CONSTRUCTI ON AND ANALYSIS OF THE SUBTRACTIVE cDNA L IBRARY OF CRUC I AN CARP INDUCED BY HYPOXI A

Crucian carp (Carassius auratusL.) is one of the most anoxia-tolerant vertebrates known. It has been found to survive many days or, at low temperatures, even several months of anoxia in the field and the laboratory. Its adaptive mechanisms surviving anoxia/hypoxia include the ability of promoting anaerobic metabolism to produce ATP, increasing respiratory surface area to boost oxygen uptake, and so on. However, the molecular basis of its response to hypoxia stress has not yet been clarified. In order to analyze the changes of gene expression and isolate those genes induced by hypoxia in blastulae embryonic cells of crucian carp (CAB), a subtrative cDNA librarywas constructed by using suppression sub-trative hybridization techniques. CAB cellswere maintained at 27℃ in a humidified incubator containing-0% O-. For the hypoxia treatment, cells were placed in a hypoxia chamber containing 1% O-, 5% CO-and 94% N-respectively and treated for one hour. Total RNAs were extracted from hypoxia-treated and untreated CAB cellswith SV-Total RNA Isolation System. Double-stranded cDNAswere prepared from 0. 5 μg of total RNA by using S MART cDNA synthesis technique. The subtrative cDNA library was constructed by using suppression subtrative hybridization technique. The subtractive cDNAs were ligated into the pGEM-T vector and transfected into competent E. coli cells. A housekeeping gene, β-actin, was used to estimate the efficiency of subtractive cDNA and found to be subtracted at appropriate 28folds. 2200 colonieswere selected from the plas-mid library, and PCR analysis showed that the length of the subtractive cDNA fragments cloned into pGEM-T vector ranged from 100 bp to-000 bp. Two hundred and sixty-seven genes (e≤0.001; Identity>40%) were obtained by se-quencing and bioinformatics. Our results showed that the subtractive cDNA library is successful, which will be very useful for the understanding of the response to hypoxia and essential for rapid isolation of differentially expressed genes induced by hypoxia.