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以‘玫瑰香’葡萄着色期的果肉为试材,通过反转录技术克隆了ABA合成相关酶β–葡萄糖苷酶基因VvBG1的1 518 bp编码区核苷酸序列,其编码1个含有505氨基酸的多肽。生物信息学分析表明,VvBG1含有1个跨膜区和糖基水解酶1超家族保守域。通过BamHⅠ和XhoⅠ限制性内切酶将除去信号的编码区克隆到原核表达载体pET28a(+)中,并转化大肠杆菌表达菌株BL21(DE3),成功诱导并纯化了VvBG1融合蛋白。经过透析复性和超滤浓缩得到了0.8 mg.mL-1可溶的融合蛋白。通过纯化蛋白葡萄果肉均浆液孵育试验和GC–MS ABA含量分析证实了葡萄果实中β–葡萄糖苷酶VvBG1具有较高的生理活性。
The nucleotide sequence of the 1 518 bp coding region of ABA biosynthesis-related enzyme β-glucosidase gene VvBG1 was cloned by reverse transcription technology, which encoded 1 fragment containing 505 amino acids Of the polypeptide. Bioinformatics analysis showed that VvBG1 contains a transmembrane region and a glycosyl hydrolase 1 superfamily conserved domain. The coding region of the signal was cloned into prokaryotic expression vector pET28a (+) by BamH I and Xho I restriction endonucleases and transformed into E. coli expression strain BL21 (DE3). The VvBG1 fusion protein was successfully induced and purified. After dialysis refolding and ultrafiltration concentration 0.8 mg.mL-1 soluble fusion protein was obtained. Through the purification of grape pulp homogenate test and GC-MS ABA content analysis confirmed grape fruit β-glucosidase VvBG1 has a higher physiological activity.