Cloning And Expression Analysis Of Some MAPK Family Members In Large Yellow Croaker

Temperature alteration limits the survival,distribution and reproduction of fish,and induces to massive outbreaks of desease,resulting in a bad influence on fish farming industry.Large yellow croaker(Larimichthys crocea),as an important member in mariculture industry,suffers from frenquncy pathogen infection induced by temperature alteration,restricting the yield of large yellow croaker.We start the research from temperature stimuli and pathogen infection to clarify the mechanism inresponse to environmental stress and pathogen infection for large yellow croaker,which is significant in improving the yield of large yellow croaker.In the present study,the full length of cDNA sequences of JNK1,JNK2,JNK3,p38,ERK2 and ERK5 were cloned from large yellow croaker by RACE-PCR and RT-PCR.Their molecular characterization and structures were analyzed.The expression profiles in untreatment tissues and in immune challenge,including LPS,Poly I:C,PGN and flagellin treatment LCK(Larimichthys crocea kidney)cell line were also detected.The expression profile in temperature stimuliated LCK cells was also described.The combinant vectors with pEGFP were built to investigate the subcellular localization of the MAPK members in LCK cell.The phosphorylation of p38 in temperature stimulated cells and inhibitor incubated cells and the down-stream factor of p38 MAPK signal pathwy Hsp27 transcripts alteration in above cells was also analyzed.The obtained results can described as follows:(1)The JNK subfamily genesThe full-length cDNA sequence of LcJNK1 was 1970 bp,containing a 386 bp 3' untranslated region(UTR),a 249 bp 5'UTR,a 1335 bp open reading frame(ORF)which could encode 444 amino acids.The predicted molecular weight of JNK1 amino acids was 49.57 KD,with isoelectric point of6.57,suggesting an acidic protein.JNK1 amino acids were containing a conserved S_TKc domain and dual phosphorylation sites TPY,which were key catalyze structures in JNK.The expression of LcJNK1 were detected in most tissues of large yellow croaker,with the most predominant expression in gill,stomach,instestine and muscle,and the weakest expression of LcJNK1 in spleen,headkidney and liver.The expression levels of LcJNK1 after LPS,PGN,poly I:C and flagellin challenge and temperature stress were investigated in LCK cells.After immune challenge,significant up-regulations of LcJNK1 transcripts were detected after poly I:C and flagellin challenge(p<0.05),whereas LcJNK1 transcripts increased significantly after LPS and PGN challenge(p<0.01).However,after temperature stress,no significant chagne of LcJNK1 expression were detected at most time points after both cold and heat stress.Subcellular localization revealed that LcJNK1 expressed in the cytoplasm and cell nucleus.Our results showed that LcJNK transcripts enhanced after poly I:C and flagellin challenge and no significant alteration after temperature stess.These findings indicated that LcJNK1 might play an important role in fish immune response.The full-length cDNA sequence of LcJNK2 was 2436 bp,containing a 983 bp 3' UTR,a 193 bp 5'UTR and a 1260 bp ORF encoded 444 amino acids.The predicted molecular weight of JNK2 amino acids was 47.59 KD,with predicted isoelectrict point of 5.07,suggesting an acidic protein.JNK2 amino acids were containing a conserved S_TKc domain and dual phosphorylation sites TPY,which were key catalyze structures in JNK.The expression of LcJNK2 were detected in most tissues of large yellow croaker,with the most predominant expression in blood,brain,kidney and skin,and the weakest expression of LcJNK2 inmuscle,intestine and heart.The expression levels of LcJNK2 after LPS,PGN,poly I:C and flagellin challenge and temperature stress were investigated in LCK cells.After immune challenge,significant up-regulations of LcJNK2 transcripts were detected after LPS and PGN challenge(p<0.01)whereas LcJNK2 transcripts increased significantly after poly I:C challenge(p<0.01).No significant chagne of LcJNK2 expression were detected at most time points after flagellin.However,tiny up-regulation chagne of LcJNK2 expression were detected after cold stress,but no significant.Tiny down-regulation chagne of LcJNK2 expression were detected after heat stress(p<0.05).Subcellular localization revealed that LcJNK2 expressed in the cytoplasm and cell nucleus.Our results showed that LcJNK2 transcripts enhanced after LPS and PGN challenge and down-regulation after heat temperature stess.These findings indicated that LcJNK2 might play an important role in fish immune response and heat stress.The full-length cDNA sequence of LcJNK3 was 1669 bp,containing a 126 bp 3' UTR,a 122 bp 5'UTR,a 1422 bp ORF encoded 473 amino acids.The predicted molecular weight of JNK3 amino acids was 53.63 KD,with isoelectric point of 6.86,suggesting an acidic protein.JNK3 amino acids were containing a conserved S_TKc domain and dual phosphorylation sites TPY,which was the unique catalyze structure in JNK.The expression of LcJNK3 was detected with the most predominant expression in brain,skin and gill.The expression levels of LcJNK3 after LPS,PGN,poly I:C and flagellin challenge and temperature stress were investigated in LCK cells.After immune challenge,significant up-regulations of LcJNK3 transcripts were detected after poly I:C and flagellin challenge(p<0.01)whereas LcJNK3 transcripts decreased significantly after LPS and PGNchallenge(p<0.05).However,up-regulation chagne of LcJNK3 expression were detected after cold stress(p<0.05).No significant chagne of LcJNK3 expression were detected after heat stress.Subcellular localization revealed that LcJNK3 expressed in the cytoplasm and cell nucleus.Our results showed that LcJNK3 transcripts enhanced after poly I:C and flagellin challenge and up-regulation after cold temperature stess.These findings indicated that LcJNK3 might play an important role in fish immune response and cold stress.(2)p38 geneThe full-length cDNA sequence of Lcp38 was 1716 bp,containing a 488 bp 3' untranslated region(UTR),a 143 bp 5'UTR,a 1086 bpORF which encoded 361 amino acids.The predicted molecular weight of p38 amino acids was 41.76 KD,with isoelectric point of 5.4,suggesting a acidic protein.p38 amino acids were containing a conserved S_TKc domain and dual phosphorylation sites TGY,ED site,substrate binding site,which were the key catalyze structure in p38.The expression of Lcp38 was detected with the most predominant expression in intestine,with the weakest expression in gill.The expression levels of Lcp38 after LPS,PGN,poly I:C and flagellin challenge and temperature stress were investigated in LCK cells.After immune challenge,significant up-regulations of Lcp38 transcripts were detected after LPS,PGN poly I:C and flagellin challenge(p<0.01).Both cold stress and heat stress can significantly up-regulate the Lcp38transcripts(p<0.01).Subcellular localization revealed that LcJNK3 expressed in the cytoplasm and cell nucleus.Both cold and heat stress can activate the phosphorylation of p38 in LCK cell(p<0.01),whereas inhibitor can significantly decrease the phosphorylation of p38(p<0.05).The transcripts of p38 MAPK signal pathway down-stream Hsp27 was detected in temperature stress and inhibitor incubated LCK cells.Cold stress can up-regulate the expression of Hsp27 in LCK,and inhibitor can significanty decrease the expression of Hsp27(p<0.01).However,heat stress and inhibitor have no significant influence on Hsp27.Both cold and heat stress can up-regulate the expression of caspase3(p<0.05),and inhibitor also could suppress the expression of caspase3 stimulated by cold and heat stress(p<0.01).Our results showed that Lcp38 transcripts enhanced after immune challenge and temperature stess.These findings indicated that Lcp38 might play an important role in fish immune response and temperature stress.(3)ERK2 geneThe full length cDNA sequence of LcERK2 was of 1910 bp,including an ORF of 1110 bp encoding a polypeptide of 369 amino acids.The predicted molecular weight of LcERK2 amino acids was 42.1 KD,with isoelectric point of 6.19,suggesting a acidic protein.LcERK2 contained highlyconserved TEY motif,HRD catalytic site,SPS,Gly-rich region and S_TKc domain in MAPK family.The expression of LcERK2 was detected in most tissues of large yellow croaker,with the most predominant expression in brain and the weakest expression of LcERK2 in liver.The expression levels of LcERK2 after temperature stress and poly I:C and flagellin challenge were investigated in LCK cells.Significant up-regulation of LcERK2 transcripts was found after immune challenge(p<0.05).Significant down-regulations of LcERK2 transcripts were detected after 10 oC stress(p<0.05)whereas LcERK2 transcripts increased significantly after 35 oC stress(p<0.05).Subcellular localization revealed that LcERK2 expressed in the cytoplasm and cell nucleus.Our results showed that LcERK2 transcripts enhanced after heat stress and LcERK2 transcripts could be induced by immune challenge.These findings indicated that LcERK2 might be more important in fish response to high temperature stress and in fish immune response.(4)ERK5 geneThe full length cDNA sequence of LcERK5 was of 3720 bp,including a 5'UTR of 103 bp,a3'UTR of 264 bp,an ORF of 3375 bp encoding a polypeptide of 1124 amino acids.The predicted molecular weight of LcERK5 amino acids was 123.27 KD,with isoelectric point of 6.6,suggesting a acidic protein.LcERK5 contained highly conserved TEY motif,HRD catalytic site,NLS,MEK5 binding site and S_TKc domain in MAPK family.The expression of LcERK5 was detected in most tissues of large yellow croaker,with the most predominant expression in gill and the weakest expression in intestine.The expression levels of LcERK5 after temperature stress and poly I:C and flagellin challenge were investigated in LCK cells.Significant up-regulation of LcERK5 transcripts was found after immune challenge(p<0.05).However,significant down-regulations of LcERK5 expression were detected at most time points after both cold and heat stress(p<0.05).Subcellular localization revealed that LcERK5 expressed in the cytoplasm and cell nucleus.Our results showed that LcERK5 transcripts could be induced by immune challenge and be inhibited by temperature stress.These findings indicated that LcERK5 might be more important in fish response to immune challenge.