Cry Genes From Bacillus Thuringiensis And Engineered Strains Toxic To Scarabaeidae And Chrysomelidae Pests

Bacillus thuringiensis (Bt) is the most widely used insecticidal microorganism. Its activity is attributed largely to insecticidal crystal proteins (ICP). Though Bt toxins are used as biopesticides, there are some drawbacks such as narrow host range and low toxicity to target pest, therefore novel genes and strains improvement were needed for effective pest control. The study of novel genes, the structure of ICP and the mechanism of action of these toxins are areas of high interest. In this study, we investigated B.thuringiensis Cry8-type ICPs toxic to coleopteran pests. This work includes the cloning and characterization of cry8-type genes toxic to scarab, the gene engineered strains and the insecticidal mechanisms of Cry8 ICPs.1,The cloning of cry8-type genes toxic to Scarabaeidae pests:The gene, cry8Ga2 whose name was assigned by Btδ-endotoxin nomenclature committee, was cloned from wild-type strain Bt145. The gene was cloned into the acrystalliferous mutant strain HD-73- and the resulted engineered strain was named HD8G2. HD8G2 and Bt145 were toxic to Coleopteran pests Holotrichia parallela with an LC50 1.25×108 cfu/g and 5.59×108 cfu/g, respectively.Two genes, cry8Ha1 and cry8Ia1, were cloned from wild-type strain BtSU4. The two genes were cloned into the acrystalliferous mutant strain HD-73- and two engineered strains named HD8H and HD8I were obtained, respectively. Bioassay results showed that HD8H and HD8I were highly toxic to H. parallela, the LC50 s were 2.19×1010 cfu/g and 8.50×109 cfu/g, respectively. The activated toxin of Cry8H digested by chymotrypsin showed toxicity towards neonate larvae of a class of leaf beetle--Colaphellus bowringi (Coleopteran) with an LC50 of 18.00μg/ml. The results of bioassay, RT-PCR and Western-blot showed that the cry8Ha1 and cry8Ia1 genes were all expressed in BtSU4. The two genes were all applied for national patent, which lays a foundation for their application.2,Construction of engineered Bt strains with high toxicity to Scarabaeidae and Chrysomelidae pests:A recombinant plasmid pSTK-3A containing cry3Aa7 gene encoding Chrysomelidae specific toxin was cloned into the wild strain BtSU4 and HBF-1 with high toxicity to Scarabaeidae insect pests by electroporation, and the resulted strain was named 3A-SU4 and 3A-HBF, respectively. Bioassay results showed that the two engineered strains were highly toxic not only to Scarabaeidae pests, but also Chrysomelidae. The strain, 3A-SU4 was highly toxic to Scarabaeidae pests H. parallela (LC50=3.60×108 cfu/g), Chrysomelidae pest Colaphellus bowringi (LC50=1.25μg/ml) and Leptinotarsa decernlineata (LC50 2.62=μg/ml), while 3A-HBF showed activity against Anomala carpulenta, C. bowringi and L. decernlineata with an LC50 0.73×108 cfu/g, 1.10μg/ml and 1.74μg/ml, respectively. The two engineered strains with broad insecticidal spectrum obtained here have strong potential for application as biocontrol agent.3,Study of the insecticidal mechanism of Cry8-type protein:The insecticidal mechanism of Cry8-type protein was studied on the following aspects: the structure of the protein, the protein activated by midgut juice and proteinase, and interaction between the protein and receptor.Eight chimeric proteins were obtained by Domain and Loop exchanges of Cry8Ca2 and Cry8Ea1 proteins. The eight chimeric proteins were all expressed in strain HD-73-. Biocontrol effect of the enginnered strain HD (3+15) (containing the chemiric gene composing of DomainⅠand DomainⅡof Cry8C and DomainⅢand LoopⅡof Cry8E), HD (7+11) (containing the chemiric gene composing of DomainⅠand DomainⅡof Cry8E and DomainⅢand LoopⅡof Cry8C) and HD (6+10) (containing the chemiric gene composing of DomainⅠand LoopⅠof Cry8E and DomainⅡand DomainⅢof Cry8C) to A. corpulenta was 44%, 36% and 32% with the concentration 1010 cfu /g, respectively.The four proteins Cry8Ha1, Cry8Ia1, Cry8Ga1 and Cry8Ga2, were digested by midgut juice of H. parallela and chymotrypsin. The bioassay results of the activated protein showed activated Cry8Ha1 showed toxicity against C. bowringi.The activated Cry8Ia1, Cry8Ga1 and Cry8Ga2 proteins showed no activity against C. bowringi.Binding assay with the BBMV was performed using the toxins Cry8Ha1 and Cry8Ia1 which showed toxicity against H. parallela larvae. Cry8Ha1 and Cry8Ia1 could bind to BBMV from H. parallela and H. oblita.