| Melanopsin is encoded by the opn4. As a vitamin A type photosensitive receptor in non-visual system, it’s a key element of the peripheral circadian clock light conducting pathway upstream,and it participate in a variety of physiological and biochemical functions of the organism. The purpose of the research is to reveal the Lutjanus erythopterus opn4 gene structure, tissue distribution, expression pattern of the early development, and evolutionary relationship with other snapper. The sequence of opn4 was cloned by reverse transcription polymerase chain reaction(RT-PCR). The history of L. erythopterus and other snappers were analyzed based opn4 by conventional PCR and DNA sequencing techniques. The expression patterns were analyzed in the tissues and early development stages of L. erythopterus by real-time quantitative PCR. The main results are as follows:1. A 4595 bp full-length cDNA of opn4 gene was the first successfully acquired from L. erythopterus. The sequence analysis indicated that the fragment consists of a 121 bp 5’untranslated region(UTR), a 2794 bp 3’UTR and 1680 bp open reading frame(ORF). The translated protein is composed of 559 amino acids and shares more than 83% identity in deduced amino acid sequence with other teleost species. It contains 7 transmembrane regions and 4 highly conserved G protein structure functional domains.2. After conventional PCR and DNA sequencing techniques, 8 kinds of snappers corresponding 927 bp mRNA sequence and 657 bp gene sequences(intron-containing 554bp) were obtained. We analyzed the genetic cariation of amino acid sequence and gene sequence, and found that there were not much sequence differences regardless of between the exon(927bp) or the intron(554bp) from eight snappers. There were no insertions, deletions or stop codons, in eight kinds of snappers 927 bp nucleic acid sequence. But they had 14 polymorphic locus in the corresponding encoded these amino acid sequence, in which neither L. erythopterus or L. sebae had unique amino acid residues, but other snappers all contained two or more unique amino acid residues respectively. In addition, we detected there were some insertions and deletions in eight kinds of snappers 554 bp intron sequence.3. With RT-qPCR technology, the opn4 gene expression in 11 tissue(Retina, Brain, Heart, Liver, Spleen, Stomach, Intestines, Muscle, Skin, Gill and Gonad) of L. erythopterus were respectively measured. This gene was detected in liver, retina, gonads and skin. Further, the gene expression in the retina was far higher than other tissue. The gene higher expression in skin was likely to be associated with fish body color along with the change of light environment. Due to no report ever done showed this gene expressed in fish liver and gonad, the repeating tests in the experiments was carried out to confirm this finding is true. Nevertheless, what the specific role the opn4 gene playing in fish liver and gonad, which needs further investigation.4. Taking advantage of RT-qPCR, respectively detected the opn4 gene expression in 10 early development stages(Multi-cell, 1/2 epiboly, Blastopore-closure, Optic-capsule, Eye-lens-formed, Heart-beating, Hatching, 1d-larva, 3d-larva and 10d-larva of L. erythopterus. The results showed that this gene expression can be discovered in all stage. However, the expression level of opn4 in 1/2 epiboly stage, closure of blastopore stage, eye lens formed stage and 1 day larva after hatching was significantly higher than other stages. This gene had appeared expression of volatility changes in early development period of L. erythopterus. This may be related to adjust environment light changes and meet the needs of a variety of physiological behavior. |
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