1986 Fiscal Year Final Research Report Summary
Studies on the viral DNA of the nuclear polyhedrosis virus of the silkworm, Bombyx mori
| Project/Area Number |
60480050
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
蚕糸学
|
|---|
| Research Institution | Tottori University |
|---|
Principal Investigator |
MAEDA Susumu Tottori University ・ Assistant Professor, 農学部, 助手 (30116371)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
KOBARA Ryuzo Tottori University ・ Professor, 農学部, 教授 (70032092)
|
|---|
| Project Period (FY) |
1985 – 1986
|
| Keywords | Baculovirus / Silkworm / NPV / Physical Map / Expression Vector / Gene Cloning / Mutants / アミノ酸配列 |
|---|
| Research Abstract |
A nuclear polyhedrosis virus of the silkworm, Bombyx mori, was plaque purified on BmN cells. One of the isolates, named T3, were propagated and viral DNA was purified and cloned into plasmids, pBR322 and pUC19. Physical maps of each DNA fragments cloned into plasmids were constructed using several endonucleases. By hybridization experiments of cloned plasmids, a physical map of the viral DNA was constructed. The genome size of the viral DNA was estimated as about 130 kb. Repeated sequences containing EcoRI recognition site were found in the viral genome.
The polyhedrin gene of the BmNPV T3 isolate was identified by hybridization experiments using cDNA synthesized by a reverse transcriptase from mRNA isolated from infected fat bodies at late stage of infection. The polyhedrin gene was cloned into pBR322 at an EcoRI site and the entire nucleotide sequence was determined by dideoxy chain termination procedure by Sanger et al. Comparing the sequences of Autographa californica, several nucleotide changes were found in the coding region of the polyhedrin gene. Deduced amino acid sequences of the polyhedrins were relatively conserved.
A mutant, which produces a cubic polyhedra, was recently isolated in Tanegashima. BT31 isolate was plaque purified and compared with T3 isolate by restriction endonuclease analysis. The polyhedrin gene was molecular cloned into pUC19 and sequenced. Four base changes were found in the coding region of the polyhedrin. Two of the four base changes causes amino acid changes indicated that these changes are important for the 3-D structure of the polyhedral inclusion body.
A recombinant BmNPV with insertion of the gene of chloramphenicol transferase was constructed for the studies on viral replication and infectious path way in in vivo system.
|
