Fundamental Research On The Molecular Mechanism In Response To Heat Stress In Apostichopus Japonicus (Selenka)

In order to understand the response to heat stress at the molecular level in Apostichopus japonicus, a suppression subtractive hybridization library was constructed to analyze the difference expression in the body wall of A. japonicus. The full-length cDNAs of Aphsp90 and Aphsp26 were cloned and analyzed, and the expression profiles of Aphsp90 and Aphsp26 in different tissues under different temperatures and length of times were determined by real time quantitative PCR. Further the heat tolerance of different populations was assessed.1. Suppression subtractive hybridization library was constructed to analyze the differences in gene expression in the body wall of A. japonicus induced by heat stress (28°C). The forward SSH library used the cDNA from the heat-treated group as the tester and the cDNA from the control as the driver in order to find the up-regulated genes. The reverse SSH library was used to identify the down-regulated genes, using the cDNA from the control as a tester and the cDNA from the heat-treated group as a driver.The 737 clones were proved as positive clones with bacterial colony PCR and dot hybridization, and 165 distinct sequences were obtained, with 65 sharing similarity to sequences in public databases, 100 sharing no similarity to sequences in public databases. The sequenced genes were classified into different functional categories, and it was found that increased expression of some genes in response to stress was concomitant with decreased expression of genes concerned with energy production and metabolism under heat stress in A. japonicus. It indicates that heat stress could induce the expression of some genes with the function of immunity but suppress the expression of gene concerned with metabolism.2. The genes of Aphsp90 and Aphsp26 were cloned. The full-length cDNA of Aphsp90 and Aphsp26 contains 3,458bp and 1,688bp encoding 720 and 236 amino acids separately. Multiple alignments and phylogenetic analysis indicated that the animo acid sequences of ApHsp90 and ApHsp26 shared high similarity with Hsp90 and sHSPs sequences of other species. The three-dimensional structure of ApHsp90 contains two characteristic domains of Hsp90 which indicated the same function of ApHsp90, the structure analysis of ApHsp26 showed thatα-crystallin domain which is the most conserved elements in sHSPs is presented in this sequence. It indicates the validity of these sequences.3. The expression characteristics of Aphsp90 and Aphsp26 were analyzed. The expression patterns of these two genes in different tissues of A. japonicus exposure in four temperature treatments (20, 22, 24, 26°C) were determined by real time quantitative PCR. It was found that the messenger RNA (mRNA) transcripts of these two genes varied among different tissues. Under normal condition and heat shock, the mRNA expression of these two genes was higher in intestine compared to other tissues. Heat shock could accelerate the gene expression of Aphsp90 and Aphsp26, with the increase of temperature the expression of Aphsp90 and Aphsp26 mRNA elevated, especially in the intestine of A. japonicus. It indicates that the intestine of A. japonicus is the heat-sensitive tissue.The relationship between expression level and duration of heat shock was determined, and the results showed that the level of Aphsp90 and Aphsp26 was time-dependent. The transcripts of Aphsp26 increased significantly in the first few hours, reached maximum expression after heat shock for 6h then decreased gradually and reached the pretreatment level at 48h. The mRNA level of Aphsp90 reached a peak at 4 h and remained high at 6 h under heat stress, then decreased gradually to the pretreatment level at 48 h.4. The heat tolerance of different populations of A. japonicus was analyzed. Yantai population and Zhejiang population experiencing high-temperature breeding and south breeding were chosen as research objects. The survival rate, metamorphosis rate and special growth rate of larvae in these two populations under heat stress were determined. The results showed that heat shock could influence the survival of larvae, these indexes in heat group were lower than in control group in both populations, but the larvae of Zhejiang population exhibited higher heat tolerance compared to Yantai population with higher survival rate, metamorphosis rate and special growth rate. Under heat shock the development of larvae increased, with advance into the doliolarias stage. The maximum length of larvae in Yantai population shortened under heat shock, but the variation trend was opposite in Zhejiang population. Juveniles of both populations were maintained at 32°C for 17 days to determine the survival rate. It was indicated that Zhejiang population exhibited higher survival rate than Yantai population under heat shock. Further in molecular level the difference in gene expression of Aphsp90 and Aphsp26 in these two populations under 28°C heat shock was studied. The results indicated that the transcripts of Aphsp90 and Aphsp26 mRNA in Zhejiang population were always lower than Yantai population at different time of heat shock. It indicates that the difference in heat tolerance has relationship with the expression profiles of Aphsp90 and Aphsp26 mRNA.