| In this thesis, the molecular biology of Bacillus thuringiensis Ly30 strain (patent application number: 01108274) were systematically studied. The components of insecticidal crystal proteins and parasporal crystals of Ly30 strain were firstly defined based on morphology. The insecticidal spectrum and toxicities of Ly30 strain were tested. Several ICPs(Insecticidal Crystal Proteins) genes were identified, cloned and expressed, and then the bioassay of the expression products were carried out. The major research results are as follows :The Ly30 belongs to serotype H7, Bacillus thuringiensis subsp aizawai, biochemically and serologically. Many sorts of parasporal crystals that were rhombic, irregular and mosaic-bipyramid produced by Ly30 strain were observed using scanning and transmission electron microscopes. Based on SDS-PAGE, the parasporal crystals are composed of polypeptides with molecular weight at 133 kDa, 81kDa,70kDa and 60kDa. The bioassay results showed that the Ly30 strain was highly toxic to the larvae of Plutella xylostella and Helicoverpa armigera (LC50 0.022 jig/ml and 2.252u.g/ml respectively). It was also toxic to Oedaleus Infernalis De Saussureinsect (belong to Orthoptera) with corrected mortality of 81.82%. The LC50 of Ly30 to Culex pipiens Linnaeus was 19.8 ug/ml.The Ly30 strain contained at least five types of cry genes by means of CAPS(cleaved amplified polymorphic sequences) identification system. These cry genes were cloned by using construct plasmid library and amplified by PCR. They have been deposited into GenBank the designated as novel cry genes and named as cryl Aal2 (Accession number is AF384211), cry!Ac!4 (AF492767), crylla9 (AF521013), cry2AalO (AF433645) and cry2Ab5 (AF389442), respectively, by Bt 6 -endotoxin gene Nomenclature Committee. The alignment analysis indicates that there were the highest homology with 100%, 99%, 99%, 100% and 99% respectively when comparied to the published amino acid sequences.One pair of specific primer was designed to amplify of cry!Aa!2, and the PCR products were inserted into an expression plasmid pkk233-2 with a strong promotor Ptrc induced by IPTG. Three pairs of primers were designed for the amplification of cryl Ac 14, crylla9 and cry2Ab5 genes, and the amplified products were inserted into expressionplasmid pET-21b with strong promoter of T7 RNA polymerase induced by IPTG respectively. The expression vectors were transformed into an E.coli strain. After induced by IPTG, expression products were analyzed by SDS-PAGE. The results indicated cry genes were able to be expressed normally in E.coli. When the cry2AalO gene was inserted into an E.coli-Bt shuttle vector pHT315, and transformed into Bt acrystalliferous mutant strain 4Q7, typical square crystals and a 60 kDa protein band were observed by scanning electron microscope and SDS-PAGE, respectively.Bioassay of expression products of these genes showed that CrylAal2 and CrylAcl4 were highly toxic to many kinds of insect pests belonging to Lep., for example, CrylAal2 were toxic to the second instar larvae of Plutella xylostella, Helicoverpa armigera and Spodotera exigua with LC50 0.554 u g/mK 3.504 u g/ml ^P 3.276 u g/ml respectively. It suggested that CrylAal2 and CrylAcl4 played a critical role in the toxicity of BtLySO strain against Lep. It is the first time to demonstrate the toxicity of Cry2AalO against Oedaleus Infernalis de Saussure with corrected mortality of 69.73%. The toxicities of Cry2AalO and Cry2Ab5 to Culex pipiens Linnaeus (LC50 10.7 ug/ml and 8.3 ng/ml respectively) were also reported for the first time.By using overlapping PCR techniques, cry2Ab was fusioned with or/7, or/2 and orfl+orf2 of cry2Aa respectively. The expression vectors, pFU(orfl+2Ab), pF\J(orf2+2Ab) and pFU(orfl+orf2+2Ab), were constructed by inserting the fusion fragments into pHT315. They were transformed into a Bt acrystalliferous mutant strain 4Q7. The square crystals were observed only in pFU ( Orfl+orf2+2Ab) by scanning electron microscope, and a 60 kDa protein band detected only in pFU(Orfl... |
PDF Full Text Request