Cloning And Analysis Of MAL1 Promoter Region & Construction And Expression Of α-amy Gene Vector

MAL1 promoter region can promote the gene expression from 5'-3' transprition direction in Hansenula polymorpha and Saccharomyces cerevisiae .But the promoer function of the 3'-5' transprition direction has not been understand.1.26kb DNA fragment containing the full length MAL1 promoter region was coloned from Hansenula polymorpha A16 by PCR amplification . Plasmid which contained Zeocin resistant gene was constructed with the 5'-3' directional and 3'-5'directional promoter and successfully transformed into Ecoli .DH5α.The bidirection function of the MAL1 promoter region was confirmed by the expression of Zeocin resistant gene.After removing the SacI fragment in the middle of the full MAL1 promoter region, the rest MAL1 promoter region could also promote the expression of Zeocin resistant gene from two divery directions .It could be used for a new promoter in eukaryote and prokaryote.In order to analysis of promoter function in Saccharomyce cerevisiae, the α-amylase gene was amplified by PCR using Saccharomyces cerevisiae genomic DNA as template, inserted into the vector GZAho, GMAho5'and GMAho3'.The recombinant α-amylase gene was transformed into Saccharomyces cerevisiae and the recombinant strain YZAho , YMAho5', YMAho3'containing α-amylase gene was expressed to be a protein of 52 kD by SDS-PAGE analysis.The recombinant YZAho, its optimum condition was grown up for 24 hours to OD600=1.7, the a-activity of amylase was 5.53U/100μl in the vibrating bottle.The recombinant YMAho5'and YMAho3',could express the activity of the α-amylase also, but need to be induced by sucrose. In glucose-grown transformants the α-amylase activity was no. It indicate that glucose suppressed the a-amylase activity .The conditions of the expression of α-amylase engineered strain YMAho5' induced by sucrose in the vibrating bottle was studied. The results showed that sucrose with the concentration of 1.5% was added when grown up for 24 hours and induced for 3 hours were suitable , the activity of the a-amylase was 10.73U/100μl.After sucrose induction in liquid medium bottle, the activity of the α-amylase could be detected in the YMAho3'. The optimum reaction condition was that sucrose...