Biological Function Analysis Of Stress-Induced-Phosphoprotein 1 Gene(AccSTIP1) In Apis Cerana Cerana

Apis cerana cerana is a major honeybee species in China and has many advantages over other honeybee species,such as strong adaptability,strong anti-mite and-disease resistance,cold resistance and heat resistance,and good at using sporadic nectariferous plant,etc.As one of the main pollinators of flowering plants,Apis cerana cerana made great contributions to the balance between regional ecology and agricultural development.However,due to the deterioration of the environmental conditions,the abuse of pesticides and other factors have made the survival environment of the Apis cerana cerana more and more severe.Therefore,it is becoming increasingly urgent to protect the Apis cerana cerana,a bee resource unique to China.When the Apis cerana cerana is faced with various environmental stresses,such as heat shock,heavy metals,ultraviolet（UV）radiation,and different pesticides,it induces oxidative stress responses.Heat shock protein（Hsps）,as the molecular chaperone,is commonly used to regulate cellular oxidative stress responses.To date,stress-induced-phosphoprotein 1（STIP1）is one of the most widely studied co-chaperones.Its function is as the linker molecule of Hsp70 and Hsp90 and can be directly involved in both.After the interaction,Hsp90 was directed to the Hsp70-client protein complex.It is reported that STIP1 gene plays an important role in stress defense response.So far,few studies have reported on the biological function of STIP1 in Apis cerana cerana.In this study,AccSTIP1gene was isolated from Apis cerana cerana,and sequence analysis,expression characteristic analysis and functional analysis were performed.The main results of this study are as follows:（1）The STIP1 gene was isolated from Apis cerana cerana and named AccSTIP1.The open reading frame of AccSTIP1 is 1623bp in length and encodes a protein consisting of 540amino acids.The molecular weight and isoelectric point of the protein predicted by DNAman are approximately 61kDa and 6.8,respectively.Through the amino acid sequence alignment analysis,we found that the AccSTIP1 gene is highly homologous to other honeybee’s STIP1genes.Through phylogenetic tree analysis,we found that the AccSTIP1 has the highest homology with the AmSTIP1,and the evolutionary relationship is the closest.（2）The expression characteristics of AccSTIP1 gene at different developmental stages were analyzed by quantitative real-time PCR.The results showed that the AccSTIP1 gene had the highest expression level at 6-day-old larval stage.At larval stage,5-day-old larvae expressed higher levels than 4-day-old larvae.During the pupae stage,the expression level of the white-eyed pupae was higher than that of other pupaes（pink-eyed pupae,brown-eyed pupae,and black-eyed pupae）,and the expression level of the brown-eyed pupae was the lowest.In the adult stage,the expression level of 1-day-old adult was higher than that of15-day-old and 30-day-old adults.（3）The expression characteristics of AccSTIP1 gene in different tissues were analyzed by qRT-PCR.The results showed that the AccSTIP1 gene had the highest expression level in the muscle,followed by the thorax and the lowest expression level in the midgut.（4）The expression characteristics of AccSTIP1 gene under different abiotic stresses were analyzed by qRT-PCR.The results showed that the expression level of AccSTIP1 gene was significantly induced by high temperature（42°C）,HgCl₂,H₂O₂,emamectin benzoate,thiamethoxam,hexythiazox and paraquat.However,CdCl₂,ultraviolet（UV）radiation and kresoxim-methyl inhibited the expression of the AccSTIP1 gene.These results indicate that AccSTIP1 gene is involved in abiotic stress responses.（5）The expression characteristics of AccSTIP1 gene under different abiotic stresses were analyzed by Western blot.It was found that,at high temperature（42°C）,emamectin benzoate,thiamethoxam and hexythiazox induced the protein expression of AccSTIP1.However,CdCl₂,ultraviolet（UV）radiation and kresoxim-methyl inhibited the protein expression of AccSTIP1.These results indicate that the protein level of AccSTIP1 is consistent with the expression trend of transcriptional level,further demonstrating that AccSTIP1 gene plays an important role in abiotic stress oxidative stress.（6）The function of recombinant AccSTIP1 protein was analyzed by disk diffusion method and mixed functional oxidation（MFO）system method.As a result,it was found that the recombinant AccSTIP1 protein can reduce the oxidative damage caused by paraquat and HgCl₂,and protect the plasmid DNA from the shear of hydroxyl radicals.These results indicate that AccSTIP1 gene has a protective effect in oxidative stress.（7）AccSTIP1 gene was silenced in Apis cerana cerana through RNA interference and the transcriptional level of antioxidant-related marker genes was detected after silencing.As a result,it was found that the transcriptional level of the antioxidant-related marker genes was significantly reduced in the silent group compared with the control group.These results indicate that the gene plays an important role in the defense against oxidative stress.