Cloning and characterization of heat shock protein genes from the hemocytes of black tiger prawn Penaeus monodon

Abstract

The experiment was firstly conducted on the cell maintainance of P. monodon haemocytes in 3 different media. High viability and relatively high activity of the cells were found in the haemocytes maintained in M199 and the haemocyte were maintained for 4 days with high survival rate whereas the haemocytes maintained in TC100 and Grace's insect media showed high activity but very low viability. Therefore, M199 were further used for maintaining the haemocytes in heat shock experiment. Haemocytes were treated with the temperature of 4, 30, 33, and 35 ?C for 1 and 2 h. Protein profile s of haemocyte extracts were detected using polyacrylamide gel electrophoresis. The result revealed the difference of peptide bands in samples extracted from different heat treatment. However, the consistency of the results was not satisfied. When Western blot analysis using specific antibodies was carried out, only HSP90 was detected. The sensitivity was also low therefore it was not suitable for quatitative analysis. The investigation on heat induced genes using differential display technique obtained 10 transcript markers. Nine of them were unknown genes and one was identified as vigilin gene. The EST analysis of heat induced haemocyte cDNA library provided DNA sequences of 1090 clones. Of these, 687 clones (63%) were identified genes and 132 clones (12.1%) were reported to be involved in the defense system and homeostasis. Full length sequences of HSP60, HSP70, and HSP90 genes were completed by the combination of techniques. This included RT-PCR, RACE-PCR and cDNA library screening. The results revealed that the ORF of HSP60 was 1731 bp coding for 576 amino acids, HSP70 ORF was 1959 bp coding for 652 amino acids, and HSP90 ORF was 2157 bp coding for 718 amino acids. All 3 genes contained a number of specific HSP patterns of their kinds confirming the identity of each gene. The results of this study will be basic knowledge for HSP function investigation and will be useful forbiomarker application in stress condition and breeding selection in P.monodon.