| Delta-endotoxins (-endotoxin, or Insecticidal Crystal Proteins, ICPs) from Bacillus thuringiensis (Bt) are toxic to insect pests of many orders and great biocontrol resources against natural pests. Identification of cry genes encoding ICPs is of great significance to the discovery, cloning of novel genes with specific bioactivity and research of transgenic plants and microbe.Current methods for identifing Delta-endotoxins genes from Bt, especially PCR-based method, are cumbersome. Rapid, parallel and high-throughput tools that can adapt to the era of genome are needed.Microarray, a recently developed genomic technology has been shown to be a powerful tool for studying of gene expression and regulation on a genomic scale and detecting function genes or genetic polymorphisms in both eukaryotes and prokaryotes. However, there is no report about using oligonucleotide probes directly to detect functional genes without PCR in total DNA samples.In this study, the method using oligonucleotide microarray to detect cry genes from total DNA of Bt was studied. After the bioinformatics was applied, the detection microarray of cryl , cry2, cry9 genes has been constructed and tested.1. The key techniques of Bt cry genes detection microarrayAfter the key parameters such as the length and the concentration of probes, labeling, samples, sensitivity and hybridization were studied, a new and feasible approach, which could identify cry genes from total DNA of Bacillus thuringiensis strains but non-amplified based on oligonucleotide microarray hybridization had been established and involved fluorescent labeling for high concentration of total DNA using high concentration Klenow fragment, and hybridization to the oligonucleotide probes(40-50 mer, 40 mol L-1) specific for each cry genes.The results showed that the sensitivity difference among the length of probes (P<0.01) was notable, but no significant sensitivity difference among the concentration of probes in cry genes detection microarray. The condition of hybridization was 42 C, 3 hour and the detection limit with randomly labeled total DNA under these conditions was estimated to be approximately 2.5ng, a linear quantitative relationship (r2 = 0.985 to 0.989) was observed between signal intensity and labeling sample concentration over a range of 0.625 to 20 g.2. The application of bioinformatics in the detection microarray of cry genesTo overcome the shortages of on-line BLAST, the local nucleotide database of cry genes from Bt was established, and local BLAST was run based on it to ensure the specificity for each cry genes of oligonucleotide probes. By comparing local BLAST with on-line BLAST, advantages such as veracity, removing redundant and convenience were observed in local BLAST.According to the homology of nucleotide sequence among cry and cyt genes of Bt, the specific hierarchical oligonucleotide probes of the cry1 , cry2, cry9 genes were designed by bioinformatics method.3. The fabrication and application of the detection microarray of cry I, cry 2, cry9 genes After the detection oligonucleotide microarray was fabricated, the cry/, cry2, and cryP-type genes of 8 Bt strains had been determined. As the reference strains, detection result of microarray was accorded with the result of PCR-RFLP, as unknown strains, the result was also on the whole accorded with the result of PCR-RFLP. |
