Function Of Exogenous Melatonin And Melatonin Synthase Gene T5H-1 In Cold Resistance Of Apis Cerana Cerana

Apis cerana cerana is a unique pollinator in China and has made important contributions to the maintenance of the balance of Chinese ecosystem.In recent years,the cold stress problem occurs frequently in China,which has a great impact on the survival of Apis cerana cerana.Recent studies have shown that melatonin is an amine hormone and can help organisms improve cold tolerance.However,whether melatonin can improve the cold tolerance of Apis cerana cerana has not been reported.Therefore,this study took Apis cerana cerana as the experimental material to explore the optimal feeding concentration under low temperature treatment by adding exogenous melatonin to the diet.In addition,the activity of antioxidant enzyme,content of oxide and content of cold-resistant substances in Apis cerana cerana under optimal melatonin concentration were tested to reveal the effect of exogenous melatonin on cold resistance of Apis cerana cerana from the physiological perspective.The tryptophan-5-hydroxylase-1?AccT5H-1?gene in the melatonin synthesis pathway was isolated,and its mechanism of action was studied.The effect of endogenous melatonin on the cold resistance of Apis cerana cerana was discussed at the molecular level,providing a theoretical basis for the further development of melatonin feed suitable for improving the cold resistance of Apis cerana cerana.The main results of the study are as follows:?1?It was concluded that adding 10?g/mL melatonin into the daily diet of bee resulted in the highest survival rate of Apis cerana cerana.?2?Under cold stress,the contents of glycerin and glycogen in Apis cerana cerana were increased after exogenous melatonin was added into the diet,and the cold tolerance of Apis cerana cerana was enhanced.?3?Under cold stress,exogenous melatonin can improve the activity of antioxidant enzymes and the content of Vc in Apis cerana cerana,and reduce the content of oxidizing substances?MDA and H₂O₂?,indicating that exogenous melatonin can alleviate the oxidative damage caused by cold stress on Apis cerana cerana.?4?Bioinformatics analysis of AccT5H-1 gene showed that the total length of Tryptophan-5-Hydroxylase-1?AccT5H-1?in Apis cerana cerana is 1620 bp,encoding 539 amino acids,and its predicted molecular weight is 61.22kDa,which is homologous to AmT5H-1 in Apis mellifera.Promoter sequence analysis revealed that the promoter region of AccT5H-1 contained multiple transcription factor binding sites related to stress response.?5?Analysis of expression pattern of AccT5H-1 gene showed that the expression of AccT5H-1 gene was induced by cold stress and participated in the resistance of Apis cerana cerana to cold stress response.?6?After AccT5H-1 gene was silenced,the expression level of antioxidant genes in Apis cerana cerana was analyzed.the expression levels of resistance genes such as AccTrxR1,AccGSTO1,AccGSTZ1,AccSOD1,AccTrxR-like,AccGSTO2,AccTpx4,AccGSTT1,AccCYP4G11,AccTpx5 and AccGSTD in Apis cerana cerana were up-regulated,indicating that AccT5H-1 gene plays an indispensable role in the resistance to stress.?7?After AccT5H-1 gene silencing,the antioxidant enzyme activity in Apis cerana cerana was higher than that in the control group.The content of oxidative substances MDA and H₂O₂ increased,while the content of Vc decreased,indicating that the AccT5H-1 gene plays an antioxidant role in Apis cerana cerana.