Studies On Bacillus Thuringiensis Strains Bt11 And GS8

Bacillus thuringiensis(Bt) is one of the most widely used insecticidal microbes. The toxicity of Bt mainly results from its insecticidal crystal proteins formed during sporulation. Cloning of novel cry genes will be beneficial for appilication of gene resources and construction of genetically engineered strains and transgenic plants. In this study, cry1Ia, cry1Ib and cry9Ea genes were cloned and expressed. Especially, the mechanism between Cry proteins and insects was primarily investigated.Bt strains Bt11 and GS8 were studied systematically on biological and genetic characteristics. Growth speed of Bt11 and GS8 was similar with that of HD-73 and 4Q7. The crystal shapes from Bt11 were diamondoid, spherical and squarish, and from GS8 were diamondoid, spherical and irregular. SDS-PAGE analysis of the parasporal crystal demonstrated that Bt11 strain was composed of 35, 80, 130kDa proteins, and that GS8 was composed of 60, 81, 130kDa proteins. The results of cry genes identified by PCR-RFLP method showed that cry1Aa, cry1Ab, cry1Ia, cry2Ab and cry9Ea were found in Bt11 and cry1Aa, cry1Ab, cry1Ib, cry2Ab and cry9Ba were found in GS8. Five plasmid bands from Bt11 strain were observed in agarose gel and four bands from GS8. The biochemical and morphological analyses showed Bt11 was the same as B.t subsp. kurstaki and GS8 was the same as B.t subsp. tohokuensis.The cry9Ea,cry1Ia and cry1Ib genes have been isolated from Bt11 and GS8, respectively. Sequencing analysis showed cry9Ea is 3453 bps in length and its deduced amino acid is 129.874kDa. Isoelectric point is pH4.68. cry1Ia is 2160 bps in length and its deduced amino acid is 81.202kDa. Isoelectric point is pH5.955. cry1Ib is 2160 bps in length and its deduced amino acid is 81.372kDa. Isoelectric point is pH6.215. The cry9Ea, cry1Ia and cry1Ib genes have been registered in GenBank (Accession number are EU887516, EU887515 and EU677422 , respectively) and named as cry9Ea6, cry1Ia14 and cry1Ib3 by International Nomenclature Committee of Bt-δ-endotoxin Gene , respectively.The cry9Ea6, cry1Ia14 and cry1Ib3 gene were inserted into expression vector pET-21b and recombinant plasmids pET-9Ea, pET-1Ia and pET-1Ib were abtained and transformed into E.coli BL21 (DE3), respectively. The cry9Ea6, cry1Ia14 and cry1Ib3 genes were expressed in BL21 (DE3). The bioassay results showed that Cry9Ea6 was highly toxic to Trichoplusia ni neonates(LC₅₀ 0.0639μg/mL) and plutella xylostea neonates (LC₅₀ 4.574μg/mL) , and that Cry1Ia14 was highly toxic to Trichoplusia ni neonates (LC₅₀ 2.0342μg/mL) , Bombyx mori neonates (LC₅₀ 0.0163μg/mL) and plutella xylostea neonates (LC₅₀ 0.0261μg/mL), higher toxity than to Spodoptera exigua neonates (LC₅₀ 184.7727μg/mL) and Helicoverpa armigera neonates (LC₅₀ 70.90μg/mL). Cry1Ib3 was highly toxic to plutella xylostea neonates (LC₅₀ 0.0763μg/mL).The cry1Ia14 was inserted into expression vector pQE30 and transformed into E.coli M15. The Cry1Ia14 protein could be expressed normally in M15 with 81kDa molecular weight. Cry1Ia14 was purified by using affinity chromatography. Bt-E.coli shuttle expression vector pSXY-9EA, pSXY-1IA and pSXY-1IB were constructed by cry9Ea6, cry1Ia14 and cry1Ib3 gene being inserted into pSXY422b, respectively. Recombinant plasmids were transformed into Bt acrystalliferous mutant strain HD73(cry-). SDS-PAGE analysis indicated that the cry9Ea6 gene was expressed well in the HD73(cry-), however cry1Ia14 and cry1Ib3 gene were not expressed in HD73(cry-). Cry9Ea6 crystal protein was extracted by pH dependent precipitation method. The antisera against Cry9Ea6 and Cry1Ia14 were gained by immuning rabbit. The titre of them was 1:10,000. Bioassay showed Cry9Ea6 crystal protein appeared high insecticidal activity against both Trichoplusia ni neonates (LC₅₀ 0.0532μg/mL) and plutella xylostea neonates (LC₅₀ 1.2259μg/mL).The activated Cry9Ea6 and Cry1Ia14 proteins by Trypsin, were fed to Trichoplusia ni neonates. The bioassay results showed that the toxicity of activated Cry9Ea6 was as 5 times as Cry9Ea6 protoxin, and of activated Cry1Ia14 was as 3 times as Cry1Ia14 protoxin. From the above results, it is inferred that the toxic polypeptide of Cry1Ia14 was 65kDa and 50kDa. Whereas, the activated Cry9Ea6 showed 38kDa by SDS-PAGE analysis.PMs of T.ni and S. exigua were treated with Cry1Ia14 and Cry9Ea6 proteins in vivo and in vitro.The results showed that Cry1Ia14 protein destroyed the structure of the PM and midgut of T.ni and S. exigua, however Cry9Ea6 protein only destroyed the structure of the PM and midgut of T.ni.By using Mg/EGTA method, the BBMV of T.ni and S. exigua midguts were successfully separated, by centrifuging with different speeds. The CHAPS enhanced the dissolution of BBMV. Western blot analyses showed that the polypeptide of both Cry1Ia14 and Cry9Ea6 bound to the BBMV of T.ni and S. exigua midguts.