| Project/Area Number |
04807153
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
Chemical pharmacy
|
|---|
| Research Institution | Chiba University |
|---|
Principal Investigator |
SAITO Kazuki Chiba Univ., Fac. Pharm. Sci. Associate Professor, 薬学部, 助教授 (00146705)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
YAMAZAKI Mami Chiba Univ., Fac. Pharm. Sci. Res. Assist., 薬学部, 教務職員 (70222370)
MURAKOSHI Isamu Chiba Univ., Fac. Pharm. Sci. Professor, 薬学部, 教授 (30009162)
|
|---|
| Project Period (FY) |
1992 – 1993
|
| Project Status |
Completed (Fiscal Year 1993)
|
| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1993: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1992: ¥1,100,000 (Direct Cost: ¥1,100,000)
|
|---|
| Keywords | transgenic plant / secondary metabolism / cysteine biosynthesis / alkaloid / molecular breeding / cDNAクローニング / システイン合成酵素 |
|---|
| Research Abstract |
Cysteine synthase (CSase) (O-acetylserine(thiol)-lyase, EC 4.2.99.8) in higher plants catalyxes the formation of L-cysteine from Oacetyl-L-serine and hydrogen sulfide as the substrates. This enzyme is also reponsible for the biosynthesis of secondary non-protein heterocyclic beta-substituted alamines, e.g. beta-pyrazole alanine and mimosine. The final aims of uor long-term study are to reveal molecular basis for the biosynthesis of these amino acids and to engineer the producibility. We have obtained two cDNA clones encoding cytoplasmic CSaseA and chloroplast CSaseB from spinach by means of the microsequencing of peptide fragments followed by synthetic oligonucleotide. The cDNA for CSaseB contained the transit peptide sequence of52 amino acids. The experiments of site-directed mutagenesis teplacing 12 conserved Lysresidues into Ala indentified a functional Lys residue respansible for the binding of pyridoxal phosphate cofactor. Transgenic tobacco plants, integrated with the constructs for 35S-CSaseA cDNA, were obtained.analysis of transgenic plants showed the enhanced CSase activities in the plants with the sense constructs and the decreased activity in the antisense-construct plants. Theprokaryotic high expression system was estabkished with pET vector. beta-Purazole alanine, a specific metabolite of watermelon, was produced in bibo by feeding pyrazole to E.coli cuture expressing plant CSase.
|
