Expression And Functional Analysis Of Early-response Factors Ecr And Usp Of Ecdysteroid In Different Castes Of Apis Mellifera

The ecdysone is a generic term for ecdysone and its analogues,Steroid hormone Hydroxytetracycline(20E)is its main active form.Ecdysone hormones play a central role in the regulation of insect metamorphosis,the insect relying on ecdystone titer changes in the body,regulates the development process of metamorphosis.In this study,we used the RNA interference technique to further study the expression of EcR and the USP gene in the different developmental stages of three-caste bees in the Apis mellifera.The expression mechanism of the analysis The results showed:In order to explore the temporal-spatial expression and other related biological functions of the ecdysone early response factors ecr and usp in different castes of the Apis mellifera,the study detected the expression levels of ecr and usp in different development stages and different castes of the Apis mellifera by RT-PCR.The results showed that the expression of ecr and usp were the same as the reference in the larvae,and then increased slightly,but decreased sharply to the lowest level in the metamorphosis stage,and then increased gradually in the pupal stage.The expression of the adult was maintained at a high level.The change trend of the queen was similar to that of the worker,but the expression level of the queen was significantly higher than that of the worker,and the expression in the egg-laying queen was significantly higher than that of the virgin queen.The expression of ecr in the drone was slightly higher in the larvae,then kept low expression level for the following larvae and early pupae stage,increased a little in old pupae,followed by a decreased in new emerged bee,and lastly increase a little in old adult drone.However,the expression of usp in drone bee was high expressed with level of 283 times of reference despite of development stage.This expression pattern of ecr and usp revealed that MH not only regulates the metamorphosis of honeybee,but also plays an important role in reproduction.The high expression of usp in all developmental stages of drone bee indicated that in addition to participate in activating EcR in signal transduction of MH for regulation of molting and reproduction,USP might also have other biological functions.Then,in order to further study the biological functions of EcR in three-caste bees of the Apis mellifera and reveal its expression mechanism,the study used the T7 RiboMAXTM Express RNAi System to synthesize dsRNA to interfere with ecr.A 5 ?g/?L of dsRNA was injected into the worker larvae of 3-day-old,and the number of deaths was counted at 12 hours,24 hours,36 hours and 48 hours,respectively.And in the two groups of surviving larvae for semi-quantitative run gel validation.The However,the gel electrophoresis chart on the comparison found that the test group and the control group was no significant difference.In this study,the expression of EcR and USP in the developmental stages of the three-caste bees was analyzed by fluorescence quantitative technique.EcR showed obvious temporal and spatial specificity in the three-caste bees,but in worker adults,t he expression remained essentially unchanged,the low expression of the drones from the early emerging to the mature period doubled to a level comparable to that of the workers.The expression of the queen from the early emerging to the spawning stage is also growth trend,but the higher expression,indicating that EcR is related to the reproduction of bees.And the expression of USP in the three-caste bees was very significant,and the expression of USP was high in the drones.It is a high expression of time in the body of the queen and a low expression of time in the body of the worker.This suggests that USP in addition to the composition of active complex with EcR involved MH regulation molting and reproduction,but also may participate in other growth and development regulation.In the test which using double-stranded RNA injection to interfere with ecr in the body of 3 day-old worker larva in the Apis mellifera,there was no significant difference in the expression of ecr gene between the experimental group and the control group,and the results showed that ecr may play an important role in the development of the bee metamorphosis.There is a corresponding compensation mechanism,the gene can not be significantly silent.