| The genetic diversity of loach(Misgurnus anguillicaudatus) in Poyang Lake was abundant, and the size and skin color of loach also had great differences. It provided good materials for selective breeding of loach in Poyang Lake. This study inclued two aspects. We firstly cloned melanin-concentrating hormone gene, proopiomelanocortin gene, melanin-concentrating hormone receptor 1 and ?-melanophore-stimulating hormone receptor 1. And we preliminary indicated the relationships of the four genes and the formation of dark spots. Secondly, in order to explore suitable conditions of fry rearing, we investigated the different development temperature, water transparency, and the initial feeding time impact on the survival rates of loach larvae.The main results are as follows:1. The cDNA sequence of MaPmch1, MaPomc, MaMchr1, and MaMc1 r gene were cloned by RACE-PCR. Physicochemical property, conserved domains, secondary structures, homologous analysis and phylogenetic trees of the four genes and their proteins were further analyzed using bioinformatic methods.(1) The full length of MaPmch1 gene was 570 bp which contained an open reading frame of 375 bp encoding 124 amino acid residues. A 13-AA-amidated cyclic neuropeptide named MaMCH1 was generated from MaPMCH1 after hydrolyzing. PMCH1 and MCH1 of Misgurnus anguillicaudatus were predicted to be hydrophilic proteins, and their secondary structures mainly contained random coil. The super secondary structure of MaMCH1 had an antiparallel ?-plated sheet which was connected by a disulfide bond. Homology analysis indicated that amino acid sequence of MaMCH1 was highly consistent with other bony fishes. The phylogenetic tree of PMCH1 showed that loach and Cyprinid fish were in the same branch. In vertebrate, RCM*GRVYRPCW(* as random amino acid) was a highly conserved sequence.(2) The full length of MaPomc gene was 1133 bp that contained an open reading frame of 666 bp encoding 221 amino acid residues. A 13-AA-amidated ?-MSH was generated from MaPOMC after hydrolyzing. Homology analysis indicated that amino acid sequence of ?-MSH was the same with other species, and ?-MSH had a motif of HFRW which was important for its function. The phylogenetic tree showed that MaPMCH1 and Cyprinid fish were in the same branch.(3) The full length of MaMchr1 gene and MaMc1 r gene were 1364 bp and 1529 bp encoding 328 and 316 amino acid residues, respectively. Their secondary structures mainly contained random coil, and the tertiary structures contained seven transmembrane domain structures. They also had a DRY motif, and they were the typical of G protein coupled receptors. The phylogenetic tree showed that the genetic relationship of MaMCHR1 and MaMC1 R were consistent with the traditional classification status.2. The expression of four genes in the abdominal skin, dorsal skin and whole brain tissues of the different brindled loach were detected by qPCR. The expression of MaPmch1 gene in the brain of the different brindled loach showed no apparent rule. The expression of MaPomc gene in the brain of small-brindled loach was higher than that in the big-brindled and non-brindled loach. It indicated that the high expression of MaPomc gene could help increasing the secretion of melanin at the dark spots of small-brindled loach, and producting more dark spots. The expression of MaMchr1 gene was mainly in the brain, and there was no significant difference in the brains among the three different brindled loach. The expression of MaMc1 r gene was mainly in the brain and dorsal skin. The expression in the dorsal skin and brain of the big-brindled and small-brindled loach were the same. The expression in the brain was higher than that in the dorsal skin of non-brindled loach. We speculated that the high expression of MaMc1 r gene helped the loach to form the dark spots. The values of MaPmch1/MaPomc in the non-brindled loach was higher than that in the big-brindled and small-brindled loach, and it explained that the different melanin pigmentation patterns in the different brindled loach were regulated by the proportion of MaPmch1 and MaPomc gene expression.3. This study described 24 periods of embryonic development in detail. When the water temperature was 24 ? or 27 ?, the embryonic development lasted for 31 h 20 min or 24 h. The effective accumulated temperature of embryonic development of loach was 307.65 ?·h, and the biological zero temperature was 14.18 ?. The influence of water temperature on hatching rate was not obvious, however, it had great influence on survival rate of larvae after hatched for 72 h. The survival rates of 24 ? and 27 ? was 72.42% and 65.84%, respectively. The higher the water temperature was, the shorter time of the embryonic development lasted, and lower the survival rate of larvae was. When the water transparency was 5 cm, the survival rate of larvae was 71.83%, it was significantly lower than that in 15 cm and 25 cm. we found the suspended particle in the water could stick on the external gill, so the breathing and vitality of the larvae had been affected. According to the survival rate of the larvae, the optimum time of initial feeding was about 20 h to 30 h after the larvae hatched(24?), in other word, it was about 10 h after the larvae intestinal tract formed. In combination with microtechnique, this study solved the key problems during the growth of larvae. It could greatly improve the loach seedling survival rate and scientifically guide the loach breeding work. |
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