Association Analysis Of HSP90AA1 Gene Polymorphisms With Heat Stress Response-related Phenotypes In Chinese Holstein Cattle And The Mechanism Study

Research background and purpose: Dairy cows are thermostatic animals and are susceptible to heat stress when they are in a growing environment with high temperature and humidity.Heat stress can disrupt the normal physiology of the cow,leading to a decrease in her production performance,reproductive capacity,and immune function,affecting milk quality and yield,which in turn leads to damage to the economic benefits of the pasture.Therefore,genetic selection and breeding of heat stress-resistant dairy breeds are of great importance.In recent years,with the development of molecular genetics,molecular breeding represented by molecular marker-assisted selection(MAS)can achieve rapid genetic progress and has become a breakthrough point in the selection and breeding of heat stress-resistant dairy breeds.This study aims to reveal the mechanism of the HSP90AA1 gene involved in the heat stress response of dairy cows,as well as to screen candidate molecular markers that can be used in the selection of heat stress-resistant Chinese Holstein cattle breeds,and conduct in-depth research on the functional mechanism of SNP.Research methods and content: In this study,a transmission electron microscope was used to observe the effects of heat stress on the morphology of bovine kidney cells(MDBK).Afterward,the inhibitor 17-AAG was used to reduce the expression of HSP90AA1 protein in the cells,and the level of apoptosis was detected by TUNEL staining.RT-q PCR and Western blot were performed to detect the differences in the expression of apoptosis markers(Bax,Bcl-2,Cleaved caspase-3)and autophagy markers(p62,LC3-Ⅱ/Ⅰ)in each experimental group to reveal the mechanism of the HSP90AA1 gene involved in the heat stress response of dairy cows.After primer design,mixed pool PCR,and sequencing,the single nucleotide polymorphisms(SNPs)in the HSP90AA1 gene of Chinese Holstein cattle were detected and analyzed for linkage disequilibrium.Then 1 665 Chinese Holstein cattle individuals were genotyped by using the KASP technique.Association analysis was performed using the general linear model Y = μ + G + 0)to link typing results with estimated breeding values(EBV)for heat stress-related physiological indicators and production performance to screen molecular markers that can be used for heat stress-resistant Chinese Holstein cattle breed selection.Finally,a series of bioinformatics analyses such as m RNA secondary structure prediction,miRNA prediction,thermodynamic model prediction was performed,dual-luciferase reporter system detection and molecular biology experimental validation were conducted to deeply study the functional mechanism of SNPs.Research results:(1)Heat stress upregulated the expression level of the HSP90AA1 gene,which also caused increased apoptosis and autophagy in MDBK cells.Inhibition of HSP90AA1 protein promoted apoptosis and inhibits autophagy,and this trend increased with increasing inhibitor concentration,suggested that the HSP90AA1 gene can inhibit apoptosis and promote autophagy during the cellular heat stress response.(2)A total of 13 SNPs were detected in the HSP90AA1 gene of Chinese Holstein cattle.After linkage disequilibrium analysis,SNP rs109014822(C>T),SNP rs209516308(A>G),SNP rs135053858(C>T),SNP rs109256957(A> C),and SNP rs110026192(T>C)can form a haplotype block,which includes 3 haplotypes,namely H1(TACAC),H2(CCTGT),and H3(TACGC).The population genotyping results showed that the five SNPs had good genetic polymorphism and all were in Hadwinburg equilibrium.The results of genetic association analysis showed that the 5 SNPs were significantly related to the heat stress response of Chinese Holstein cattle(P <0.05),and could be used as candidate molecular markers for the selection of heat stress-resistant Chinese Holstein cattle breeds.Among them,SNP rs109256957(A>C)and SNP rs110026192(T>C)are located in the 3’ UTR region of the HSP90AA1 gene.The results of both SNP-based and haplotype combination association analysis showed that individuals with AA genotype of SNP rs109256957(A>C)and TT genotype of SNP rs110026192(T>C)had significantly lower physiological phenotypes during heat stress(P < 0.05),indicating that these individuals were more resistant to heat stress,and therefore SNP rs109256957(A>C)and SNP rs110026192(T>C)were used as candidate SNPs for functional mechanism study.(3)SNP rs109256957(A>C)is a functional SNP that can affect the secondary structure stability of the 3’ UTR region of HSP90AA1 m RNA,and the stability of the 3’ UTR region of HSP90AA1 m RNA was diminished when it was the C allele.When SNP rs109256957(A>C)was the C allele,the targeted binding of bta-miR-1224 to the 3’ UTR region of HSP90AA1 m RNA was tighter,and the effect of inhibiting HSP90AA1 gene expression was stronger.Research conclusion:(1)Heat stress causes increased apoptosis and autophagy,and the HSP90AA1 gen can inhibit apoptosis and promote autophagy in response to cellular heat stress.(2)A total of 13 SNPs were detected in the HSP90AA1 gene of Chinese Holstein cattle,among which SNP rs109014822(C>T),SNP rs209516308(A>G),SNP rs135053858(C>T),SNP rs109256957(A>C),and SNP rs110026192(T>C)are significantly associated with heat stress response-related phenotypes.(3)The SNP rs109256957(A>C)is a functional SNP whose different alleles can regulate the expression of the HSP90AA1 gene through bta-miR-1224.