Full-length CDNA Cloning And Tissue Expression Of CRH-BP Gene From Grass Carp

Grass carp corticotrophin releasing hormone binding protein (CRH-BP) is37kda protein, the pituitary induced by CRH adrenocorticotropic hormone release the inhibition effect of stress and pregnancy reaction.This study use grass carp as the material, cloning the length of the grass carp CRH-BP gene cDNA sequence, expressing healthy grass carp’s heart, liver, spleen, kidney, head kidney, brain, skin, gill organization differentially expressed analyses, for grass carp under the condition of cold and heat stress expression of brain and kidney tissue were analyzed.In this paper, the main research results are as follows:1. According to fish CRH-BP gene sequences in GenBank conservative region of primer design, amplification of grass carp CRH-BP gene sequence of parts. After cloning sequencing in the NCBI gene identified as the purpose, design on the known sequence3’ and5’specific primers, using the end of the RACE gene cloning method quickly out of the grass carp3’and5’end, CRH-BP length gene cDNA sequence is obtained by joining together. Gene identified for the purpose, then designed according to known sequence specific primers and5’and3’end, respectively, and5’and3’RACE cloning, finally get the grass carp CRH-BP length gene cDNA sequence.Results show that the CRH-BP cDNA gene length is1324bp, open reading frame (52-1017) of966bp, encoding321amino acids. According to the online software to predict grass carp CRH-BP gene of relative molecular weight of36055.3;Theory of pi value is5.91;Unstable coefficient of45.51.Software to predict grass carp CRH-BP for hydrophilic protein, contains a lot of antigenic sites. The phylogenetic tree shows that, Grass Carp and carp and zebrafishwere clustered into one branch, the closest relationship; second is partedorbit saw damselfish, Fugu rubripes, phylogenetic relationships and Italy bee, Apis cerana cerana, Tigriopus japonicus far.2. According to the cloning of CRH-BP genetic design semi-quantitative PCR primers, housekeeping genes beta actin, for inside, by the method of semi-quantitative PCR, healthy grass carp CRH-BP gene in the heart, liver, spleen, kidney, head kidney, brain, skin, gill eight organization relative expression mRNA was analyzed.Results show that in healthy grass carp, grass carp brain and skin CRH-BP gene expression quantity highest, kidney, head kidney, gills expression of The Times, the expression of the heart, liver, and spleen lowest amount.After3. Grass carp in the cold and heat stress, CRH-BP gene expression increased significantly. This could herald that CRH-BP may play an important role in the process of stress in animals.