Variation Of The Trypsin Activities And Expression Of Trypsin MRNA During The Embryonic And Larval Development Of Odontobutis Potamophila

The Odontobutis potamophila belongs to Perciformes, Odontobutidae. It is an important freshwater economic fish in China. The aim of this theis was to systematically investigate the variation of protease activities and the expression of mRNA of tirpsin by methods of biochemistry and molecular biology. The main results are concluded as follows:The results showed that the activity of the pepsin kept at very low level during the early embryonic development. At the prehatching statge, the activity of the pepsin increase significantly, from then on, it always remained at a high relative activity and it showed a highest level at the fifth day of hatch. The activity of trypsin was exhibited in the early embryonic development. At the eye pigmentation statge the activity of trypsin increase rapidly, and then kept at a high level.At the fifth day after hatching it also showed a highest level. It is indicates the proteinic enzymes activities increase gradually during the embryonic and larval development of Odontobutis potamophila.Total RNA was abstracted from Odontobutis potamophila with Trizol reagent, and its ratio of A280 / A260 was between 1.8 and 2.0. Using RNA as a template, we conduct the RT-PCR with trypsin special primers designed by ourselves and obtained 555bp, 224bp Odontobutis potamophila trypsin cDNAs. Analysis the homology of the cDNA we have got several fish . The results proved that the cDNAs we have got are the trypsin expressing genes.Real-time PCR method was used for quantitative analysis of trypsin mRNA level in embryonic and larval development of Odontobutis potamophila using SYBR Green as reporter dye and product of PCR as standard curve template. The result showed the variation of the trypsin activities and expression of trypsin mRNA had almost the same change trend. The expression of trypsin mRNA was detected in the early embryonic development. At the eye pigmentation statge the expressin of trypsin mRNA increase rapidly, and then kept at a high level. At the fifth day after hatching also showed a highest level. It showed variation of the trypsin activities was controled by the regulation of trypsin gene expression.