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Cloning, Expression and Insecticidal Activity of Novel cry9 Genes from Bacillus Thuringiensis

Author: SuHuiQin
Tutor: GaoJiGuo
School: Northeast Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: Bacillus thuringiensis cry9 class genes High-resolution melting point Clone Biological activity
CLC: S476.1
Type: Master's thesis
Year: 2010
Downloads: 46
Quote: 3
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Abstract


Bacillus thuringiensis (Bacillus thuringiensis, Bt) is a widely used in the world of biocontrol microbes, it has a high degree of insecticidal specificity and harmless to humans and animals of advantages. The main ingredient of insecticidal insecticidal crystal proteins, cry and cyt genes coding. Wherein cry9 gene expression product to lepidopterous a variety of pests with high insecticidal activity, but due to the limitations of the detection method, the types of genes found much less high gene activity. To fully explore the the Bt strains cry9 genetic resources, this study established a PCR-based product, the use of high resolution melting curve (high resolution melting HRM) of cry9 genotyping methods. To design a pair of amplified fragment of 100 bp common identifying primer, using the high-resolution melting point apparatus to analyze the melting curve of the product, due to the different composition of the amplified product a nucleotide, different types based on the conserved region of all the reported cry9 gene gene produce different melting curve, which will sample gene classification. This method can be performed with high throughput genotyping especially can detect known, novel cry9 gene. In this study, the use of the PCR method of identification containing cry9Aa, and cry9Ea genes were strain the SC5-D2 and SC5-E8, in order to of the two cry9 class of genes as a standard sample of the method was verified. HRM platform established by the application of 72 strains of Bacillus thuringiensis standards identified for cry9 genotype 3 PCR positive. To melting curve found B.thuringiensis subsp.fukuokaensis (T03C001) the containing cry9Aa gene fragments, B.thuringiensis subsp.sumiyoshiensis (T03B001) gene fragment containing cry9Da and of two novel cry9 class, B.thuringiensis subsp.japonensis (T23001) containing cry9Da gene fragment. Standard sample applications HRM platform to above cry9 gene fragments identified 2000 isolates for cry9 genotype, PCR positive 273 melting curve of type 20 class were sequenced strain SC5-D4 strains containing cry9Ba genes. The full-length primers designed according to the full-length sequence of these genes amplified cry9 gene successfully cloned gene, and international Bt officially named as cry9Aa3 cry9Ea8, cry9Aa4 cry9Da3 cry9Da4 cry9Eb2 cry9Ee1 cry9Ba3. The expression vector pEB expression of these genes in E. coli Rosetta (DE3), expression products are about 130 kDa protein (Cry9Ba3 unexpressed). Cry9Aa3 Cry9Aa4, Cry9Eb2 Cry9Ee1 on lepidopteran pests the diamondback moth insecticidal activity, LC50, respectively 1.83μg/mL.1.90μg/mL.1.03μg/mL and 1.70μg/mL; and four protein Cry1Ac The virulence of resistant diamondback moth, LC50, respectively 1.39μg/mL, 1.74μg/mL.1.24μg/mL and 1.45μg/mL. Four Cry9 protein the diamondback moth virulence with CrylAc are no cross-resistance. And Cry9Ea8 Cry9Da3 Cry9Da4 diamondback moth activity. Cry9Aa3, Cry9Aa4, Cry9Eb2, Cry9Ee1 on another lepidopteran pests Asian corn borer insecticidal activity better, LC50 6.93μg/mL, 1.06μg/mL, 5.93μg/mL and 2.20μg/mL of bollworm There are certain weight suppression. And Cry9Ea8, Cry9Da3, Cry9Da4 None of the diamondback moth and the Asian corn borer activity. Above the seven expressed Cry9 protein two pests were not the great apes leaf beetle and black cutworm activity.

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CLC: > Agricultural Sciences > Plant Protection > A variety of control methods > Biological control > The use of microbial pathogens
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