[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.


METHODS
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.


CONCLUSIONS
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.
Zhongkang
Wang
W
Bioengineering College of Chongqing University, Chongqing Engineering and Technology Center for Fungal Insecticides, Key Lab of Genetic Function and Regulation, Chongqing 400030, China. zkwang@cqu.edu.cn
Wei
He
H
Guoxiong
Peng
P
Yuxian
Xia
X
Qiang
Li
L
Youping
Yin
Y
D000818:Animals; D001407:Bacillus / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D001517:Coleoptera / Q000187:drug effects / Q000382:microbiology*; D004731:Endotoxins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression; D005818:Genetic Engineering; D006460:Hemolysin Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D007306:Insecticides / Q000378:metabolism / Q000494:pharmacology*; D007422:Intestines / Q000382:microbiology; D007814:Larva / Q000187:drug effects / Q000382:microbiology; D008969:Molecular Sequence Data; D010572:Pest Control, Biological / Q000379:methods*; D014169:Transformation, Bacterial
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; C030956:insecticidal crystal protein, Bacillus Thuringiensis