Chloroplast Targeting Signal of a Rice Rubisco Activase Gene Enhances Transgene Expression
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摘要: 将水稻RCA基因N端预测的转运肽(TP)序列融合报告基因GFP,构建瞬时表达载体pGD-RCATP-GFP,转化农杆菌后,注射侵染烟草叶片,通过荧光显微镜观察瞬时表达情况,结果表明外源蛋白GFP定位在叶绿体上,依此预测水稻RCA基因N端48aa为叶绿体转运肽。将该序列与报告基因GUS融合,构建植物表达载体p1300-RCATP-AGS,转化水稻后,检测阳性株叶片的GUS酶活,其GUS蛋白表达效率是未连有叶绿体转运肽的转基因植物的4.2倍左右。
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关键词:
- RCA(二磷酸核酮糖羧化酶激酶) /
- 叶绿体转运肽 /
- GUS酶活分析
Abstract: N-terminal sequence of the protein encoded by rice RCA gene was predicted to be a chloroplast transit peptide.The 144bp fragment of this gene was fused with GFP to construct a transient expression vector pGD-RCATP-GFP for genetic transformation.Transient transformation of tobacco was produced by the Agrobacterium-mediated infiltration method.Fluorescence microscopy analyses demonstrated that,in cells expressing the RCATP:GFP fusion protein,the fluorescence was specifically targeted to chloroplast.Then this fragment was fused with GUS to construct a vector p1300-RCATP-AGS for genetic transformation.To analyze GUS activity for the leaves of the positive plants,we discovered that the RCA TP sequence increased GUS protein expression levels about 3 fold,when compared to without RCA TP sequence. -
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