Genetic Variation And Function Of Lipid Metabolism-related Genes ACSL1 And PTL In Sable

Sable is widely distributed in the coniferous forests of Siberia and northeastern Europe,where the long-term climate is cold and the lowest temperature in winter is generally lower than-30?.In addition,sable have lower body fat levels than other animals in cold environments(polar bears,penguins,cetaceans,pinnipeds,etc.),and cannot effectively isolate the cold air through the thickness of the fat layer.In conclusion,sable have evolved different cold adaptation strategies,but the mechanism of their cold adaptation remains unclear.In this study,based on the previous sable genome data of the research group,we used the comparative genome method to screen the positive selection genes for fat metabolism of sable.The transcriptome sequencing results of the other two sable individuals were used to determine whether the specific mutation sites of positive selection genes were SNPs.Compare the three-dimensional structure of the protein before and after the amino acid mutation at the specific mutation site of the positive selection gene,and use the conserved domain search to determine whether the specific mutation site of the positive selection gene is located in the conserved domain.Then,16 species were selected for comparison of the amino acid sequence of the positive selection gene to determine whether the specific mutation site is located in the conserved region of amino acids,in order to verify the reliability of the calculation results,we conducted in vitro gene function experiments.For the ACSL1 gene,gene expression vector construction,point mutation experiments,cell-level functional experiments and enzyme activity analysis were carried out to explore the possible functional consequences of mutations specific to sable positive selection genes.1.First,using the branch point model in PAML,the positive selection sites of ACSL1,PTL and GDPD3 genes were detected in the sable branch lines.Alignment analysis of the amino acid sequences of 17 species among these 3 genes showed that only ACSL1 and PTL genes had amino acid changes at the positive selection sites.2.To determine whether the specific mutation sites of ACSL1^{Pro219Ser and Ala266Arg}and PTL^Ser318Lys are SNPs,the transcriptome sequencing results of the other two sable individuals were compared.The results showed that the amino acid substitutions at the specific mutation sites of the positive selection genes we screened were fixed and Unified,not due to individual polymorphism.In addition,we also analyzed the transcriptome expression differences of these two genes in 12 different tissues in winter and summer.The results showed that the expression level of ACSL1 in adipose tissue,heart and liver in winter was higher than that in summer.Fat,heart,and liver are important heat-producing tissues in the body.The expression of ACSL1 gene in winter is higher than that in summer,which is an important reason for sable to increase heat production through fatty acid metabolism to resist cold.PTL genes are differentially expressed in pancreatic tissues in winter and summer,and the expression level of PTL genes in pancreatic tissues in winter is lower than that in summer.From the perspective of PTL expression levels in pancreas tissues in winter and summer,the expression differences of sable PTL are most likely related to the seasonal food resources of sable,food shortages in winter,and reduced expression of enzymes related to dietary fat absorption and decomposition.The expression of PTL gene was also higher in the winter,which improved the utilization efficiency of dietary fat intake in the sable.3.The specific mutation sites of these two genes were subsequently predicted by conservative domains,and the amino acid sequences of 17 species were compared to determine whether the specific mutation sites were located in the conservative regions of the amino acid sequence.The results showed that ACSL1 and PTL specific mutation sites were located in the conserved amino acid domains,ACSL1 219 and 266were located in the AMP-binding enzyme conserved domain,PTL 318 was located in the Lipase conserved domain.The ACSL1 positive selection site is located in a conserved region of 20 amino acids in 17 species,and the PTL positive selection site is located in a conserved region of 21 amino acids in 17 species.By comparing the three-dimensional structure of the protein before and after the positive selection gene mutation,it can be found that the three-dimensional structure of the protein has changed.In summary,the changes in amino acids at specific mutation sites of positive selection genes are very likely to cause changes in their spatial structure,thereby affecting enzyme activity.4.In order to verify the possible functional consequences of the specific mutation site of ACSL1 gene,in vitro gene expression vector construction,point mutation experiment and cell function experiment were carried out.The results of vector construction showed that we successfully constructed the wild-type and mutant-type expression vectors of ACSL1 gene in vitro.The results of transfection of293T cells and Western Blot showed that the two expression vectors constructed by us could be successfully expressed in vitro,and there was no expression difference.Subsequent enzyme activity experiments showed that the wild-type of ACSL1 had stronger enzyme activity than the mutant-type.In conclusion,amino acid changes at the specific mutation site of ACSL1 gene enhanced the biological activity of this enzyme in sable,which made sable have stronger adaptability in fatty acid metabolism for heat production and cold resistance.In summary,the specific mutation site of the fatty acid metabolism gene ACSL1gene enhances the ability of fatty acid metabolism and heat production of sable,and the specific mutation site of the dietary fat absorption gene PTL gene is speculated to improve the utilization efficiency of sable's limited food resources.Therefore,the two genes of sable ACSL1 and PTL promote its cold adaptability in fat metabolism.