| The nuclear receptors constitute a large family of proteins and can be considered as ligand-inducible transcription factors, which can bind directly to DNA and regulate expression of downstream target genes. In this group of protein superfamily, however, there are some putative receptor molecules whose ligands are not identified and these are classified as orphan nuclear receptors. The estrogen-related receptors(ERRa, ERRβ, and ERRγ) belong to a subfamily of such orphan nuclear receptors, share the similar structure with the ERs and can bind to the steroid receptor coactivator family without any ligands and drive transcription activity of the target genes. The results from various laboratories have suggested mammalian ERR receptors collaborate with the estrogen signaling, involving in many physiological and developmental processes, as well as the proliferation and differentiation of cells. The Polyrhachis vicina roger and the Apis mellifera, as the eusociality insect, are important resources insects because they have close relationship with human. It possesses important significance to investigate the reproduction and development of them. In this paper, the cloning and expression of ERRs gene was investigated, with the Polyrhachis vicina roger and the Apis mellifera, by RT-PCR, 5'and 3'Race, real-time quantitative PCR and in situ hybridization methods, in order to establish theories foundation for further understanding the functions of insect ERR. The main results are offered as following:1. The full length of pvERR gene is 1918bp, including an open reading frame of 1305bp, 5'-UTR of 245bp and 3'-UTR of 368bp, code the protein that is constituted by 434 amino acidses, the molecular weight is 49.0989 KDs, and theoretical pI is 6.62. The pvERR gene is composed of eight exons and seven introns. The nucleotide sequence of pvERR gene was submitted to GenBank and assigned the accession number EF474463.2. The full length of amERR gene is 1760bp, including an open reading frame of 1305bp, 5'-UTR of 197bp and 3'-UTR of 258bp, code the protein that is constituted by 434 amino acidses, the molecular weight is 49.131 KD, and theoretical pI is 7.43. The amERR gene is composed of eight exons and seven introns. The nucleotide sequence of amERR gene was submitted to GenBank and assigned the accession number EF506198.3. The homologous analysis suggest that the pvERR and the amERR proteins share 89.9% identities in amino acids sequence, and their homology is the highest in all known insect ERR; The sequence identities of the pvERR and amERR with the ERR(aaERR, asERR and dERR) of diptera insect are all in 50.2%-53%, and with human ERRa, ERRB and ERRγ, are range from 37.6% to 44%. The phylogenetic tree of all known insect ERR and human ERRs based on NJ method, show that the pvERR and the amERR clustered as expected with all insect ERR; The phylogenetic relationgship of the pvERR and amERR with human ERRs is closer compare with which of the dERR with human ERRs.4. The prediction of the secondary structure of pvERR and amERR proteins, using APSSP method, show that there are fourβ-strands and three a-helixs in DNA binding domain (105-107), and twoβ-strands and eleven a-helixs in ligand binding domain (206-429). The DBD is characterised by two Zinc-finger structures, the first is composed of cysteines located in the sequence of 110, 113, 117, 120, and the second is composed of cysteines located in the sequence of 136, 142, 152, 155. But the LBD of ERRs is formed by twelve a-helixs in general, there are in the absence of H2 in LBD of the pvERR and amERR protein, which is very similar to the LBD of mammalian ERRγof known crystal-structure.5. The results of tertiary structure prediction, using SwissModel First Approach, show that there are high similarity in DBD among the proteins of pvERR, amERR and human ERRβ; The difference of Spatial Structure in the LBD between the pvERR and the amERR is that in amERR, the last a-helix domain stretches to the model inner part, but in pvERR it stretches out outwardly, which may be related to their hydrophile. Ligand-dependent transactivation is mediated by activation function-2 (AF-2) in the LBD H12 and involves interactions between a hydrophobic surface and LXXLL sequence motifs from coactivator proteins, which may indicate pvERR and amERR have different ligands.6. The relative quantification expression of the mRNA level of pvERR and amERR genes in different developmental periods and the different castes were analyzed by the method of 2-△△CT, the results illustrate the expression of pvERR and amERR increases gradually with the development; As a result, the queen (ant and honeybee) has the highest expression, but the males' dose is the lowest. The expression patterns of the pvERR and the amERR are of the similar state, which indicate amERR and pvERR correlate to the insect development. Remarkable increase of the relative expression of amERR and pvERR from pupa to adult may indicate they have important roles in differentiation of tissues. Abundance expression of amERR and pvERR in adults may suggest they have a broad expression pattern as mammalian ERRs, and the different expression in castes are relevant with their functions in social community.7. The results of in situ hybridization show that pvERR mRNA has broad expression pattern in the brain of different castes, which can be detected in mushroom body, accessory lobe, optic lobes, deutocerebrum, suboesophageal ganglion and kenyon cells. Strongly positive expression was detected in mushroom body and Kenyon cells in brain of Queen ant, which indicates pvERR involved in the visual learning and memory and Courtship control, corresponding their functions in social community. Compare with Queen ant, the expression degree of pvERR mRNA is stronger in the deutocerebrum of worker ant, but other parts are equivalent, which indicates pvERR relate to the olfactory discrimination and the learning and memory. Compare with Queen and worker ants, the degree of pvERR expression is weaker in mushroom body, accessory lobe and suboesophageal ganglion of male ants, but stronger expression found in optic lobes, which may suggest pvERR involve in the visual information integration, further to control the courtship behavior.8. The results of in situ hybridization show that, the positive expression of pvERR mRNA has been found in the cytoplast of follicle cells during the oogenesis and the oocytes in period of the growth and the yolk formation. Expression of pvERR mRNA in the follicle cells during differentiation stage may be related to the proliferation and differentiation of follicle cells. During growth period, the expression pattern of pvERR mRNA indicate that the follicle cells may provide the developmental information to oocytes, and the expression in oocytes during yolk formation stage, which may suggest the pvERR involve in the formation of the yolk formation. The positive expression of pvERR mRNA also been found in the cytoplast of spermatid cell and the head of mature sperm, it may suggest the pvERR involved in the developmental control and sperm mature.For the first time, we demonstrated estrogen related receptor exist in eusociality insect, and submitted their sequence to the GeneBank. And found that the pvERR and amERR genes are more evolutional than dERR in phylogenetic relationship. It is also find firstly that ERR gene of eusociality insect involve in insect development using real time PCR, and the expression of pvERR and amERR genes were detected in brain and gonad of insect by in situ hybridization. These results may be useful in further discussing the important functions of estrogen related receptor of insect, and hlpe to lay the theory foundation to make clear the relationship of insect steroid hormone receptors in the development and others physiological aspects.
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