Digging And Mosquito Activities Of The Toxic Proteins Of Mosquitocidal Strains Bt S2160-1 And Bt S3580-1

Bacillus thuringiensis is a gram-positive,spore-forming soil bacterium,characterized by the presence of parasporal crystal proteins which are very specific insecticidal activity to certain insect larva species as well as invertebrate pests during sporulation,and is broadly used in control of agricultural and forestry pests and mosquitoes as the biological pesticides.Mosquitocidal isolate of Bacillus thuringiensis S2160-1 was isolated from soil samples in the Dawangling Forest Nature Reserve(Guangxi,China)and maintained by Hainan Institute of Tropical Agricultural Resources(HITAR)and was considered as a potential alternative to mosquitocidal model strain Bti.In this research,the whole-genome sequence of Bt S2160-1 strain was performed through De Novo sequencing(Genome Gen Bank accession number:NSKZ00000000),in virtue of bioinformatics method,which was assembled into one nucleoid genome and two plasmids pS2160-1p1 and pS2160-1p2.Via constructing the BLAST alignment local database,16predicted toxic proteins were found in these two plasmids.10 predicted toxins on plasmid pS2160-1p1 concentrated distribution in four adjacent scaffolds,within a small range of 59.675 kb;while the distribution of other 6toxins on plasmid pS2160-1p2 was relatively scattered and distributed in 6scaffolds with arange of 252.658 kb.Obviously,these toxin genes were clustered distribution,forming a gene cluster like the bacterial pathogenicity island,we speculated that it should be a mosquitocidal toxin island.Subsequently,a whole-proteomics analysis of Bt S2160-1 strain was carried out by using the LTQ-Obitrap MS/MS mass spectrometry technology.As a result,159 250 raw mass spectra data were obtained.Using the predicted ORF protein data of the Bt S2160-1 genome as the searching database,26 866 PMFs(Peptide Mass Fingerprinting,PMF)9 282 PSMs(Peptide Spectrum Match,PSM)and 9 017 unique peptides were retrieved.Based on the obtained spectra,1 959 proteins with high scores were identified.And it was confirmed that 13 of 16 predicted toxic proteins were expressed during the growth of the strain.Meanwhile,p ET30a expression vector was carried out for all the 16 candidated toxins in vitro in E.coli BL21(DE3)prokaryotic expression,and 15 of them were successfully expressed in vitro and identified again by MALDI-TOF MS.Finally,biological activity of the 15 predicted toxins of Bt S2160-1 strain against three-instar larvae of Culex pipiens pallens were performed,it was found that all these 15 predicted toxic proteins were toxicity to three instar larvae of Culex pipiens pallens,but the mosquitocidal activity of each protein is quite different.In addition,we found that the individual protein mosquitocidal activity on the pS2160-1p1 plasmid was generally higher than that of pS2160-1p2.Nevertheless,even if the highest mosquitocidal activity of the individual protein pS2160-1p1?gene?id?2(LC₅₀:5.373?g/m L)was far less than that of the strain itself.The bioassay 15 predicted toxic proteins in pairs and their synerger to the third instar larvae of mosquitoes,and the results showed that pS2160-1p1?gene?id?2(Cry54Ba)combined with pS2160-1p1?gene?id?22(Cry4Cc*),pS2160-1p1?gene?id?41(Cry4Ca)and pS2160-1p1?gene?id?55(Cry4Cb),respectively,is able to enhance the latter's mosquito larvicidal toxicity with its synergy factor of 1.75,1.60 and 1.67;while PS2160-1p1?gene?id?40(Cry71Aa),pS2160-1p1?gene?id?48(Cry4Ca)and pS2160-1p1?gene?id?54(Cry30Ea4)were combined with pS2160-1p1?gene?id?41(Cry4Ca),respectively,are able to enhance the latter's mosquito larvicidal toxicity with its synergy factor of 1.37,1.28 and 1.23.The combination of pS2160-1p1?gene?id?8(Cry73Aa)and pS2160-1p1?gene?id?55(Cry4Cb)has a synergy factor of 1.25,and the combination of pS2160-1p1?gene?id?48(Cry4Ca*)and pS2160-1p1?gene?id?49(Cry72Aa)has synergy factor 1.47.Simultaneously,biological activity of the mixture of expressed toxins with equal molar ratio on plasmid pS2160-1p1 and pS2160-1p2 were carried out.The result showed that the mortality rate of mosquito larvae for the 9expressed proteins in pS2160-1p1 plasmid reached 50%after 48 h of co-culture,and 100%after 72 h of co-culture,LC₅₀ was 2.4703?g/ml(95%FL:1.843?3.185?g/ml),indicating the mosquito larvicidal toxicity of the mixed all 9 expressed toxic proteins in pS2160-1p1 plasmid.While all the six toxic proteins in pS2160-1p2 plasmid,after 48 h of co-culture,mosquito larvae had not yet started to die,but the physilogical activity of mosquito larvae was lower than normal,and the mortality rate of mosquito larvae after72 h of co-culture had not reached 50%,LC₅₀ was 66.879?g/ml(95%FL:37.879?451.976?g/ml),all these bioassay data fully support that plasmid pS2160-1p1 is the dominant plasmid contributing to the major toxicity,the pS2160-1p2 plasmid might be a helper plasmid that is conducive to mosquitocidal toxicity.Finally,the 15 expressed toxins on plasmid pS2160-1p1 and pS2160-1p2were performed for further bioassay with the same molar ratio,and it caused100%larval death within 24 h infected.Obviously,the mosquitocidal activity of all in vitro expressed toxins of the strain Bt S2160-1 is very high(LC₅₀:1.589?g/m L and 95%FL:1.166?2.048?g/m L),but it is still lower than that of the total crude protein of the strain itself(LC₅₀:135.873 ng/m L).In addition,we cloned and identified two new insecticidal toxin proteins Epp and Cry80Aa1 in another mosquitocidal strain Bt S3580-1.Epp protein exhibits toxic activity against Spodoptera litura and Culex pipiens pallens;Cry80Aa1 has mosquitocidal activity to Culex pipiens pallens(LC₅₀:71.9?g/m L;95%FL:59.5?122.7?g/m L).Summary,our investdigation revealed the presence of the mosquitocidal toxin island in Bt S2160-1 mosquitocidal strain,which possessed at least 16toxic proteins and their synergistic effect to produce highly mosquito toxicity.Bt S2160-1,it is a common mosquitocidal strain that has obvious difference with generally accepted Bti mosquitocidal mode strain;therefore,it is very vital significance to perform further more in-depth and meticulous research.The application of Bti alternative strain Bt S2160-1 to control mosquitoes and other diptera pests would bring more benefits to human health and agriculture.