Construction Of A Protein Pesticide Producing Strain Against Plant Soft-rot Disease And Exploration Of Its Basic Fermentation Conditions

In this study,we firstly constructed a protein pesticide produce engineering strain by molecular biological method,which could inhibit the plant soft-rot disease.The sequence of the antibacterial gene?submitted to NCBI?was obtained from our group.The recombinant plasmid?named as LSD-P1?containing the antibacterial gene was constructed based on a shuffle vector.LSD-P1 was transferred and expressed in Escherichia coli and Bacillus subtilis,the two engineering strains was named LSD-PEC1and LSD-PBS1 respectively.And LSD-PBS1 could be used for industrial pesticide produce.The APC protein was effective against Pectobacterium carotovorum subsp.Carotovorum,however the antibacterial mechanism is still unknown.Secondly,the characteristics of APC protein was studied by bioinformatics methods and microbial biochemical methods.The physical and chemical properties of gene apc were predicted through a software for bioinformatics analysis named ExPASy.Results showed that APC protein was composed of 174 amino acids,molecular formula might be C₈₄₀H₁₃₅₁N₂₄₉O₂₆₅S₁₄,molecular weight was 19.6 kDa,and pI was 6.2.APC Protein was effectively expressed in Escherichia coli,and then purified with Ni column affinity chromatography.The stability of APC protein under different temperatures and pH conditions was also tested in this experiment.The data showed that APC protein remained stable under 28oC and degraded little at pH 6.0⁸.0.In the meanwhile,APC showed good antibacterial activity,the rate of antisepticise reached up to 48%.Finally,LSD-PBS1's fermentation conditions was optimized by single factor experiment,orthogonal experiment and response surface analysis with cell growth curve or numbers of viable bacteria as indicator parameter.The results demonstrated that the optimum culture medium formula for flask fermentation was:1.5%sucrose,1.5%yeast extract and 0.5%MgCl₂;optimization of flask fermentation conditions were as follows:the temperature was 33.5oC,start pH was 7.2 and liquid volume in flask was 56.24mL/500 mL.In order to be more close to the actual production,fermentors in the factory were used for optimization of fermentation conditions.The experiment data indicated that0.75%sucrose?0.75%corn starch?0.75%soybean powder?0.75%yeast extract?0.3%MgCl₂?0.3%KH₂PO₄ and 0.3%K₂HPO₄ were the best culture medium formula for fermentor fermentation;while 35oC,pH 7.2 and 300 r/min were the optimal fermentation conditions.Above all,LSD-PBS1 was aerobic,suitable for neutral medium,and grew best between 33.5oC³5oC.This study combined the APC antibacterial protein and plant probiotics,with the advantages of no waste in producing and no environmental pollution in application,which created a new method for the control of plant soft rot in the field.