1. Whole transcriptome sequencing reveals photoreceptors mRNA expression in nocturnal cutlass fish during the midnight
Ji-Yeon Hyeon1, Jun-Hwan Byun1, Seong-Rip Oh2, Mun-Kwan Kim2, Su-Hyun Park2, Sung-Pyo Hur1 and Hyeong-Cheol Kang2*
1Jeju International Marine Science Research & Logistics Center, Korea Institute of Ocean Science & Technology
2Ocean and Fisheries Researches Institute, Jeju Special Self-Governing Provence
Introduction
The opsin family are the universal photoreceptor molecules of all visual systems in the vertebrates including teleost. They can change their conformation from a resting state to a signaling state upon light absorption, which activates the G-protein coupled receptor, thereby resulting in a signaling cascade that produces physiological responses. However, the species is poorly characterized at molecular level with little sequence information available in public databases.
Materials and Method
Results
Table 1. Primer set for full-length cloning
Cutlass fish, Trichiurus lepturus
Gene
Primer
Sequences
Sequence
RH1
GSP1
5’- TCCAGGTGAAGACCAAGCCCATGAT -3’
VA opsin
5’- ACAGTGGCTGTCTTGGAAAAGAACG -3’
NGSP1
5’- AGGTGAAGACCAAGCCCATGATCGC -3’
5’- GTGGCTGTCTTGGAAAAGAACGCGG -3’
GSP2
5’- CTGTGGTCACTGGTCGTTCTGGCTA -3’
5’- TGGTGATGATCGTGGCGTTCATGGT -3’
NGSP2
5’- TGGTCACTGGTCGTTCTGGCTATTG -3’
5’- TGATGATCGTGGCGTTCATGGTGTG -3’
RH2
5’- CCAACTGTGCTGAGCATGCAGTTAC -3’
Peropsin
5’- CATCTTGGTGACGTCCATCTGGTCC -3’
5’- ACTGTGCTGAGCATGCAGTTACGGA -3’
5’- CTTGGTGACGTCCATCTGGTCCGAC -3’
5’- TCCTGATGGTGCTTGGCTTCCTGGT -3’
5’- GCGGTGATTGCCGTGAACTTCGTGG -3’
5’- TGATGGTGCTTGGCTTCCTGGTAGC -3’
5’- GTGATTGCCGTGAACTTCGTGGTGC -3’
Opn3
5’- GGCGCTCTTTGAGAGTTTGCTGCAG -3’
Melanopsin-A
5’- ATGCCCAGCCCAGGAAATCAGTGTG -3’
5’- GCTCTTTGAGAGTTTGCTGCAGACA -3’
5’- CCCAGCCCAGGAAATCAGTGTGACA -3’
5’- TCTCTCCCACAATGGCCATCATCCC -3’
5’- GGTACAACGGCTGGGAACTCAGGTG -3’
5’- CTCCCACAATGGCCATCATCCCCTC -3’
Table 3. The deduced photoreceptors sequence of cutlass fish.
Collected at nighttime
Photoreceptors
Nucleotide sequence
(bp)
Amino acid sequence
(aa)
RH1
1668
354
RH2
2036
355
Opn3
1790
385
Peropsin
1400
461
VA opsin
3187
381
Melanopsin-A
3557
400
B.W.: ~131.0 g
B.L.: 61.5 ~69.0 cm
Table 2. Primer set for in mRNA expressions
Gene
Primer
Sequences
EF1α
Forward
5’- TCACCCTGGGAGTAAAGCAG -3’
Reverse
5’- TCCATCCCTTGAACCAGGAC -3’
RH1
5’- GGTGAAGACCAAGCCCATGA -3’
5’- GTGATTCTCGGTGAAGCGGA -3’
RH2
5’- CTGGTGGTCACAGCTCAGAA -3’
5’- GGACTCCAATTCCTGCATGT -3’
Opn3
5’- GCTTCTGCAACAACGTCGTT -3’
5’- GTAGCGCTCATAGGCCAGAG -3’
VA opsin
5’- AGACTCGCGCCTTGTAAACA -3’
5’- AGGCTACTTGCTTGGCTGAG -3’
Per
5’- GGCTGGATACCTCATCACCG -3’
5’- GTGAGGTAGCGGTCTATGGC -3’
Melanopsin-A
5’- TGGAGCTCGTACATCCCTGA -3’
5’- TCGCCAACTTCCACTCACTC -3’
Figure 3. Distribution of RH1, RH2, Opn3, VAopsin, Peropsin and Melanopsin-A mRNA expressions in the organ and tissue of cutlass fish, Thunnus orientalis. Organ and tissue samples (n= 13) were collected from the fish. They were analyzed with RT–PCR. The expression of Elongation factor 1-alpha (EF1a) mRNA was used as reference. Samples without cDNA templates were loaded as negative control. Marker; 100 bp DNA ladder.
Figure 2. Comparison of the predicted amino acid sequences of Melanopsin-A, Peropsin, Opn3, VA opsin, RH1 and RH2. The seven transmembrane domains are labeled TM1 to TM7 above the sequence.
Figure 1. Phylogenetic tree of opsins including .One thousand bootstrap repetitions were performed, and values are shown at the inner nodes. The scale bar is calibrated in substitutions per site.
Figure 4. Histological observation of retina layer in Trichiurus lepturus (A), Paralichthys olivaceus (B), Pagrus maior (C).
Conclusion and Discussion
The cutlass fish opsin genes contained the seven presumed transmembrane domains that are characteristic of the G-protein-coupled receptor family. However, middle wavelength sensitive pigment (MWS) long wavelength sensitive pigment (LWS) were not detected in this species. RT-PCR revealed that cutlass fish all of opsin genes was detected in retina, while rodopsin2 mRNA were detected only in the retina. The levels of rhodopsin1 mRNA were high expressed than other opsin genes during the nighttime. These results suggested that retina and rhodopsin1 (498nm) may play important role in detecting light and produces physiological responses.