Study Of Cloning And Expression In Escherichia Coli Of Mulberry Borer Cellulase Gene CDS

There is 7 million tons of straw per year in china,but only20 %~30 % of straw was used for animial feed. Most animals have limited digestive ability toward cellulose,except ruminants and some herbivores, therefore, they can not use cellulose effectivelly. Then it is improtmant to pay for attention to improve the efficiency of cellulose animals. Usually,the digestion ability of animal can be improved by adding cellulose into feedstuff. This study focus on the cellulose activity of mulberry borer, obtained the sequences of CDS domain of cellulase gene, and expressed in E. coli.The purpose of this study is to supply the theory base for constructing engineering bacteria which can decompose cellulose effectively.1 The sodium carboxymethyl cellulose (CMC-Na) was used as substrate, the cellulase activity of mulberry borer was measured by 3,5-dinitrosalicylic acid method,the result showed: optimum pH of the cellulase of mulberry borer was 5.0,the cellulase activity was 1.057 U/g; optimum temperature was 50℃,the cellulase activity was 1.349 U/g;after treated under 37℃for 30 min, the cellulase activity almost remained, treated under 50℃for 30 min, the cellulase activity turned into 70.64 % of the maximum activity(1.349 U/g)。2 Designed a primer based on the sequence of Apriona germari cellulase III mRNA in GenBank, amplified cellulase gene from intestinal mRNA and fat genomic DNA of mulberry borer, respectively, ligated this gene with T-Vector for sequenceing. The sequence result showed this gene is from mulberry borer itself, and the length was 978 bp as a complete ORF, initiation codon was ATG, stop codon was TAA, the contents of A,G,T,Cwas27.81 %, 21.88 %, 24.34 % and 25.97 % respectively; the contents of A plus T was 52.15 %, the contents of G plus C was 47.85 %, cording 325 amino acid residues, Protein molecule weight is about 36.368 KD, Isoelectric point(pI) is about 4.30. BLAST results showed this sequence has most identity with GHF5(glycosyl hydrolase family 5) which belongs to cellulase superfamily.3 Redesigned the primers in which added BamHⅠr estriction site in upstream primer and SacⅠrestriction site in downstream primer. Amplified the cellulase gene of mulberry borer by PCR, after cutting by restriction enzymes of BamH I and SacI,ligated the sequence with PET-32(a) which treated by the same restriction enzymes, named the recombinant vector as PET-AG, transformed into E. coli. BL21(DE3) to incuce expression of cellulose gene. When the final concentrations of IPTG was 0.5 mmol/L, 1 mmol/L, 1.5 mmol/L, 2 mmo/L respectively, the result of SDS-PAGE electrophoresis showed that different final concentrations of IPTG had no effect on induced expression; when the final concentration of IPTG was 0.5 mmol/L, and the durations of inducing was 2 h, 4 h, 5 h, 6 h respectively, the expression of cellulose gene was lower at 2 h and keep the same level at 4 h,5 h,6 h respectively.