Isolation And Identification Of The Amylase-producing Bacteria In Silkworm Intestine Then Cloning And Expression Of Their Amylase Genes

Silkworm is an important economic insect which has a great economic and social benefit.Microorganisms in silkworm gut have an important function for their growth and micro-ecological balance.Most reports about silkworm gut bacteria focused on the structure of their micro-ecosystem and nosogenesis while less reports about their application in enzyme-producing bacteria at present.Amylase producing intestinal bacteria can help the host digest starch or other nutrients more effectively.And these bacteria have some other probiotic functions.In recent years,probiotics application in animal microbial preparation concentrated in the production of aquatic and poultry production.Yet few study was about silkworm probiotics.Problems on low leave silk changing rate and high silkworm disease incidence in Sericulture always existed,so make research about silkworm bacteria and their enzyme production ability then make a prokaryotic gene expression analysis about enzyme gene may have some significance in improving the silkworm gut microenvironment and in development of silkworm artificial diet.Some culturable bacteria in larval guts of the Bombyx mori at5th year phase were isolated and purified by traditional method.Two amylase-producing strains of them was screened though beef extract peptone starch medium.Enzyme-producing bacteria’s16SrDNA sequences were amplified and cloned with bacteria identification universal primers respectively.Blasted these cloning sequences with sequences existed in GenBank,and the two strains both have99%homology with Bacillus cereus.Identified these strains we got as Bacillus cereus combining with observation of their colony shape and observation of their individual shape by microscope.Determining strains’ amylase activity by DNS method.Results showed,enzyme produced by the silkworm gut bacteria had a certain activity,and strains’16SrDNA sequences were also different from sequences existed in GenBank,and thus has some significance.Primers of α-amylase genes(Ec.3.2.1.1) were designed according to known genes after identification of isolated strains.Amylase gene sequences were amplified from genomic DNA of the two strains.Complete open reading frames sequences were got after cloning,sequencing and taking analysis of the Bacillus cereus.Sequences analysis of the amylase genes showed that the cloned sequences of the two strains were3414bp and3378bp respectively.They both contained complete open reading frames1761bp encoding586amino acid residues.Isolated these amino acid residues as α-amylase superfamily,but sequences of the two strains were diffieret.Among them,the793th base of the strain DZ-a was T,while the strain DZ-h was C.The1381th base of the strain DZ-a was A,while the strain DZ-h was G.So the relevant amino acid of them,the265th aa of the strain DZ-a was S,while of the strain DZ-h was P.The461th aa of the strain DZ-a was I,while of the strain DZ-h was V.α-amylase gene of the strain DZ-a were expressed in Escherichia coli prokaryotic expression systems.The target protein about66kD was got,and this was matching the size of its predicted protein product by DNAStar software.Primers containing enzyme restriction sites according to the coding sequence were designed to amplify target gene sequences.Then linked them to the expression vector pET-28a (+) and constructed a prokaryotic expression vector.The vector was digested and identified then was transformed into the Escherichia coli host cell Transetta (DE3) which containing infrequent codons.Induced their expression by different concentrations of IPTG.SDS-PAGE electrophoresis detection indicated that the target protein all had a stable expression.These results will provide the foundation for further eukaryotic expression of the strains’ amylase gene and for further function study of them.