Cloning, Expression And Application Of β-1,3-1,4-glucanase Gene From Bacillus Subtilis J18

Pear black spot caused by Alternaria alternate (Fr.) Keissler is one of the mainpostharvest diseases which affect the storage time and quality of pear and cause economicloss seriously. Using pear black spot as indicator，an antagonistic microbe named Bacillussubtilis J18was screened from soil samples of pear orchard. The research results show J18can secrete β-1,3-1,4-glucanase with resistance activity against pear black spot. In this study,the gene encoding β-1,3-1,4-glucanase is cloned and expressed in E. coli. The activity ofthe recombinant enzyme in controlling the black spotis detected. This study provides atheoretical basis for biological control.According to the reported sequences of β-1,3-1,4-glucanase, the primers for bothterminals are designed. The full sequence of β-1,3-1,4-glucanase gene from Bacillussubtilis J18is amplified with PCR technique and is cloned into pMD-18T vector. Thegene’s open reading frame contains648bp in total length, encodes215amino acids with acalculated molecular weight24kDa and contains the conserved sequence EIDIEF ofglycosyl hydrolase family F16. Its theoretical isoelectric point is5.77. Comparing with thesequences of other reported Bacillus β-1,3-1,4-glucanase, the sequence homology fornuclear acids residues are99%. The phylogenetic tree shows that the gene β-1,3-1,4glucanase of J18strain has the closest relationship with the same source of Bacillus subtilis.The gene β-1,3-1,4-glucanase of J18strain is inserted into the expression vector ofpET28a(+) and transformed into E. coli BL21(DE3) to express the recombinant proteininduced by IPTG.The antifungal experiments of recombinant β-1,3-1,4-glucanase shows that thisenzyme can significantly control the germination and germ tube’s elongation of Alternariaconidia, and destroy the mycelial growth. With the methods of damage vaccination anddirectly spraying recombinant β-1,3-1,4-glucanase and pathogens on pear, the prophylacticeffect of recombinant enzymes is conducted. The results show that the lesion diametercaused by co-inoculation pathogens and recombinant enzyme is less than the lesiondiameter with pathogens inoculation only. Moreover, the level of decay is significantlylower than the control. After treated by enzyme, the incidence of pear is37.61%and theprophylactic effect is66.25%. It is the first report for the function of β-1,3-1,4-glucanase to control pear black spot.The characteristics of the recombinant enzyme are investigated. The inhibitory activityof recombinant enzyme has no significant difference compared to the control when it isincubated30min in the temperature below55℃, but it begins a slow decline when above55℃. The recombinant enzyme is with highest inhibitory activity when the pH is6.0andstill in high antibacterial activity between5.0-8.0. Light has little impact on the antibacterialactivity of recombinant enzyme. After treated48h, the activity of recombinant enzymereduces only14.91%. Metal ions (1mmol/L) have little influence on the antibacterialactivity of recombinant enzyme, only some ions such as Ca2+, Mn2+and Cu2+can enhance,while Fe2+and Zn2+can inhibit the antibacterial activity of the recombinant enzyme.