Cloning Of Grass Carp MAPKAPK2,Hsp27,ACTA1 Gene And Effects Of Feeding Broad Bean On P38MAPK-Hsp27-Actin Signaling Pathway

Exp.1: Fragment Cloning,Bioinformatics Analysis And Tissue Differential Expression Of MAP Kinase-Activated Protein Kinase 2(MAPKAPK2)Gene In Grass CarpMitogen-activated protein kinase-activated protein kinase 2(MAPKAPK2)encoded by MAPKAPK2 gene,is an important phosphorylation substrate for p38 MAPK.The sequence of MAPKAPK2 gene was cloned from the grass carp(Ctenopharyngodon idellus),and the protein encoded by MAPKAPK2 gene fragment was constructed by bioinformatics method.The phylogenetic tree was constructed and the expression of MAPKAPK2 m RNA in grass carp was detected by real-time fluorescent quantitative PCR.The results showed that the grass carp MAPKAPK2 gene fragment was 968 bp long and the start codon was ATG.The amino acid sequence of grass carp MAPKAPK2 had 99%,99%,94%,78%,and 78%,respectively,homology with carp,zebrafish,large yellow croaker,mouse and human.Real-time quantitative PCR showed that MAPKAPK2 gene was expressed in liver,spleen,brain,kidney,gill,muscle,heart,skin,foregut,and blood of grass carp.It was highly expressed in heart,blood,and brain,while relatively high in spleen,muscle,and kidney,and low expression in liver,skin,foregut and sputum(P<0.05).Exp.2: Full-length cloning,bioinformatics analysis and tissue differential expression of heat shock protein 27(Hsp27)gene in grass carpHeat shock protein 27(Hsp27)is a small molecule heat shock protein with molecular chaperone properties that can bind to actin and regulate actin cytoskeletal reorganization.In this paper,the full-length c DNA sequence of grass carp Hsp27 was obtained by rapid amplification of c DNA ends(RACE).The characteristics of the protein encoded by Hsp27 were analyzed and predicted by bioinformatics methods.The quantitative expression of Hsp27 m RNA in different tissues of grass carp was detected by quantitative real-time PCR.The results showed that the full-length Hsp27 gene of grass carp was 1271 bp.The sequence contained a 5' non-coding region of 417 bp,a 3' non-coding region of 317 bp and a 537 bp open reading frame encoding 178 amino acids.The amino acid sequence of grass carp encoded by Hsp27 gene had 83%,76%,75%,and 70%,respectively,homology with crucian,zebrafish,channel catfish,and Mexican tetra.Hsp27 was expressed in liver,spleen,brain,kidney,gill,muscle,heart,skin,foregut,and blood.It was highly expressed in heart and muscle(P<0.05),and low expression in other tissues such as liver.Exp.3: Full-length cloning,bioinformatics analysis and tissue differential expression of grass skeletal muscle actin(ACTA1)gene?-skeletal muscle actin,encoded by ACTA1 gene,is one of the six identified actin isoforms.Actin is a highly conserved protein involved in cell movement,structure,and integrity,and ?-actin is a major component of muscle contraction.In this paper,the full length sequence of grass carp ACTA1 gene was obtained by rapid amplification of c DNA ends(RACE),and bioinformatics analysis and protein character analysis were performed on the obtained sequence and predicted amino acid sequence accordingly.The differential expression of ACTA1 gene m RNA in tissues of grass carp was detected.The results showed that the grass carp ACTA1 gene was 1553 bp in length,and the sequence contained a 443 bp 5' non-coding region,a 111 bp 3' non-coding region,and a 999 bp open reading frame encoding 332 amino acids.The amino acid sequence of grass carp ACTA1 was 99% homologous with that of carp,zebrafish,rainbow trout,Uganda red marmoset and house mouse.Real-time quantitative PCR showed that the ACTA1 gene was expressed in liver,spleen,brain,kidney,gill,muscle,heart,skin,foregut and blood of grass carp,and the expression level in heart was significantly higher than that in other tissues(P<0.05),followed by a relatively high expression level in muscle and skin.Exp.4: Effects of feeding broad bean on p38MAPK-Actin signaling pathway and expression of myofibrillar assembly genes in muscle and heart tissues of grass carpIn this research,two experimental groups were set up,each group consisted of three parallels,each parallel contained 15 grass carp.The grass carp of broad bean group were fed with soaked broad bean(dried broad bean immersed in fresh water for 24 hours),and the control group was fed with normal compound feed for 12 weeks.Samples of muscle and heart of grass carp were collected at 0,4,8,and 12 weeks,respectively.Relative m RNA expression levels of p38 MAPK,MAPKAPK2,Hsp27 and ACTA1 genes in these tissues were detected.The results showed that the relative m RNA expression of each gene in the broad bean group was higher than that in the control group,indicating the relative expression of p38MAPK-Hsp27-Actin signaling pathway in muscle could be enhanced to different degrees.In the tissue of muscle,the relative expression of p38 MAPK and MAPKAPK2 m RNA peaked at 8 week,and decreased at 12 week.The relative expression of Hsp27 and ACTA1 increased throughout the feeding period.In cardiac tissues,the relative expression levels of p38 MAPK,MAPKAPK2,Hsp27 and ACTA1 peaked at different weeks.P38 MAPK and MAPKAPK2 peaked at 4 week,while Hsp27 and ACTA1 peaked at 12 week.The above results indicated that the feeding of broad bean to grass carp could enhance the relative expression of m RNA of p38MAPK-Hsp27-Actin signaling pathway in muscle tissue and heart tissue,which increased the synthesis of actin and enhanced the assembly of myofibrils.