The Preliminary Study Of The Molecular Mechanism Of Feeding Inhibition Induced By High Plant Protein In Japanses Sea Bass(Lateolabrax Japonicus)

The teleostean species Japanese seabass(Lateolabrax japonicus),also known as seven perch and classified as serranidae of perciformes,is an important aquaculture higher bony fish species in the world. The protein source of Japanese seabass depends on fishmeal. The present study aimed to study molecular mechanism of food intake suppression and regulation of feeing mechanism in Japanese seabass feeding total plant protein. This paper includes three parts, the summarized results respectively as follows:1. Cloning and Sequence Analysis of Full-Length cDNAs of Factors in Control of Food Intake of Ghrelin/Leptin-NPY/AGRP and mTOR-NPY signaling pathway in Japanese Seabass(Lateolabrax japonicus)The full-length cDNAs of the factors of control of food intake,including serine/threonine-protein kinase (mTOR), ribosomal protein S6 kinase beta-1(S6K1), neuropeptide Y precursor (NPY),preproghrelin and agouti-related protein (AgRP) were cloned from Japanese Seabass (Lateolabrax japonicus) using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods. The results showed as follows:the full-length mTOR cDNA of Japanese Seabass(GenBank accession No.KJ746670) was 8296 bp, consisting of an open reading frame (ORF) of 7551 bp encoding a polypeptide of 2516 amino acids with a theoretical molecular weight of 286.14K.U, and shared 62.0% to 98% similarity with other genes. The full-length S6K1 cDNA of Japanese Seabass(GenBank accession No.KJ746671) was 2232 bp, consisting of an open reading frame (ORF) of 1539 bp encoding a polypeptide of 512 amino acids with a theoretical molecular weight of 56.87Ku, and shared 80.4% to 84.9% similarity with other genes. The full-length NPY cDNA of Japanese Seabass (GenBank accession No.KJ850326) was 676 bp, consisting of an open reading frame (ORF) of 300 bp encoding a polypeptide of 99 amino acids with a theoretical molecular weight of 11.26Ku, and shared 53.6% to 99.0% similarity with other genes. The full-length preproghrelin cDNA of Japanese Seabass (GenBank accession No.KJ850327) was 1201 bp, consisting of an open reading frame (ORF) of 324 bp encoding a polypeptide of 107 amino acids with a theoretical molecular weight of 12.03Ku, and shared 19.6% to 85.0% similarity with other genes;The full-length AgRP1 cDNA of Japanese Seabass (GenBank accession No.KJ825853) was 739 bp, consisting of an open reading frame (ORF) of 429 bp encoding a polypeptide of 142 amino acids with a theoretical molecular weight of 15.86Ku, and shared 28.9% to 88.0% similarity with other genes;The full-length AgRP2 cDNA of Japanese Seabass (GenBank accession No.KJ825854) was 1357 bp,consisting of an open reading frame (ORF) of 351 bp encoding a polypeptide of 116 amino acids with a theoretical molecular weight of 13.46Ku, and shared 22.4% to 93.1% similarity with other genes.The results of this study will provide a scientific basis for further study to explain regulatory mechanism of the factors of control of food intake in Japanese Seabass.2. The effects of total fishmeal substitution by plant protein blend on Ghrelin/ Leptin-NPY/AGRP and mTOR-NPY signaling pathway during the feeding inhibition in Japanese SeabassThe test was divided into two groups, one group for the whole fishmeal, another group of plant protein blend completely replacing fishmeal for high protein group, which had the same nitrogen and energy. Samples were collected at 3,6,9,12 and 24h post-feeding,when feeding of high plant protein group was obviously suppressed,in order to research the impact of high protein feed on regulation factors of food intake.The results showed that:in stomach, mRNA expression of mTOR at 3,6,9 and 12h post-feeding in fishmeal group were significantly higher than in the high plant protein group (P<0.05);The mRNA expression of S6K1 at 6 and 24h post-feeding in stomach of fishmeal group and at 9 and 12h post-feeding in liver of the fishmeal group were significantly lower than in stomach and liver of high plant protein group (P<0.05).However, the mRNA expression of S6K1 at 12h post-feeding in stomach of the fishmeal group was significantly higher than in high plant protein group (.P<0.05);In the liver, the mRNA expressions of leptin at postprandial 6 and 9h post-feeding in fishmeal group were significantly higher than in the high plant protein group (P<0.05);In hypothalamus, No significant differences of the mRNA expressions of NPY at each time points between two groups were also founded(P>0.05);In stomach, the mRNA expressions of preproghrelin at 6h post-feeding in fishmeal group was significantly lower than in the high plant protein group(P<0.05); In hypothalamus, No significant differences of the mRNA expressions of AgRP1 at each time points between two groups were also founded (P>0.05) the mRNA expressions of AgRP2 at 6h post-feeding in the fishmeal group was significantly higher than in the high plant protein group (P<0.05);mTOR and leptin levels decreasedand preproghrelin increased,suggesting Japanese seabass in total plant protein group was in negative energy because of refusing to feed high plant protein diet.But the lack of the central appetite regulation system in response to feed in feeding inhibition induced by total plant protein was maybe the reason why Japanese seabass didn’t feed. These results provide a theoretical basis for the further study of molecular mechanism involved in feed intake suppression of carnivorous fish fed total plant protein diet.3. The influences of total plant protein during food intake suppression on Japanese seabass transcriptome in hypothalamus and liverThis experiment mainly studyed the molecular mechanism of significant inhibition phenomenon in feeding carnivorous Japanese seabass after a significant inhibition consumption of high plant protein diets occurs, according to changes of feeding and growth performance in the event of postprandial feeding points for sample collection.RNA-Seq technology was used to detect the differences between feeding regulation center (hypothalamus) and peripheral tissue(stomach) of the control groups (fish meal as the sole protein source) and whole plant proteomics. The transcriptome results of postprandial 3h in feeding inhibition showed that:there were five and ten regulatory factors respectively during the annotated genes differentially expressed in stomach and hypothalamus. Down-regulation of predicted:gastrin-releasing peptide-like, Glucagon-like peptide 2 receptor, G-protein coupled estrogen receptor 1, Thyrotropin subunit beta, somatotropin, prolactin, somatolactin, pro-opiomelanocortin and up-regulation of Glucagon-1, Glucagon-2, Growth hormone receptor suggested Japanese seabass was in negative energy.However up-regulation of Peptide YY-A, Corticotropin-releasing factor receptor 1 and melanin-concentrating hormone-like protein might induce a reduction of feeding in Japanese seabass. In the test,our date will provide clues for further probing of molecular mechanism of feeding inhibition on a high total plant protein feed in Japanese seabass.