Polyrhachis Ants Ecdysone Receptor (EcR) And Ultraspiracle Protein (USP) Of CDNA Sequencing, MRNA Expression And Correlation Analysis With Estrogen-related Receptor (ERR) Function

The ecdysone receptor (EcR) belongs to the superfamily of nuclear receptors (NRs) and is the only ligand-depended nuclear receptors known in insects. Its ligand is ecdysteroid hormones (EH),which plays critical roles in control insect growthing, developmenting, molting as well as metamorphosising. EcR takes part in the signal path of EH, also, EcR is in the key possion of cascade process including insect metamorphosising, molting and reproduction.As a member of the second nuclear receptors subfamily,Ultraspiracle protein (USP) possesses a affinity with Juvenile hormone(JH),and is thought to be the candidate receptors of JH. During insect molting and metamorphosising,EcR must bind USP to form heterologous dimmers, then combine with EH,activate downstream of relation gene and cause cascade response in molt, metamorphosis as well as reproduction.The estrogen receptor-related receptors (ERRs) are a group of nuclear receptors that were originally identified on the basis of sequence similarity to estrogen receptors. The three mammalian ERR genes have been implicated in diverse physiological processes ranging from placental development to maintenance of bone density, but the function and regulation of ERRs in invertebrates are not well understood.Polyrhachis vicina Roger belongs to the genus Polyrhachis (Hymenoptera:Formicidae). As a typical kind of eusocial insects, P.vicina possesses the characteristic of castes differentiation, sophisticated behaviors, behavioral plasticity and highly complex nervous system.In this study, the full-length cDNAs of P.vicina EcR and USP gene is coloned by RT-PCR and RACE methods. The bioinformatics method is used to analyze characteristics of full-length cDNAs and predict functional motifs in the ORF. The mRNA expression levels of EcR、USP and ERR in the ants were investigated by real-time RT-PCR and in situ hybridization methods. The functional relationship among the three genes were investigated by RNAi.The major experiment results are as followed:1. The full-length cDNA of EcR in P.vicina is2235bp, containing an open reading frame of1737bp, which encodes a deduced579-amino acid peptide with a predicted molecular mass of63.4kDa and with the theoretical p1=7.79,which contains a5’-untraslated region (5’-UTR) of20bp and a3’-UTR of478bp. The nucleotide sequence of EcR in P.vicina, named PvEcR, is submitted to GenBank and assigned the number JX028426.1. The results of homologous analysis showed that the full length cDNA of EcR in P.vicina is similar to Camponotus japonicus, Pheidole megacephala and Apis mellifera at99%,the similarity to other insects is over70%. Bioinformatics analysis showed that PvEcR protein belongs to a non-secreted member. In this study, a fluorescent real-time quantitative RT-PCR is used to analyze the relative quantification expression of the mRNA level of PvEcR in different developmental periods, different castes adults and different adults’brain. The results showed that PvEcR is expressed in each tested sample. The expression level of PvEcR in lavar is higher than other stages, the highest expression level is found in the third instar, and declined until the pupal. Among three caste adults, the expression of PvEcR is almost the same. The PvEcR mRNA expression analysis among the heads of the three casts showed that the expression level from height to low was male, worker and female ants in turn. The distribution of PvEcR mRNA was analyzed by in situ hybridization to cryosections of the brain of adult ants. The results showed that the hybridization signals are present extensively in the brains of all adult’s heads.The hybridization signals are highest expression in the mushroom bodies of worker and females.In male ants,the highest hybridization signals are found in the optic lobes and deutocerebrum. From these results,we speculate that the functions of PvEcR are involved in obtaining and integrating the visual and olfaction information in the nervous system.. Using RNAi technology to silence the expression of PvEcR mRNA in different developmental periods, different castes adults, the results showed that, in embryo and first instar stages, the expression of PvEcR has no obvious changes,from the second instar,the expression began to decline, especially in the stages of third and fourth instar, when the expression of PvEcR is much higher than others. The expression of PvEcR was significantly decreased, and leading to insect phenotypic changes containing larval weight loss, the residual wings of male ants, illustrate that in this study,the dsRNA solution we used can reduce the expression of PvEcR mRNA, also prove the important function of PvEcR during the process of development in ants.2. The full-length cDNA of USP in P.vicina is1553bp, containing an open reading frame of1275bp, which encodes a deduced424-amino acid peptide with a predicted molecular mass of47.7kDa and with the theoretica1=8.63,which contains a5’-untraslated region of78bp and a3’-UTR of200bp. The nucleotide sequence of USP in P.vicina, named PvUSP, is submitted to GenBank and assigned the number KC188780.The results of homologous analysis showed that the full length cDNA of USP in P.vicina is similar to bees at over80%, and to other insects is over70%. Bioinformatics analysis showed that PvUSP protein belongs to a non-secreted member. In this study, a fluorescent real-time quantitative RT-PCR is used to analyze the relative quantification expression of the mRNA level of PvUSP in different developmental periods, different castes adults and different adults’brain. The results showed that PvUSP is expressed in each tested sample. During the development stage, the highest expression level is found in embryo and pupa. Among three caste adults, the highest expression of PvUSP is male ants. The PvUSP mRNA expression analysis among the heads of the three casts showed that the expression level from height to low was worker, male and female ants in turn. From these results we speculate that the high mRNA levels in both the embryo and adults may suggest the essential role of PvUSP in ant development and the nervous system.3. In this study, a fluorescent real-time quantitative RT-PCR is used to analyze the relative quantification expression of the mRNA level of PvERR in different developmental periods, different castes adults and different adults’brain. The results showed that PvERR is expressed in each tested sample. During the development stage, the expression level of PvERR is gradually increased, and the highest levels are found in different castes, expecially in female ants. Among the heads of the three casts, the results showed that the expression level from height to low was female, worker and male ants in turn. The distribution of PvERR mRNA was analyzed by in situ hybridization to cryosections of the brain of adult ants. The results showed that the hybridization signals are present extensively in the brains of all adults’ heads.The hybridization signals are highest expression in the mushroom bodies of worker and females, in male ants, the highest hybridization signals are found in the optic lobes and deutocerebrum. From these results, we speculate that the functions of PvERR are involved in the visual information integration, further to control the courtship behavior. Using RNAi technology to silence the expression of PvERR mRNA in different developmental periods and different castes adults, the results showed that, in embryo, first instar and second instar stages, the expression of PvERR has no obvious changes, from the third instar to adults, the expression declined significantly, in each tested samples, the expression of PvERR mRNA may decreased from50%to60%. the weight of larval and adults was lost, some larval were dead in three and fourth instar, all the results illustrate that in this study the dsRNA solution we used can reduce the expression of PvERR mRNA, and also prove that the PvERR plays an important role in the reproductive and developmental stages of the ants.4. In this study, RNAi is used to analyze the functional relationship among the three genes during ants’ development. The results showed that when the expression level of PvEcR was silent or low, the expression level of PvUSP would be immediately increased to cover the lack of PvEcR’s expression; when the expression level of PvERR was silent or low, the expression level of PvUSP and PvEcR would be decreased during the larva stage,but in pupal and adult,both of the two genes’ expression would be increased, expecially the expression of PvEcR.From these results, we speculate that, ERR can regulate the expression of EcR and USP during the development of ants,but more information is needed to be further explored. In conclution we have cloned the full-length cDNAs of EcR and USP genes firstly from the ants, P. vicina, and submitted the cDNA sequences to GenBank. The results of real-time quantitative RT-PCR and in situ hybridization methods revealed that the PvEcR, PvUSP and PvERR mRNA were found in all developmental stages, different castes and heads. The functional relationship among the three genes ware analyzed by RNAi.This may indicate that three genes have the main physiological functions and involved in regulating of growth, development and reproduction in insects. These results may provide the theory foundation for further investigation on the concrete function of the three genes in insects.