Molecular Basis Of The ACSS2 Gene In Chinese Cattle Breeds To Adapt To Plateau Hypoxia

Domesticated cattle,as one of the“Six Domesticated Animals”,not only provides a significant source of food for human beings,but also represents a scientific resource for understanding the genetics of complex traits since domestication.Due to the complexity of geography and diversity of climate environment in China,cattle living in different regions and environments gradually reshaped and formed a set of genomes adapted to survive in extreme climate in the long-term process of natural and artificial selection,population events and introgression.Animals living in the plain for a long time will produce altitude response when they are transferred to high altitude areas,while Tibetan cattle,Apeijiaza cattle and Shigatse cattle distributing in the plateau（3500m-4500m altitude）have well adapted to the hypoxia environment in the long-term adaptation and acclimatization process.Such plateau cattle breeds underwent a series of adaptation changes at the physiology,morphology and genome levels,as well as left signatures at the severe high altitude environment.In view of this,the present study was conducted in-depth to investigate the altitude adaptability of cattle(Bos taurus taurus,Bos taurus indicus,hybrid of Bos taurus taurus and Bos taurus indicus,Bos grunniens distributed at different altitudes,especially the local cattle at high altitudes,and to explore the molecular genetic mechanism of altitude hypoxia adaptation.The main results are as follows:Using the bovine 777K high-density SNP chip and integrating FLK,hap FLK,XPEHH three genomic selection signal analysis methods and GEMMA whole genome association analysis method to screen genomic signatures in high-altitude and low-altitude cattle breeds,we identified that the SNP rs110793511 A>G showed the strongest positive selective signal and the other 6 SNPs also exhibited significant signals which harbors the ACSS2 gene,an key synthetase for the alternative pathway for Acetyl COA synthesis under extreme environments,indicating a promising candidate gene for plateau hypoxia adaption.To confirm and identify the candidate specific genetic variants for high-altitude breeds,we further analyzed the molecular genetic variation characteristics of high altitude adaptability of bovine ACSS2 gene by sequencing the 5’-flanking region,exons and 3’-UTR of ACSS2 in five Chinese local Bos taurus taurus,Bos taurus indicus and their hybrid cattle breeds and two Bos grunniens（Yak）breeds located at different altitudes,and identified 58 SNPs in the above 4 cattle breeds.We screened the high altitude hypoxia specific haplotype（CAGTCT）that is composed of six highly linked SNPs.Of which,the SNP g.-473 T>C（rs23）is located in the flanking region of ACSS2 and present with a high frequency within all of high-altitude breeds.In addition,we also found a unique 12 bp base insertion in the 5-flanking region of ACSS2 in the yak breeds.To further determinate the potential regulatory elements,we identified the core promoter of ACSS2 by constructing a recombinant plasmid combined with luciferase activity analysis method into FFB and Hep G2 cells.The result showed that the core promoter region of bovine ACSS2 is located between g.-682～g.-264 and the rs23 SNP was just located in the core promoter region in high-altitude cattle breeds,which suggests an interesting functional genetic variant of ACSS2.To corroborate whether the candidate SNPs and haplotypes affect the promoter activity,we constructed the recombinant plasmids with the wild and mutant genetic variants transfected into FFB and Hep G2 cells.Luciferase activity analysis results showed that when the T mutation of the SNP rs23 was changed to C,the activity of the ACSS2 promoter was significantly increased（P<0.05）.However,we found that the yak-specific 12 bp base insertion phenomenon significantly reduced the promoter activity（P<0.05）,suggesting a common and unique regulatory pattern of ACSS2transcription within various cattle breeds.To analyze whether ACSS2 is high expressed or differentially expressed in plateau adaptation-related tissues,the RT-q PCR and immunohistochemical experiments were performed in liver tissues of four cattle breeds at high-and low-altitudes.We found that ACSS2 gene was highly expressed in bovine liver tissue,and that the level of m RNA and protein of ACSS2 in Tibetan cattle located at 3600m above sea level was significantly higher than that of Holstein cattle distributed at 200m above sea level（P<0.05）.The expression of ACSS2 increased with the elevation of cattle,suggesting that the bovine ACSS2 gene plays a role in the adaptability of hypoxia in plateau.To further verify how the ACSS2 gene plays a role in altitude hypoxia adaptability,we used CRISPR-Cas9 gene editing technology to knock out the ACSS2 gene in fetal cattle fibroblasts,and used RT-q PCR to verify that ACSS2^-/-cells significantly reduced the m RNA expression of the hypoxia-responsive genes EPAS1 and EGLN1.The finding suggests that ACSS2 may regulate the expression of genes related to hypoxia adaptation through metabolism and thus play a role in high-altitude hypoxia adaptation in cattle.