Screening Of Cellulase-producing Microorganisms And The Expression Research Of Cellulase Genes In Silkworm, Bombyx Mori L.

Cellulases catalyze the hydrolysis of cellulose which include mainly three types:endoglucanases, exoglucanases andβ-glucosidases. It can be used in converting cellulosic biomass to glucose that can be used in different applications such as fuel ethanol production, animal feed production, waste water treatment, and brewing industry. Usually, the cellulase enzyme activities are higher under acid conditions, but lower under neutral or alkaline conditions. This research isolated a new cellulase-producing E. coli by natural screening whose characteristic of cellulase was very good, got several T. viride mutants mutated by microwave and ultraviolet whose cellulase productions were increased significantly and very stable, cloned several endoglucanase genes from wild T. viride and expressed them in silkworm bioactively, which provided a theoretical foundation to produce a cellulase transgenic silkworm, and generated a cellulase transgenic vector, which provided an experimental basis to generate a cellulase transgenic silkworm. The main results were listed as follows:1. Screening, identification and characteristic analysis of cellulase-producing microorganismsA cellulase-producing bacterium strain was isolated from soil that produced novel thermoalkalotolerant cellulases after growth on CMC-Na agar screening plate at 37℃. It was identified as Escherichia coli using the method of 16S rRNA and ITS gene analysis combined with grams staining. The activities of CMCase, FPase, and (3-glucosidase produced by the E.coli cultured in the LB broth with 1% CMC-Na were measured, finding the maximal activities (0.23,0.08 and 0.15 IU/ml, respectively) and maximum specific activities (4.13,0.56 and 0.50 IU/mg total protein, respectively) were after 72 h,96 h, and 120 h growth, respectively. The maximum CMCase activity was measured at 50℃and pH 6.0, respectively.It retained more than 60% of its maximal activity after incubating at 50～70℃for 20 min or at 80℃for 10 min, and retained approximately 50% of its maximal activity after incubating at 90℃for 10 min.2. Enhanced cellulase production of the Trichoderma viride mutated by microwave and ultravioletCellulase-producing fungi Trichoderma viride were cultured and fermented on the solid-state wheat bran fermentation medium. The characteristics of its carboxymethyl cellulase (CMCase) under the condition of this solid-state fermentation were evaluated, that the optimum culture time, optimum pH, and optimum temperature for CMCase activity of T. viride fermented in this solid state was 60 h,5.0, and 50℃, respectively. Carboxymethyl cellulose sodium (CMC-Na) and Congo red were used to screen the strains that had stronger ability to produce enzymes. After the compound mutagenesis by microwave and ultraviolet, seven mutant strains (M-B1 to M-B7) were selected and their CMCase activities were assayed. Five of them (M-B1, M-B2, M-B3, M-B5, and M-B7) had significantly stronger ability to produce enzymes than the wild type, and they were also very stable for a period of 9 generations to produce cellulase. Molecular studies showed that there were some base mutations in the mutants' EGⅠgenes, suggesting that some amino mutations in EGⅠproteins caused by base mutations were related with their enhanced cellulase productions.3. Cloning and sequence analysis of endoglucanase genes from Trichoderma virideBased on the DNA sequences of EGⅠ(Accession no. AY343986), EGⅢ(Accession no. AY343987), EGⅣ(Accession no. Y11113), and EGⅤ(Accession no. AY343989), we designed special primers for PCR amplification and exon splicing using PrimeSTARTM HS DNA Polymerase with the genomic DNA of Trichoderma viride AS 3.3711 as the template. The exon splicing products were cloned into T vectors followed by terminal adding A reaction. The sequencing results showed that in terms of both nucleotide sequence homology and amino acid sequence homology, EGⅠ, EGⅢ, and EGⅣshowed 99% similarity to those genes in Genbank and EGⅤshowed 100%. Possibly, the cloned EGⅣgene of T. viride AS 3.3711 was a new gene, which has been registered in Genbank database with the accession no. HM222525.4. Expressing of endoglucanase genes from Trichoderma viride in silkwormThe mature peptide genes of EGⅠ, EGⅢ, EGⅣ, and EGⅤwere cloned by PCR technique, and expressed in both silkworm BmN cells and silkworm larvaes with a newly established BmNPV/Bac-to-Bac mutant baculovirus expression system, which lacks the virus-encoded chitinase (chiA) and cathepsin (v-cath) genes of Bombyx mori nucleopolyhedrovirus (BmNPV). The results of western blotting showed that,49-kDa,45-kDa,42-kDa, and 36-kDa recombinant proteins were visualized after silkworm BmN cells were infected by recombinant baculovirus mBacmid/BmNPV/EGⅠ, mBacmid/BmNPV/EGⅢ, mBacmid/BmNPV/EGⅣ, and mBacmid/BmNPV/EGⅤ, respectively. The maximum expression of EG I in silkworm larvae was at 84 h postinfection, and its enzyme activity was determined at this time point. The enzyme activities of other recombinant proteins were determined at the same time point. The ANOVA results showed that, the enzymes of silkworms infected by recombinant baculoviruses mBacmid/BmNPV/EGⅠ, mBacmid/BmNPV/EGⅢ, mBacmid/BmNPV/EG Ⅳ, and mBacmid/BmNPV/EGⅤexhibited significant maximum enzyme activities at the environmental condition of pH 7.0 and temperature 50℃, pH 8.0 and temperature 50℃, pH 6.0 and temperature 50℃, and pH 5.0 and temperature 50℃, respectively. They increased 24.71% and 22.84%,20.94% and 19.13%,48.84% and 46.61%, and 39.86% and 37.76%, compared with those from wild mutant baculoviruses mBacmid/BmNPV infected silkworms and normal silkworms, and were stable at the condition of pH 5.0～10.0 and temperature 40～60℃, pH 5.0～9.0 and temperature 40～60℃, pH 5.0～10.0 and temperature 40～60℃, and pH 5.0～10.0 and temperature 50～60℃, respectively. The availability of large quantities of cellulase that the silkworm provides maybe greatly facilitate the future research and the potential application in industries. It provided a possibility to generate transgenic silkworms, which can produce bioactive cellulase especially in the digestive tract and catabolize dietary fibers more efficiently. It might be of great significance for sericulture industry.5. Construction of cellulase transgenic vector and cell transfectionIn order to construct cellulase transgenic silkworm, it is necessary to construct a cellulase transgenic vector at first. Through primer design and PCR technique, firstly, several gene sequences, i.e. IE promoter, SV40 polyA, GFP, and Neo which connected with IE promoter and SV40 polyA, were cloned from plasmid pigA3-IE-Neo, and the EGⅣgene was cloned from plasmid pMD18-T/EGⅣe.. Then, both EGⅣand GFP were connected with IE promoter and SV40 polyA. Finally, the three genes, i.e. EGⅣ, GFP, and Neo, all of which were connected with IE promoter and SV40 polyA, were sequentially cloned into the plasmid pXL/BacⅡ, and the produced recombinant plasmid was named pXL/EGⅣ/GFP/Neo. The recombinant plasmid pXL/EGⅣ/GFP/Neo, which is extracted using a endotoxin-free plasmid extracting kit, was used to transfect silkworm BmN cells, and the result showed that the GFP and Neo were expressed successfully.