Response Of ADSL In Lasiopodomys Mandarinus To Hypoxia And Its Structure Analysis And Functional Verification

AMP-activated protein kinase(AMPK)is a sensor of energy status.It is critical in maintaining cellular energy homeostasis via regulation of a series of biological processes,including glucose metabolism,lipid biogenesis,and protein synthesis.ADSL promotes and maintains the activation of AMPK through its metabolites AMP and AICAR,and associates the de novo purine biosynthesis with the AMPK signaling pathway.Previously,our group found that the Mandarin voles(Lasiopodomys mandarinus)ADSL gene have been affected by positive selection pressure based on single-copy homologous gene analysis.We speculated that ADSL gene may participate in the energy regulation of body through AMPK signaling pathway under hypoxia.At present,there are few reports about the ADSL mediated regulational pattern in different organs under hypoxia.In this study,the Mandarin voles(L.mandarinus)was used as the research object,and the Kunming Mice was used as the control.On the one hand,we used RT-q PCR technology to explore the ADSL gene responses in the heart,liver,spleen and kidney of the Mandarin voles and Mice under the condition of 10% oxygen treatment.On the other hand,sanger sequencing technology was used to obtain the coding sequence of ADSL gene in Mandarin voles.And then we analyzed the systematic evolution,selection pressure,domain and amino acid modifications of ADSL gene based on its CDS sequence.In addition,the ADSL gene of the Mandarin voles and Mice were overexpressed in HEK293 T cells to explore their effect on cell viability.This thesis aims to reveal the different responses of the Mandarin voles ADSL gene between different organs upon hypoxia,and to provide new insights for hypoxia adaptation in subterranean rodents.Major results are as follows:1 Effects on ADSL gene expression in four tissues of the Mandarin voles under hypoxiaAfter exposure to 10% oxygen for 48 hours,the expression of ADSL gene significantly changed in the Mandarin voles liver and the Mice kidney,but not in others.Upon hypoxia,the expression of liver ADSL in the Mandarin voles was significantly upregulated(P = 0.010)but not in Mice(P = 0.820).While hypoxia induced significantly down-regulation of ADSL gene in the kidney of Mice(P = 0.043),not significant for the Mandarin voles(P = 0.880).The differential responses of ADSL gene in four tissues of the Mandarin voles and Mice reflects its tissue specificity and species differences.What is more,hypoxia had no significant effect on the expression of PPAT,GART,FGAMS,PAICS and ATIC genes in the Mandarin voles and Mice liver.2 Sequence analysis of ADSL gene in the Mandarin volesThe sequence of the ADSL coding region of the Mandarin voles was obtained by Sanger sequencing.The ADSL CDS in the Mandarin voles is 1455 bp in length,encoding 484 amino acids.The multiple sequence alignment revealed that the ADSL coding sequence of the Mandarin voles had unique non-synonymous variants p.V247 M.Phylogenetic analysis found that the ADSL gene did not undergo convergent evolution in hypoxia adaptive species.Selection pressure analysis found that ADSL in five hypoxia adaptive rodent species occurs purification selection in this study.PROVEAN results showed that compared with the Mice ADSL gene,the Mandarin voles had two variation sites,p.D151 A and p.M181 V,that might significantly affect the function of the protein.The results of SMART showed that the Lyase?1 domain,257 amino acids,of ADSL protein is the shortest in the Mandarin voles.The subcellular localization result by Target P 1.1 showed that the probability of mitochondrial localization of ADSL in Mice was 42.2%,which was higher than that the 24.8% in the Mandarin voles.The results of post-translational modification sites by Net NGlyc 1.0,Net OGlyc 4.0 and Net Phos 2.0 showed that ADSL had more phosphorylation sites,but no glycosylation and phosphorylation sites specific to ADSL were found in the Mandarin voles.3 The effect of of ADSL gene on cell viability in HEK293 T cellsOverexpression of the Mandarin voles and Mice ADSL gene had a significant inhibitory effect on the cell viability in HEK293 T when exposed to 1% oxygen.But overexpression of the Mandarin voles ADSL gene had less inhibitory effect on the cells viability in HEK293 T.Overexpression of the Mice ADSL gene had a significant inhibitory effect on HEK293 T cells viability after it exposed to 1% oxygen for 48 hours(P <0.001)and 72 hours(P = 0.043).Overexpression of the Mandarin voles ADSL gene had a significant inhibitory effect on HEK293 T cells viability after it exposed to 1%oxygen for 48 hours(P <0.001),but exposure for 72 hours(P = 0.083)had no significant effect on HEK293 T cells viability.CONCLUSION:The liver is an important organ for ADSL gene to respond to hypoxia in the Mandarin voles.ADSL may be the main gene involved in hypoxia response in the de novo purine biosynthesis.PPAT,GART,FGAMS,PAICS and ATIC genes are not regulated by hypoxia.There are specific variant sites p.V247 M and two variant sites p.D151 A,p.M181 V that significantly affect protein function in the Lyase-1 domain in the Mandarin voles ADSL protein.Cellular experimental result indicates that overexpression of the Mandarin voles ADSL gene has a significant inhibitory effect on renal cell viability under hypoxic.