Molecular Mechanism Of Yak(Bos Grunniens) Ghrelin Adaption To Its Extreme Environment

The yak(Bos grunniens) is the only one large herbivorous mammal which could survive, reproduce and manufacture in the environment of the extreme cold, hypoxia and lengthy grass withering period of the Qinghai-Tibet plateau. Ghrelin is the only endogenous ligand of growth hormone secretagogue receptor(GHSR), and is also the only appetite-stimulating hormone in the periphery. As a lipid-sensor, the ghrelin participated in the regulation of appetite, weight, gastrointestinal tract, fat, glucose and lipid metabolize through the neuronal synapse, vagus nerve and circulation, which finally regulated the energy metabolism synergistically in the hypothalamus-pituitary-alimentary canal system. Therefore, in order to investigate the adaption mechanism of plateau animals to the extreme environment of the Qinghai-Tibet plateau, we discussed that the molecular mechanism of the yak ghrelin adaption to its extreme environment systematically by gene cloning techniques and analysis of sequence alignment, quantitative analysis of mRNA and protein, positioning analysis of ghrelin, as well as constructed the regulatory network of ghrelin involved in energy metabolism by bioinformatics techniques and Literature mining.Results:1. In this paper, we first cloned the full-length sequence of the yak ghrelin gene CDS using hypothalamus, which contains only a 351 bp open reading frame through encoding 117 amino acids. The homologous of the amino acid sequence the ghrelin gene CDS of the yak and cattle, sheep, goat and Tibetan sheep were highly, which was 100%, 91.38%, 92.24% and 92.24%, respectively. However, the homologous of the yak and people rat and mice are lower, which was 69.83%, 70.09% and 68.97%, respectively. And the phylogenetic distance consistent with their genetic relationship.2. The Real-time quantitative PCR results showed that the ghrelin mRNA was expressed in the hypothalamus, pituitary, abomasum and duodenum of the yak and cattle, but the expression trends were not identical; in which the expression levels of the yak abomasum and hypothalamus were significantly lower than that of cattle(p <0.001), which was 0.5 times and 0.7 times that of cattle, respectively; however, the expression levels in the yak pituitary gland and duodenum were significantly higher than those of cattle(p<0.001), which was 8 and 2 times that of cattle, respectively.3. The immunohistochemistry results showed that the ghrelin immunoreactive products were distributed into the yak duodenum, jejunum, ileum and cecum, and the large number of the positive products were mainly distributed into the intestinal glands secreting cells, while only a small amount were distributed into the basilar membrane of the small intestine acinar. However, the positive products were mainly distributed into the basilar membrane of the small intestine acinar of the cattle. In addition, the immunofluorescence results show that the distr ibution and expression characteristic the ghrelin protein of the red fluorescent marker in the duodenum was consisted with the immunohistochemical in the yak and cattle.4. The ELISA analysis showed that the expression tendency of the ghrelin protein total levels(TL) in the hypothalamus, pituitary, abomasum and duodenum of the yak and cattle was the same, which was TL abomasum> TL duodenum> TL pituitary> TL hypothalamus; but the expression level of the ghrelin protein in the above-mentioned organizations of the yak was significantly higher than that of the cattle(p <0.001), respectively.5. In the gene regulatory network model of the yak ghrelin involved in the energy metabolism, in which the ghrelin and NPY, AgRP, GOAT, Insulin, GHRH, Leptin, Motilin, Orexin, OX1 R, OX2 R, GH, MC4 R, POMC, CCK and PYY total of 15 genes were directly or indirectly involved in energy metabolism through interacting with its receptor gene GHSR.6. We successfully constructed the eukaryotic fluorescent expression plasmid pEGFP-Ghrelin of the yak ghrelin CDS coding sequences.Conclusions:1. The evolutionary characteristics of amino acid sequences the yak ghrelin was closely related to its feeding habits, but was not closely related to its habitat characteristics.2. Compared with the low-altitude cattle, the expression level of the ghrelin protein in hypothalamus-pituitary-digestive tract axis of the yak was higher, which could help the yak adapt to the extreme environment by improving appetite to increase food intake, increasi ng fat accumulation, regulating body temperature, and reducing energy consumption in the regulation of energy metabolism.3. In the gene regulatory network model of the yak ghrelin involved in the energy metabolism, in which the ghrelin and NPY, AgRP, GOAT, insulin, GHRH and leptin total of 6 genes were directly involved in energy metabolism through interacting with its receptor gene GHSR, and the former 5 regulate positive homeostasis, but the other one regulate negative homeostasis; in which the ghrelin and Motilin, Orexin, OX1 R, OX2 R, GH, MC4 R, POMC, CCK and PYY total of 9 genes were indirectly involved in energy metabolism through interacting with its receptor gene GHSR, and the former 5 regulate positive homeostasis, but the later 4 regulate negative homeostasis. The results has laid a theoretical foundation to further explore the gene regulatory networks of the plateau herbivore ghrelin involved in its energy metabolism, in particular the yak.