核盘菌(Sclerotinia sclerotiorum)arom基因3′-末端的克隆与分析

Cloning and characterization of 3′-terminal of arom gene from sclerotinia sclerotiorum

根据五功能AROM蛋白保守区域设计简并引物,从核盘菌基因组中获得了一段大小为1626bp的DNA片段。结合Southern杂交结果,用反向PCR克隆了arom基因3′-末端4104bp的序列。序列分析结果表明,该DNA片段推测的氨基酸序列与烟曲霉、粗糙脉孢霉、构巢曲霉和粟酒裂殖酵母等的AROM蛋白同源,包含了部分5-烯醇丙酮酰莽草酸-3-磷酸合成酶(EPSP合成酶)结构域、莽草酸激酶结构域、脱氢奎尼酸脱水酶(脱氢奎尼酸酶)结构域和脱氢莽草酸脱氢酶结构域,其中含有EPSP合成酶模式2(255—273)、莽草酸激酶模式(426—453)和Ⅰ型脱氢奎尼酸酶活性位点模式(659-687)。

A 1636hp DNA fragment was cloned from Sclerotinia sclerotiorum genomic DNA with a pair of degenerate primers designed according to conserve sequences of pentafunctional AROM protein. Then the 4104bp sequence at the 3＇-terminus of arom gene was attained with inverse PCR based on the result of Southern blot. Analysis of the sequence show that the putative amino acid sequence is homologous to AROM protein from Aspergillus fumigatus, Neurospora crassa, Emericella nidulans, schizosaccharomyces pombe etc. and contains partial 5-enolpyruvylshikimate-3-phosphate synthase domain, shikimate kinase domain, 3-dehydroquinate dehydratase domain and shikimate 5-dehydrogenase domain. Three signatures, EPSP synthase signature Ⅱ, Shikimate kinase signature and Dehydroquinase class Ⅰ active site, were also found in it.