| The specific insecticidal proteins synthesized by Bacillus thuringiensis（Bt）make Bt strains to be the most successful microorganisms in the field of agriculture and forestry,but the function against disease of Bacillus thuringiensis is rarely reported.Some studies have transferred the cry genes into Bacillus subtilis with disease control functions,and constructed Bacillus engineering bacteria with both disease prevention and insecticidal functions to expand the function of biocontrol microorganisms,but the application of genetically modified microorganisms is still limited.Therefore,it has broad application prospects to study Bacillus with natural insecticidal and disease prevention functions and develop it into biological pesticides.In addition,as a biological pesticide,Bacillus is often used in crop seed treatment to control pests and diseases,and the temperature during sowing in early spring or late autumn is low.Whether Bacillus can tolerate low temperature is one of the important limiting factors for its control effect.Our lab isolated and identified many Bacillus strains with cold-adaptation ability and good bacteriostatic effect from the rhizosphere soil of plants on the Qinghai-Tibet Plateau in the early stage.Among them,Bacillus thuringiensis GBAC46 strain has the application potential of controlling plant diseases and insect pests.In this paper,the cold tolerance of the strain and the effect of cold shock proteins on the cold tolerance of the strain were studied,as well as their functions of controlling diseases and insect pests.In addition,the regulation effect of RapA4-PhrA4 system on the biocontrol active substance Cry protein synthesis and spore formation was studied.The above researches can provide a theoretical basis for the application of the Bacillus thuringiensis strain in the control of plant diseases and insect pests.The main findings are as follows:1.When focusing on the biological characteristics of GBAC46 strain,we found that the strain has good low temperature tolerance and can grow at 10°C.The strain also has good high alkali resistance and could still grow at p H=10.However,the strain doesn’t have the ability to tolerate high salt and high oxidative stress.Considering the outstanding low temperature tolerance of GBAC46 strain,we screened and identified the cold shock proteins of the strain,and found that this strain has 9 genes encoding cold shock proteins,including 7typical cold shock proteins Csp A,Csp B1,Csp B2,Csp C1,Csp C2,Csp D1,Csp D2 and two unknown cold shock proteins Csp1,Csp2.When analyzing the amino acid sequence,we found that the amino acid sequence homology of 7 typical cold shock proteins of GBAC46strain and 3 cold shock proteins of OKB105 strain ranged from 64.18%to 86.57.Phylogenetic tree of the cold shock protein showed that Csp1 and Csp2 had the closest homology to Csp H from E.coli,but the amino acid sequence homology was 9.72%and 10%,respectively.When studying the biological function of cold shock protein,we found that the overexpression of cold shock protein from GBAC46 strain could help OKB105 strain adapt low temperature environment more quickly.The possible target genes regulated by Csps were screened by RT-q PCR experiments,and we found that mae N（sodium ion,malate transporter）,rbsa（ribosome binding protein）,narg（nitrate reductase）liah and ywsb（small molecule stress protein）were down-regulated when the Csps was overexpressed at 37℃,while the expression of these genes were upregulated when the Csps was overexpressed at14℃,which means that cold shock proteins may regulate the expression of these genes or adjust the secondary structure of these m RNA.Finally,we proved that the cold shock proteins Csp A,Csp B1,Csp C1 and Csp D1 can interact with the promoters of genes liah and rbsa in vitro by MST experiments,which indicates that the cold shock proteins may activate the transcribe of target genes by targeting the promoters of liah and rbsa genes,resulting more Lia H protein was synthesized to protect the stability of cell membrane and more ATP was synthesized,thereby promoting the adaptability of OKB105 strain under low temperature.The separate interaction between cold shock proteins Csp A,Csp B1,Csp C1 and Csp D1 and the promoters of genes liah and rbsa also indicates the functions redundancy of cold shock proteins.2.It was found that the GBAC46 strain shows high pathogenic to Aphelenchoides besseyi.In order to further identify the nematicidal active substances of the strain,we screened the whole genomes of the strain by protein-nucleic acid alignment.Three novel Cry proteins were found in GBAC46 strain,namely Cry31Aa,Cry73Aa and Cry40ORF proteins.By comparing with all the reported insecticidal proteins（Bacterial Insecticidal Protein Database,BPPRC）,we found that the Cry31Aa protein has the closest evolutionary relationship with the Cry70Ba1 protein with 65.48%amino acid sequence homology,and the Cry40ORF protein and the Cry48Ab1 protein are closely related with 14.26%amino acid homology.Cry73Aa protein and MPP46Aa1 protein are closely related,and the amino acid homology is only 11.62%.It means that the three Cry proteins from GBAC46 strain are novel and unknown Cry proteins.The Cry31Aa protein only shows insecticidal activity against Aphelenchoides besseyi,and its EC50 value is 131.80ug/ml,while the Cry31Aa protein don’t have insecticidal activity against Caenorhabditis elegans and other nematodes.It was proved that Cry31Aa protein could be uptaken by Aphelenchoides besseyi by labeling the protein with rhodamine,and propidium iodide experiments further showed that Cry31Aa could damage the nematode intestinal cell membrane,which indicated that the protein was a pore-formation toxin..In addition,we tested the growth-promoting effects of GBAC46 strain on rice.The statistics results from plant root scanner machines showed that the strain could significantly promote the growth and development of rice root length,total root surface area,root tips and root forks.Our RT-q PCR experiments also showed that both strains were able to activate the expression of rice growth-related genes.Moreover,GBAC46 strain can significantly induce the expression of rice pathogen-related genes（PR genes）,especially the relative expression of PR1a gene was up-regulated by about 22 times.Consistent results were also obtained when detecting hydrogen peroxide and malondialdehyde（indicators of plant defense responses）in rice leaves.These results indicate that GBAC46 strain can not only promote rice growth,but also induce high expression of rice defense-related genes and activate systemic resistance of rice.3.The Rap-Phr system in Bacillus could regulate the synthesis of some metabolites.We hope to discover a new and powerful Rap-Phr system to increase the expression of biocontrol factors.Bioinformatics analysis found that GBAC46 strain contained 7 Rapproteins,respectively,which were all present in their genomes.When analyzing the amino acid sequence homology of Rapprotein,a novel RapA4-PhrA4 system was identified in B3 strain,which is absent in GBAC46,NMTD81,FZB42 and 168 strains and has low similarity with known Rap-Phr systems.By overexpressing in GBAC46,NMTD81 and OKB105 strains,we found that the RapA4-PhrA4 system can regulate the formation of spores in these strains.RT-q PCR results showed that the RapA4-PhrA4 system could regulate the expression of three cry genes in GBAC46 strain.At the same time,we also found that the system can regulate the antibacterial activity of GBAC46 and NMTD81 strains against Sclerotinia sclerotiorum,indicating that the system can regulate the synthesis of antibacterial active substances of the two strains.In addition,the results of hemolysis analysis,expelling oil and HPLC experiments showed that the system could also regulate the synthesis of surfactin in OKB105 strain.RT-q PCR results further proved that the system could regulate the expression of genes related to sporulation and surfactin synthesis.4.When biological controlling Zea may diseases and insect pests caused by Fusarium verticillium and Spodoptera frugiperda using GBAC46 strain,we found that GBAC46 strain has the ability to control diseases and insects at the same time.The experimental results showed that the phenomenon of Zea may seedlings being eaten by Spodoptera frugiperda was significantly reduced when the GBAC46 strain solution was sprayed,and the plant height,fresh weight and dry weight were significantly increased.The yellowing and shrinking of leaves caused by Fusarium verticillium were reduced,and plant height,fresh weight and dry weight and other indicators also increased significantly,indicating that GBAC46 strain can simultaneously control Spodoptera frugiperda pests and disease caused by Fusarium verticillium.RT-q PCR results showed that GBAC46 strain could significantly induce the expression of key genes of salicylic acid,jasmonic acid and ethylene pathway,which indicated that GBAC46 strain could induce defense response of Zea may.In addition,the feeding of Spodoptera frugiperda on Zea can significantly up-regulated the expression level of defense-related genes in Zea.In addition,when Spodoptera frugiperda and F.verticillata shared the same host plant,we found that the feeding of S.frugiperda could promote the infection of F.verticillate on Zea may.The Zea may seedling plants were dwarfed as a whole,and the leaves and stems were severely shrunk,when Fusarium verticillium and Spodoptera frugiperda coexisted.The above-ground height,fresh weight,dry weight,and root length,fresh weight,and dry weight of the plants further indicated that Spodoptera frugiperda feeding could promote the infection of Fusarium verticillium on Zea may. |
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