Cloning Of Insecticidal Cry Genes To Coleopteran Pests And Construction Of Genetically Engineered Bacteria

Coleopteran pest is very important in agriculture and fOrestry. C1oning ofinsecticidal novel cry' genes to Coleopteran pest tYom Bacillus lhuringiensisisolates will be beneficial fOr app1ication of pesticidal novel gene resources andwill lessen the gap betWeen domestic and abroad. In this study, cloning ofcrJ'3Aa and cryiBa, construction of expression vectors. geneticaIIy engineeredbacterial strains and bioassay have been perfOrmed based on research results ofbiology and genetic characterization of Bt isolates Bt22 and UVl7, isolated fromChina, highly toxic to Coleopteran and Coleopteran / Lepidopteran pestsrespectively.1. Bt isolates Bt22 and UV17 were studied systematical1y on biology and geneticcharacterization in this thesis. Growth speed of Bt22 was the same as that of Bttand slightly slower than that of UV17. Parasporal crysta1 from Bt22 was fiat inshape (2~5um in size), composed of 73.1kDa protein, and that the crystalshape from UVl7 was bipyramidal (2um), molecular weight of crystal proteinwas l40kDa. Isoelectric point of Cry3Aa and CrylBa is pHi5. l65 and pHi4.835separately. Results of cry gene identification by PCR-RFLP showed that CI:1)3.4awas found in Bt22 and cryiBa found in UVl7. and they were unique c):1'-typegene strains. Four plasmid bands from Bt22 isolate were observed in agarose geland 8 bands from UV 1 7. Results of biochemical reaction and esterase pattern fOrstrain identification showed that Bt22 was the same as Bt subsp. Ienebrioinis andUV l7 was identical with Bt subsp. thuringiensis.2. Sollthern Blotting showed that cry3Aa gene was located on 3.0kb plasmidDNA-HindIII fragment of Bt22, furthermore, this fragmellt colltaining cry3AQgene was inserted into cloning vector pBlueScript (SK+). Subcloning andsequencing analysis showed that this gene was comPosed of l932 bps, and theamino acid sequence of Cry3A was deduced from its nucleotide sequence, itsmolecular mass was 73. lkDa. This gene had been registered in EMBL/ GenBank(Accession number is AJ237900) and named cry3Aa7 as a novel cry gene byInternational Nomenclatllre Committee of Bt- 8 -endotoxin Gene. cry3Aa7 wasthe first cloned cry gene highly toxic to coleopteran order in China.DNA hybridization analysis showed cry1Ba gene was located on aboutl0kb plasmid DNA BamHI, SacI, SalI digested fragments. A pair of specificprimers was designed for cry]Ba 5'-end DNA fragment, and 2058 bps amplifiedPCR product was cloned into pGEM-T Easy vectoL Sequencing result of thisfragment illustrated that one base had been changed compared with knowncry1Ba1(A-G) and resulted in one amino acid change, His-Arg. 686 aminoacids were deduced from this DNA fragment. It indicated that UVl7 contained anovel cryIBa, its isoelectric point was pHi 5.705. Although one amino acidchanged, toxicity, solubility was stable. According to the result of DNAhybridization, plasmid 1ibrary had been constructed. Two positive recombinants,containing cryiBa 9.5kb and l0kb DNA fragments respectively, were screenedand obtained by PCRRFLP method.3. In order to research cloning, transformation and expression of cry gene, onebroad-host-range pIasmid was constructed in this study l .2kb repIication proteingene (reP) from Pseudomonas was inserted into AatII site of Bt-Ecoli shuttlevector pHT3 l5, and Bt Xcoli- Pfshllttle vector pGMl l05 was gained. It can bereplicated and inherited stably in Bl. Ecoli and Pfcell. During 48hrs stability ofpGM1 l05 in Pf and Bt was more than 90%.Expression vectors pHY5l2 (BI-Ecoli) and pLF3l l05 (Bt-E.coli-Pn,constructed by cry3Aa7 inserted into pHT3l5 and pGMl l05 separately. wastransformed into Bt acrystalliferous mutant strain BE20 and UVl7 isolatesrespectively, two genetically engineered strains BiotIII205 and BiotIIIl75 wereobtained, and stability of them was over 90% in BE20 and UVl7 after 48hr.cry3Aa7 could be expressed normally and steady in natural strain UVl7. Effecton cry3Aa7 transformed...