【摘要】 为研究家蚕Fhx/P25基因在时空上的调控机制,通过PCR扩增获得家蚕丝素蛋白Fhx/P25基因的启动子序列并进行克隆和序列分析。进一步构建了由Fhx/P25启动子驱动报告基因DsRed的表达载体pSK-P25-DsRed-PolyA,并通过家蚕BmN细胞进行瞬时表达。结果显示:Fhx/P25基因的启动子序列符合真核生物启动子特点,具有丝腺特异性表达启动子的特征,TATA框的保守序列为TATAA,位于-28—-32处,CAAT基序有3个,其中-110—-117和-90—-87处的2个CAAT基序可能具有活性;二级结构分析显示:Fhx/P25启动子区域具有复杂的茎环结构,这可能与蛋白表达的组织特异性、时间性以及活性有关。基因启动子可以驱动红色荧光基因DsRed在家蚕BmN培养细胞中的瞬时表达。更多还原

【Abstract】 In order to better understand the model of the molecular mechanisms governing spatially and temporally programmed transcription,the Fhx/P25 gene promoter of the fibroin protein from Bombyx mori was cloned and sequenced.An expression vector named pSK-P25-DsRed-PolyA was constructed,in which the reporter gene DsRed was driven by Fhx/P25 promoter.The promoter’s activity was then characterized by transient expression assays in BmN cells of B.mori.The results of a sequence analysis showed that the Fhx/P25 promoter possesses the characteristics both of a eukaryotic promoter and a silk gland-specific expression promoter.A conserved TATA box sequence was located at position-28--32 and had the sequence TATAA.There were three CAAT motifs,of which the two CAAT motifs located at positions-110--117 and-90--87 may be active.Secondary structure analysis indicated that the sequence of the promoter region forms a complicated stem-loop structure.This may relate to the tissue speciality or the timing and activity of the protein expression.The transient expression product of the red fluorescent gene,driven by the promoter Fhx/P25,can be observed in cultured BmN cells of B.mori.更多还原