纤维堆囊菌So9731热休克蛋白HSP60基因的克隆与分析.DOC

大学生科技创新基金项目 PAGE 15 纤维堆囊菌So9733-1热休克蛋白HSP60基因的克隆与分析 吴凡 刘鑫 吴志红 摘 要：【目的】克隆获得纤维堆囊菌So9733-1热休克蛋白的完整基因，并对其进行序列分析。【方法】 根据HSP60保守区设计合适的PCR引物，以So9733-1基因组DNA为模板，通过PCR扩增、片段克隆和测序分析，筛选获得HSP60相关的基因片断。以该片断序列为基础，通过Tail-PCR扩增获得该片断两侧的未知基因序列，最终获得So9733-1热休克蛋白HSP60的完整基因。【结果】利用H279和H280高兼并度引物，首先成功地在纤维堆囊菌So9733-1获得HSP60相关的基因片段。以该片段序列为基础，利用Tail-PCR方法成功地获得了该序列上游的未知基因序列。通过Tail-PCR方法克隆该片段下游的未知基因序列的相关工作正在进行中，期望在近期完成相应的工作。纤维堆囊菌So9733-1 热休克蛋白HSP60完整基因序列克隆及其序列特征的解析将为后续研究和应用奠定良好的基础。 关键词：纤维堆囊菌 ；热休克蛋白HSP60 ；Tail-PCR ；基因克隆 ；序列分析 Cloning and Analysis of Heat-Shock Protein from Sorangium cellulosum So9733-1 WU Fan, LIU Xin， WU Zhi-Hong Abstract: 【Aim】 To obtain the full length gene of 60kDa heat-shock protein (HSP60) from Sorangium cellulosum So9733-1 and analysis its characters. 【Methods】Using the primers developed complementary to the conserved regions of HSP60 or the primers selected from references,PCR amplification of the DNA fragment related to HSP60 gene was carried out with the genomic DNA from So9733-1 as a template. The flanking sequences upstream and downstream the obtained HSP60 related DNA fragment was further gained by Tail-PCR. The full length HSP60 gene from S. cellulosum So9733-1 was expected. 【Results】 A pair of high degeneracy primer was applied in the PCR anplification of HSP60 gene fragment and successfully resulted in a HSP60 gene related DNA fragment in S. cellulosum So9733-1.Based on the cloned DNA sequence, 2 sets of specific primers for the upstream and downstream flankings in the Tail-PCR were developed, respectively. The upstream of the cloned DNA fragment was obtained with the combination of the specific primers and the AD primers using Tail-PCR. The further amplification and cloning of the downstream of the cloned DNA fragment by Tail-PCR was in progress. The cloning and analysis of full length HSP60 gene were expected in the near future. The results would supply a wise step for the consequent research. Key words: Sorangium cellulosum; heat-shock protein; Tail-P