Cloning Of Heat Shock Proteins And The Response Of Heat Shock Proteins And Immune Genes On Heat Stress In Rhynchocypris Oxycephalus

Fish as the low-grade hydrothermal vertebrate,are easily affected by water temperature.Rhynchocypris oxycephalus is a typical small fish in cold water,which is sensitive to temperature changes.Previous studies have predicted that the distribution range of this fish will shrink because of global warming,and it’s survival will be a serious problem.The study on the stress response of R.oxycephalus and temperature adaptation of the fish is of great significance.In this study,the cDNA sequences of HSP60,HSP70 and HSP90 were respectively cloned and obtained in R.oxycephalus,We analyzed the molecular characteristics of each cDNA sequence.Meanwhile,We studied the expression responses of HSPs and some related immune genes after heat stress treatment.The main experimental results are as following:1.In this study,the full length cDNA sequences of HSP60,HSP70,HSP90β1 and HSP90 β2 were obtained using the RT-PCR and RACE techniques.The full-length HSP60 cDNA is 2300 base pairs(bp),97 bp of 5 ’noncoding region,475 bp of 3’ noncoding region,1728 bp of open reading frame(ORF),encoding 575 amino acids.Theorical isoelectric point was 5.91,and the molecular weight was 61.25 kDa.Amino acid sequence analysis showed that there was a mitochondrial HSP60 tag sequence AAVEEGIVPGG(amino acid location 430-441),and GGM repeat sequence was situated in the C terminal.The total length of HSP70 cDNA is 2238 bp,including 43 bp of 5’ noncoding region,272 bp of 3’ noncoding region,1923 bp of open reading frame encoding 640 amino acids.Theorical pI is 5.35,and molecular weight is 70.27 kDa.The label sequences of IDLGTTYS,IFDLGGGTFDVSIL,IVLVGGSTRIPKIQK(9-16,197-210,334-348)in amino acid sequence were detected in HSP70 family.The cytoplasmic HSP70 characteristic motif EEVD(637-640),and a signal sequence KRKHKKDISDNKRAVRRL(246-263)were found in HSP70,and a GGMP(612-615)of four peptide sequence was found in C terminal.The full length cDNA sequences of HSP90β1 is 2657 bp,including 86 bp of 5 ’noncoding region,381 bp of 3’ noncoding region,2190 bp of open reading frame,which encodes 729 amino acids,and the predicted isoelectric point is 4.89,and molecular weight is 83.78 kDa.One label sequence of YSNKEIFLRE(32-41)HSP90 family and with double parting RRAPFDLFENKKKKN approved a signal sequence(340-354),cytoplasmic characteristic motif MEEVD(725-729)were detected in protein sequences.The full length cDNA sequences of HSP90β2 is 2674 bp,containing 86 bp of 5’noncoding region,413 bp of 3’ noncoding region,2175 bp of open reading frame,encoding 724 amino acids.The predicted pI is 4.89,and the molecular weight is 83.2 kDa.One label sequence YSNKEIFLRE(32-41),a signal of RRAPFDLFENKKKKN(335-349),and the cytoplasmic motif MEEVD(719-724)were detected in HSP90β2.2.The experimental fish was randomly divided into 4 groups(11℃,16℃,21℃,and 26℃),and the 11℃ group was set as the control,sampling the tissue of liver,gill,brain,and spleen in each group of three parallel in 0,1,3,6,12,24,48 and 96 h respectively,After the heat stress of different treatment groups was 96 h,the heat stress recovery of 72 h was also tested,determination mRNA expression level of HSP60,HSP70 and HSP90β1,HSP90β2,C3,NF-κB2 and MHCIIB.The results are fiowing:Heat shock protein gene HSP60,HSP70,HSP90β1,and HSP90β2 were presented with different expression changes after heat stress treatment.Under the 16℃ stress,all genes are expressed with a certain degree of increase,under mild heat stress,with extended stress time,the expression quantity rising of heat shock protein gene.In the 21℃ and 26℃ heat stress groups,the expression level trend of the HSP60,HSP70,HSP90β1 and HSP90β2 in the different tissues is rising in the initial stage and decline later,these genes are involved in protecting the body from harm and play an important role in the protection mechanism,especially the HSP70 expression.The expression of different tissues and genes is specific.In the 26℃ heat stress,the highest expression of HSP60β is spleen,HSP70 has the highest expression in the liver,and the highest expression of HSP90β1 and HSP90β2 is gill.HSP60’s expression peak first appeared in the spleen and the brains,expression of HSP70 in brain came first,the members of two HSP90β and the earliest appear highest expression in the spleen,but the four genes expressed in liver peak appeared relatively late;Recovering after heat stress,each treatment group,the group of 26℃ HSP60,HSP70 expression in the liver and HSP90β1 and HSP90β2 expression in the brain has not been recovered to the untreatment stage level.These specific manifestations are related to the tolerance of heat stress and the physiological functions of these tissues.Under in 16℃ heat stress group,the expression of C3,NF-κB2 and MHCⅡB,there is no obviously changed,but in 21℃ and 26℃ heat stress group,the three genes are raised in a certain degree,especially of 26℃ group obviously increased was detected,the results illustrate that these genes are involved in the body’s defense under heat stress process,strengthen the body’s immunity.