Cloning of a shrimp (Metapenaeus ensis) cDNA encoding a nuclear receptor superfamily member: an insect homologue of E75 gene1

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Abstract
Degenerate primers were derived from the amino acid sequence in the DNA binding domain of the Drosophila ecdysone receptor (DmEcR). Several partial cDNAs were amplified from the shrimp epidermis by reverse transcription polymerase chain reaction (RT-PCR). One of these fragments shows the highest amino acid sequence homology to the insect ecdysone inducible gene E75. This partial cDNA was used as a probe to screen the swimming leg cDNA library of the shrimp, Metapenaeus ensis. A 3.6 kb cDNA clone was obtained. The longest open reading frame of this cDNA consists of 606 amino acids and its deduced amino acid sequence has all five domains typical of a nuclear receptor. The putative polyadenylation signal is located at about 400 bp 3′ to the stop signal. The deduced amino acid sequence of this cDNA shows the highest identity to that of the E75A reported in Manduca sexta, Galleria melonella, Drosophila melanogaster, and Choristoneura fumiferana. Based on the amino acid sequence comparison, the shrimp nuclear receptor is considered the insect homologue of E75A. Northern blot analysis shows that the shrimp E75 is expressed in the epidermis, eyestalk and the nerve cord of the pre-molt shrimp. Moreover, E75 transcripts can be detected in the epidermal tissues of early pre-molt shrimp by in situ hybridization. To determine whether the shrimp could also express other E75s like the insects, 5′ end RACE and RT-PCR were performed on epidermal cDNA of a single shrimp. Subcloning and DNA sequence determination of the PCR products confirmed the presence of two other forms of E75 (tentatively called E75C and E75D) in shrimp. By RT-PCR, different levels of E75 expression can be detected in the epidermis, nerve cord and the eyestalk of early pre-molt shrimp. In addition to the different levels of expression of the shrimp E75s in the epidermis, the pattern of their expression is also different during the molting cycle. This is the first report on the cloning of a shrimp nuclear receptor superfamily member.