Partial CDNA Cloning And MRNA Expression Of Growth Hormone In Rock Carp, Procypris Rabaudi

Rock carp,Procypris rabaudi(Tchang),which belongs to Cypriniformes,Cyprinidae, Cyprininae,Procypris Lin,is a vulnerable species only distributed in the main stream and tributary of upper reaches of the Yangtze River.In recent years,it's wild resource has been seriously destroyed and it has been listed as vulnerable species in China Red Data Book of Endangered Animals.It has also been listed as protected species in Chongqing.It is well known for delicious fleshiness and tame temper,it is worthy of being cultured and studied.But problems such as low growth rate exist in the culturing of Procypris rabaudi,no matter in nature or in artificial environment.Growth hormone(GH)is a very important protein hormone which is secreted by the anterior pituitary cells and plays many important roles on animals,such as regulating somatic growth and stimulating appetite,affecting various metabolic activities and improving the food transformation rate,etc.In this research,the sequence of partial GH cDNA was first cloned from Procypris rabaudi,and then we used the RT-PCR to research the expression of GH in multiple tissues of Procypris rabaudi and how the estradiol effects the expression of GH mRNA in the pituitary. Main researchs are as follows:1.Partial GH cDNA sequence of Procypris rabaudi and its homology analysisTwo special primers were designed after multiple comparisons among GH genes in many kind teleosteans.GH segment sequence of Procypris rabaudi was acquired through amplification from the mRNA of its puituity.The partial GH cDNA sequence of Procypris rabaudi was 315bp. Comparision of the acquired GH of Procypris rabaudi with those of other 25 species by the DNAMAN soft shows 81.41%nucleotide consistency.In comparision with other fishes shows high consistency,being 95.77%.Comparision with other species such as Xenopus laevis,Gallus domestiaus,Anas platyrhynchos,Homo sapiens,Mus musculus,Bos taurus,Felis catus shows 47.32%,50.79%,49.53%,48.13%,52.37%,53.63%,51.42%nucleotide consistency respectively.2.Homology analysis of deduced amino acid sequence of Procypris rabaudiComparison of the amino acid sequence of Procypris rabaudi GH from cDNA to those of other 25 species shows 76.75%consistency,and to those of other species such as Xenopus laevis,Gallus domestiaus,Anas platyrhynchos,Homo sapiens,Mus musculus,Bos taurus,Felis catus shows 33.33%,35.24%,35.24%,32.08%,36.19%,39.05%,37.14%similarity respectively.But the counterpart between mammalian is 88.46%.3.Multiple tissues expression of GH in Procypris rabaudiA semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR)assay was developed to estimate tissue-specific expression of GH gene of the Procypris rabaudi.Two pairs of special primers were designed for studying of tissue expression.Procypris rabaudiβ-actin cDNA was amplified in order to confirm the steady-state expression level of a housekeeping gene as an inner control reference for GH gene expression.Primers were designed based on zebrafishβ-actin mRNA sequence.After the condition of semi-quantitative RT-PCR was established,6 tissues of Procypris rabaudi were detected.The results showed that GH gene was expressed in pituitary gland,muscle,gill,eyes,kidney in diferent level,but no GH mRNA expression was detected in liver.4.The effects of 17 beta-estradiol on the synthesis of GH mRNAIn order to investigat the effects of 17 beta-estradiol on the synthesis of GH mRNA,Procypris rabaudi were administrated with 1mg/kg,5mg/kg,10mg/kg 17 beta-estradiol and normal saline by intraperitoneal injection.And then acquired the templet of mRNA after 48h,72h,96h.GH mRNA expression level was measured by using of RT-PCR.The results showed that the intraperitoneal injection of 17 beta-estradiol could not affect the expression of GH mRNA.