1989 Fiscal Year Final Research Report Summary
Analysis of insecticidal -endotoxin protein genes and the modification of insecticidal activity by mutagenesis
| Project/Area Number |
62470120
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
応用生物化学・栄養化学
|
|---|
| Research Institution | Kyoto University |
|---|
Principal Investigator |
KOMANO Tohru Kyoto University Department of Agriculture Professor, 農学部, 教授 (30026413)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
YAMANO Yoshiaki Kyoto University Department of Agriculture Instructor, 農学部, 助手 (00182593)
UEDA Kazumitsu Kyoto University Department of Agriculture Instructor, 農学部, 助手 (10151789)
SAKAI Hiroshi Kyoto University Department of Agriculture Associate Professor, 農学部, 助教授 (60089117)
|
|---|
| Project Period (FY) |
1987 – 1989
|
| Keywords | BT toxin / delta-endotoxin / BT toxin genes / 130 kDa protein genes / Insecticidal domain / Recombinant plasmid / ISRH3 / ISRH4 |
|---|
| Research Abstract |
Bacillus thuringiensis var. israelensis carried seven plasmids, and the insecticidal protein &endotoxin) genes were located in one of the large plasmids. Two 130 kDa protein genes, ISRH3 and ISRH4, were isolated from the plasmid, and the base sequences were determined. An open reading frame (ORF) consisting of 3408 bases was present in ISRH3, and an ORF consisting of 3543 bases was present in ISRH4. When amino acid sequences of ISRH3 and ISRH4 proteins were compared, about 40% length proteins from the C-terminus were identical, while about 60% length proteins from the N-terminus were different.
About 50% length fragment from the C-terminal region of ISRH3 corresponding 575 amino acid residues was cloned into pUC13 and pKK223-4, and the cloned genes were expressed in E. coli cells. Protein extracts from the host cells showed insecticidal activity.
ISRH3 and ISRH4 were fused with lacZ' respectively on a plasmid, pUC19, and the fused genes were expressed in E. coli cells. Protein extracts showed strong insecticidal activity. The fused genes were sequitially deleted from C-termtnus or N-teminus to construct a series of deletion mutants. These mutants were transformed into E. coli cells, and insecticidal activity of protein extracts was examined. The results showed that there were two regions showing insecticidal activity: the one was located at about 40% position from the N-terminus, (N-terminus insecticidal domain), and the other was at about 50% position from C-teminus (C-terminus insecticidal domain). These insecticidal domains were located in the middle of the 130 kDa protein, and had a common amino acid sequence.
When the DNA fragment corresponding to the insecticidal domains was deleted, no insecticidal activity was found in the protein produced.
|
