| As one kind of steroid hormones, estrogen plays an important role in animals, and it mainly performs respective functions by mediation of estrogen receptor. Gonadal development is mediated by many regulatory factors, and estrogen together with its receptor has been recognized as the significant part of these factors in ovarian development. Estrogen receptor (ER) was not found in invertebrates and another gene existed with high homology, so it was named estrogen related receptor (ERR). There has not been any research during growth and development in crustacean until now. Therefore, the research of nuclear receptor can help us to elaborate the signaling pathway of estrogen.In this paper, Macrobrachium rosenbergii was used as experiment object to study the estrogen related receptor (ERR). The full-length cDNA of ERR was cloned by using homology cloning method and RACE technology. Then the tissue distribution of ERR was detected by semi-quantitative reverse transcription PCR. Effect of NP exposure under different concentration of distinct time on ERR mRNA expression in female ovary were examined using quantitative real-time PCR (qPCR). In addition, the open reading frame of ERR and its homology or conserved regions was analysised by bioinformatics software. After constructing the prokaryotic expression vector pET-32a-ERR, the expression product was purified by Ni-affinity. The main achievements are as follow:1. Molecular cloning and sequence analysis of ERR gene in Macrobrachium rosenbergiiERR gene of Macrobrachium rosenbergii has only one subtype, and it is 1897 bp in full-length including a 1374 bp ORF region,143 bp of 5’-UTR and 380 bp of 3’-UTR, which encodes for a deduced protein of 457 amino acids, and its molecular mass is about 50.75 kDa. The sequence analysis of amino acid showed that Macrobrachium rosenbergii estrogen related receptor contained the typical domain of nuclear receptor family.2. Tissue distribution research of ERR in M. rosenbergiiThe distribution results showed that the ERR mRNA mainly expressed in heart and ovary, and weakly expressed in cranial nerve and hepatopancreas in female without the signal in eyestalk, muscle and gill; ERR mRNA mainly expressed in heart, and weakly expressed in cranial nerve, hepatopancreas, testis and gill without the signal in eyestalk and muscle. We speculate that ERR may play an important role in ovarian development.3. Effects of NP on ERR mRNA expression in M. rosenbergiiAfter NP exposure under different concentration (5,25 and 125μg/L) for two weeks, all treatment groups could significantly increase the expression of ERR mRNA (P< 0.05). When the concentration of NP arrived 25 μg/L, the estrogenic effects became stable. So 25 μg/L was its appropriate concentration and used in the subsequent experiment. The data showed that with the increase in processing time, ERR mRNA expression level all remarkably improved, but the effect of estrogen reduced after 18 days.4. Prokaryotic expression and purification of ERRThe ORF of ERR was cloned into prokaryotic expression vector pET-32a to construct the recombinant expression vector pET-32a-ERR. The best expression condition was 37 ℃,0.1 mmol/L IPTG,4 h. After purification and detection, the 70 kDa recombinant protein was obtained as expected.In summary, the ERR gene was preliminarily studied and the regulation of environmental endocrine disruptors was explored. The construction of prokaryotic expression system and purification of target protein help to creat the foundation for the subsequent research, such as verification of the fusion protein, preparation of polyclonal antibody and related functional research. It also can provide the basic data for the regulation mechanism of energy metabolism during growth and development in M. rosenbergii. |
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