Study On The Isolation, Identification Of Bacillus Thuringienis Resources And Cloning, Expression Of Novel Pesticidal Crystal Genes From Sichuan Ecology

Bacillus thuringiensis(Bt) is currently one of the most extensive studied and widely used pesticidal microorganism.To futher dig abundant resource of Bt in our country,screen high toxicity and specific strains,analyse their cry gene types,isolate and clone novel pesticidal genes will have important meanings in theories and practices for developping microbial insecticides,constructing high toxicity and broadspectrum engineering strains and breeding insect resistant transgenic plants.This study described a systematic study of Bt resources in different ecological regions in Sichuan.Several novel pesticidal protein genes were cloned and expressed.The concrete results are as follows:1.In totle,791 B.thuringiensis isolates have been screened from 2650 soil samples with an average rate as 13.2%,which were collected from different ecological regions in Sichuan.Observed by electron microscope,these B.thuringiensis parasporal crystal shapes were long bipyramid,short bipyramid,big bipyramid,small bipyramid, cuboidal,round and abnormity,which showed the diversity of Bt resources in Sichuan ecology.The cry gene-types of 791 B.thuringiensis isolates were identified by use of PCR-RFLP.522 isolates harbored cry1 genes,312 isolates harbored cry2 genes,20 isolates harbored cry3 genes,33 isolates harbored cry9 genes,28 isolates harbored cry4/10 genes,33 isolates harbored cry3 genes and 3 isolates harbored cry40 genes.In addition,80 isolates did not produce any PCR products when assayed with the primers. However,SDS-PAGE assay indicated that these isolates produced crystal inclusions, suggesting that they may contain potentially novel Cry toxins.2.This paper systematically investigated the biological characteristics of Bt strains Rpp02 and Rpp39 isloated from Sichuan ecology.Based on their morphologic characteristics,culture characteristics,biochemical reactions and cell wall compounds analysis,the strain Rpp02 was approved to be a kind of Bacillus thuringiensis subsp. morrisoni while Rpp39 was Bacillus thuringiensis var entomocidus.After growing three hours,the strain Rpp02 entered logarithmic growth phase.The growth velocity of Rpp02 was quicker slightly than Rpp39(four hours) whose growth velocity was the same as that of the standard strain HD-1.The strain Rpp02 produced big bipyramid, small bipyramid,round and abnormity parasporal crystal while Rpp39 produced diamond,cuboidal and round parasporal crystal.The strain Rpp02 contained cry1Ab, cry1Ac,cry1Ca and cry1Ia genes by use of PCR-RFLP method and Rpp39 contained cry1Aa,cry1Ab,cry1Ac,cry1Ia and cry2Aa genes.SDS-PAGE analysis showed that two kinds of molecular mass of insecticidal crystal proteins,one was about 130kDa and other 40kDa,were expressed in Rpp02 while 130kDa and 60kDa proteins in Rpp39.3.According to the whole length of cry1Ac gene published on GenBank,a pair of primers was designed to amplify the genomic DNA of Rpp02 and a fragment of about 3.7kb was obtained.The sequence analysis showed that the novel gene cry1Ac20, named by B.thuringiensis Pesticidal Crystal Protein Nomenclature Committee, contained an open reading frame of 3534 nucleotides encoding a protein of 1177 amino acids with a predicted molecular mass of 133.144 kDa and isoelectric point of 4.952. Compared with other known cry1Ac genes,cry1Ac20 has shown as high as 99% nucleotide sequence homology.Bioassay showed that the toxic protein appeared high insecticidal activity against Pieris rapae L.with LC₅₀ as 9.01μg / mL4.One cry2Aa-type gene of Rpp39 was cloned and designated as cry2Aa12 by Bt Insecticidal Crystal Proteins Nomenclature Committee.Sequence analysis revealed this gene contained an open reading frame of 1902 nucleotides encoding a protein of 634 amino acids.Compared with Cry2Aa1 protein,Cry2Aa12 protein has shown as high as 99.7%amino acid homology.The full open reading frame sequence of the cry2Aa12 gene was amplified with a pair of PCR primers P3/P4 designed according to its DNA sequence,and inserted into the Ndeâ… / BamHâ… site of E.coli expression vector pET-30a to obtain the recombinant plasmid pET-2Aa.The result of SDS-PAGE proved that Cry2Aa12 could be expressed as 65kDa protein in E.coli BL21(DE3) strain induced by IPTG Bioassay of the expressed product of the cry2Aa12 gene showed that Cry2Aa12 was highly toxic to the larvae of Plutella xylostella and Chilo supperssalis, with LC₅₀ as 5.4μg / mL and 22.3μg / mL,respectively.5.One novel holetype genes was found from the strain BtMC28 by the method PCR-RFLP.The sequence analysis revealed that the partial sequence of one had 57% and 60.5%identical to cry4Aa and cry10Aa,respectively.Furthermore,the full-length sequence of the novel gene was obtained by Tail-PCR and Son-PCR.The sequence analysis showed that the novel 8ene cry54Aa1,named by B.thuringiensis Pesticidal Crystal Protein Nomenclature Committee,contained an open reading frame of 2022 nucleotides encoding a protein of 673 amino acids with a predicted molecular mass of 76.32 kDa and isoelectric point of 7.535.Compared with other known proteins, Cry54Aa1 has shown 38%amino acids homology.The full open reading frame sequence of the cry54Aa1 gene was amplified with a pair of PCR primers designed according to its DNA sequence,and inserted into the Ndeâ… / EcoRâ… site of E.coli expression vector pET-30a to obtain the recombinant plasmid pET-54Aa.The result of SDS-PAGE proved that Cry54Aa1 could be expressed as 76 kDa protein in E.coli BL21(DE3) strain induced by IPTG.Bioassay of the expressed product of the cry54Aa1 8ene showed that cry54Aa1 was highly toxic to the larvae of Laphygma exigua and Aedes aegypti with LC₅₀as 5.08μg / mL and 6.02μg / mL,respectively.