| 摘要: |
| 采用抑制性消减杂交技术(SSH)研究刺参高温胁迫下差异表达的基因。分别以高温实验组为检测组(tester)、常温对照组为驱动组(driver), 进行正向抑制性消减杂交; 以常温组为 tester、高温组为 driver, 进行反向消减杂交, 成功构建了刺参正反双向差异消减文库。从两个文库随机挑选 384个白斑克隆进行斑点杂交进一步筛选, 对其中差异显著的 50 个克隆进行测序分析。测序结果经BLAST 比对分析得出: 44 个克隆与已知基因序列高度同源, 主要包括 3 个上调表达基因和 2 个下调表达基因; 另外 5 个克隆为未知新基因序列。该消减文库的成功构建对刺参耐高温相关基因的深入研究以及阐述耐高温的分子机理有非常重要的意义。
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| 关键词: 刺参, 高温胁迫, 抑制性消减杂交, 消减文库 cDNA |
| DOI：10.11693/hyhz201101009009 |
| 分类号: |
| 基金项目:山东省良种工程项目“速生抗病耐高温刺参良种选育”, 2008—2010; 山东省优秀中青年科学家奖励基金(博士基金)项目, 2008BS06004 号; 山东省科技攻关项目“海参新品系选育及构建”, 2008GG10005023 号 |
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| CONSTRUCTION AND ANALYSIS OF THE SUBTRACTED cDNA LIBRARY OF APOSTICHOPUS JAPONICUS (SELENKA) UNDER HIGH TEMPERATURE STRESS |
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JI Cheng-Long1,2, SUN Guo-Hua1, YANG Jian-Min1, SONG Zhi-Le3, LIU Xiang-Quan1, WANG Wei-Jun1
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1.Shandong Marine Fisheries Research Institute;2.School of Fishery and Life Science, Shanghai Ocean University;3.Zhifu Fishery Technical Extension Station
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| Abstract: |
| Suppression subtractive hybridization (SSH) was used to study the differentially expressed genes of Apostichopus japonicus under high temperature stress. The cDNA of A. japonicus underwent heat stress was used as tester and the same cDNA treated under room temperature was used as driver to construct a forward subtractive library. A reverse subtractive library was generated by using the same cNDA but underwent heat stress was the driver and untreated was tester. 384 positive clones were randomly picked from each library and used for dot blotting. 50 significant differentially expression clones were sequenced and analyzed using BLAST. The results show there are 44 sequences have high identities with the known genes. There are 10 major differentially expressed genes: three up-regulated known genes, two down-regulated known genes, and 5 are unknown genes. The constructed libraries in this study play an important role in studying the heat-resistant genes in A. japonicus and the molecular genetic mechanism of its ability in heat tolerance.
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| Key words: Apostichopus japonicus, High temperature stress, Suppression subtractive hybridization, Subtracted cDNA library |
