The Effect Of Tyrosine Hydroxylase (TH) On The Growth And Development Of P. Striata And Its Correlation With Estrogen-related Receptor (ERR) And Ecdysone Receptor (EcR)

Tyrosine hydroxylase(TH),a initial enzyme and rate-limiting enzyme of dopamine and other catecholamines in the neurotransmitter biosynthesis,is widely expression in animal bodies.Previous research showed that TH highly conserved in both invertebrates and vertebrates,modulating various of biological functions,and played important roles in insect melanism,tanning,immune response as well as nervimotion.Some data also reported that TH may participate in the regulation of food intake and body weight.Abnormal expression of tyrosine hydroxylase was often closely related to the neurological diseases and retinal diseases.Estrogen-related receptors(ERR),an orphan nuclear receptor,belongs to one of the nuclear receptor family members,while hasn't found the corresponding natural ligand yet.Although ERR was not combined with estrogen,but previous data showed that ERR was involved in ER signal pathway,regulating of mammalian various life activities and played very impotant roles in coordinating insect normal metabolism,growth and development.Ecdysone receptor(EcR)also belongs to member of the first subfamily of the nuclear receptor superfamily,and as a nuclear receptor is only known ligands in insects.EcR and ultraspiracle(USP)constitute dimer,which as ecdysone action target can be started a series of cascade expression of transcription factors,regulating the growth and development of insect molting,abnormal condition,reproductive and immune response activities.Polyrhachis vicina Roger is the representative of social insect in hymenoptera insects of insect,which life cycle is divided into four main stages:egg,larva,pupa and adult.According to different morphology,behavior and social division of labor,adult is divided into three grades:queens,males and workers.In addition to extensive polymorphism and the highly complex nervous system,P.vicina has short development cycle,easy to feed as well as different stages easy to be distinguished.So it is a good material to study physiology and biochemistry of social insects.In this experiment,we investigated the PvTH mRNA expression and PvTH protein level in P.vicina through real-time fluorescent quantitative PCR,Western blot,in situ hybridization and immunohistochemical technique.The relationship of PvTH,PvERR and PvEcR in mRNA and protein levels were explored by food mediated RNAi,to reveal those genes whether or not modulating the caste of differentiation,growth and development in P.vicina.The results are as follows:1.The expression levels of PvTH mRNA was analysed by in situ hybridization technique in the different adult brain and abdomen of P.vicina.The results showed that PvTH mRNA widely spread in the brain of females,males and workers.The expression levels were influenced by the differences of organizational and castes.In the brain of the females and workers,PvTH mRNA strongest positive reactions were concentrated in the Keyron cells,pedunculus and optic lobe.PvTH mRNA in the entire regions brain of males has a strong positive reaction.PvTH mRNA widely distributed in females,males and workers abdomen,especially the gonads,and the positive reaction intensities were different because of effection of tissue differences.2.The expression of PvTH protein levels positing were detected by immunohistochemical method in the brain and abdomen of different caste adults.The results showed that the localization trend of expression of PvTH protein levels were similar to PvTH mRNA levels in the adult brain and abdomen,especialy in the gonads.In general,PvTH protein strongly expressed in the Keyron cells,pedunculus and calyx of adult brain in the various castes.PvTH protein positive expression quantity of the male brain was still very strong.PvTH protein widely expressed in each caste adult abdomen especially in the gonads,which expression degrees were effected by tissue specificity.3.PvTH protein expression levels quantities were detected by Western blot technique in the different development stages and caste adults of P.vicina.Results showed that the PvTH proteins expression levels were similar to PvTH mRNA's in the P.vicina.The results of specific expression pattern implied that PvTH may play different roles in different development stages of P.vicina at transcription and translation levels.4.Importing segments of dsRNA into insect body based on RNAi food mediated technology.The data showed that after PvTH-RNAi,the PvTH mRNA levels were dropped significantly in 2,3,4 instar larva,pupa,workers,males and females,the PvERR and PvEcR mRNA levels were also dropped in different degrees in 3,4 instar larva,pupa,different caste adults.Those results were effected by caste difference.The expression level of PvTH,PvERR and PvEcR mRNA and protein in the head and abdomen of different caste adults were detected by in situ hybridization and immunohistochemical techniques after PvTH-RNAi.The results showed that the expression of protein and mRNA level of PvTH,PvERR and PvEcR were significant reduction.Using ZEISS anatomical lens observed the phenotypes of 2,3,4 instar larva,pupa and adults after PvTH-RNAi.Results showed that the PvTH-RNAi mainly influence the phenotypes of larvae,and have a small effect on the phenotype of adults.This research firstly detected the expression of protein level of PvTH.The roles of PvTH on growth and differentiation caste of P.vicina were discussed in this paper.Using RNAi explored the correlation with PvTH,PvERR and PvEcR for the first time,which can provide a theoretical data for further know physiological activities and development of P.vicina and other insects.