鲫鱼低氧相关基因差减cDNA文库的构建与分析
CONSTRUCTI ON AND ANALYSIS OF THE SUBTRACTIVE cDNA L IBRARY OF CRUC I AN CARP INDUCED BY HYPOXI A
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摘要: 鲫鱼对低氧具有极强的耐受性.在低氧状态下鲫鱼鳃瓣表面积增加,无氧代谢增强,能量消耗降低.但是人们对鲫鱼产生这些低氧反应的分子机理还缺乏了解.本研究以1%低氧处理24h的鲫鱼囊胚细胞(CAB)作为检测子(Tester),常氧条件下培养的CAB细胞作为驱赶子(Driver),分别提取总RNA,利用SMART cDNA技术合成双链cDNA,经差减杂交和抑制性PCR扩增获得差减PCR产物.然后将差减PCR产物连接到pGEM-T载体上,构建差减cDNA文库.以管家基因β-actin作为指标检测差减效率,发现该文库差减效率达28倍.随机挑选阳性克隆进行PCR检测,显示差减片段在0.1-2kb之间.挑取含有插入片段的1300个克隆进行测序,通过生物信息学分析获得267个基因序列(e≤0.001;Identity>40%).该差减cDNA文库的成功构建对克隆鱼类耐低氧相关基因和深入认识鱼类耐受低氧的分子机制有非常重要的意义.Abstract: 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.
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