| Our country is the largest producer of grain,and also the largest producer of straw.Straw can produce over 700 million tons per year,about 20%-30% of the world tota amount.Straw is wrapped with cellulose,hemicellulose and lignin.Hemicellulose and lignin are in the form of a covalent bound,in which the cellulose molecules are coated to form a natural barrier,which leads to the main reason of the low utilization of straw cellulose.Currently,in the treatment with improving utilization of straw as the purposes,microorganisms and biological degradation enzyme treatment atracts most attention.The main components of the structure of straw hemicellulose is xylan,which is a kind of complex polysaccharide.Straw hemicellulose can be degraded into xylose and xylooligosaccharide so as to be efficiently used.Saccharomyces cerevisiae is one of the important microorganisms in the fermentation industry because of its excellent growth characteristics,easy cultivation,high biomass and well-recognized safety.According to the characteristics of homologous recombination,rDNA sequences with 100 ~ 200 repeat units in the yeast genome as the homologous recombination site are effective ways to increase the copy number of the foreign gene in the yeast genome.In this research,we put the β-1,4 xylanase gene which was cut from aspergillus Niger CICC2462 into Pyes2.After that we insert the rDNA gene from saccharomyces cerevisiae into the pYES2-xynB.As a result,we got a recombinant plasmid with gene sequences of pYES2-xynB-rDNA and transform the vector into saccharomyces cerevisiae INVSc1.We screen the positive transformation and use them to do the ddPCR and fermentation experiments.We can find the relationship between gene copy number and enzyme activity by comparing the xylanase activity with different copy numbers.The results are as followings:1.The pYES2-xynB-rDNA vector was successfully biult and transformed into saccharomyces cerevisiae INVSc1.A plenty number of transformations were obtained by medium without uracil.2.The copy numbers obtained from screening were identified by ddPCR.The ATC1 was used as reference gene and the copy number is : 1、2、3、5、9、10、14、15、16、17、18.3.We did fermentation experiments with different copy numbers and detected xylanase activity at 72 nd hours.As a result,best activity show with the copy number 9.The activity is 308U/ml which is 5.6 times as single copy. |
