Crucian carp (Carassius auratus L.) is one of the,lost 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 (in. 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 ( CAR a subtrative cDNA library was constructed by using suppression subtrative hybridization techniques.CAB cells Were maintained at 27 degrees C in a humidified incubator containing, 20% O-2 For the hypoxia treatment, cells were placed in a hypoxia chamber containing 1% O-2, 5% CO2 and 94% N-2 respectively and treated for one hour. Total RNAs were extracted front hypoxia-treated and untreated CAB cells with SV-Total RNA Isolation System. Double-stranded cDNAs were prepared front 0.5 mu g of total RNA by using SMART 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, beta-actin, Was used to estimate the efficiency of subtractive cDNA and found to he subtracted at appropriate 2(8) folds. 2200 colonies were selected front the plasmid library, and PCR analysis showed that the length Of the subtractive cDNA fragments cloned into pGEM-T vector ranged from 100 bp to 2000 bp. Two hundred and sixty-seven genes (c <= 0.001; Identity > 40%) were obtained by sequencing and bioinformatics. Our results showed that the subtractive cDNA library is successful, which will he very useful for the understanding of the response to hypoxia and essential for rapid isolation of differentially expressed genes induced by hypoxia.
Zhong Xue-Ping (firstname.lastname@example.org) ; Wang Dan; Zhang Yi-Bing; Gui Jian-Fang (email@example.com).CONSTRUCTION AND ANALYSIS OF THE SUBTRACTIVE cDNA LIBRARY OF CRUCIAN CARP INDUCED BY HYPOXIA,Acta Hydrobiologica Sinica,2009,33(1):113-118