Yellow Faces Oil Gourd Estrogen-related Receptor (err) And The Ecdysone Receptor (ecr) Gene Cloning And Its Expression During Development

Teleogryllus emma (Gryllusmitratus Burmeister), belongs to the genus Teleogryllus (Orthoptera:Gryllidae). They are possess a big body and easy to dissect, so they have been used as a experimental animal to study gene expression in the polyphagous insects.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. Ecdysteroids are steroid hormones that play an important role in development, growth, molting of larva, and reproduction in the Arthropoda. The effect of ecdysteroids is mediated by ecdysteroid receptor (EcR). The ecdysone receptor (EcR) belongs to the superfamily of nuclear receptors (NRs) that are ligand-dependent transcription factors. Ecdysone receptor is present only in invertebrates and plays a critical role in regulating the expression of a series of genes during development and reproduction.In this thesis the cloning and expression analysis of estrogen receptor-related receptor (ERR) and ecdysteroid receptor (EcR) from T.emma was studied for the first time. The full-length cDNA of T.emma ERR (TeERR) and EcR (TeEcR) were cloned using RT-PCR and RACE methods; the mRNA expression levels of TeERR and TeEcR in the cricket were investigated by real-time RT-PCR and in situ hybridization methods. The results of investigations are offered as follows:1. The full-length cDNA of TeERR is 1618 bp, containing a 5'-untraslated region (5'-UTR) of 140 bp and a 3'-UTR of 272 bp. The open reading frame (ORF) of TeERR is 1206 bp, which encodes a deduced 401-amino acid peptide with a predicted molecular mass of 45.6 kDa and with the theoretical pI of 8.94. The results of sequence alignments analysis showed that this protein shares 84% identity with Nasonia vitripennis, Apis mellifera, Polyrhachis vicina, Aedes aegypti, and even high identity with mammalian, amphibian including human ERR. Sequence analysis by bioinformatics showed that ERR protein in T.emma belongs to a non-secreted member and contains many function motifs which are conservative during evolution.These results indicate that the newly isolated cDNA sequence indeed encodes the cricket ERR protein. The full-length cDNA sequence of the TeERR was submitted to GenBank and assigned the accession number FJ770332.2. The full-length cDNA of TeEcR is 2558 bp, containing a 5'-untraslated region (5'-UTR) of 555 bp and a 3'-UTR of 407 bp. The open reading frame (ORF) of TeEcR is 1596 bp, which encodes a deduced 531-amino acid peptide with a predicted molecular mass of 59.7 kDa and with the theoretical pI of 8.57. The results of sequence alignments analysis showed that this protein shares 92% identity with Locusta migratoria, and 89%-87% identity with Blattella germanica, Tribolium castaneum and Pediculus corporis. Sequence analysis by bioinformatics showed that EcR protein in T.emma belongs to a non-secreted member and contains many function motifs which are conservative during evolution. These results indicate that the newly isolated cDNA sequence indeed encodes the cricket EcR protein. The full-length cDNA sequence of the TeEcR was submitted to GenBank and assigned the accession number HQ131915.3. Real-time quantitative RT-PCR analysis was performed to quantify the relative expression levels of TeERR transcripts at different developmental stages of the whole body and gonads. The results displayed that TeERR was expressed in all samples at different levels in both the bodies and gonads. In the whole body of different developmental stages, the TeERR gene was found to be expressed at the highest level in the embryo, then significantly less in the larvae, and least in fourth instar; later the expression is slightly increased in the adult. In the gonad of different developmental stages, the TeERR gene was found to be expressed at the highest level in adult male testicle and lowest in sixth instar female ovary, but there was no statistical difference between them. Interestingly, the level of expression in the testicle was always higher than the ovary at each developmental stage of the gonad.4. Real-time quantitative RT-PCR analysis was performed to quantify the relative expression levels of TeEcR transcripts at different developmental stages of the whole body and gonads. The results displayed that TeEcR was expressed in all samples at different levels both the bodies and gonads (Fig.4). In the whole body of different development stages, TeEcR gene was found to be expressed gradually decreased from embryo to third instar and then significantly increased from fourth instar to sixth instar, but the expression was decreased again in the adult. Compared with male, the expression of TeEcR gene was higher in female during every development stage. In the gonad of different development stages, both the testis and the ovary, the expression of TeEcR gene was found to be gradually decreased from fourth instar to adult. The expression was significantly higher in ovary than in testis during fourth instar and fifth instar larvae development stage.5. The distribution of TeERR mRNA was analyzed by in situ hybridization to cryosections of the brain of adult crickets. The results showed that the hybridization signals are present extensively in the brains of adult crickets both male and female. The calyx and collaret of the mushroom bodies are weakly stained. And the prominent positive reactions appeared in the Keyron cells, the optic lobes and deutocerebrum, which may reflect the functions of TeERR involved in obtaining and integrating the visual and olfaction information in the nervous system.6. The distribution of TeEcR mRNA was analyzed by in situ hybridization to cryosections of the brain of adult crickets. The results were similar to the TeERR mRNA expression patern. The hybridization signals are also present extensively in the brains of adult crickets both male and female. The calyx and collaret of the mushroom bodies are weakly stained. And the prominent positive reactions appeared in the Keyron cells, the optic lobes and deutocerebrum, which may reflect the functions of TeERR involved in obtaining and integrating the visual and olfaction information in the nervous system.For the first time, we have cloned the full-length cDNAs of ERR and EcR genes from the cricket, T.emma, and submitted the cDNA sequences to GenBank. The results of real-time quantitative RT-PCR and in situ hybridization methods revealed that the TeERR and TeEcR mRNA was found in all developmental stages brain and gonads. This may indicate that two genes have the main physiological functions and involved in regulating of development, nerol and reproduction in insects. These results may provide the theory foundation for further investigation on the concrete function of ERR and EcR in insects.