Characteristics Of Hatching Enzymes And Egg Envelope In Cross Progenies From Crucian Carp And Zebrafish

Fish hybridization has a long history,which provides more possibilities for breeding excellent varieties.At present,in the production and reproduction of freshwater fish,the selection of hybrid dominant species has become one of the important means to improve fishery yield and quality.However,in distant hybridization,the mortality rate of hybrid progeny is higher,especially in the case of low hatching rate.Some studies had shown that the hatching of fish was affected by factors such as hatching enzyme and embryo body movement.The formation of egg envelope after fertilization in fish prevents the embryo from forming a membrane at a later stage of embryonic development.This phenomenon is common in fish hybrids,which may be one of the reasons why the mortality rate of fish hybrids is so high.The hatching enzyme and egg envelope are the key substances to determine the hatching of embryos.Therefore,the research on the characteristics of hatching enzymes and egg envelope in hybrid fish provides a new direction to explain the high mortality of hybrid fish.In this dissertation,orthogonal experiments and anti-crossing experiments were carried out using crucian carp and zebrafish as parents,and the characteristics of hatching enzymes and egg envelope of hybrids of crucian carp and zebrafish were studied.Results are as follows:Hybridization of crucian carp and zebrafish:(1)The self-fertilization rate of crucian carp and zebrafish were respectively 88.22% and 92.66%,while that of cross combination zebrafish(♀)x crucian carp(♂)(ZC)and crucian carp(♀)x zebrafish(♂)(CZ)were respectively 93.30% and 79.40%.(2)The hatching rate of hybrid combination(17.83%)was lower than that of self-crossbreeding(96.53%),and the hatching rate of orthogonal combination(35.67%)was greater than that of reverse crossbreeding(0%).Hatching enzyme’s characteristics of hybrid progenies of crucian carp and zebrafish:(1)The cDNA sequences of hatching enzymes of crucian carp and hybrid progenies of crucian carp and zebrafish were obtained by cloning.Then we did a sequence alignment analysis,found that hybrid progenies have a higher rate of similarity with their mother in hatching enzyme genes;(2)We analyzed the expression of three hatching enzyme genes(he1a,he1 b and he2)at different stages of embryo.The he1 a and he1 b genes were expressed mainly after gastrula stage,not only in CC and ZF,but also their hybrids.In ZF and the ZC,he1 a and he1 b genes were highly expressed in the embryos of muscle differentiation stage,then,significantly reduced before hatching.However,in CC and the CZ,the expression level of he1 a and he1 b gene increased from gastrula to pre-hatching stage.The he2 gene was only expressed in ZF and the ZC from optical vesicle stage to eye pigmentation stage embryo,and no obvious expression in embryos of CC and the CZ.(3)We constructed a vector named p-HE-EGFP-N1 plasmid,which contains the enhanced green fluorescent protein(EGFP)under the control of he1 CDS cloned from CC.The expression of the hatching enzyme gene was tracked by microinjection of the plasmid into one cell stage fertilized egg of the crucian carp.Experimental results show that the expression of the hatching enzyme gene started from the optic base stage and was mainly distributed in the trunk and the surface of the yolk sac.Study on the characteristics of egg envelope of hybrid offspring of crucian carp and zebrafish:(1)The morphology and structure of ZF,CC and their hybrid offspring were observed by scanning electron microscopy at six stages: blastocyst stage,protointestinal embryo stage,optic vesicle stage,muscle response stage,eye pigmentation stage,and somatic pigmentation stage.The results showed that the egg envelope of hybrid offspring was similar to the female parent in morphology and structure.(2)The SDS-PAGE electrophoresis was used to analyze the protein composition of the self-crossing and hybrid offspring in different development stages.The results showed that the protein composition of the self-crossing and hybrid offspring of zebrafish and crucian carp(ZC)was highly similar in different development stages,while the protein composition of the hybrid offspring of crucian carp and zebrafish(CZ)was significantly reduced compared with that of crucian carp.In summary,we conducted self-crossing and back-crossing experiments between crucian carp and zebrafish,and found that the hybrid offspring of zebrafish and crucian carp can hatch out the egg envelope,while the hybrid offspring of crucian carp and zebrafish can not.The characteristics of hatching enzymes in crucian carp,zebrafish and their hybrid offspring were studied at the molecular level.Scanning electron microscopy was used to observe the egg envelope of hybrids and their parents at different development stages.SDS-PAGE electrophoresis was also used to further analyze the proteins of egg envelope of hybrids and their parents at different developmental stages.The results showed that the hatching enzyme gene and egg envelope structure of hybrid offspring of zebrafish and crucian carp were similar to that of the mother,but the protein composition of the hybrid offspring of crucian carp and zebrafish were quite different from that of the mother,which may be the reason why they could not hatch the egg envelope.The relevant research results will lay a certain theoretical foundation for the in-depth study of the biological characteristics and physiological functions of hatching enzymes,and provide a basis for the further establishment of distant hybridization breeding strategies.