The Effect Of DAO And Its Mutation Loci On Heat Stress Injury In Sheep Kidney

Heat stress is an important factor affecting sheep production in southern China.It is of great significance to analyze the regulation mechanism of heat stress and select heat-tolerant breeds to reduce the economic loss caused by heat stress in sheep breeding.Hu sheep is a kind of characteristic sheep breed in jiangsu province,which has many characteristics such as high fecundity,early sexual maturity,heat and humidity tolerance and so on.Blackbone sheep originated from Nanping,Yunnan province,is sensitive to heat stress.In the preliminary work of the project,transcriptome sequencing was performed on the hypothalamus of Hu sheep in the heat stress sensitive and resistant group,and the results showed that D amino acid oxidase gene(DAO)expression was significantly different.On this basis of this study,firstly,the DAO gene was cloned,tissue expression profile was analyzed,and the expression localization in kidney was analyzed in Hu sheep.Then DAO expression,oxidative stress and apoptosis in kidney were identified between heat stress sensitive and resistant group.The SNPs of DAO gene in Hu sheep and blackbone sheep population were screened,and the effects of DAO gene and its mutation on oxidative stress in renal cells were discussed by gene transfection.The study will provide clues for analyzingthe regulation mechanism of heat stress in sheep and provide candidate molecular markers for the breeding of heat stress varieties.The main results were as follows:(1)DAO gene cloning,expression characteristics and tissue expression profiling:The complete sequence of DAO gene coding region was cloned by PCR amplification,and the length was 1044 bp,encoding 347 amino acid residues.Bioinformatics analysis showed that the DAO was conservative among mammals.The molecular weight of DAO protein in sheep was 39238 Da and it was no signal peptide,the isoelectric point was 6.82,the extinction coefficient was 78755,and it was a hydrophilic protein.The DAO protein had 3 serine,9 threonine and 2 tyrosine,14 phosphorylation sites,3 N-glycosylation sites and 2 O-glycosylation sites.The protein secondary structure consisted of 54.2%amino acid random coil,27.4%amino acid alpha helix and 18.4%amino acid beta fold.The DAO gene had tissue expression specificity,which was highest in sheep kidney,followed by liver and hypothalamus Immunohistochemistry showed that DAO was highly expressed in proximal tubules,distal tubules and thin segments of renal tubules,but not in renal corpuscles(2)The correlation between DAO expression,oxidative injury and heat stress sensitivity in kidney:The continuously changes of rectal temperature in the high temperature season was monitored for 240 hours at a period of 24h,and 5 heat-sensitive and heat-resistant Hu sheep were selected,and the rectal temperature change was the maximum three as heat stress sensitive group,the minimum 3 three as heat stress resistant group.After 10 days feeding in suitable environment,two groups sheep were acute heat stress at 42? for 4 hours.The kidney tissue of sheep was collected and the expression of DAO and oxidative damage were determined.The result showed that DAO mRNA expression,H2O2 content of heat stress sensitive group were significantly higher than heat stress resistant group(p<0.05)and unheat control group(p<0.05)DAO protein,Caspase 3 activity of heat stress sensitive group were significantly higher than heat stress resistant group(p<0.05)and unheat control group(p<0.01).Bax/Bcl-2 mRNA expression in the heat stress sensitive group were significantly higher than those in the resistant group(p<0.05)and unheated control group(p<0.01).These results suggested that heat stress may lead to oxidative damage and apoptosis in renal tissue by up-regulating DAO expression The heat stress resistance of Hu sheep may be related to low expression of DAO,oxidative damage and apoptosis.(3)Effect of DAO overexpression on heat stress injury in renal cell lines:The eukaryotic overexpression vector pcDNA3.1(+)-DAO was constructedand transfected the renal cell line,and was treated with heat stress.The results showed that the expression of ROS and Hsp70 gene increased in renal cells.The expression of ROS and Bax/Bcl-2 mRNA in the overexpressed DAO group was significantly higher than that in the control group(p<0.05),and the expression of Hsp70 mRNA was significantly higher than that in the control group(p<0.01),which was suggested that the overexpressed DAO aggravated the heat stress injury by increasing oxidative damage and apoptosis(4)Effect of DAO mutation on heat stress injury in renal cell lines:Using the heat tolerant Hu sheep group and the heat sensitive Blackbone sheep as the research object,the single nucleotide polymorphisms of DAO gene was screened in the two sheep populations.It was found that the genotype frequency of 640 locus was significantly different between Hu sheep and Blackbone sheep.The main genotype of Hu sheep was GG type,while the main genotype of Blackbone sheep was AA type.pcDNA3.1(+)-DAO-AA and pcDNA3.1(+)-DAO-GG overexpression vectors were constructed and transfected kidney cell lines and treated with heat stress.It was found that the ROS of renal cells increased under heat stress,and the ROS of transfected with A A type vector was higher than GG type.The mRNA expression of Hsp70 and Bax/Bcl-2 in renal cells increased after heat stress treatment,and the expression of Hsp70 and Bax/Bcl-2 mRNA in transfecting AA type vector was significantly higher than that of GG type after heat stress treatment(p<0.01).These data suggested that 640 GG-type of DAO gene had better tolerance to heat stress in sheep.