Regulation Of Caspase-3 Gene On Abnormal Development Of Apostichopus Japonicus

Metamorphosis is the most important stage of marine invertebrates’ growth and development.The ontogeny of A.japonicus must undergo metamorphosis from planktonic life to bent hic life,and the normal metamorphosis will directly affect its yield,distribution and economic benefit.It has been found that caspase-dependent apoptosis plays an important role in the early development of marine bent hic invertebrates such as shellfish,in which caspase-8 and caspase-3 genes,as the most important promoters and effectors in the family,respectively,play an indispensable role in cell apoptosis.In order to further understand the regulatory role of caspase-3 gene in the abnormal development of A.japonicus,this paper first obtained the full length sequence of caspase-3 gene by RACE technology.Then,real-time PCR was used to detect the expression of caspase-8 and its upstream activating gene in the early development stage and adult tissues of A.japonicus.Finally,the spatial localization of the two genes in the morphosis stage was studied by global in situ hybridization and immunofluorescence techniques.The main results were as follows :(1)a full-length caspase-3 gene was cloned,with a total length of 2007 bp,a 5’-end untranslated region of 309 bp,a 3’-end untranslated region of 171 bp,a coding region(ORF)of 1527 bp,encoding 508 amino acids,and a classical CASc domain.(2)The results of real-time PCR showed that the expression levels of caspase-3 and caspase-8 genes were very low and the difference was not significant in the cleavage of the development of A.japonicus.The expression levels were gradually increased from the uricularia to the juvenile stage,and reached the highest level in the juvenile stage(P<0.05).The expression trends of caspase-3 and caspase-8 genes were similar,suggesting that there was an upstream and downstream cascade regulation relations hip between caspase-3 and caspase-8 genes and they played an important regulatory role in the metagenesis of A.japonicus.(3)The results of w hole-mount in situ hybridization showed that both caspase-3 and caspase-8 genes had purple signal near the stomach in late-stage auricularia,and increased signal was detected in and around the globular body in the doliolaria,and the signal area reached the maximum in the juvenile.Immunofluorescence showed the same results.In latestage auricularia,both caspase-3 and caspase-8 genes had weak fluorescence signals,and increased fluorescence signals in doliolaria,almost the whole individual had fluorescence signals in juvenile.(4)In adult tissues of A.japonicus,caspase-3 and caspase-8 were expressed in different degrees.Caspase-3 was highly expressed in coelomere cells,tube feet and respiratory tree,while caspase-8 was highly expressed in coelomere cells,tube feet and body wall.The expression levels of gonads were significantly lower than those of other tissues(P<0.05).Coelomic cells are the core of the immune system of echinoderms,so it can be speculated that caspase-3 and caspase-8 genes also play a role in the immune defense process of adult A.japonicus.Based on the above results,it is preliminarily found that caspase 3 and caspase-8are involved and play an important role in the metamorphosis process and immune defense process of A.japonicus.These results provide research direction for further study on metamorphosis mechanism of sea cucumbers and the regulation mechanism of immune defense and theoretical foundation for larval breeding of marine economic invertebrates such as echinoderms.