The Expression Of Bacillus Thuringiensis Cry5Ba3 And Its Lethal Effect On Pine Wood Nematode

The pine wood nematode(Bursaphelenchus xylophilus) is a destructive disease for pines. The insecticidal crystal protein encoded by cry5Ba3, previously isolated from Bacillus thuringiensis zjfc85, has a significantly lethal effect on the nematode B. xylophilus. To define the lethal effect and the mechanism of cry5Ba3 on B. xylophilus, codon optimization of this gene was executed to enhance the expression of cry5Ba3 in Botrytis cinerea. The expression vector pTFCM-cry5Ba3 was constructed, and transformed into Botrytis cinerea using Agrobacterium tumefaciens-mediated transformation(ATMT). The effect of Cry5Ba3 on B. xylophilus was determined. The results was listed as following.1. The new coding gene was designed(567 bp was changed) to enhance the expression effect in fungi, and named as cry5Ba3Φ,according to codon preference in filamentous fungi,.2. The promoter and terminator of trpC and cry5Ba3 were synthesized and inserted into Puc57 vector to obtain Puc57-cry5Ba3Φ vector. These three flanking sequences were then digested, ligated and introduced into pTFCM vector to construct pTFCM-cry5Ba3Φ for ATMT.3. ATMT was performed to obtain the transformant. A. tumefaciens AGL-1 containing pTFCM-cry5Ba3Φ vector and Bo. cinerea were co-cultured in induction medium. The transformant was obtained through hygromycin resistance screening. The transformant was verified by PCR amplification. Southern blot indicated that the insertion of cry5Ba3Φ was a single homologous recombination event in this transformant. cry5Ba3Φ and the hygromycin resistance gene were genetically stable after several subcultures.4.For the wild tpye strain of Bo. cinerea, there was no expression of cry5Ba3Φ using real-time qRT-PCR. When cry5Ba3Φ was transferred into the wild tpye strain, the expression level of cry5Ba3Φ was 5.6±1.1fold, compared to the expression level of actin.5. B. xylophilus was fed using Bo. cinerea which containing cry5Ba3Φ gene to test the lethal effect. Compared to the wild type strain of Bo. cinerea, Bo. cinerea with cry5Ba3Φ gene has great successfully controlled the reproduction of B. Xylophilus(growth rate, 48%-65%), and has a lethal effect on B. xylophilus(death rate, 1.7-2.5 fold).In summary, this research modified a new insecticidal crystal protein encoded by cry5Ba3Φ, and achieved the high-level expression of cry5Ba3 in Bo. cinerea. It will lay a theoretical foundation for the exploitation and application of insecticide Bt.