Ecdysteroid-responsive Genes: E75 And Retinoid X Receptor (RXR), Essential Molt-regulating Genes In The Shrimp, Fenneropenaeus Chinensis

The study presented in this thesis is an attempt to explore the potential involvement of two important molt-regulating genes: generally called ecdysone responsive genes, E75 and RXR in the molting process of Chinese shrimp, Fenneropenaeus chinensis. Full-length cDNAs encoding FcE75 and FcRXR were obtained using PCR technique. The FcE75 cDNA sequence (3.6Kb) contained the complete ORF encoding a 798-amino acid protein and the sequence was submitted to GenBank (accession number EU599574). The estimated mass of the deduced amino acid sequence of FcE75 was 87.9 kDa and the calculated theoretical pI was 7.96. On the otherhand, the full-length cDNA of FcRXR was 1715 bp revealed a 1315 bp open reading frame (ORF) encoding a protein of 442 amino acids. The FcRXR cDNA sequence was submitted to GenBank (Accession Number: 1130559). The estimated mass and the calculated theoretical pI of the deduced amino acid sequence of FcRXR were of 109.05 kDa & 4.98 respectively.In addition, the genomic structure of FcRXR was acquired from the genomic library constructed from the testis DNA of F. chinensis. The genomic sequence of FcRXR was about 4.57 kb with seven introns which depicted the existence of several isoforms in shrimp and two isoforms (FcRXR-1 and FcRXR-2), have been identified in F. chinensis. Quantitative Real time PCR confirmed the prominent expression of FcE75 and FcRXR mRNAs in the late pre-molt period (D3) of shrimp. Among the two isoforms, FcRXR-2 appeared in a considerably high level in all the seven molt stages in contrast to the FcRXR-1. Moreover, a marked tissue-specific difference in the expression of FcE75 and FcRXR transcripts were also seen which revealed that the expression of FcE75 & FcRXR could be regulated in a tissue-specific manner.In order to investigate the role of E75 and RXR in shrimp molting, we have used double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) technique to elucidate the function of both these genes in shrimp. Interestingly, the application of double stranded RNA specific to the E75 & RXR into the juvenile Chinese shrimp efficiently decreased the FcE75 and FcRXR expression levels. The efficiency of FcE75 silencing by double stranded E75 (dsE75) was examined by varying the amount of injected dsE75 and the result clearly indicated that dsE75 exhibited dose-dependent effect in vivo. Injection of 7μg dsE75 into shrimp (approximately 1.8 - 2gm body weight) resulted in a maximum silencing effect (81%) at 48 h post injection. The level of E75 expression in all control shrimp that received no dsE75 showed normal level of expression. Shrimp injected with PBS served as control. Observations suggested that FcE75 silencing in vivo completely arrested the molting process and caused death of the shrimp. Analysis of the animals which failed to molt showed defective epidermal retraction, poor development of setae and new cuticle. Similarly, injection of 10μg double stranded RXR (dsRXR) into juvenile shrimp (3 - 4 gm body weight) resulted in a maximum silencing effect of about 74.5% at 48 h post injection. An unrelated long dsRNA corresponding to a green fluorescence protein (GFP) gene, injected into the shrimp served as negative control for both the above silencing experiment.In addition, we examined the temporal expression of two chitinase genes: FcChitinase (FcChi) & FcChitinase-1 (FcChi-1) during the molt cycle of F.chinensis. The FcChi & FcChi-1 transcripts were detected in all stages of molting, although considerable fluctuations observed through the molt cycle. Subsequently, to determine whether or not FcChitinase was down-regulated by silencing the ecdysone responsive genes E75 and RXR, we analyzed the expression levels of FcChi & FcChi-1 genes in samples of both dsE75 and dsRXR treated shrimp. As expected, significant reduction in levels of both FcChi & FcChi-1 transcripts occurred in the silenced shrimp. This correlation suggests that FcE75 and FcRXR might involve in the downstream regulation of chitinase gene transcription in ecdysone signaling.Furthermore, FcE75 silenced samples revealed reduced level of FcRXR expression and vice versa. From these results, we propose that FcE75 & FcRXR may exert a feedback control of ecdysteroidogenesis (ecdysteroid syntheis) which in turn, down regulate the ecdysteroid responsive genes including E75, RXR and enzymes responsible for exoskeleton degradation (Chitinase).And at last, we conclude that the ecdysteroid responsive genes FcE75 & FcRXR may play a prominent function during the molting process and might be essential for proper molting and survival of shrimp. This is the first report elucidating the function of these genes in the molting process of decapod crustaceans.