Preliminary Study On Origin And Development Of Primordial Germ Cells During Early Embryonic Developmental Stages And Expression Patterns Of Pluripotent Stem Cells Related Genes Of Haliotis Discus Hannai

Haliotis discus hannai is the dominant species of abalone culture in China.In recent years,new cell engineering technologies such as "pregnant by belly" have appeared in the field of aquatic genetics and breeding,which are based on the in-depth study of germ stem cells,especially primordial germ cells(PGC).In addition,the process of abalone culture is often accompanied by germplasm degradation,diseases and other problems,and induced pluripotent stem cell(IPSC)technology may be an effective strategy to fundamentally solve these problems.Explore the functions of PGC and iPSC-related genes in H.discus hannai can facilitate cell engineering breeding of H.discus hannai and provide theoretical basis for realizing iPSC in H.discus hannai.In this study,ORF of PGC related genes and iPSC related genes in H.discus hannai were cloned and obtained.Quantitative PCR and WMISH were used to study the expression of these genes in adult tissues and embryo development stages,and function analysis of these genes were conducted.The results of this study are expected to provide basic information for further revealing the genesis and differentiation mechanism of PGC and pluripotent stem cells in abalone.The main research results are as follows:1.Origin and development of PGC during early embryonic developmental stages1)Cloning and structural characteristics of related genesThe ORFs of vasa,PL10,nanos and piwi homologous genes in Haliotis discus hannai were cloned successfully.The ORF of Hdh-vasa is 2562bp and can encode 853 amino acids.Hdh-vasa has 9 conserved domains of the DEAD-box family,and RG duplication,RGG duplication and 4 glycine rich regions were also found at the N terminal.The ARFK frame of vasa subfamily protein and PL10 subfamily protein and the residues of tryptophan(W),glutamate(E)and aspartic acid(D)specific to the Cterminal of vasa subfamily protein were also found.The ORF of Hdh-PL10 is 2250bp and can encode 749 amino acids.Hdh-PL10 has 9 conserved domains of the DEADbox family,and N-terminal RG repeats,RGG repeats and ARFK frames have also been found.The ORF of Hdh-nanos is 888bp and can encode 295 amino acids.Hdh-nanos has two CCHC zinc finger domains peculiar to the nanos protein family near the C terminal.The ORF of Hdh-piwi1 is 2835bp and can encode 944 amino acids while that of Hdh-piwi2 is 2169bp and can encode 722 amino acids.They all have the typical characteristics of the piwi subfamily proteins,namely the PAZ domain near the Nterminal and the PIWI domain near the C-terminal.2)Spatial and temporal expression and function analysis of related genesPGC related genes in H.discus hannai were studied.Among all tissues,only Hdhnanos is expressed relatively specifically in the gonad,and only Hdh-nanos can specifically denote PGC during embryonic development.In H.discus hannai,Hdhvasa and Hdh-nanos are probably also typical maternal genetic factors,which are similar to those reported in other species.According to the results of WMISH of Hdhnanos,before 64-cell stage,Hdh-nanos was only expressed in micromeres.Hdh-nanos appeared to be expressed in a cluster of cells in the mesoderm,with a stronger signal in the middle and a weaker signal in the periphery during gastrula stage.In trochophore,Hdh-nanos appeared to be expressed in the posterior ciliary region of mesoderm cells and was symmetric along the ventral midline.In early veliger,positive signal of Hdhnanos appeared in the ventral foot region.In metaphase veliger,Hdh-nanos positive signal appeared only in a small area at the tip of the visceral mass.The formation mode of PGC in H.discus hannai might belongs to epigenesis,and the migration of PGC may happen between early veliger and metaphase veliger.vasa,PL 10 and piwi homologues should have other functions in H.discus hannai.2.Expression patterns of pluripotent stem cells related genes1)The ORFs of Oct4,KLF4 and c-MYC homologous genes in Haliotis discus hannai were cloned successfully.The ORF of Hdh-Oct4 is 1266bp and can encode 421 amino acids.Hdh-Oct4 has a POU domain typical of the Oct4 protein family.The ORF of Hdh-KLF4 is 1221 bp and can encode 406 amino acids.Hdh-KLF4 has three(C2H2)zinc finger domains at the C-terminal,which are typical of the KLF4 protein family.The ORF of Hdh-c-MYC is 1188bp and can encode 395 amino acids.Hdh-c-MYC has the Myc_N domain and the bHLHzip domain.The Myc_N domain is a typical feature of the MYC protein family,while the bHLHzip domain is a typical feature of the cMYC protein subfamily.2)The expression of iPSC related genes in adult tissues and embryonic development stages were explored and their different expression patterns were revealed.Hdh-KLF4 and Hdh-c-MYC are expressed in different tissues,suggesting that they are widely involved in the maintenance and renewal of pluripotent stem cells in each tissues.Hdh-KLF4 may be related to the formation of sperms in testis,while Hdh-Oct4 should only be related to the renewal of cells in foot and mantle.Hdh-Oct4,Hdh-KLF4 and Hdh-c-MYC all show typical zygotic expression patterns during embryonic development,and they are all highly expressed after gastrula stage.We speculate that Hdh-Oct4 is expressed in PGC during early veliger and metaphase veliger and is involved in the development of foot and mantle.Hdh-KLF4 is only expressed in a small cluster of cells in the mantle,suggesting that this region of mantle may poccess a specific developmental biological status.According to the localization of Hdh-c-MYC,ectoderm cells proliferates fastest during gastrula stage and trochophore stage,while cells of cephalopod region proliferate fastest during veliger.