New Insecticidal Gene Excavation And Functional Verification Of Bacillus Thuringiensis From High Altitude Regions

Bacillus thuringiensis(referred to as Bt)is a gram positive bacteria,which are widely distributed in nature.The main ingredients of insecticidal activity is coded by the insecticidal protein gene(cry/cyt)gene,and produce insecticidal crystal protein at stage of spore.In recent years,many domestic scholars successive isolated high virulence of new Bt strains from the soil in different provinces and different regions.However,it's rarely reported that Bt resources are from high altitude area and different ecological environment.The west of Sichuan,including the different ecological environment,such as primeval forest,grassland,wetland and glacier;climate areas show significant difference.This experiment described a systematic study of resources in different ecological regions and filtrated new insecticidal genes on high altitude area in west of Sichuan,the results are as follows:1.In total,351 Bt strains have been isolated from 912 soil samples collected from the high altitudes in the west of Sichuan.And identified 161 crystal Bt bacteria strains by using optical microscope,the average separation rate was 38.49%(the ratio of Bt bacteria accounted for all of coli)and crystal Bt bacteria separation rate is 17.65%.These Bt strains produced bipyramidal,spHerical,square,and grain crystal inclusions observed under scanning electron microscopy,which indicatied the diversity of Bt strains harboured in the west of Sichuan.With the increase of altitude,Bt separation rate began to fall;meanwhile,the separation of forest rate is higher than the snow mountain,grassland separation rate.2.The analysis of the cry and cyt genes of the 161 Bt strains was based on the method of Polymerase Chain Reaction(PCR)-Restriction Fragment Length PolymorpHism(PCR-RFLP).cry1,cry1l,cry2,cry4,cry5,cry6,cry9,cry12,cry14,cry15,cryl8,cry28,cry30,cry54,cry57 genotypes were found in these Bt strains.Strains containing cry1 genes were the most abundant in our collection(50%).followed by containing cry16 class and cry9 class gene,46.36%and 46.36%,respectively.and the strains containing cryll,cryl5,cry30 and cry4 genes were found in 29.09%,27.27%,21.81%,17.27%,respectively.and other Bt strains contained fewer kinds of genotypes.3.The SDS-PAGE analysis of the producing crystal protein results showed these protein were expressed almost at size of 30-130 kDa.Expressed protein of the 161 Bt strains were tested against Ostrinia fumacalis,Helicoverpa armigera,Laphygma exigua,Plutella xylostella,Agrotis ypsilon Rottemberg and Celegans.The bioassay results indicated that,insecticidal activities of 18.6%Bt strains had high activity against Plutella xylostella;19.87%Bt strains had high activity against Helicoverpa armigera;31.7%Bt strains strains had high activity against Laphygma exigua;13%Bt strains had high activity against Agrotis ypsilon Rottemberga;17.39%Bt strains had high activity against Ostrinia fumacalis;17.4%Bt strains had high activity against C.elegans.whereby,insecticidal activities of the bipyramidal and square crystal Bt strains had higher toxicity against lepidoptera pests,compared to the insecticidal activities of grain shape crystal Bt strains.In addition,part of the spHerical crystal strains had insecticidal effect on the Helicoverpa armigera and Laphygma exigua,but the 50%lethal concentration(LC50)is higher.4.Chosed a bipyramidal crystal Bt strain which had effect on C elegans and two spHerical crystal strains which had effect on on the Helicoverpa armigera and Laphygma exigua from 161 strains for next research.Through the complete genome sequencing,four new gene were found and named as cry32Y-like,cry32X-like,cry9H-like and cry72A-liike.By the method of PCR amplification with the degenerate primers and clone sequencing,the full-length of 4107bp,1893bp,2094 bp and 2127bp,respectively,were obtained.The four encoded protein shared 55%,45%,48%and 95%sequence homology with cry32Hb1,cry32Sa1,cry9Ha4 and cry72Aal.The gene cry32Y-like was inserted into the shuttle expression vector pSTK to obtain the recombinant plasmid pSTK-32Y,then transformed into Bt mutant HD73-and produced spHerical crystal.The gene cry32X-like,cry9H-like and cry72A-like were inserted into E.coli prokaryotic expression vector pET-28a to obtain the recombinant plasmid pET-32X,pET-9H and pET-72A,and then transformed into E.coli BL21(DE3)cell.The protein expression induced by Isopropyl ?-D-1-thiogalactopyranoside(IPTG)at low temperature.New gene expression protein was tested against Ostrinia fumacalis,Helicoverpa armigera,Laphygma exigua,Celegans and Meloidogyne incognita.Biological activity results showed that Cry32Y-like had high insecticidal activity against C.elegans and Meloidogyne incognita,the corrected mortality were 72%and 64%72h later,with LC50 of 803 and 920?g/mL,respectively.Compared to Ostrinia fumacalis and Helicoverpa armigerav,Cry32X-like expression product had higher adjust mortality ratio(65%)against Laphygma exigua,with LC50 of 710 ?g/mL.Cry32X-like expression product also had high insecticidal activity against C.elegans and Meloidogyne incognita,the corrected mortality was 76%and 70%72h later,with LC50 of 0.421 and 603?g/mL,respectively.Cry9H-like expression product had higher insecticidal activity against Helicoverpa armigerav with the adjust mortality ratio of 45%and the inhibition growth rate of 55%7 days later.Cry72A-like expression product have higher insecticidal activity aginst Laphygma exigua with LC50 of 961?g/mL,and the adjust mortality ratio of 95%7 days later.5.The gene cry71Aal and cry72Aal was inserted into the shuttle expression vector pSTK to obtain the recombinant plasmid pSTK-71A and pSTK-72A,then transformed into Bt mutant HD73-and produced spHerical crystal.Biological activity results showed that Cry71Aa1 and Cry72Aa1 had insecticidal activity against Meloidogyne incognita,and the corrected mortality were 76%and 53%72h later,with LC50 of 542 and 872 ?g/mL.6.The Biological activity results of known genes from our lab showed that Cry1Hc1?Cry1Aj1 had high insecticidal activity against C.elegans,and the corrected mortality were 84%and 82%72h later.These genes expression products showed insecticidal activity against lepidoptera and nematodes respectively,which not only enriched the variety of insecticidal genes,also provided experimental basis for new genes insecticidal spectrum.