Functional Analysis Of AccCDK20 And AccCDKN1 Genes In Apis Cerana Cerana Under Pesticide And Heavy Metal Stress

Apis cerana cerana plays an important role in agricultural production and ecological balance.However,the development of Apis cerana cerana is limited by the existence of biological and abiotic environmental pressures.In mammals,cell cycle regulators not only regulate cell proliferation and differentiation,but also participate in important processes such as DNA damage repair,immune regulation and antioxidant response.However,so far,there are few reports on the regulation of cell cycle regulators on the resistance of Apis cerana cerana to oxidative damage.Therefore,it is of great theoretical and practical significance to study the regulation of cell cycle regulators in oxidative stress caused by environmental stress in bees by molecular biology methods,which is of great theoretical and practical significance for improving the healthy and sustainable development of bee colonies.In this study,the homologous genes of CDK20 and CDKN1 were isolated from Apis cerana cerana and named as AccCDK20 and AccCDKN1.It was found that they played an important role in resistance to pesticide and heavy metal stress.The main results are as follows:（1）Sequence alignment and phylogenetic tree analysis of AccCDK20 and AccCDKN1revealed that AccCDK20 protein had the STKc＿CCRK conservative domain and AccCDKN1protein had the CDI conservative domain,showing high homology to the amino acid sequences of CDK20 and CDKN1 of Apis mellifera,respectively.This suggests that AccCDK20 and AccCDKN1 are conserved in evolution.（2）The promoter sequences of AccCDK20 and AccCDKN1 were analyzed by TFBIND.It was found that there were many cis-acting elements related to tissue development and environmental stress,such as P53,HSF,STAT,AP-1 and NFKB.The interaction protein network of AccCDK20 and AccCDKN1 proteins was predicted by String,and KEGG pathway analysis showed that AccCDK20 and AccCDKN1 were involved in the regulation of various stress responses and DNA damage repair pathways.This indicates that AccCDK20and AccCDKN1 genes may have potential functions in resisting external stresses.（3）The spatio-temporal expression levels of AccCDK20 and AccCDKN1 were detected by RT-q PCR.The results showed that the expression levels of AccCDK20 and AccCDKN1 in pupal stage were higher in different developmental stages.In different tissues of adult bees,the transcription level of AccCDK20 is the highest in the head,and that of AccCDKN1 is the highest in the thorax.This indicated that AccCDK20 and AccCDKN1 participated in the growth and development of Apis cerana cerana and their expressions in different tissue parts had certain specificity.（4）The expression patterns of AccCDK20 and AccCDKN1 under different pesticides and heavy metal stresses treatments were analyzed by RT-q PCR,and the results showed that the transcription levels of AccCDK20 and AccCDKN1 were changed.This indicated that AccCDK20 and AccCDKN1 responded to the stress responses of pesticide and heavy metal stress.（5）Using RNAi technology,silencing of AccCDK20 and AccCDKN1 resulted in changes in the expression levels of antioxidant-related genes as well as enzyme activities in Apis cerana cerana.It was shown that silencing AccCDK20 and AccCDKN1 reduced the antioxidant capacity of Apis cerana cerana.（6）Using RNAi technology,the survival of Apis cerana cerana under glyphosate stress was observed after silencing AccCDK20 and AccCDKN1.It was found that the bees in the silencing group showed increased sensitivity to glyphosate and decreased survival rate compared to the control group.This suggests that AccCDK20 and AccCDKN1 play an important role in the resistance of Apis cerana cerana to pesticide stress.（7）The inhibition assay revealed that the resistance to Hg Cl₂ and cumene hydroperoxide was enhanced after overexpression of AccCDK20 and AccCDKN1 proteins in E.coli.The results suggest that AccCDK20 and AccCDKN1 can help E.coli to resist oxidative damage.In summary,this study shows that AccCDK20 and AccCDKN1 genes play an important role in the resistance of Apis cerana cerana to pesticide and heavy metal stress.Silencing AccCDK20 and AccCDKN1 genes affected the antioxidant system of Apis cerana cerana,resulting in a decrease in antioxidant capacity and increased sensitivity to pesticides.