家蚕丝蛋白Fhx/P25基因启动子区域的克隆及序列分析
Cloning and Analysis of Fhx/P25 Gene Promoter of Bombyx mori Fibroin Protein
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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。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 stemloop 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.
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