Constructing Of Trichoderma Reesei Engineering Bacteria For Soybean Dietary Fiber Modification

Dietary fiber on human health has special physiological functions of dietary fiber foods so welcomed by consumers, while the soluble dietary fiber has been demonstrated to have better physical function and functional characteristics. Currently, soybean soluble dietary fiber is prepared by enzymatic degradation residue. However, enzymes used in components with a variety of enzymes, each enzyme modified soy dietary fiber may play a different role. Trichoderma reesei endoglucanase EGII, endo-xylanase XYNⅠand XYNⅡfavor the formation of soluble dietary fiber, and xylosidase XYND,β-glucosidase BGLI, endoglucanase EGI, CBHI and other foreign endoglucanase component is not conducive to the formation of soluble dietary fiber. Enzymatic production of soybean resulting low yield of soluble dietary fiber, poor quality, process instability and other issues. Therefore, the use of genetic engineering technology training engineering bacteria to increase the beneficial enzyme components, reduce the negative enzyme components, soluble dietary fiber to improve the yield and quality, has important research value.The subject of using Agrobacterium mediated transformation of Trichoderma reesei QM9414, knocking out its genes xynd xylosidase andβ-glucosidase gene bglⅠ, trying to soybean production of soluble dietary fiber to provide enzyme engineering strains. results were summarized as follows:1.optimization Agrobacterium tumefaciens-mediated transformation of filamentous fungi The factors influencing the transformantion efficiency of A.tumefaciens mediated transformation of QM9414 were studied.The results showed that the transformation efficiency was correlated with the 3'5'-Dimethoxy-4-Hydroxy Acetophenone(AS)concentration in induced medium and co-cultivated medium, the induced time of A.tumefaciens, co-cultivation time, co-cultivation temperature,and the initial concentration of A.tumefaciens and the initial concentration of QM9414 conidia.According to these results, we established optimal conditions for transformation of QM9414 as follows:A.tumefaciens cultured to OD660 0.25 in induced medium with 200μM AS for 42hours incubating 48h at 24℃.Under these conditions,the transformation efficiency of ATMT on QM9414 is very well.2. Screeing△ura3 discrepancy strainConstruction of knocked-out vector of pEMT-△ura3 for deletion of ura3 gene, pEMT-△ura3 was transferred into GV3101 Agrobacterium Tumefacien, The Agrobacterium Tumefacien withpEMT-△ura3 plamsids and QM9414were cocultured in medium and to select transformants, By using this transformation system, QM9414 strain was successfully transformed.PCR analysis showed that the△ura3 gene was integrated into the genome, homgous recombination rates was 16.6%.3. Screeing△ku70 discrepancy strainConstruction of knocked-out vector of pEMT-pyrA for deletion of ku70 gene, pEMT-pyrA was transferred into GV3101 Agrobacterium Tumefacien, The Agrobacterium Tumefacien with pEMT-pyrA plamsids and△ura3 auxotrophic strain were cocultured in medium and to select transformants, By using this transformation system,△ura3 auxotrophic strain was successfully transformed.PCR analysis showed that the pyrA gene was integrated into the genome, homgous recombination rates was25%.4. Screeing xynd,bgll discrepancy strainxynd5, bglI3, xynⅡgenes were linked thought overlap extension PCR. Construction of knocked-out vector of pEMT-△xynd-pyrA for deletion of xynd-bgll-xynll gene, pEMT-△xynd-pyrA was transferred into GV3101 Agrobacterium Tumefacien, The Agrobacterium Tumefacien withpEMT-△xynd-pyrA plamsids and and△ura3 auxotrophic strain were cocultured in medium and to select transformants, By using this transformation system,△ura3 auxotrophic strain was successfully transformed. PCR analysis showed that the xynd gene was knocked out the genome.