The Expression And Function Of Honeybee (Apis Mellifera) 19 KDa Haemolymph Protein

Insect 19kDa haemolymph protein(HP 19)mainly participate in regulation the metamorphosis development.The honeybee is a social insect,colony in three different sizes,weights,and different development stage and biological function of worker bees,drones and queen for the research of development and its regulation mechanism provides a good material.A gene encoding the 19 kDa haemolymph protein(hp19)in honeybee were found by genomic DNA blasting.The function of the gene and its closely related in time and space expression characteristics of organisms.To explore the expression and function of the gene hp19 in honeybee Apis mellifera,its expression in different developing stage of worker,queen and drone were determined by using Real-time PCR technology.The results showed that the hp19 was highly expressed in small worker larvae,with an expression of 103-10~4 times of reference.the expression was significantly reduced to 10-10~2 times of reference in fifth instar worker larvae,and further decreased to almost the same level of reference when the larvae in prepupae stage.The expression of hp19 in drones was also decreased from 10~4-10~5 times in young larvae to 10~2 times of reference in last instar larva.hp19 was stably expressed in level of 10~2 times of reference in young queen larvae,gradually increased in caped larvae,and reached to 10~4-10~5 times of reference pupae stage.While in adult bees,hpl9 was stable expressed in breed or foraging workers with level of 10~4 times of reference whatever,but significantly high expressed in new emerged queen or drone with an expression of 107 times of reference,then decreased to 10~4 times of reference in old drone or egg laying queen.Taking together,drones and the queen,because of the large individual material energy demand is greater than the worker bees,so the hp19 expression quantity is far higher than the worker bees.The queen as the only female with reproductive ability in normal colony just for reproduction,the drones only undertake the task of the mating,and males in the late of pupa to feather out shortly after the reproductive system fully developed and generate germ cells,queen began expands further development after the feather the ovary,and the matching is hp19 in drone and the queen and corresponding high expression of developmental stage,suggesting that hp19 may also be related to the reproductive regulation of bees.In addition,in the process of membrane protein phosphorylation tests of different developed worker fat cells.Results show that,in the age of 3,5 instars fat cell membrane protein was not detected on protein phosphorylation,Starting from the 1-day prepupa until eclosion phosphorylated proteins can be detected,and gel electrophoresis after coomassie brilliant blue staining at the same time,the kits to about 120 kDa protein strip,in accordance with the size of the storage protein receptors.As the amount of after eclosion hp19 express again rise,at the protein level,also failed to detecte phosphorylation protein on adult bees fat cell membrane protein.These results prove that the HP 19 has receptor inhibition effect for storage protein phosphorylation.To further explore hp19 function and mechanism of action,this research use kit in vitro synthesis of a large number of pure hp19 dsRNA,after microinjected 2 ug dsRNA to 3-days worker bees,test interference effect every 12 h.The results show that there are no significant differences between the experimental group and the control group.The purpose gene still have higher expression level,the method failed to silence hp19 expression.To sum up,this experiment tested hp19 transcription level in different development period in Apis mellifera,It finds that this genes didn’t has completely same transcription mode in three different types,which reveals this is not only a proteins which play an important regulatory role in abnormal pubertal,but also a proteins which closely related with the reproductive regulation,type level development and the corresponding biological functions of honeybee.These results enrich the content of development and regulation biology study,also provide a certain theoretical basis and experimental basis for the further research on hp19 function,class type development and reproductive.