| Blunt snout bream(Megalobrama amblycephala),due to its short reproductive cycle,two age to achieve sexual maturity,which act as good materials for the study of muscle growth,breeding new varieties.Myostatin(MSTN),also known as growth differentiation factor 8(GDF 8),belongs to the transforming growth factor ?(TGF-?)family.This gene inhibits skeletal muscle growth and development.In this study,full-length c DNA of myostatin was cloned in blunt snout bream(Megalobrama amblycephala)by rapid amplification of c DNA ends(RACE).We also explore the expression of MSTN gene in embryos,adult tissues and the overexpression of MSTN gene.The results showed that the full length of MSTN gene is 2187 bp,its open reading frame is 1128 bp and encodes 376 amino acids.The isoelectric point of MSTN precursor protein is 5.02 and the relative molecular mass is 92.34 KDa.The MSTN gene encodes a protein containing two TGF-? protein domains,as well as the RXXR protease hydrolysis site RIRR;nine conserved cysteine residues.RT-PCR analysis demonstrated that MSTN are extensively expressed in tissues of blunt snout bream.Its expression level in the muscle,brain and testis is the highest;secondly in the liver,spleen,and ovary;lowest in intestine,gills,heart,eye,and kidney.During embryos development,the expression of MSTN m RNA is low from 0hpf to 44 hpf,whereas the expression level increases gradually from 48 hpf to 52 hpf.Using whole mount in situ hybridization demonstrated that MSTN m RNA was transcribed at different tissues of blunt snout bream's embryos.MSTN m RNA was detected at the Notochord at 16 hpf.In 28 hpf and 55 hpf embryos,the MSNT m RNA signal was strong in brain.Overexpression of MSTN m RNA in embryos can cause embryo abnormal,which results in elongated anterior-posterior axis,shorter dorsal-ventral axis,slightly distorted notochord,and strongly inhibited somites.The aim of this study is to research the MSTN gene expression in the organization,embryo and MSTN gene expression effect on embryonic development,for subsequent blunt snout bream MSTN gene function research and provide reference for molecular breeding of blunt snout bream.Heparin-binding growth factor(Midkine,Mdk)belongs to the PTN family,which consists of Mdk and PTN(pleiotrophin).Mdk is a growth factor that plays an important role in cell growth,differentiation and migration.Mdk also involved in the growth and repair of different tissues,especially nerve tissue.The full length of Mdka gene was 1409 bp,its open reading frame is 441 bp and encodes 146 amino acids,including 22 amino acid signal peptide and 124 amino acid mature peptide.The isoelectric point of Mdka precursor protein is 9.57 and the relative molecular mass is 15.73 KD.The full length of Mdkb gene was 1408 bp,its open reading frame is 448 bp and encodes 147 amino acids,including 21 amino acid signal peptide and 126 amino acid mature peptide.The isoelectric point of Mdka precursor protein is 9.41 and the relative molecular mass is 16.23 KD.Similar to humans,mice and zebrafish,blunt snout bream Mdka/-b mature polypeptides also contain 10 conserved cysteine residues,one arginine residue,two glutamine residues,one highly conserved of the hinge region and two base residues.Blunt snout bream Mdka and Mdkb genes share a sequence identity of 64% in the mature peptide.The amino acid sequence identities between blunt snout bream Mdka,Mdkb and human Mdk were 57% and 55%,respectively.The amino acid sequence(87%)of Mdka between blunt snout bream and zebrafish was markedly lower than that(95%)of Mdkb between blunt snout bream and zebrafish.Real-time quantitative PCR(q RT-PCR)showed that Mdka m RNA was highly expressed in the brain,gonads and intestines,while Mdkb m RNA was highly expressed in all adult tissues except the skin.The results of q RT-PCR showed that Mdka m RNA is detected for 12 h after fertilization for the first time.Mdkb m RNA is first detected 8h after fertilization,4h earlier than that Mdka m RNA expression.After that,both Mdka and Mdkb m RNAs rose steadily,reached their maximum at 28 h after fertilization,and then decreased and remained stable.Whole-mount in situ hybridization demonstrated that Mdka m RNA was ubiquitous expression and the Mdkb m RNA was detected at the brain and dorsal neural at 16 hpf.At 28 hpf embryos,Mdkb m RNA was only detected in eyes,brain and tailbud,while the Mdka m RNA was also detected ubiquitous expression.At 55 hpf embryos,both Mdka and Mdkb were restrictedly expressed in the brain.During starvation,both Mdka and Mdkb m RNAs were significantly downregulated in the liver and intestine after treatment with 50 ?g of h GH per gram body weight.Mdka and Mdkb m RNAs were also downregulated in the intestine after treatment with 10 ?g of h GH,but the changeable level was different.In contrast,both Mdka and Mdkb m RNAs in the brain were found significantly up-regulated with 10 ?g of h GH.Mdka m RNA in the brain was also found significantly up-regulated with 50 ?g of h GH,while no significant difference was found in Mdkb m RNAs injected with same dose.These results suggest that duplicated Mdk may play important but divergent roles in regulating the growth and development of blunt snout bream.The triploid blunt snout bream has advantage in the growth,population yield,resistance and so on,which can quickly and efficiently be obtained from the tetraploid blunt snout bream blends with the diploid blunt snout bream,that can be create a new method for breed breeding.In this study,we investigated the effect of heat shock on the first mitochondria of blunt snout bream,in order to obtain the tetraploid blunt snout bream which can be able to stabe gentic.The results showed that the highest seedling rate was 8.33% at 30 min post fertilization with heat shock at 40.5?,the highest seedling rate was 5.65% at 36 min post fertilization with heat shock at 41?,the highest seedling rate was 3.76% at 24 min post fertilization with heat shock at 41.5?,the highest seedling rate was 4.02% at 30 min post fertilization with heat shock at 42?,all induction groups were treated for 2 min.Ploidy analysis of DNA content in the blunt snout bream which were treated by heat shock result show that,only diploid and triploid blunt snout breams are obtained,there are no tetraploid blunt snout bream.Chromosome analysis results also show that the right of ploidy analysis,the probably reson is that the loss of chromosome which induced by the process of heat shock iduced the blunt snout breams,the deep reason need further study.In the four groups,the rate of triploid blunt snout breams was the highest at 2.307% in the heat shock induced by heat shock at 42 ?.The DNA content of the triploid blunt snout bream was about 1.5 times higher than that of the normal diploid blunt snout bream.The chromosomes of the triploid blunt snout bream were 72,and the chromosomes of the triploid blunt snout bream were more than 24 chromosomes of the diploid bream.In this study,we studied the triploid blunt snout bream by the heat shock method,and provided some reference for the research of the polyploid breeding of the bream. |
PDF Full Text Request