The Development Of Hatching Gland And Cloning And Spatiotemporal Expression Of Hatching Enzyme Genes In Pikeperch(Sander Lucioperca)

Hatching is the most critical period in the fish incubation,and hatching enzymes play a key role in the process of fish hatching.Pikeperch(Sander lucioperca)is a sub-cooled water fierce fish species with great potential for aquaculture.In its artificial reproduction,we found that the embryo incubation period of pikeperch was long and part of the embryos in the incubation could not hatched out timely and normally,resulting in these embryos death.It is speculated that the failure of the hatching-out for these embryos is closely related to the occurrence and expression of hatching enzymes,In this study,the histogenesis and migration of the hatching glands(HG)in pikeperch were revealed using microsection and ultrasection techniques.The hatching enzyme gene of pikeperch were cloned and analyzed,and the RNA molecular probe and antibody of hatching enzyme(HE)were prepared.The spatiotemporal expression pattern of hatching enzyme gene during embryo hatching were explored.The purpose of this study was to elucidate the occurrence of hatching glands and the expression pattern of hatching enzymes in the embryonic development,to lay a foundation for the further research on the hatching enzyme of pikeperch.The specific research results are as follows:1 Histological observation of embryos and larave of pikeperchThe development of the hatching gland and the secretion of hatching enzyme in pikeperch were examined with microscopy and scanning electron microscopy(SEM).The results showed that hatching gland cell(HGC)were firstly found in the joint region between head and yolk sac during the stages of body segment appearance,and were stained pink with eosin.With the development of the embryo,the HGCs had significant increase in number and volume,and still mainly distributed on outer surface of embryonic body and yolk sac.In heart pulsation stage,HGCs were 4?6?m in short diameter and 8?10 ?m in long diameter.It was widely distributed in the head,back,tail,near the eye capsule,and yolk of the embryo.On the surface of the capsule,a large number of HGCs were also observed to secrete granular HE,and these HEs stick together as a white mass.When the embryo of pikeperch developed to hatching-out stage,HGCs began to decline and disappear,and the color of HGCs was found being lighter under the light microscope,and the intracellular material had been found discharged under the scanning electron microscope.The vacuoles left after the disappearance of HGC at 1 day larvae after hatching could be observed under the light microscope,and the holes left after the secretion of HE could be found under the scanning electron microscope.These holes were found to have been repaired and only intact epithelial cells could be found there at 4 day after hatching.2 Cloning and characterization of high choriolytic enzyme(HCE)genes in pikeperchThe full-length cDNA sequence of HCE was isolated from the liver of pikeperch by 5'and 3'rapid-amplification of cDNA ends(RACE).The cDNA is 1189 bp,which contains an 42 bp 5'-UTR,a 271 bp 3'-UTR including a polyA tail,and a 876 bp open reading frame(ORF)that encodes 291 amino acids,including the characteristic sequence of the astacin family(HE X X H X X GF X HE X X R X DR)HCE protein sequence of pikeperch contains 21 signal peptides,53 propeptide sequences and 217 mature peptides.The theoretical mass is 33138.47 Da and the theoretical isoelectric point is 9.50 and the calculated instability index is 43.03,According to the high-level structure prediction,the HCE protein of pikeperch contains three ?-helixes and four ?-sheets.The characteristic structure "HExxH" is an important region for binding to zinc ions.The phylogenetic tree based on HCE amino acid sequences of pikeperch and other animals,showed that pikeperch and Perca flavescens form an evolution branch and then merged with other fish,amphibians,birds to form a vertebrate evolutionary branch.3 Cloning and characterization of low choriolytic enzyme(LCE)genes in pikeperchThe full-length cDNA sequence of HCE was isolated from the liver of pikeperch(Sander lucioperca)by 5'and 3'rapid-amplification of cDNA ends(RACE).The cDNA is 1147 bp,which contains an 200 bp 5'-UTR,a 149 bp 3'-UTR including a polyA tail,and a 798 bp open reading frame(ORF)that encodes 265 amino acids,including the characteristic sequence of the astacin family(HE××H××GF××HE×× R × DR).LCE protein sequence of pikeperch contains 21 signal peptides,45 propeptide sequences and 199 mature peptides.The theoretical mass is 29851.40 Da and the theoretical isoelectric point is 8.09 and the calculated instability index is 41.22.According to the high-level structure prediction,LCE protein of pikeperch contains three ?-helixes and four ?-sheets.The characteristic structure "HExxH" is an important region for binding to zinc ions.The phylogenetic tree based on LCE amino acid sequences of pikeperch and other animals,shows that pikeperch and Perca flavescens form an evolution branch and then merged with other fish,amphibians,birds to form a vertebrate evolutionary branch.4 Expression patterns of hatching enzyme genes during embryonic development of pikeperchBased on the cloned HCE and LCE cDNA sequences,real-time fluorescent PCR primers and in situ hybridization probes were designed.Embryos and larvae of pikeperch at different developmental stages were sampled,and the expression levels and transcriptional positions of HE genes at different periods were detected.Transcription was not detected in early incubation,such as cleavage and blastocyst stages.By the time of body segment appearance stage,HCE and LCE expression levels increased,and HCE peaked rapidly.HCE and LCE maintained high expression from body segment appearance stage to heart pulsation stage,and LCE reached the maximum level during heart pulsation stage.The expression levels of HCE and LCE decreased during hatching period,and both remained at low level in the larval stage.The results of in situ hybridization showed that the initial transcripts of HCE were evident in the mid region of the blastoderm and the surface of the yolk sac at body segment appearance stage.HCE was expressed throughout the whole body during development to hatching stage,and then it was no longer expressed in the larval period.LCE mRNA was initially detectable in the head region at body segment appearance stage.From the beginning of the embry development to the optic vesicle stage,the transcripts of LCE were expressed in the whole body fromhead to the tail.The transcripts of HCE and LCE could not be detected at larval stage.5 Preparation of polyclonal antibody to HCE and LCE of pikeperchBased on the cloned HCE and LCE cDNA sequences and bioinformatics analysis,two polypeptides consisting of 15 amino acid sequences were designed,and synthesized by traditional solid-phase peptide synthesis method.Immunized New Zealand rabbits were used to obtain HCE and LCE polyclonal antibodies.Enzyme-linked immunosorbent assay(ELISA)method was applied to detect antibody titer and verify its specificity.The molecular weights of the HCE and LCE synthetic peptides measured by ESI-MS spectra were close to the calculated relative molecular weights.The results of HPLC analysis showed that HCE and LCE synthetic peptides have higher purity.The ELISA titers of the prepared HCE and LCE polyclonal antibodies were higher than 1:1,000,000.