【作者】 潘海芳；

【导师】 陈由强；

【作者基本信息】 福建师范大学 ， 植物学， 2006， 硕士

【摘要】 白藜芦醇以游离态和糖苷结合态两种形式存在，是一种重要的植物抗毒素，主要存在于虎杖、葡萄等植物中。β-葡萄糖苷酶不仅能够水解白藜芦醇苷为白藜芦醇，还能够作用于细胞壁碎片或其它细胞组成，从而生成更多的白藜芦醇。本实验实现了木醋杆菌β-葡萄糖苷酶基因的原核表达，目的是为了生产粗酶制剂，用于提高从植物中提取白藜芦醇的得率；分别在DNA和cDNA水平从黑曲霉中克隆β-葡萄糖苷酶基因，为下一步在酿酒酵母中实现分泌表达以提高葡萄酒的白藜芦醇含量的研究奠定基础。 本实验通过降落PCR得到β-葡萄糖苷酶基因，然后将目的基因亚克隆到原核表达载体pQE30，转化大肠杆菌M15。测序表明，表达载体构建成功。用IPTG诱导表达，目的蛋白表达量达到菌体总蛋白的23.8％，但表达的蛋白主要以包涵体形式存在。 本实验利用种属相似性设计了一对引物。先后以黑曲霉基因组DNA、mRNA为模板，利用PCR与RT-PCR技术得到β-葡萄糖苷酶基因的DNA序列和cDNA序列，将目的基因分别克隆在pMD18—T和pMD19—T上。测序表明，克隆得到的DNA序列全长2924bp，cDNA序列全长2583bp，编码860个氨基酸。与Genbank中已登录的黑曲霉β-葡萄糖苷酶的氨基酸序列比较，相似性达97％-99％。更多还原

【Abstract】 Resveratrol, existed in two forms, glycosylated isomers and free isomers, is an important phytoalexin, mainly produced in plants such like Polygonum cuspidatum and grape. β-glucosidase can hydrolyse glycosidically-bound derivatives and release the corresponding resveratrol ismor, besides, β-glucosidase provides more substrate for the release of resveratrol from cell wall debris or other cellular components so as to increase the amount of resveratrol. The purpose of the experiment is to realize the prokaryotic expression of β-glucosidase gene from Acetobacter xylinum ATCC 23769, which will be used to produce enzyme preparation so as to develop the efficiency of extracting resveratrol from plants. Aspergillus niger β-glucosidase gene is cloned both at DNA lever and cDNA lever, which will lay a foundation for further research about its expression in Saccharomyces cerevisiae in a secretory form with the aim of increasing resveratrol contents in wines.In the experiment, touch-down PCR was used to obtain beta-glucosidase gene. The target gene was then sub-clone to prokaryotic expression vector pQE30 and the recombinant plasmid was transformed to E.coil M15. Sequencing the construct showed that the plasmid pQE30-bgln is constructed successfully. The target gene was expressed by induction of EPTG and the expression product accounted for 23.8% of the total proteins. However, most of the expressed protein exited in the form of inclusion body.In the experiment, a pair of primers was designed by species similarity. Genomic DNA and mRNA isolated from Aspergillus niger were successively used as PCR template. The methods of PCR and RT-PCR were used respectively to obtain the DNA and cDNA of beta-glucosidase gene. The two target genes were then cloned to pMD18-T vector and pMD19-T vector respectively. Seqence analysis indicated that DNA sequence was 2924 bp, cDNA sequence was 2583 bp, which is encoding 860 aa. The identity of the derived protein with Aspergillus niger β-glucosidase registered in Genbank is 97%-99%.更多还原