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

Hirlhira (histone regulation ) gene is one of the negative regulators of histone 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. In this thesis, the integrated cDNA sequences of hira genes have been isolated from full-matured ovary SMART cDNA library of gynogenetic gibel carp (Carassius auratus gibelio) and bisexual coloreded crucian carp (Carassius auratus) by PCR and RACE-PCR. Compared with the identified HIRA family, their identities are quite high and the newly acquired integrated sequences of genes have been proved to be the members of HIRA family. The gene cloned from gibel carp is named Carassius auratus gibelio hira (Caghira) and that from coloreded crucian carp is named Carassius auratus hira (Cahira) . The complete length of Caghira gene is 3828bp containing a 3033bp open reading-frame (ORF) and encoding a protein with 1011 amino acids. The complete length of CargvHIRA gene is 3722bp and the ORF locates from 187bp to 3220bp encoding 1011 amino acids. The both amino acid sequences contain 7 WD domains and their high identities are up to 99%. HIRA proteins are characterized by 7 conserved WD repeats in their amino-terminus, which are predicted to assemble into a β-propeller structure known to mediate protein-protein interactions. It is inferred that HIRA proteins of gibel carp and coloreded crucian carp raise the possibility of a conserved function in the regulation histone gene expression because CagHIRA and CaHIRA also have 7 conserved WD repeats in their amino-terminus which found in a number of eukaryotic proteins that cover a wide variety of functions including adaptor/regulatory modules in signal transduction, pre-mRNA processing.The expression characterization of gibel carp Caghira gene in different tissues and stages of embryogenesis were analyzed by RT-PCR and whole mout in situ hybridization (WISH). In the tissues of mature fishes, hira mRNA can be detected in ovary and brain. Based on the expression characterization, it may play a definitive role in maintaining the function of ovary and brain. Caghira gene is expressed in the unfertilized eggs and all stages of embryogenesis and the quantity of Caghira gene expression didn't changeobviously, which could be inferred that hira gene had an effect on gynogenetic development of gibel carp. The result of WISH showed that the trascription of hira gene distributed widely in the early stages of embryos development and centralized to head, heart and the end of tail. Hira gene might affect the development of brain and heart. The expression characterization of colored crucian carp Cahira gene in different tissues and different stages of embryogenesis were also investigated by RT-PCR. In the tissues of mature fishes, hira mRNA was strongly expressed in ovary and liver, while weakly in brain, heart, spleen and kidney, hira mRNA was not detected in testis and muscle. This indicates that CaHIRA plays a definitive role in the development of ovary or oogenesis. In coloreded crucian carp, Cahira gene was expressed in the unfertilized eggs. However its expression decreased dramatically after fertilization. The expression increased again and kept stable from gastrulation till hatching. This suggests that CaHIRA plays a role in the early embryo development and the late stages. The different expression characters of hira gene in these fish species may be related to the different reproduction models. 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. However, the sperm nucleus is not decondensated during the fertilization and is only to activate egg nucleus in gynogenetic gibel carp. We infer that hira gene is related to sperm nucleus decondensation in fishes. The high identity of hira gene ORF in gibel carp and colored crucian carp indicates that the function distinction may be caused by the expression distinction regulated by regulation region of gene.