Identification And Expression Analysis Of Differentially Expressed Genes Of Apostichopus Japonicus (Selenka) Under High Temperature Stress

Temperature is one of the most important environmental factors influencing the grown and physiological activities of Apostichopus japonicus. In order to study the molecular regulatory mechanism of the responses to high temperature stress of Apostichopus japonicus, suppression subtractive hybridization (SSH) was used to construct forward and reverse subtractive cDNA libraries of Apostichopus japonicus under high temperature stress, some differentially expressed responsive genes were identified from the libraries and studied using real-time quantitative PCR with 2-ΔΔCT method.1 Construction and analysis of the subtracted cDNA library of Apostichopus japonicus (Selenka) under high temperature stressThe cDNA of Apostichopus japonicus underwent heat stress was used as tester and the cDNA of Apostichopus japonicus under room temperature was used as driver to construct a forward subtractive library. A reverse subtractive library was generated by using the same cDNA but underwent heat stress was the driver and untreated was tester. There were 384 positive clones randomly picked from each library and used for dot blotting. Fifty clones with significantly different signals were sequenced and analyzed by using BLAST. The results showed 44 sequences had high identities with the known genes and 3 up-regulated known genes such as heat shock protein 70, mitochondrial large subunit ribosomal RNA and protein lethal (2) essential for life and 2 down-regulated known genes such as major yolk protein 1 and proprotein convertase subtilisin/kexin type 9 were important functional genes of which. Other five clones were unknown gene sequences.2 Different expression analyses of five genes among different tissues, under different temperature stresses and at different timesReal-time quantitative PCR and the 2-ΔΔCT method were used to study the expressions of 5 differentially expressed genes among the different tissues (body wall, longitudinal muscles, alimentary tract and respiratory tree) of Apostichopus japonicus, using housekeeping geneβ-actin as control. Comparing the expressions of the genes among different tissues under room temperature, the results showed that 5 differentially expressed genes were expressed in all the above tissues, the highest expression level of HSP70, Protein 1(2)efl, mt LSU rRNA and PCSK9 was detected in body wall, the highest expression level of MYP1 was detected in alimentary tract. Expressions of the five genes changed obviously and the variations of tissues were different after high temperature stimulation, the results showed that the differential expressions of HSP70, Protein 1(2)efl and MYP1 were significant in the tissue of respiratory tree, while mt LSU rRNA and PCSK9 were found changed most obviously in body wall and longitudinal muscles respectively after heat stress.The same method and technology were employed to study the differentially expressed genes of Apostichopus japonicus under different temperature stresses. Same time of 5 different temperature treatments 18℃,21℃,24℃,27℃,30℃were set and the expression levels of differentially expressed genes at 18℃were used as standards. The results showed expression levels of HSP70, mt LSU rRNA and Protein 1(2)efl increased with increasing temperature and the change of HSP70 was most significant. HSP70, mt LSU rRNA and Protein 1(2)efl achieved their maximum levels with 3.32,2.89 and 2.58 at 30℃, respectively. While the expression levels of MYP1 and PCSK9 reduced with the rise of temperature, MYP1 and PCSK9 achieved their minimum expression levels with 0.66 and 0.54 at 30℃and 27℃, respectively.At last, expression profiles of the differentially expressed genes at different times in the whole heat stress phase were studied. During the course of heat stress, the expression differences of the differentially expressed genes at 0 h,0.5 h,1 h,1.5 h,2 h,2.5 h and 3 h were compared, setting the expression levels of differentially expressed genes at 0 h as standards. The expression level of HSP70 was up-regulated at first time point and then down-regulated, at last up-regulated to 4.59 at 3 h; mt LSU rRNA was slightly up-regulated and achieved to its maximum level with 2.27 at 3 h; Protein 1(2)efl achieved to its maximum level with 2.1 at 2.5 h; MYP1 was slightly down-regulated to minimum level with 0.56 at 3 h; PCSK9 achieved to its minimum level with 0.5 at 2 h.