Preparation Of A Double Effect Wettable Powder Of Bacillus Subtilis And Heterologous Expression Of Its Anti Insect Protein

Bacillus subtilis represents a plant growth-promoting rhizosphere bacterium(PGPR),which is ubiquitous in soil and rotten organic substance.As a GRAS strain awarded by the US Food and drug administration,B.subtilis is widely used in agriculture,animal husbandry,medicine and other fields.Owing to aforementioned characteristics,it is desirable to exploit B.subtilis for benefiting us.Plant hormones are a class of organic compounds with low content in plants.Of reported plant hormones,auxin(IAA)and brassinosterol(BR)can effectively promote plant growth.In this study,B.subtilis and plant hormones were investigated for the following aspects.1.The purpose of this section aims to determine the compatibility of B.subtilis with plant hormones,and thereby provide basis for their combination.The experiment was carried out by micro broth dilution method.There was no cytotoxicity in the range of soluble concentrations of auxin and brassinosterol.In addition,we systematically investigated the effects of auxin and brassinosterol on the growth of B.subtilis.Results indicateed that B.subtilis could grow normally in the presence of plant hormones.Hence,they can be mixed for use.2.Based on aforementioned experimental results,subsequent experiments optimimized the concentration of B.subtilis and plant hormones.Taking wheat as indicator plant,plant hormone solution and B.subtilis fermentation broth diluent were sprayed regularly.In this section,we examined the chlorophyll content,growth index,and soil microbial diversity of wheat.Based on above results,we optimized the growth-promoting concentration of plant hormone and the inhibitory concentration of B.subtilis.Results showed that 5 μg/mL of auxin and 0.04 μg/mL of brassinosterol led to excellent results.Furthermore,all dilutions of composition showed bacteriostatic effect,and the bacteriostatic effect of 10 times dilution resulted in higher bacteriostatic effect relative to other dilutions.3.In this section,we determined the formulation of wettable powder with B.subtilis and plant hormones as the main effective components.In doing so,we screened and optimized the carrier and various additives,and determined the composition as follows:1%auxin/0.08%brassinosterol,10%adjuvant(wetting agent:dispersant=9:1),2%stabilizer,1%UV protective agent,and the rest was master powder(The master powder was subjected to freeze-drying with 10%carrier in 100 ml bacterial solution).4.We successfully constructed the recombinant expression vector pBE-cm-Cry2Aa in gram negative bacteria,which can be used to express the target gene.Unfortunately,the recombinant strain B.subtilis could not be obtained.The reason behind might be that the replicator of pBE shuttle plasmid cannot work properly.The recombinant expression vector of B.subtilis pBE-cm-Cry2Aa can be constructed by replacing plasmid or altering resistance concentration.If this holds true,B.subtilis may have a wider application prospect in bacteriostasis and insect resistance.This will deeply tap the advantages of B.subtilis,and therefore pave the way for subsequent genetic engineering of platform bacteria.