【摘要】 实验室已经克隆了金龟子绿僵菌中性海藻糖酶基因(NTL)的编码序列(GenBank登录号:AY557612),为了解该基因的上游调控信息,采用Panhandle Polymerase Chain Reaction Amplification方法获得了长为982bp的中性海藻糖酶基因上游序列。序列分析表明,该序列含有5个CAAT启动子元件和一个压力反应元件(CCCCT).经PCR和Southern杂交验证表明,利用panhandle PCR法成功地获得了金龟子绿僵菌CQMa102中性海藻糖酶基因上游序列,并且该基因在金龟子绿僵菌基因组中以单拷贝形式存在.更多还原

【Abstract】 Based on the coding sequence of neutral trehalase gene(NTL) of Metarhizium anisopliae which has been cloned in the authors laboratory previously,Panhandle Polymerase Chain Reaction strategy was used to amplify the flanking sequences adjacent to the known sequence of the neutral trehalase gene.The genomic DNA was first digested with XbaⅠ to create a 5’overhang and then treated with calf intestinal alkaline phosphatase.Next,a 34-nucleotide single-stranded 5’phosphorylated oligonucleotide was ligated to the 3’ends of XbaⅠ-digested DNA.After denaturation,intra-strand annealing and polymerase extension,a panhandle was formed and then nested PCR performed.A 1121bp panhandle PCR product was subsequently subcloned and the sequence analysis shows that it contains one stress response element(STREs),which may mediate transcriptional activation of the neutral trehalase gene in response to various stress.Southern analysis indicated that the neutral trehalase gene was present as a single copy in M.anisopliae.The upstream sequence of neutral trehalase gene has been accessed by GenBank(Accession: AY557613).更多还原