| Mastacembelus armatus belongs to the genus Mastacembelus in the family mastacembeles.In recent years,due to overfishing,the wild population of M.armatus gradually shrank.The research on the artificial breeding of M.armatus has made slow progress,and the research on the internal molecular mechanism of the growth of M.armatus is rare.In order to further improve the application value and increase the basic molecular data for the subsequent artificial breeding of the M.armatus.In this experiment,homologous cloning and RACE method were used to clone the growth related genes(ghrh,gh,igf-1,igf-2,mstn)of M.armatus.The expression of these genes in the tissue,embryo and postembryonic development period was studied.At the same time,the effects of in vivo injection of different concentrations of GHRH polypeptide,LHRH-A2 and RNA interference ghrh on the expression of ghrh,gh,igf-1,igf-2,and mstn m RNA were also studied..It laid a theoretical foundation for elucidating the regulatory mechanism among the growth genes of M.armatus.The main results of this paper are as follows:1.The full-length c DNA of ghrh,gh,igf-1,igf-2 and mstn genes of M.armatus was 1090 bp,815 bp,2989 bp,1446 bp and 2690 bp.The development reading frame(ORF)was 429 bp,615 bp,558 bp,648 bp and 1331 bp.They encoded 142,204,185,215 and 376 amino acids.5 '-utrs are 121 bp,33 bp,305 bp,117 bp and 200 bp.The 3 '-utrs are 540 bp,167 bp,2126 bp,681 bp and 1359 bp.By software analyzed GHRH 19 amino acid to amino acid sequences of signal peptide,27 amino acid composition of domain Hormone?2 mature peptide family structure.GH amino acid has a signal peptide composed of 17 amino acids and a conserved 4 cysteine residues(Cys).IGF-1 amino acid has a signal peptide composed of 43 amino acids and an IIGF family domain composed of 56 amino acids.IGF-2 amino acid has a signal peptide composed of 52 amino acids,an IIGF family domain composed of 58 amino acids and an IGF2?C domain composed of 56 amino acids.MSTN has a signal peptide composed of 22 amino acids and 9 Cys.The results of homology analysis showed that the sequence consistency of GHRH,Micropterus salmoides,Epinephelus coioides and Perca flavescens were more than 90%.The homology of M.armatus GH was higher with Siniperca scherzeri,Lateolabrax japonicus and Monopterus albus.They were 95.59%,95.07% and 92.16%.The sequence homology of IGF-1 from other vertebrates was 48.37% ? 94.05%,and the sequence consistency with Acanthopagrus latus,E.coioides and Scatophagus Argus was over 90%.The sequence homology of IGF-2 from other vertebrates was 44.44% ? 92.09%,among which the sequence consistency of S.Argus was the highest(90.09%).The homology of MSTN was higher than that of Lates calcarifer,Larimichthys crocea and S.scherzer.They were 94.68%,93.09% and 92.55%.Phylogenetic analysis of all amino acids showed that the M.armatus was clustered in a large branch with M.albus and Perciformes.The results showed that the M.armatus was closely related to M.albu and Perciformes.2.The expressions of ghrh,gh,igf-1,igf-2,and mstn m RNA in brain,muscle,heart,liver,kidney,spleen,stomach,intestine,eyes,gill,ovary,and testis of M.armatus were detected by real time q PCR.The results showed that ghrh m RNA was only highly expressed in brain tissues,and the other tissues were extremely low.gh m RNA expression was highest in the brain,followed by liver,muscle,gonad and heart,and lowest in the intestine.Both igf-1 m RNA and igf-2 m RNA were highly expressed in the liver of M.armatus,and a small amount was also expressed in other tissues.mstn m RNA expression was the highest in muscle,followed by eyes,brain,gonads and heart,and the lowest in gills.3.The expressions of ghrh,gh,igf-1,igf-2 and mstn m RNA in the embryonic and postembryonic development of M.armatus were detected by real time q PCR.The results showed that the expression of ghrh m RNA was very low in each stage of embryonic development,and almost no expression.gh m RNA expression was detected in every stage of embryo development,and the expression level was higher in blastocyst stage,followed by that in protoenteric embryo stage,and expression levels were lower in sarcomere stage and membrane stage.gh gene expression in postembryonic development was relatively stable with no significant difference.The expression of two kinds of igf m RNA in the embryo of M.armatus was expressed in each developmental stage,and the expression of igfs m RNA in each postembryonic development stage was gradually increased.The expression of mstn m RNA was detected in embryo and postembryonic development and the expression level was higher in blastocyst stage,followed by that in protoenteric embryo stage,and the expression level of mstn m RNA was lower in sarcomere stage and membrane stage.Postembryonic development showed a gradually increasing trend.4.The injection of GHRH polypeptide had an inhibitory effect on the expression of ghrh m RNA in the brain and an obvious promoting effect on the expression of gh m RNA in the brain.The expression of igf-1 m RNA and igf-2 m RNA in liver was significantly enhanced by medium and high concentration of GHRH polypeptide.GHRH polypeptide had little effect on mstn m RNA expression level of M.armatus.The above results indicate that the influence of GHRH on gh and igfs expression is obvious in a short time.5.LHRH-A2 injection had no significant effect on the expression of ghrh m RNA in the brain of M.armatus.LHRH-A2 injection had a significant effect on the expression of gh m RNA in the brain.LHRH-A2 injection promoted the expression of igf-1 m RNA in the liver s,with no significant difference(P > 0.05).The injection of LHRH-A2 promoted the expression of igf-2 m RNA in the liver.LHRH-A2 had little effect on mstn m RNA expression level of muscle.The above results indicate that LHRH-A,as A Gn RH analogue,has A certain influence on the expression of gh and igfs.6.After the interference of GHRH si RNA the transcriptional level of ghrh gene was decreased.Which had a significant inhibitory effect on the expression of gh gene.The expression of gh gene was lower than that of the control group,and the expression levels of igf-1 m RNA and igf-2 m RNA were lower than that of the control group.There was no significant difference between the expression levels of mstn m RNA and the control group after the injection of GHRH si RNA in the M.armatus for 3 hours to 12 hours. |
PDF Full Text Request