水性、非跨膜蛋白,共有53个磷酸化位点,分别为23个Ser位点、12个Thr位点、18个Tyr位点;MEGF6氨基酸多序列比对发现,刺参MEGF6与其他物种的氨基酸序列一致性为38.59%~49.07%,其中与紫球海胆Strongylocentrotus purpuratus的一致性最高,为49.07%,其次为长棘海星Acanthaster planci(48.71%);基于MEGF6氨基酸序列的系统进化树分析显示,刺参与紫球海胆、长棘海星聚为一支,该结果符合刺参的分类和进化地位,MEGF家族成员间比对分析显示,MEGF家族成员间具有一定的分化;实时定量PCR(qPCR)结果显示,在刺参肠再生过程中megf6 mRNA表达量呈先升高后降低的趋势,其中再生6 d时的表达量达到峰值,为对照组的126.47倍,再生18 d时降至正常水平。研究表明,刺参megf6基因的表达与刺参肠管形态的变化趋势相一致,推断megf6可能在刺参肠再生过程中发挥重要作用。The full-length cDNA sequence of multiple epidermal growth factor 6(megf6)gene was cloned in sea cucumber Apostichopus japonicus with body weight of(92.0±4.0)g by RACE(rapid amplification of cDNA ends)technology,and the informatics characteristics and expression level of the megf6 gene were analyzed during intestine regeneration in the sea cucumber to explore the information and function of megf6 gene.The results showed that the sea cucumber megf6 gene(Aj-megf6)had total length of 5665 bp,encoding 1604 amino acids and 37 EGF domains.Aj-MEGF6 protein had relative molecular weight of 172500,with the isoelectric point of 4.74,and secretory and hydrophilic putative protein,which had signal peptide,and no transmembrane region.megf6 gene was shown to have 53 phosphorylation sites,including 23 Ser sites,12 Thr sites and 18 Tyr sites.By comparing with amino acid sequences of MEGF6 from 18 species,it was found that the sequences of Aj-MEGF6 showed 38.59%-49.07%similarity with that of other species,with maximal similarity(49.07%identity)between Aj-MEGF6 and that in sea urchin Strongylocentrotus purpuratus,followed by that in starfish Acanthaster planci(48.71%identity).Sea cucumber A.japonicus,sea urchin S.purpuratus and starfish A.planci were found on the same branch of phylogenetic tree constructed using amino acid sequences of MEGF6 from 19 species.These were consistent with the taxonomic and evolutionary status of sea cucumber A.japonicus.The comparative analysis among MEGFs showed that there was a certain differentiation among the members of MEGF family.The real-time quantitative PCR(qPCR)revealed that there was first increase in expression level of Aj-megf6 gene and then decrease during the intestine regeneration in the sea cucumber,the peak value in 6 days,126.47 times of that in the control group,and decreased to the normal level in 18 days.The findings indicated that the expression trend of Aj-MEGF6 gene was consistent with the change in intestinal morphology during intestine regeneration.It was concluded that Aj-

关 键 词：刺参 多功能表皮生长因子6基因(megf6) 克隆 再生 表达 

分 类 号：S917[农业科学—水产科学]