Mining And Functional Identification Of Efficient New Insecticidal Genes Against Laodelphax Striatellus From Bacillus Thuringiensis

Bacillus thuringiensis?Bt?,is a gram-positive bacteria,possess highly specific insecticidal activity.Decades of application show that Bt production including transgenic plants with Bt insecticidal gene is the most effective means of biological control of agricultural pests.Planthopper has always been an important pest of rice,recurrent outbreaks have brought serious disaster to rice production.The control of the rice planthopper mainly rely on the chemical pesticides,laeding to an enhanced resistance and resurgence damage,resulting in serious environmental pollution and food safety issues.Developing an efficient biological control measures to prevent the damage caused by rice planthopper is a hot and challenging problem urgent needed to be solved.However,to date no Bt gene has been found show high insecticidal activity to the planthoppers,which seriously impeded the expansion of Bt insecticidal spectrum and large-scale applications.In this thesis,we started from the establishment of standard bioassay system,extensive screening large number of Bt strains to the rice planthopper.After the screening we obtained a Bt strain which show high toxicity against Laodelphax striatellus,small brown planthopper,SBPH.And we mined new insecticidal gene from the Bt strain and then verified the gene function.1.Screening of the insecticidal activity Bt strains against SBPH.Improvement of traditional planthopper bioassay method.SBPH is very sensitive to humidity,the air in north of China is dry,the control of humidity do not meet the require of SBPH ever in a light incubator.Mortality rate of CK would reach above 20%frequently.We use plastic bottles with lids,at the bottom of the bottle 4 mL 2%agarose gel was added,it can maintain a suitable humidity.After the upgrade of the method,mortality rate of CK decline to less than 10%.The strategy which separated the lids and bottles,conducive to replace the diet,convenient for numerous operations,has laid a solid foundation to high-throughput screening of insecticidal genes.Screening of high insecticidal activity strains.Using the modified bioassay system,feed the SBPH Bt crystal protein extracted form different strains which dissolve into artificial diet.A total of 68 strains candidates preserved in biotechnology group,institute of plant protection,Chinese academy of agricultural sciences were chosen for the screening.Finally,we got a Bt strain,IPPBI0TSUC1012,show extremely high toxicity to the SBPH.2.Mining and verification of new insecticidal Bt gene.Purification and identification of insecticidal protein.The protein sample which extracted from IPPBI0TSUC1012 was separated by gel filtration.Differed fraction were collected whose insecticidal activity were tested while SDS-PAGE analysis were conducted simultaneously.Combine the concentrate of the protein?read by the gray value of the protein band on gel?and the mortality rate caused by different fraction,a correlation coefficient has been calculated.Two protein bands were in high correlation with the insecticidal activity?r=0.83?,and there is another one has moderate correlation?r=0.66?.Bt genome sequence and new gene mining.Sequence the genome DNA of IPPBI0TSUC1012 with the 454 and illumina platform.Assembled the reads,obtained a 5.51 M draft genome,a total of 39 scaffolds.The genome sequences were predicted by GeneMark.hmm PROKARYOTIC generated 6063 protein coding sequences.All those gene were annotated by uniport protein database,and 5846 out of them get an annotation information accounted for 96.4%of the predicted sequences.Based those information three key words?Cry,Cyt,Vip?used for retrieval the sequences.Only two sequences named Translation5511312..5512181?direct?,290 amino acids;Translation5512497..5513384?direct?,296 amino acids correspondence to the key word 'Cry'.Coincidentally,the two sequences share the same annotation,whose primary accession number in Uniport is D4QGQ7?Cry64Aa1?.Collected the annotated sequences format a Mascot local database,retrieved with the LC-MS/MS data.The aforementioned two proteins high correlated with the insecticidal activity match the two sequences annotated the D4QGQ7 in Uniport.Two protein coding sequences were named Small Brown Plant Hopper Toxin,two genes were short for ST-1 and ST-2 respectively.The amino acid sequence identity between ST-1 and ST-2 is 51%.Take ST-2 for example it share 45%,36%,and 31%identity with Cry64Aa1,Cry33Aal and Cry15Aa.3.Insecticidal activity assay of the genes which show tocixity to SBPHHeterologous prokaryotic expression,purification and activity assay of ST-1 and ST-2.PHT-1A plasmid was occupied,three recombinant plasmid were constructed containing ST-1,ST-2,and ST-12?ST-1+ST-2?respectively.The recombinant plasmids were electrotransformated into Bt crystalliferous strain HD73-.ST-1 and ST-2 do not express the target protein,while the ST-1 and ST-2 co-expressed successfully.A 6 d bioassay were conducted with the two expressed protein?concentration is 5.27 and 5.13?g/mL respectively?,cause a 90%corrected mortality.Purify the two proteins by gel filtration followed by ion-exchange.Desalted and concentrated purified proteins,used to test the insecticidal activity.The purified ST-1 and ST-2 show extremely high toxicity to the tested two insects.The ST-1 and ST-2 virulence regression equation to SBPH is y=3.67x+3.62,and to white-backed planthopper?Sogatellafurcifera,WBPH?is y=3.95x+2.99.LC50 is 2.38?g/g for SBPH,3.22?g/g for WBPH which was calculated by SPSS Probit regression.95%confidence interval were 2.12-3.80?g/g,1.18-2.57?g/g.Among all the Bt proteins,the insecticidal activity of ST-1 and ST-2 second only to the Vipl/Vip2 binary toxin,furthermore ST-1 and ST-2 possess the highest insecticidal activity against rice planthopper.ST-1 and ST-2 do not show any insecticidal activity to Chilo suppressalis,Plutella xylostella,and colaphellus bowringi at the concentrate of 300?g/g,indicate a high specific insecticidal activity to rice planthopper.ST-1 and ST-2 contain ETX-MTX2 domain.According to previous research,proteins which contain ETX-MTX2 or Toxin₁0 domain may share the same?-sheet structure similar to the aerolysin.Based on the mimic structure we believe that they may be pore-forming toxin lead to high insecticidal activity against hemiptera insects.In this research we fill the blank that no Bt protein show high insecticidal activity against rice planthopper,expand the insecticidal spectrum of Bt proteins,make a great advance for the biocontrol of hemiptera pests using Bt proteins,meanwhile provid a powerful tool for biological control of pests on rice.4.Analysis of Bt receptors and related functional genes based on the midgut transcriptomeSequencing and analysis the SBPH midgut transcriptome.After removal of low-quality reads,produced a total of 26,404,470 pieces of the average length of 90 bp clean reads.After Trinity splicing produces a total of 31,435 scaffold,of which 15,792?50%?items share a significant similarity(E-value<10^-5)with known NCBI protein sequences and submitted to NCBI SRA database with an accession number of SRX274939.GO and COG analysis been done to the transcriptome after annotation.And a comparison between midgut transcriptome and whole body transcriptome of SBPH has been carried out.GO analyzed the up regulated genes in midgut,a considerable number of genes categorized into two items,"Binding" and"Catalytic activity".This consistent with the physiological functions of the intestine,the digestion of the food and absorption of the nutrition,closely related the catalytic activity and binding.The common receptors of Cry protein were also found,including Aminopeptidase N?APN?,Alkaline phosphatase?ALP?,and Cadherin?CAD?.Provide the molecular biology basis for the insecticidal activity Bt protein screening and the mechanism model.Ryanodine receptor?RyR?gene mining.Take advantage of five agricultural pests?L.striatellus,Bemisia tabaci,C.suppressalis,P.xylostella,Cnaphalocrocis medinalis?transcriptome to mine RyR fragments.Based on the location information of each fragments,primer were designed to amplify longer transcripts to cover the gaps between different fragments.Rapid-amplification of cDNA end?RACE?used to get the terminal sequences.Finnally,integrate all the obtained sequences into a full-length RyR gene.Three out 5 species RyR gene got the full length sequnces,one of the remaining two got a complete ORF,and one got a partial sequence.Northern Blot conformed that those five genes exist in mRNA transcripts.Analyze the sequences of those RyRs,2 EF-Hands,6 transmembrane regions were identify.And 7 lepidopteran pest exclusive sites?N4968,N4970,N4981,S5004,L5027,N5059 and R5076?were found near the pore-forming sequence in the C-terminal of those RyRs.Given the fact that diamide pesticide is highly toxic to lepidopteran insects,and RyR receptor act as the insecticide target,it is reasonable to believe that the seven lepidopteran pest exclusive sites is directly related to the specificity of diamide insecticides.The cloning of the RyR sequences is an orderly advance to the resistance management of rice planthopper.The successfully cloning of RyR would also provide a useful reference and technical support for the mining of Bt receptors.