The Genetic Diversity Of Orexin And Ghrelin Gene In Cattle And The Construction Of High-Level Prokaryotic Expression Of Ghrelin Gene

Growth velocity is an important economic character of domestic animals. The appetite is related to the growth velocity intimately. In general, the animals with honey stomach will grow faster to majority body weight earlier and make the breeding costs reduced. Orexin and Ghrelin are the two appetite regulation neuropeptides, and the former belongs to central neuropeptide and the latter is one of peripheral peptides. They play important roles through the appetite regulation network in the hypothalamus. They can stimulate animals'appetite and increase feed intake and regulate energy balance, so they will be of great value for research. At present, much research work of them is about the human and mouse and rare about cattle.The genetic polymorphism research work of the genes conding the two neuropeptides will provide molecular genetic information for cultivating breeds with honey stomach and high energy transformation. The clone and prokaryotic expression of the Ghrelin gene will help the microbiol ecological agents'developer. In the research, the single nucleotide polymorphism was studied on the 3 cattle populations, including 142 Jiaxian cattle, 74 Nanyang cattle and 67 Qinchuan cattle and the association of genetic variations with growth traits was analyzed. And the high-level prokaryotic expression system was constructed for the Ghrelin gene. The results were summarized as follows:1. The genetic diversity of Orexin gene and association of genetic variations with growth traits on the 3 populationsIn 3 populations including 293 individuals, 3 genotypes (A, B, C) and 4 SNPs were detected in -178～-818 on the Orexin gene . The 4 SNPs were found at -572 bp (T→C), -468 bp (C→T), -463 bp (A→T) and -440 bp (A→G.).2 genotypes were obtained in the O-5 fragment (-1758bp～-1400bp) and there were 6 SNPs discovered. The SNPs of A genotype were -1539 bp (C/C) and -1537 bp (C/C) and the B genotype were -1599 bp (C/G.), -1574 bp (G/A.), -1427 bp (T/C) and -1420 bp (C/A.).The Hardy-Weinberg equilibrium analysis revealed that 3 populations at -1599 bp, -1574 bp, -1427 bp and -1420 bp showed genetic equilibrium and the other 6 were not. The prediction results of the transcription factor binding sites of the promoter indicated that the SNPs of -572 bp, -468 bp and -463 bp were at the core sequence of the sites (Evil1, RREB1 and NFY ).Only B and C genotypes were detected on the O-2 fragment of Nanyang cattle. The interact effects of O-2 and O-5 was significant on the body weight of 6 months age and average day gain of 12 months age. O-5 had no effects on the other growth traits. The O-2 had the significant effects on the body weight and average day gain of indivduals of 6 months and 12 months age. The body weight and average day gain of 6 months age of B genotype was larger than that of C genotype of O-2. The cattle of C genotype could be used as a marker for body weigh selecting for abatage.2. The genetic diversity of Ghrelin gene and association of genetic variations with growth traits on the 3 populations3 genotypes and 4 SNPs were found on the G-1 fragment (-544~+35bp). The 4 SNPs were -415 bp (A→G), -414 bp (T→C), -321(A→C) and -129 bp (A→G). From -1037 bp to -509bp (G-2), one SNP was discovered at -826bp (A→T). The sequence of A genotype was same to the one of GeneBank and the SNP of B genotype was -826 bp (A/T). 2 SNPs were detected (+205 bp, C→T; +253 bp, T→C) in the G-4 (exon1, exon2 and intron1).There were no effects among G-1, G-2 and G-4 on the growth traits of Nanyang cattle. Only G-1 had significant effection on the hucklebone width of 18 months. The hucklebone width of B genotype individuals was greater than C ones'. Least square analysis between G-2 of Ghrelin and growth traits in Qinchuan cattle showed that on the G-4, the heart girth of Qinchuan cattle of A genotype was bigger than B ones. B genotype individuals could be regarded as a marker for body weight for which heart girth is a contributor.3 The cloning of Ghrelin and its construction of high-level prokaryotic expression The total RNA was extracted from the cattle abomasums fundic glands tissue. With the RT-PCR the Ghrelin gene CDS was gained and cloned into the pMD-18T vector and the positive clone plasmid was named pGh-T1. And at the same time the CDS was gained through the Overlap-PCR and the positive clone plasmid was named pGh-T2. The sequencing of the two plasmids results showed that the two 351 bp CDS-DNA sequences had 100% homology, which proved that the two methods were feasible for cloning the gene's CDS. Digested the pGh-T1 or pGh-T2 with BamHⅠand HindⅢand the 351 bp of CDS was obtained. The obtained fragment was ligated with the longer fragment digested with the same restriction enzyme from the expression vector, pET32a (+). Induced by IPTG, the fusion protein, His-Ghrelin, was expressed highly. And the major fusion protein was dissoluble and little portion was inclusion body. The fusion protein was isolated and purified and was used to inject the rabbits to obtain antibody. The resulted polyclonal antibody titer (blood serum) was 1:12800. Ghrelin is expressed in hypothalamus arcuate nuclei and we used the arcuate nuclei as the target tissue for immunohistochemistry test. Western-bloting and immunohistochemistry test (hypothalamus arcuate nuclei) showed that the fusion protein had biological activity.Bacillus subtilis is a non-pathogenic Gram-positive bacterium and is generally regarded as safe organism (GRAS). In order to express the Ghrelin in B. subtilis, with shotgun method and using the heat stableβ-galactosidase as reporter in the promoter probe vector, 103 clones were gained and a new strong promoter fragment isolated from B. subtilis was identified and characterized. The promoter fragment, P3.4.23, exhibited high expression strength both in Escherichia coli and B. subtilis. The typical prokaryotic promoter conservation regions were found in the promoter fragment and the putative promoter was identified as the control element of yxiE gene via sequencing assay and predication of promoter. To further verify and characterize the cloned strong promoter, the putative promoter was sub-cloned andβ-Gal directed by the two promoters showed weaker and stronger activities, which definited that its important region for transcription was 5～333 bp. The promoter sub-cloned vector was named pYG78. Using the pGh-32 as template, the Ghrelin CDS was amplified through PCR with the two primers (EcoRI and SacI). And the amplified Ghrelin CDS was ligated into the pMD-18T, yielding pGh-B-T. Digested the pGh-B-T and pYG78 with EcoRI and SacI, and the digested Ghrelin CDS fragment was introduced into the corresponding sites of pYG78, resulting the expression vector pYG-Gh for Ghrelin in B. subtilis. SDS-PAGE assay showed that there was the distinct target of Ghrelin protein bands which was about 11 kDa. The research work will offer data for microbiol ecological agents'developer of regulating appetite for cattle.