Functional Analysis Of Apis Cerana Cerana Genes AccPDIA1,AccPDIA3 And AccPDIA6 In The Oxidative Stress Response

The Chinese honeybee(Apis cerana cerana)is a unique pollinating insect in our country,which has important social and ecological values.Recent years,environmental problems have seriously threatened the survival of A.cerana cerana.Environmental stress factors such as heavy metals,extreme temperatures,ultraviolet(UV)radiation,and various pesticides can cause oxidative damage to A.cerana cerana.Therefore,it is necessary to study the antioxidant genes of A.cerana cerana.The protein disulfide isomerase(PDI)family has many biological functions,including regulating cell apoptosis,participating in immune response,regulating the proliferation and invasion of cancer cells,and also playing a role in responding to oxidative stress.In this study,AccPDIA1,AccPDIA3 and AccPDIA6 genes were isolated from A.cerana cerana,and their bioinformatics,expression characteristics and functions were analyzed.The main results are as follows:(1)AccPDIA1,AccPDIA3 and AccPDIA6 were isolated from A.cerana cerana.Bioinformatics analysis showed that AccPDIA1,AccPDIA3 and AccPDIA6 have higher homology with PDIA1,PDIA3 and PDIA6 of Apis mellifera,and their evolutionary relationship is closer.Both AccPDIA1 and AccPDIA3 have four thioredoxin-like domains: ab-b’-a’,and AccPDIA6 has three thioredoxin-like domains: a’-a-b.(2)Using quantitative real-time PCR(qRT-PCR)method to study the expression patterns of AccPDIA1,AccPDIA3 and AccPDIA6 genes in different tissues of A.cerana cerana,we found that the expression of AccPDIA1 in the midgut,wing and head was higher,the expression of AccPDIA3 was higher in the feet and head,and the expression of AccPDIA6 was the highest in the epidermis.(3)The qRT-PCR was used to analyze the expression levels of AccPDIA1,AccPDIA3 and AccPDIA6 under a variety of abiotic stress conditions,and the results showed that the above three genes were induced by hydrogen peroxide,extreme temperature,and multiple pesticides.These results indicated that the above three genes may be involved in the process of oxidative stress.(4)Overexpression of AccPDIA1,AccPDIA3 and AccPDIA6 proteins in E.coli showed that E.coli was more resistant to hydrogen peroxide,high temperature and tert-Butyl hydroperoxide(t-BHP)stress,which indicated that AccPDIA1,AccPDIA3 and AccPDIA6 can help E.coli protect against oxidative damage caused by hydrogen peroxide,high temperature and t-BHP.After mutating the cysteine residues of the CXXC motif of AccPDIA6 to alanine residues using site-directed mutagenesis,the ability of AccPDIA6 to help E.coli resist the oxidative damage caused by hydrogen peroxide basically disappeared,which indicated that the cysteine residue of the CXXC motif plays an important role in the catalytic activity of AccPDIA6.(5)After silencing AccPDIA1,AccPDIA3 and AccPDIA6 respectively,it was found that the expression levels of some antioxidant-related genes changed,and the enzyme activities of antioxidant enzymes(CAT,POD and CAT)also changed,and the survival rate of A.cerana cerana decreased under high temperature stress,which showed that AccPDIA1,AccPDIA3 and AccPDIA6 play important roles in the process resisting oxidative stress of A.cerana cerana.