cDNA cloning and expression of multiple epidermal growth factor 6(megf6)gene in sea cucumber Apostichopus japonicus during intestine regeneration
CHEN Lei1,2, YAO Feng1, TANG Shuang1, GUO Liangyong3, WANG Shan2, FANG Lei2,QIN Yuxue2, YIN Zengqiang2, HOU Lin1*
1.School of Life Science, Liaoning Normal University, Dalian; 2.School of Marine Science and Environment, Dalian Ocean University, Dalian; 3.Liaoning Fishing Port and Aquatic Seedling Center, Dalian
Abstract: The full-length cDNA sequence of multiple epidermal growth factor 6(megf6)gene was cloned in sea cucumber Apostichopusjaponicus 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 172 500, 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 Strongylocentrotuspurpuratus, followed by that in starfish Acanthasterplanci(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-MEGF6 might play an important role in the intestine regeneration process of sea cucumber.
2020, Vol. 35 
