Safety And Efficient Expression Of β-1,4-endo-xylanase Gene In Aspergillus Niger

Filamentous fungi has excellent ability of expression and secretion.As well as similarto mammalian glycosylation modification system.Its security is widely accepted bypeople.It is an ideal system for expression of eukaryotic genes.So far,in the world,researching and developing expression system of filamentous fungi has become a hot spotsrecombinant enzymes.Currently,although using of Trichoderma,Aspergillus fermentationproduct xylanase has reached a very high level in domestic,but it have a gap from theinternational advanced level.Meanwhile,the xylanase of production cannot meet somespecial needs.Such as,because of its high xylosidase activity,the xylanase is not applicableto the production oligosaccharide.By means of genetic engineering express endo-xylanaseis an effective way to solve this problem.The study take production glucoamylase’s Aspergillus niger CICC2462as therecipient strain.With high expression of glucoamylase gene locus as the gene integrationtargetpoint,Construction of Aspergillus niger expression vector of xylanase.Taking ofAgrobacterium-mediated transformation of Aspergillus niger,And through design forwardrepeat sequences on both sides of the hygromycin gene expression cassette,so thateliminate hygromycin marker gene transformants posterity,Obtain food-grade engineeringstrain.The main results obtains are as follows:1Cloning of gene and homology armThe xylanase gene xyn2and xynB,the Aspergillus niger glucoamylase gene upstreamfragment5’GLA and downstream fragment3’GLA were gained by using PCRamplification.2Construction of Aspergillus niger expression vectorSplicing of homologous recombination expression cassettes G2G and GBG with5’GLA,xyn2and xynB,3’GLA fragments by overlap extension PCR technique,Connecting itwith Aspergillus niger expression vector pSZH to construct xylanase Aspergillus nigerexpression vector pSZH-xyn2and pSZH-xynB.3Agrobacterium-mediated transformation of Aspergillus niger and screening oftransformants Taking of freeze-thaw method make Aspergillus niger expression vector pSZH-xyn2and pSZH-xynB transformed into Agrobacterium AGLI,Agrobacterium-mediatedtransformation of Aspergillus niger CICC2462.Identification by PCR,the genetransformation positive rate of xyn2is34.78%,the gene transformation positive rate ofxynB is70.15%.4Screening of homologous recombinants of xynBIdentification of12positive strains with homologous recombination primers byPCR,as a result of9strains can amplified about1701bp target fragment,it’s homologousrecombinants,The homologous recombination rate of transformation pSZH-xynB is75%.After five generations of continuous passage in PDA liquid medium which contain200mmol/L hygromycin B,Making of liquid culture paint in PDA solid medium whichcontain200mmol/L hygromycin B.Picking of26single colonies for identification byPCR,All of is homozygous homologous recombination strains.5Eliminating of hph gene in xynB gene homologous recombination strainMaking homozygous homologous recombination strain B10-5after continuouspassage five generations in PDA liquid medium which absence hygromycin B,painting it inPDA solid medium which absence hygromycin B,Picking up534colonies in PDA solidmedium which contain200mmol/L hygromycin B,as a result of2colonies did notgrow,Extracting of genomic DNA of these2colonies,Identification of its withglucoamylase gene primers and homologous recombination primers respectively by PCRwhich can amplified about3000bp target fragment,but cannot amplified about1701bptarget fragment,The results illustration of the target gene xynB in these2colonies remainintegrated in glucoamylase gene locus.while its chain hygromycin gene has beendeleted,the elimination rate of hygromycin gene is0.3745%.The study also illustrate thegenetic stability of the target gene is100%after several passages.6Optimization of fermentation conditions of recombination strainsThe glucoamylase gene has been replaced by xylanase gene in homozygoushomologous recombination strain,which lost the ability to use liquefied starch,sooptimization of fermentation conditions of glucoamylase.The results show that when theculture medium contain100g glucose,40g starch,20mL corn steep liquor,20g soybeanpowder in per liter,pH5.5~6.0,The expression level of homologous recombination strain ishigher,the enzyme activity is4495.8975u/mL.The results of this study provide a new way for production of low-xylosidase activityendo-xylanase,and provide a new expression system for safe and efficient productionenzyme preparation and recombinant protein.