[Transformation and expression of specific insecticide gene Bt cry3A in resident endogenetic bacteria isolated from Apriona germari (Hope) larvae intestines].

OBJECTIVE: Transforming the specific insecticidal gene Bt cry3A into the dominant resident endogenetic bacteria in intestines of Apriona germari (Hope) larvae to construct transgenic bacteria that can colonize and express the insecticidal gene Bt cry3A perfectly in intestines of Apriona germari (Hope) larvae. METHOD: We isolated and identified the dominant resident endogenetic bacteria by traditional methods and molecular method based of 16S rDNA analysis. Two Escherichia coli--Bacillus thuringiensis shuttle plasmid pHT305a and pHT7911 which contained specific insecticidal gene Bt cry3A were transformed into two resident endogenetic bacteria Brevibacillus brevis Ag12 and Bacillus thuringiensis Ag13 isolated from A. germari larvae intestines respectively by electro-transformation. RESULTS: Eighteen species of bacteria have isolated and identified from Apriona germari larvae intestines and two of them (Brevibacillus brevis Ag12 and Bacillus thuringiensis Ag13) were selected as starting bacteria to recieve the Bt cry3A. The 4 transgenic engineering strains Ag12-7911, Ag12-305a, Ag13-7911 and Ag13-305a were obtained successfully and validated by testing the plasmid stability in recombinants, transformants vegetal properties, crystal poisonous protein observation, expressional protein SDS-PAGE. The Bt cry3A gene had been transformed into Brevibacillus brevis and Bacillus thuringiensis. Both bioassay and examination of the engineering strains in intestines after feeding them to larvae showed that all these transformant strains (Brevibacillus brevis Ag12-305a, Bacillus thurigiensis Ag13-305a, Brevibacillus brevis Ag12-7911 and Bacillus thurigiensis Ag13-7911) could colonize and express 65 kDa protoxin in intestines of A. germari larvae and had insecticidal activity. CONCLUSION: We obtained four transgenic bacteria that can colonize and express the target insecticide gene Bt cry3A in A. germari larvae. They may be developed as a new insecticide.

Hybridization monitor: a method for identifying differences between complex genomes.

We have developed a method to identify and amplify differential fragments between two complex genomes. This technique, named hybridization-monitored genome differential analysis (HMDA), incorporates a monitor system into a PCR-based solid subtraction hybridization that tracks the entire hybridization process. This is achieved by monitoring the subtraction progress using PCR analysis of the conserved sequence of 18S rDNA in the tester sample after each round of subtraction. Homologous fragments can then be eliminated when bound to the driver DNA immobilized on a solid membrane. The hybridization continues until the conserved DNA sequence of 18S rDNA can no longer be detected, and most of the unbound DNA fragments left in the liquid were mainly the tester-specific fragments, thus greatly decreasing the complexity of DNA template of PCR amplification, increasing the amplification efficiency of differences accordingly, and ensuring high positive efficiency and coverage across the tester genome. We have applied the technique in a comparison between the genomes of Saccharomyces cerevisiae and Schizosaccharomyces pombe, which are two completely sequenced organisms. Results indicated that 95% of the subtracted clones have been confirmed to be different to the driver analyzed using the BLASTN homology alignment. With this technique, 240-fold enrichment of differences is obtained, and the coverage of the difference is up to 79%. These results indicate that HMDA can efficiently identify sequences that differ between two complex genomes.

ISOLATION AND IDENTIFICATION OF THE CELLULOSE-UTILIZINGBACTERIA FROM THE GUT OF APRIONA GERMATI(HOPE) / 微生物学通报

In order to clarify the cellulose-utilizing mechanism of the Cerambycidae, the microorganism in the Apriona germari(Hope) larvae's gut was cultured and identified. A strain of facultative anaerobic cellulose-utilizing bacteria was isolated from the midgut fluid of A. germari larvae with the cellulose-congo red agar medium. The colonies of the bacteria were white and round with regular margin. Clear zones of cellulolysis surrounding the colonies might reach 10m～20mm. The size of the bacteria was 0.5μm～0. 8μm× l～3μm approximately. The cell was gram-positive rod with polar flagella. It hadn't spores. With the results of biochemical tests, the bacteria were tentatively identified as Cellulomonas.

ISOLATION AND IDENTIFICATION OF THE CELLULOSE-UTILIZING BACTERIA FROM THE GUT OF APRIONA GERMATI(HOPE) / 微生物学通报

In order to clarify the cellulose utilizing mechanism of the Cerambycidae, the microorganism in the Apriona germari(Hope) larvae’s gut was cultured and identified. A strain of facultative anaerobic cellulose utilizing bacteria was isolated from the midgut fluid of A. germari larvae with the cellulose congo red agar medium. The colonies of the bacteria were white and round with regular margin. Clear zones of cellulolysis surrounding the colonies might reach 10mm～20mm. The size of the bacteria was 0.5?m～0.8?m?1～3?m approximately. The cell was gram positive rod with polar flagella. It hadn't spores. With the results of biochemical tests, the bacteria were tentatively identified as Cellulomonas.