The Molecular Mechanism Of P53 In The Yak Brain Adaptation To The Plateau Hypoxia

The yak(Bos grunniens),which is called "a boat of the plateau",is the only large mammal that is currently known to adapt to the severe hypoxic environment of the low temperature,low oxygen and lengthy grass withering period in the Qinghai-Tibet Plateau(>4,000 m above sea level).The brain,whose weight is only 2-5% of total weight,is the most sensitive organ to the environment oxygen and consumes 20% oxygen of the total oxygen consumption.As a tumor suppressor,p53 plays a crucial role in regulating cells proliferation,growth,DNA repair,cell death,cell survival,aging,differentiation,stem cell reprogramming,and cell metabolism,etc.But so far,no research has been reported on the yak brain p53 at home and abroad.Therefore,in order to study the molecular mechanism of the animal adapted to their extreme low oxygen habitat in the Qinghai-Tibet Plateau.In this paper,the yak brain as the research object,the structure,expression and function characteristics of p53 as the breakthrough point,the molecular mechanism of p53 adaptation to the extreme environment were analyzed systematically using gene cloning,carrier construction,cell culture,gene over-expression,RT-PCR,immunohistochemistry,immunofluorescence,and Western blotting technology.Results:1.The results of gene cloning and bioinformatics analysis showed that the yak p53 CDS sequence contained 1161 bp and encoded 386 amino acids,which was the same as that of the cattle.The homology of the p53 gene sequence of the yak and cattle was 99.47%,the result represented that there was only a single amino acid difference in the p53 amino acid sequence between the yak and cattle,namely,the amino acid of yak p53 was histidine,but the cattle was glutamine at the 106 th site.The yak p53 amino acids sequence had high homology in comparison with other species,and the homology was 99%,95%,74% and 73% to cattle,pantholops hodgsonii,rat and mice,respectively.At this site of p53 amino acids sequence,the amino acid of the yak and pantholops hodgsonii was histidine,however,that of the cattle,rat and mouse was glutamine.2.The results of real-time fluorescent quantitative PCR analysis showed that the p53 mRNA of the yak cerebral cortex and hippocampus were 1.04 and 1.18 times of the cattle,respectively.The results of immunohistochemistry and immunofluorescence analysis showed that p53 protein expressed both in cerebral cortex and hippocampus of the yak and cattle,p53 positive immunoreaction products were mainly distributed at the cytoplasm of neuron and glial cells in cerebral cortex of the yak and cattle,and in hippocampus,the products mainly expressed in the cytoplasm of the pyramidal cells.The results of immunohistochemistry and western blotting analysis showed that the p53 protein in the cerebral cortex and hippocampus of the yak was higher than those of the cattle(P cerebral cortex <0.05,P hippocampus >0.05).3.The modeling analysis of the gene regulatory network showed that the activated p53 involved in the regulation of protein synthesis,cell growth eventually autophagy,apoptosis,angiogenesis,and glucose metabolic processes through regulating the multiple signaling pathways of AMPK,mTOR,p21,and so on multiple target genes,under the hypoxia stress.4.The results of real-time fluorescent quantitative PCR analysis of p53 related gene showed that the mRNA of the GLUT1,GLUT3,TIGAR,PGAM1,HK2,SCO2,G6 PD,HIF-1?,ET-1,VEGF,AMPK and mTOR in the yak cerebral cortex was 0.73,1.20,1.03,1.18,0.70,0.49,0.41,0.25,0.75,3,1.25 and 0.75 times to that of the cattle,respectively.The results of immunohistochemistry and immunofluorescence analysis showed that the distribution characteristics of VEGF,ET-1,mTOR,AMPK and HIF-1? immunoreactive product in the cerebral cortex and hippocampus of the yak were similar to those of the cattle,nevertheless,the statistical analysis showed that the area(immunostaining area)and IOD(integral optical density)of the yak were different with cattle.The IOD of VEGF and ET-1 in the yak cerebral cortex and hippocampus were higher than those of the cattle(Pcerebral cortex<0.05,Phippocampus<0.001).The IOD of HIF-1? in the yak cerebral cortex and hippocampus was higher than those of the cattle(Pcerebral cortex<0.05,Phippocampus<0.01).The IOD of mTOR and AMPK in the yak cerebral cortex was higher than those of the cattle(PmTOR<0.05,PAMPK<0.01),and the IOD of the hippocampus had no difference.5.The functional experiments showed that the overexpression of the yak and cattle p53 decreased expression of HIF-1?,and reduced the Bcl-2/Bax ratio under the normal oxygen;The Bcl-2/Bax ratio reduced more apparent under hypoxic condition,but the decrease of the yak is smaller.Conclusions:1.In the plateau hypoxia adaptation,the unique amino acid structure characteristics of the 106 th in the CDS sequence(the 106 th amino acid of the yak and pantholops hodgsonii p53 are histidine,and than that of the cattle,human and mouse is glutamine)is one of the important mechanisms of the yak brain p53 adaptation to its environment.But,its specific features still need to be deeply explored.2.In the plateau hypoxia adaptation,the high expression of p53 in the yak brain is one of the important mechanisms of the gene adaptation to its environment.But,the regulation mechanism needs to be deeply explored.3.In the plateau hypoxia adaptation,the high expression of p53 in the yak brain may be against the low-oxygen-induced cell apoptosis through lowering Bcl-2/Bax value;the high expression may maintain the lower sugar metabolism levels of the yak brain cell through suppressing the expression of SCO2 and G6 PD under the low oxygen stress;the p53 may maintain the normal protein synthesis and cell growth by p53-Akt-mTOR pathway in the yak brain.The regulation mechanisms need to be deeply explored.