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为了研究梨小食心虫Grapholita molesta化学感受蛋白(chemosensory proteins,CSPs)在化学感受系统中的作用,本研究利用RT-PCR和RACE技术克隆到一条梨小食心虫化学感受蛋白的全长cDNA序列,命名为GmolCSP(GenBank登录号:JQ821389)。序列分析表明,GmolCSP开放阅读框序列为384bp,编码127个氨基酸残基,预测N末端含有18个氨基酸组成的信号肽序列,其成熟蛋白的预测分子量为12.80kD,等电点为8.33。该基因编码的氨基酸序列与其他鳞翅目昆虫化学感受蛋白的氨基酸序列具有较高同源性。RT-PCR结果显示,GmolCSP在梨小食心虫成虫触角、去触角的头、胸、腹、足和翅中都有表达。将GmolCSP重组到表达载体pET-32a中,转入大肠杆菌Escherichia coli BL21(DE3)进行表达。SDS-PAGE和Western印迹检测结果显示,梨小食心虫化学感受蛋白基因在大肠杆菌中成功地表达出一个分子量约为29kD的融合蛋白,与预测的融合蛋白分子量大小一致。本研究结果为进一步研究该蛋白的分子结构和功能奠定了良好基础。
In order to study the role of chemosensory proteins (CSPs) in Grapholita molesta chemosensory system, we cloned a full-length cDNA sequence of chemosensory proteins from Pichia pastoris using RT-PCR and RACE techniques, named as GmolCSP (GenBank Accession No. JQ821389). Sequence analysis showed that the open reading frame of GmolCSP was 384bp and encoded 127 amino acid residues. The signal peptide of 18 amino acids was predicted at the N terminus. The predicted molecular weight of the mature protein was 12.80kD and the isoelectric point was 8.33. The amino acid sequence encoded by this gene has high homology with the amino acid sequences of other lepidopteran insects chemosensitive proteins. The results of RT-PCR showed that GmolCSP was expressed in the antennae, antennae, thorax, abdomen, foot and wing of the antennae of adults. GmolCSP was recombined into expression vector pET-32a and transformed into Escherichia coli BL21 (DE3) for expression. The results of SDS-PAGE and Western blotting showed that the gene of chemotherapeutic protein of Bombyx mori was successfully expressed in E. coli with a molecular weight of about 29 kD, which was consistent with the molecular weight of the predicted fusion protein. The results of this study lay a good foundation for further study on the molecular structure and function of this protein.