Molecular Cloning, Expression And Single Nucleotide Polymorphisms Of Protein Phosphatase 1(PP1) In Mandarin Fish(Siniperca Chuatsi)

Mandarin fish(Siniperca chuatsi) is one of the main valuable economic fish in China,and be widely loved. In the wild, mandarin fish only feed on live prey fish, refusing dead prey. When reared in ponds, training will result in some mandarin fish accepting artificial diets. However, little is currently known about the molecular mechanism of the individual difference. Through the analysis of the transcriptome of the feeders and nonfeeders mandarin fish, we found that the acquisition of novel food preference of mandarin fish might associated with pathways of vis ual, appetite, circadian phase, learning and memory. Serine/threonine protein phosphatase 1(PP1) is a suppressor of learning and long-term memory(LTM) in mammals. In the present study, the relationship between PP1 and the individual difference in accepta nce of artificial diets in mandarin fish was investigated.The complete CDS(coding sequence) of four PP1 isoforms(PP1caa, PP1 cab, PP1 cb and PP1cc) were cloned in mandarin fish by RT-PCR and RACE methods. The length of PP1 caa, PP1 cab, PP1 cb and PP1 cc was 3131 bp, 1336 bp, 2108 bp, 1794 bp, and the deduced amino acid sequence was 325 aa, 290 aa, 327 aa, 323 aa, respectively. Multiple alignment was carried out based on amino acid sequence of PP1 s from mandarin fish, other fish species, frog(Xenopus tropicalis), mouse(Mus musculus) and human(Homo sapiens). The amino acid sequences of these PP1 isoforms are highly conserved in different species. Phylogenetic analysis indicates that the PP1 caa and PP1cab(PP1α), PP1cb(PP1β) and PP1cc(PP1γ) of vertebrates including mandarin fish are grouped into three clusters, respectively. Mandarin fish PP1 cb clustered with mammalian PP1 cb, and PP1 cab clustered with amphibian PP1 ca. The PP1 caa and PP1 cc of mandarin fish and other fishes(Oryzias latipes, Oreochromis niloticus, Takifugu rubripes) were grouped in the same cluster. The results suggest that the four cloned mandarin fish PP1 genes were classified into PP1α, PP1β and PP1γ, respectively.The tissue expression levels of PP1 mRNA are detected by real time PCR, we found that four PP1 subtypes of mandarin fish were wide expression, such as in brain, liver, intestines and heart. While the expression abundance of different subtypes of PP1 were very different in different tissues. The expression levels of PP1 cab and PP1 cc were most abundance in mandarin fish brain. The expression levels of PP1 caa and PP1 cb were most abundance in mandarin fish liver, which might be associated with the metabolism of mandarin fish. Followed by high expression in brain tissue, which might be related to long-term memory and synaptic plasticity of mandarin fish.The mRNA expression levels of PP1 caa, PP1 cab, PP1 cb and PP1 cc gene were analyzed by real-time PCR. The PP1 cab and PP1 cc were no significant difference observed in expression between feeders and nonfeeders. PP1 caa and PP1 cb mRNA levels of the feeders group were significant(P < 0.05) increased compared to nonfeeders group in the brain. After the training, the feeders may exhibit an imbalance between phosphorylation and dephosphorylation in brain, which may influe nce the regulation of gene transcription involved in LTM and synaptic plasticity. Based on the conservative nature of PP1, we hypothesized that the feeders may also regulate PP1 to adapt to the new feeding environment. The mandarin fish feeders with increased expression of PP1 caa and PP1 cb in brain might be able to accept artificial diets due to a defect in the maintenance of the original memory of its natural food habit. In summary, these results suggest that a possible association between PP1 and the individual difference in acceptance of artificial diets may exist in mandarin fish. The PP1 cb mRNA level was also significant increased compared to nonfeeders group in the hepatic. This phenomenon may be caused by metabolic changes in the organism, according to food intake.Polymorphisms of PP1 genes were examined in nonfeeder and feeder populations of mandarin fish by direct sequencing and restriction digestion(n = 248). W e found one SNP(G1416A) in PP1 caa, one SNP(C1285G) in PP1 cb, while no SNP in PP1 cab or PP1 cc. SNPs loci in PP1 caa and PP1 cb genes were significantly associated with the individual difference in acceptance of artificial diets in mandarin fish. These results suggest that PP1 caa and PP1 cb could be involved in the genetic mechanism of individual difference in acceptance of artificial diets in mandarin fish.In order to ascertain the relationship between the SNPs and mRN A expression of PP1 caa and PP1 cb, the transcription factor-binding sites(TFBS) were predicted online. The results show that the mutations in PP1 caa and PP1 cb may also give rise to binding transcription factors and miRNA changes, which may relate to varying expression and translation in feeders and nonfeeders.In conclusion, different mRNA expression and SNPs of PP1 caa and PP1 cb genes in feeders and nonfeeders of artificial diets might contribute to understanding the molecular mechanism of individual difference in acceptance of artificial diets in mandarin fish. The SNPs of PP1 caa and PP1 cb genes could also be useful makers for gene-associated breeding of mandarin fish.