Functional Analysis Of Thioredoxin Peroxidase, Thioredoxin And Glutaredoxin Associated Genes In Apis Cerana Cerana

As the important antioxidant enzymes in organism, thioredoxin peroxidases (Tpxs), thioredoxins (Trxs) and glutaredoxins (Grxs) play an important role in maintaining redox homeostasis and in protecting organisms from the accumulation of toxic reactive oxygen species (ROS). Researches on the genes of Tpxs, Trxs and Grxs are mainly concentrated in the model species, such as mammal and Drosophila melanogaster. However, there is little research about these genes in the Chinese honeybee, Apis cerana cerana. As a special germplasm resource, A. cerana cerana can resist cold and disease and play an important role in maintaining the ecological balance of natural systems. However, the survival environment of A. cerana cerana is becoming more and more deteriorated due to various environmental stressors including indiscriminate use of pesticides, infectious diseases and global warming. So it is becoming more and more urgent to protect A. cerana cerana, which is the important germplasm resource. To better understand the mechanism of resistance, the new genes, which are closely related to antioxidant, were cloned from A. cerana cerana in this study and multiple amino acids alignment, expression profiles and functional analysis were performed. The following are the specific results:(1) The genes of Tpx-3, Trxl, Trx2, Grxl and Grx2were identified from A. cerana cerana and named AccTpx-3, AccTrxl, AccTrx2, AccGrxl and AccGrx2, respectively. The analysis of multiple amino acids alignment, phylogenetic tree and genomic structure revealed that the antioxidant genes in A. cerana cerana are close to those in Apis mellifera. Moreover, they have highly conserved enzymatic active sites.(2) qRT-PCR was performed to examine the expression profiles of above five genes during different developmental stages in A. cerana cerana and the results showed that AccTpx-3expression was highest in fourth-instar larvae, and the next highest expression level was found in the15day post-emergence adult; AccTrx2mRNA was mainly detected in the15 day post-emergence adult, followed by the fourth day instar larvae and dark-eyed pupae; AccGrx1and AccGrx2were highly concertrated in dark-eyed pupae; AccTrx1expression is the highest in15day post-emergence adult.(3) qRT-PCR was also performed to analyse the expression profiles of above five genes during different tissues in A. cerana cerana and the results showed that AccTpx-3, AccTrx2, AccGrx1, AccGrx2and AccTrx1were the highest in muscle, brain, fat body, muscle and epidermis, respectively.(4) In addition, we also used qRT-PCR to analyse the transcriptional profiles of the above five genes in response to various oxidative stresses and the results revealed that they were induced by temperature, hydrogen peroxide, pesticide, etc., revealing that the above five genes might participate in antioxidant to prevent A. cerana cerana from ROS-induced damage.(5) AccTpx-3and AccTrx2, which was expressed in E.coli, were purified and SDS-PAGE was employed to detect the efficiency of purification. Then the mixed-function oxidation system was employed to examine the function of purified protein and found that AccTpx-3and AccTrx2could protect DNA from ROS damage. In addition, overexpression of AccTpx-3in E.coli BL21could prevent oxidative damage caused by paraquat. The purified AccTrx2could reduce insulin, indicating AccTrx2is the typical Trx.(6) dsRNA, which was synthesized in vitro, was employed to interfere AccGrx1, AccGrx2, AccTpx-3and AccTrx1, respectively, and found that the enzymatic activities of CAT and POD; the contents of hydrogen peroxide, carbonyls, and ascorbate; and the ratios of GSH/GSSG and NADP+/NADPH were elevated, revealing that some other antioxidant genes may compensate for the knockdown of AccGrx1, AccGrx2, AccTpx-3, and AccTrx1.(7) qRT-PCR was also performed to evaluate the expression levels of other antioxidant genes when AccGrx1, AccGrx2, AccTpx-3, and AccTrx1were interfered and the results showed that AccCAT, AccGSTS4, AccMsrA etc., were induced, indicating that they might be involved in compensating the function of above interfering genes, revealing that AccGrx1, AccGrx2, AccTpx-3, and AccTrx1might play an important role in resisting environmental adverse stresses.