Role Of AccsHSP21.7 In Resistance To Glyphosate Stress In Apis Cerana Cerana

Apis cerana cerana is a special honeybee species in our country and plays an important role in maintaining ecological balance and promoting agricultural economic development.In recent years,with the development of agriculture,glyphosate has been widely used causing great harm to the survival and reproduction of Apis cerana cerana.Therefore,it is of great significance to improve the defense ability of Apis cerana cerana against glyphosate stress for the healthy development of bee colonies.small Heat Shock Proteins（s HSPs）play an important role in regulating cell growth and development,oxidative stress and immune response.However,there are few studies on the regulation of s HSPs on resistance to pesticide stress in Apis cerana cerana.In this study,Apis cerana cerana was used as experimental material to study the application of Accs HSP21.7 on resistance to glyphosate stress in Apis cerana cerana.The results are as follows:（1）The Accs HSP21.7 was isolated from Apis cerana cerana and its sequence was analyzed.The results showed that Accs HSP21.7 has an open reading frame is 558 bp,encoding 185 amino acids and has isoelectric point of 6.35,a predicted molecular weight of21.739 k Da.The amino acid sequence alignment and phylogenetic tree analysis showed that Accs HSP21.7 had high homology with the proteins of Ad L2EFL,with theα-crystallin domain（ACD）and IXI conserved motif,indicating that Accs HSP21.7 was a typical s HSP.（2）The Accs HSP21.7 promoter sequence was analyzed by TFBIND online software.The results showed that several binding sites of transcription factors involved in environmental oxidative stress and immune response such as CREB,HSF and AP-1 were found in the promoter sequence of Accs HSP21.7,as well as binding sites of transcription factors involved in early embryonic development and cell differentiation（cdx-A and GATA）.It is speculated that Accs HSP21.7 is involved in the growth and development and oxidative stress response of Apis cerana cerana.（3）The temporal and spatial expression characteristics of Accs HSP21.7 were analyzed by Real-time quantitative PCR（RT-q PCR）.The results showed that Accs HSP21.7 was expressed in all developmental stages and tissue parts of Apis cerana cerana,and the expression level was higher in 15-day-old worker bees and epidermal tissue.（4）The expression pattern of Accs HSP21.7 under abiotic stress was analyzed by RT-q PCR.The results showed that the expression of Accs HSP21.7 was significantly induced by pesticides,heavy metals and H₂O₂.These results indicated that Accs HSP21.7 of Apis cerana erana was involved in abiotic stress response.（5）The function of Accs HSP21.7 recombinant protein was analyzed by bacteriostatic experiment.The results showed that under the treatment of Hg Cl₂ and cumene hydroperoxide,the ability of Escherichia coli（E.coli）overexpressing Accs HSP21.7 to resist stress was significantly stronger than that of the E.coli without Accs HSP21.7 expression.These results indicated that E.coli overexpressing Accs HSP21.7 had increased resistance to abiotic stress.（6）The function of Accs HSP21.7 on resistance to glyphosate in Apis cerana cerana by RNA interference（RNAi）technique.After paraffin section and Hematoxylin-eosin（HE）staining,it was found that the midgut cells of silenced Accs HSP21.7 Apis cerana cerana were seriously damaged under glyphosate treatment,and the survival rate was significantly lower than that of wild-type Apis cerana cerana.These results indicated that Accs HSP21.7silencing decreased the resistance of Apis cerana cerana to glyphosate.（7）The Accs HSP21.7 was silenced in Apis cerana cerana to detect the transcriptional level of antioxidant-related genes and antioxidant enzyme activities.The results showed that the transcription levels of antioxidant-related genes and the activity of antioxidant enzymes had significant changes.These results indicate that Accs HSP21.7 plays an important role in the antioxidant activity of Apis cerana cerana.