Research Of Molecular Mechanisms Of Heat Shock Response And Heat-resistance SNP In Sea Cucumbers Apostichopus Japonicus

The sea cucumber Apostichopus japonicus has been exploited as a very important economic species in China for its nutritional value and medical properties. Water temperature is a key environmental factor for A. japonicus, and high temperature restricts their normal growth and metabolism. In recent years, rapid temperature increase or extreme high temperature in summer has led to widespread mortality of A. japonicus in the northern coasts and many aquacultural ponds of China, resulting in dramatic resource decline and enormous economic loss. Therefore, it has significant meaning to understand the heat shock response in A. japonicus and breed heatresistance variety.In our study, we used i TRAQ sequencing, Western blot, Real-time PCR et al. to study the heat shock response in sea cucumbers, and screened SNP markers associated with heat-resistance trait from the sequences of hsp90 in A. japonicus. We investigated histological, ultrastructural and HSP70 responses to heat stress in the intestine of A. japonicus. We applied the i TRAQ technique to investigate the global protein expression profile under an acute short-term(48 h) heat stress. Furthermore, we cloned the c DNAs of key genes(hsf1, hsbp1, hsp10 and hsp60) involved in heat shock response, and analyzed their expression under heat stress. We also screened SNP markers associated with heat-resistance trait from the sequences of hsp90, which had been used in marker assisted selection of A. japonicus. Main results are as follows:1. Histological, ultrastructural and heat shock protein 70(HSP70) responses to heat stressHistological, ultrastructural and HSP70 responses to heat stress were investigated in the intestine of A. japonicus. Tissue degradation was observed in muscular, submucosal and mucosal layers, with significant decrease in plicae circulares of the mucosal layer. Ultrastructural damage intensified with increasing stress time, and indicators of cell apoptosis were evident after 192 h heat stress. Immunostaining showed HSP70 mainly in mucosa and serosa, with faint staining in non-stressed individuals(the control group) and denser staining under stress(the 6, 48 and 192 h groups). Western blot detection confirmed ocurrence of HSP70 in all groups and significant up-regulation under stress. The rapid and persistent response of HSP70 implies its critical role in the heat shock response of A. japonicus.2. Understanding the heat shock response using i TRAQ-based proteomicsWe applied the i TRAQ technique to investigate the global protein expression profile under an acute short-term(48 h) heat stress. Totally 3,432 proteins were identified, and 127 proteins showed significant heat stress responses, with 61 upregulated proteins and 66 downregulated proteins. Our results suggest that heat stress influenced the expression of proteins involved in various biological processes, such as tissue protection and detoxification, lipid and amino acid metabolism, energy production and usage, transcription and translation, cell apoptosis, and cell proliferation. These findings provide a better understanding about the response and thermo-tolerance mechanisms of A. japonicus under heat stress.3. Molecular cloning of key genes c DNAs and their expression analysis under heat stress(1) We obtained full-length c DNAs of hsf1 and hsbp1 in A. japonicus for the first time. Analysis by real-time PCR showed the expression of hsf1, hsbp1 and hsp70 in the intestine responded significantly at 26 oC. The m RNA expression of hsf1, hsbp1 and hsp70 increased at first, and all peaked at 6 h with a peak value of 11.67-fold, 4.71-fold and 955.30-fold repectively. Our results showed that the heat shock response(HSR) in A. japonicus is a rapid and transient gene-expression program. We assumed that hsf1 played an important role in the regulation of HSR, and hsbp1 and hsp70 repressed HSR by negatively affecting the activity of hsf1.(2) We obtained full-length c DNAs of hsp10 and hsp60 in A. japonicus for the first time. Analysis by real-time PCR showed that their m RNA expression showed a time-dependent response with a similar tendency. Their m RNA expression both showed a significant increasement at 2 h, and reached the peak at 8 h. The peak value of hsp10 and hsp60 expression was 193.3-fold and 60.0-fold respectively, compared with that in control group. The results suggested hsp10 and hsp60 were involved in the heat-shock response in A. japonicus.4. Polymorphisms of hsp90 and their association with heat-resistanceWe identified 12 exons and 11 introns in the sequences of gene hsp90 in A. japonicus. Twenty-two single nucleotide polymorphisms(SNPs), including three nonsynonymous mutations, were detected in the exons. Susceptible and resistant individuals were distinguished using a high-temperature(32 ?C) challenge experiment. Five of the twenty-two SNPs were shown to be significantly associated with susceptibility/resistance to high temperature by correlation analysis(χ2 test). To confirm the importance of these five SNPs, a heat-resistance strain(HRS) was selected through three generations. It was shown that the distributions of genotypes and alleles of SNP e10-1 and e11-6 were significantly different between the two groups. SNP e10-1 was trimorphic, with three alleles(A, C and T) and five genotypes(AA, CC, AT, CT and AC). The allele frequency of SNP e2-3 was also significantly associated with this trait. This is the first demonstration of SNPs related to heat-resistance in A. japonicus and supports the use of SNP markers in the selective breeding of sea cucumbers.