|
|
|
Abstract Targeting-induced local lesions in genomes (TILLING), as an important research technology in reverse genetics has been widely applied to study in molecular biology of many model organisms. CELⅠ is the key enzyme of this technology. In this study, we extracted the endonuclease CELⅠ with high activity from celery by (NH4)2SO4 deposition, affinity chromatography, anion and cation chromatography. Using heteroduplex DNAs as the substrates, we further investigated the various activities of CELⅠ under different conditions, the various combinations of different purified levels, digestion temperatures, treatment time, presence or absence of Taq DNA polymerases. The results indicated that the CELⅠin our test is much purified, the optimum temperature is between 40℃ and 50℃, well special digestion for mismatch bases in a long time, and that the activity of CELⅠ would be higher when Taq enzyme is present in the reaction system. In conclusion, our results will provide technology supports for application of TILLING in silkworm with large scale and low cost.
|
|
Received: 27 March 2007
|
|
|
|
|
|
