The Expression Analysis Of Hira Gene In Gibel Carp And Colored Crucian Carp

Hir/Hira(histone regulation)gene is one of the negative regulators ofhistone gene expression.It has been confirmed that HIRA contains a conserved family of proteins found in various species including yeast,Drosophila,fish,Xenopus,chicken and mammalian mouse,human,but it's function is still enigmatic.The research in Drosophila indicates that Hira gene may plays an important role in the decondensation of sperm nucleus and the formation ofpronucleus during fertilization.After cloned the full-length cDNA of Hira homologs from gynogenetic gibel carp (CagHira)and gonochoristic colored crueian carp(CaHira)respectively in the previous work,We used the semi-quantitative RT-PCR analysis to generate the temporal expression pattern of CagHira and CaHira in the unfertilized eggs and all stages of embryogenesis.The result showed that CagHira gene is expressed in the unfertilized eggs and all stages of embryogenesis and the quantity of CagHira gene expression didn't change obviously,which could be inferred that Hira gene had an effect on gynogenetic development of gibel carp.In colored crueian carp,CaHira gene was expressed in the tmfertilized eggs.However its expression decreased dramatically in the four stages after fertilization(p＜0.05).The expression increased again and kept stable from gastrulation till hatching.This suggests that CaHira plays a role in the early embryo development at the late stages.The different expression characters of Hira gene in these fish species may be related to the different reproduction models.The result of WISH showed that the trascription of CagHira gene distributed widely in the early stages of embryos development and centralized to head,heart and the end of tail.It suggests that CagHira gene might affect the development of brain and heart.We also generated the anti-CAGHIRA polyelonal antibody to get further information about the role of CagHira gene in fish development and in gynogenesis.We designed a pairs of special primers base on the sequence of the Caghira gene,used the cDNA reversed from total RNA extracted from mature overy of gynogenetie gibel carp as the template,cloned a special cDNA fragment.Then the cDNA fragment was cloned and inserted into a prokaryotic expression vector pET-32a.The recombinant protein approximately similar to the expected size was induced to express and was purified.The anti-CAGHIRA polyclonal antibody was prepared by immunization of mice using this purified fusion protein.It showed good efficiency and speciality being tested by western blot.In the tissues of mature gibel carp fishes,result of western blot showed that CAGHIRA was specifically expressed in ovary.No obvious band was observed in brain, heart,liver,spleen,kidney,muscle and testis tissues.Based on the expression characterization,CAGHIRA may play a definitive role in maintaining the function of ovary.In the tissues of mature coloreded crucian carp fishes,HIRA protein was strongly expressed in ovary and liver,while weakly in brain,heart,spleen kidney,testis and muscle.This indicates that CAHIRA plays a definitive role in the development of ovary or oogenesis.The temporal expression patterns of HIRA generated from western blot are consistent with the detection by semi-quantitative RT-PCR.It have stable expression in gibel carp,but have fluctuate expression in colored crucian carp,representing a distinct decrease in following four stages after fertilization.The research on Drosophila has indicated Hira gene related to sperm nucleus decondensation during fertilization.The high expression of Hira gene before fertilization is prepared for the sperm nucleus decondensation and the formation of male prenucleus because colored crucian carp is bisexual reproduction.But the Carassius auratus gibelio which process the special reproductive mode,do not need sperm nucleus decondensation and the formation of male prenucleus,thus could be the reason the CagHira do not change its expression during fertilization.So we conclude that Hira gene plays an important role in sperm nucleus decondensation.Activation of zygotic CaHira and expression of CagHira in all stages of embryogenesis indicate that Hira gene may also play a role in the late stage of fish embryogenesis.In fact,the result from WISH that the signal focus on the nerve system and heart at the late stages of embryogenesis suggests that CagHira could effect the development of nerve system and heart.