Studies On Cloning And Expression Of Beta-glucosidase Gene

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%.