Molecular Cloning Of Three Heat Shock Protein Genes And Their Physiological Adaption Characteristics To Environmental Stresses

In the present study,the full-length c DNA sequences encoding hsp 70,hsp 90α and hsp 90β genes in Seriola lalandi were obtained by using homologous cloning and RACE methods.The tissue distribution of the hsp 70,hsp 90α and hsp 90β genes and their temporal expression patterns during early life history stages were determined.Furthermore,the response of these three genes to heat stress,LPS induction stress and culture density stress were investigated.The present results could help understand the possible physiological functions of hsp 70,hsp 90α and hsp 90β genes in Seriola lalandi.1.Cloning and spatial expression patterns of hsp70,hsp90α and hsp90β genes in Seriola lalandiThe full-length cDNA sequences encoding hsp 70,hsp 90α,and hsp 90β genes were cloned from Seriola lalandi.The c DNA sequence of the hsp 70 gene is 2332 bp in length,with 5 ′ UTR is 187 bp,open reading frame(ORF)is 1920 bp and 3 ′ UTR is225 bp,encoding 639 amino acids,the protein molecular weight of 70.1 k Da and an isoelectric point of 5.16.The c DNA sequence contains three typical hsp70 family tag motifs including IDLGTTYS,IFDLGGGTFDVSIL and IVLVGGSTRIPKIQK,with a non-organelle-characterized protein motif of RARFEEL and a characteristic cytoplasmic localization motif of EEVD located at the C-end.The cDNA sequence encoding hsp 90α gene is 3246 bp in length,wherein the 5 ′UTR is 732 bp in length,the ORF is 2193 bp and 3 ′ UTR is 321 bp in length,encoding730 amino acids.The protein has a molecular weight of 84.3 k D a and an isoelectric point of 4.62.As for hsp 90β,the c DNA sequence is 3270 bp long,with the 5 ′ UTR is567 bp long,the ORF is 2178 bp long and the 3 ′ UTR is 525 bp long,encoding 725 amino acids,with a protein molecular weight of 83.29 k Da,and an isoelectric point of4.64.Both hsp 90α and hsp 90β sequences contain a MEEVD motif at C-end,and a conserved motif of YSNKEIFLRE,LGTIAKSGT,IGQFGVGFYSAYLVAE,IKLYVRRVFI,and GVVDSEDLPLNISRE.The spatial expression of hsp70 m RNA exhibited sex dimorphism pattern,wherein the significant high expression levels of hsp70 m RNA were found in gill,heart,spleen and ovary in female with the maximum expression level in ovary,whereas in male,the significant high expression levels of hsp70 m RNA were found in pituitary,gill,head kidney and testis with the maximum expression level in testis.The hsp 90α and hsp 90βexhibited similar expression patterns.In female Seriola lalandi,both hsp 90α and hsp90β expression levels were significantly higher than other tissues,followed by gills,kidney,head,kidney and intestine tissues,and the lowest expression level was observed in muscle tissue.In male Seriola lalandi,both hsp 90α and hsp 90β had the highest expression in testis,followed by kidney,head kidney,intestine tissue,and the lowest expression level was observed in muscle tissue.The hsp 90α and hsp 90β both exhibited sex dimorphism patterns.2.The temporal expression patterns of hsp 70,hsp 90α and hsp 90β during early life stages in Seriola lalandiDuring the embryonic development of Seriola lalandi,the hsp70 m RNA was detected in fertilized eggs before cleavage,indicating that hsp70 gene has the characteristic of parental inheritance.Meanwhile,the hsp70 m RNA could be detected at all stages of embryonic development,wherein the low expression levels were observed before low blastula stage,and significantly increased at early gastrula stage and maintain high level until hatching stage.The significant high levels of hsp70 m RNA were observed at 1d larva,then a significant decrease at 4 d larva,followed by a significant increase in 15 d larva but significantly decreased at 20 d larva and maintained until 60 d juveniles.The results from the present study could provide insights into the origin and physiological function of hsp70 during early life stages of Seriola lalandi.The hsp 90α m RNA and hsp 90β m RNA showed similar expression trend during embryonic development,maintain relatively low expression levels before the blastocyst stage,and then significantly upregulated expressed after late gastrulation,and both peaked in the embryos hatching stage.The expression changes of hsp 70,hsp 90α and hsp 90β m RNA levels in larvae and juveniles were also examined.hsp 90α significantly upregulated in 7d larvae(P <0.05),then it began to gradually decline,and then began to rise in 15 d larvae until peaked at 25 d juveniles;hsp 90β m RNA also significantly upregulated at 7d larvae(P <0.05),and maintained at high expression levels until 30 d juveniles,then the expression level began to decline gradually.The results indicated that these three hsp gene all play important roles during early life stages of Seriola lalandi.3.The adaptive expression characteristics of hsp70、hsp90α and hsp90β of Seriola lalandi and the effects on SOD,ACP and AKP enzymes in response to heat stressThe acute and gradual high-temperature stress experiments were designed to investigate the responses of hsp70,hsp90α and hsp90β of Seriola lalandi to heat stress.High temperature exposure significantly induced the expression of hsp70,hsp90α,and hsp90β in the Seriola lalandi,but it was time-dependent and tissue-specific.Under high temperature stress,the expression levels of hsp70、hsp90α and hsp90βm RNA in liver and kidney all firstly sharp increased and then decreased.However,liver hsp70 and hsp90β m RNA levels showed more sensitive responses to acute heat stress,whereas the kidney hsp70 and hsp90β m RNA levels showed less sensitive responses.When under gradual high-temperature stress,the kidney hsp70 and liver hsp90β showed more sensitive responses.However,as for hsp90α m RNA level,it increased sharply in short time and then decreased soon,which indicated that it is less sensitive to heat stress.The trend of SOD enzyme activity in yellow liver under heat stress was to first increase and then decreased,while the trend of ACP and AKP enzyme activity was decreased.4.The adaptive expression patterns of hsp70,hsp90α and hsp90β in response to LPS stimulation and density stress in Seriola lalandiAfter LPS stimulation,the expression levels of hsp70,hsp90 α and hsp90 βm RNA in head kidney all significantly increased at 6h post stimulation,then the hsp70 m RNA level decreased to control level at 12 h.However,the hsp90α and hsp90βm RNA levels maintained high expression levels,and decreased to control level until48 h.In liver of Seriola lalandi juveniles,the hsp70 m RNA level showed no significant difference between low-and mid-density groups,but was significant high in high-density group,and the significantly higher hsp90β m RNA level in high-density group was observed than low-density group.In the spleen tissue,the significant high expression level of hsp70 m RNA was observed in high-density group than both lowand mid-density groups.In the gill tissue,both hsp90α and hsp90β m RNA levels showed no significant difference between low-and mid-density groups,whereas both were obviously higher in high-density group.In the kidney tissue,the hsp70 m RNA level decreased with the density,and the hsp90α and hsp90β m RNA levels were exclusively high in high-density group.