Difference between revisions of "Os04g0178400"
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===Expression=== | ===Expression=== | ||
Please input expression information here. | Please input expression information here. | ||
| − | Rice plants (O. sativa L. ssp. Nipponbare) were cultivated in growth chambers under 12 h light | + | Rice plants (O. sativa L. ssp. Nipponbare) were cultivated in growth chambers under 12 h light and 12 h dark cycles to the sixth leaf stage, and CYP99A3’s metabolite analysis was carried out for each time point (and the control) using approximately 2 g rice plant leaf tissue. |
| − | Recombinant baculoviruses were constructed starting from pDEST8 constructs, and used to express CYP99A2 and CYP99A3 in Sf21 insect cells. Microsomes or lysates were isolated from these recombinant cell cultures and used for in vitro assays.After incubation at | + | Recombinant baculoviruses were constructed starting from pDEST8 constructs, and used to express CYP99A2 and CYP99A3 in Sf21 insect cells. Microsomes or lysates were isolated from these recombinant cell cultures and used for in vitro assays.After incubation at 28℃ for 6 h, the reaction mixture was extracted three times with an equal volume of ethyl acetate. The organic extract was dried under a gentle stream of N2 gas, and dissolved in hexane for GC-MS analysis.CYP99A2 and CYP99A3 were recombinantly expressed in E. Coli using modular diterpene metabolic engineering system . Specifically, we co-expressed these CYPs from the OsCPR1 co-expression constructs , with a GGPP synthase and CPS carried on co-compatiblepGGxC vectors, and OsKSL expressed from the additionally co-compatible pDEST14 or pDEST15 (i.e. for expression as a fusion to glutathione-S-transferase, GST).Enzymatic products were extracted from 50 ml cultures with an equal volume of hexane, then ethyl acetate, and the pooled, concentrated, and the methylated extract analyzed by GC-MS . In every case, the expected diterpene olefin product (i.e. given the co-expressed diterpene synthases) was easily observed, indicating that all potential substrates were present at sufficient levels for further transformation (i.e. by CYP99A2 or CYP99A3). |
CYP99A3 gene-specific primers (forward: 5'-GAGCCTCCTCGTCTCGGA-3'; reverse: 5'-AATTGCCTTGACGTGTGTTGA-3'). | CYP99A3 gene-specific primers (forward: 5'-GAGCCTCCTCGTCTCGGA-3'; reverse: 5'-AATTGCCTTGACGTGTGTTGA-3'). | ||
Revision as of 07:10, 5 June 2014
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Contents
Annotated Information
Function
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File:The function of CYP99A3.jpgThe rice genome contains a biosynthetic gene cluster for momilactone production, located on rice chromosome 4, which contains two cytochrome P450 (CYP) mono-oxygenases, CYP99A2 and CYP99A3. More specifically, CYP99A3 catalyzes consecutive oxidations of the C19 methyl group of the momilactone precursor syn-pimara-7,15-diene to form, sequentially, syn-pimaradien-19-ol, syn-pimaradien-19-al, and syn-pimaradien-19-oic acid, which are presumably intermediates in momilactone biosynthesis, as a C19 carboxylic acid moiety is required for formation of the core 19,6-clactone ring structure. In addition, CYP99A3 also oxidized syn-stemod-13(17)-ene at C19 to produce, sequentially, syn-stemoden-19-ol, syn-stemoden-19-al, and synstemoden-19-oic acid, albeit with lower catalytic efficiency than with syn-pimaradiene. The corresponding full length of the CYP99A3clones can be obtained from the rice cDNA database[1].
Expression
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Rice plants (O. sativa L. ssp. Nipponbare) were cultivated in growth chambers under 12 h light and 12 h dark cycles to the sixth leaf stage, and CYP99A3’s metabolite analysis was carried out for each time point (and the control) using approximately 2 g rice plant leaf tissue.
Recombinant baculoviruses were constructed starting from pDEST8 constructs, and used to express CYP99A2 and CYP99A3 in Sf21 insect cells. Microsomes or lysates were isolated from these recombinant cell cultures and used for in vitro assays.After incubation at 28℃ for 6 h, the reaction mixture was extracted three times with an equal volume of ethyl acetate. The organic extract was dried under a gentle stream of N2 gas, and dissolved in hexane for GC-MS analysis.CYP99A2 and CYP99A3 were recombinantly expressed in E. Coli using modular diterpene metabolic engineering system . Specifically, we co-expressed these CYPs from the OsCPR1 co-expression constructs , with a GGPP synthase and CPS carried on co-compatiblepGGxC vectors, and OsKSL expressed from the additionally co-compatible pDEST14 or pDEST15 (i.e. for expression as a fusion to glutathione-S-transferase, GST).Enzymatic products were extracted from 50 ml cultures with an equal volume of hexane, then ethyl acetate, and the pooled, concentrated, and the methylated extract analyzed by GC-MS . In every case, the expected diterpene olefin product (i.e. given the co-expressed diterpene synthases) was easily observed, indicating that all potential substrates were present at sufficient levels for further transformation (i.e. by CYP99A2 or CYP99A3).
CYP99A3 gene-specific primers (forward: 5'-GAGCCTCCTCGTCTCGGA-3'; reverse: 5'-AATTGCCTTGACGTGTGTTGA-3').
Evolution
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Labs working on this gene
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References
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Structured Information
| Gene Name |
Os04g0178400 |
|---|---|
| Description |
Similar to Cytochrome P450 CYP99A1 (EC 1.14.-.-) (Fragment) |
| Version |
NM_001058707.1 GI:115457143 GeneID:4335091 |
| Length |
2112 bp |
| Definition |
Oryza sativa Japonica Group Os04g0178400, complete gene. |
| Source |
Oryza sativa Japonica Group ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
| Chromosome | |
| Location |
Chromosome 4:5324744..5326855 |
| Sequence Coding Region |
5324944..5325573,5325907..5326785 |
| Expression | |
| Genome Context |
<gbrowseImage1> name=NC_008397:5324744..5326855 source=RiceChromosome04 preset=GeneLocation </gbrowseImage1> |
| Gene Structure |
<gbrowseImage2> name=NC_008397:5324744..5326855 source=RiceChromosome04 preset=GeneLocation </gbrowseImage2> |
| Coding Sequence |
<cdnaseq>atgatggagataaactcagaagccaccgtcaccctcgtctccgtcgtcaccctgcccattcttctcgcgttgctgacccgcaagtcgtcgtcaaagaagagacggcctccggggccatggaacctccccttggtcggcggcctcctccacctcctccggtcgcaaccgcaggtcgcactccgcgacctcgccggcaagtacggcccggtgatgttcctgcggacggggcaggtggacaccgtcgtgatctcctcgccggcggcggcgcaggaggtgctccgcgacaaggacgtcaccttcgcgtcgcggccgagcctcctcgtctcggagatcttctgctacggcaacctcgacatcggcttcgcgccctacggcgcctactggcggatgctgcgcaagctctgcacggtggagctcctcagcaccaagatggtgcggcagctcgcgccgatcagggacggcgagacgctggcccttgtcaggaacatcgaggccgccgccggcggcaagaagccgttcaccctcgccacgctgctcatctcgtgcacgaacacgttcaccgcgaaggcggcgttcgggcaggcgtgcggcggcgagctccaggagcagttcttgacggctctggacgaagcgctcaagttcagcaatgggttctgcttcggcgacctcttcccgtcgctgcggttcatcgacgccatgaccgggctgaggagccgcctggagcgactgcgcttgcagctggacaccgtcttcgacaagatcgtcgctcagtgcgagtcgaatccgggtgattcccttgtcaacgtcctgctcaggatcaaggaccagggcgagcttgacttcccctttagctcaacacacgtcaaggcaattatactggatatgttcacgggagggacagagacgacatcatctaccaccgagtggctcatgtcggagttgatgaggaacccagaggtgatggccaaggtgcaggctgaggtgcgaggagtattcgacaacaagagcccacaagaccacgagggcctgcttgaaaacctatcatacatgaagttggtgatcaaggagaccctgaggctgaatccggtgttgccgctgctactcccccatttatgtcgggagacctgcgaaatcggagggtatgagattgtggagggcaccagagtgttgatcaattcgtgggcgatggcgaggagccccgagtattgggacgatgcggagaagttcataccagagaggtttgaggatggcacagcggacttcaaaggctcccgttttgagtacttgccatttgggaccgggagaaggaggtgccctggtgatatatttgcaatggccacgctggaactcatcgtggcgcgccttctatactacttcgactggagcctcccggatgggatgcagccaggtgatatcgacatggagctcgtcgttggtgcaacggcgaggagaaagaatcatctccagctagtggcatcaccatataagccgatttccatgcaaagttga</cdnaseq> |
| Protein Sequence |
<aaseq>MMEINSEATVTLVSVVTLPILLALLTRKSSSKKRRPPGPWNLPL VGGLLHLLRSQPQVALRDLAGKYGPVMFLRTGQVDTVVISSPAAAQEVLRDKDVTFAS RPSLLVSEIFCYGNLDIGFAPYGAYWRMLRKLCTVELLSTKMVRQLAPIRDGETLALV RNIEAAAGGKKPFTLATLLISCTNTFTAKAAFGQACGGELQEQFLTALDEALKFSNGF CFGDLFPSLRFIDAMTGLRSRLERLRLQLDTVFDKIVAQCESNPGDSLVNVLLRIKDQ GELDFPFSSTHVKAIILDMFTGGTETTSSTTEWLMSELMRNPEVMAKVQAEVRGVFDN KSPQDHEGLLENLSYMKLVIKETLRLNPVLPLLLPHLCRETCEIGGYEIVEGTRVLIN SWAMARSPEYWDDAEKFIPERFEDGTADFKGSRFEYLPFGTGRRRCPGDIFAMATLEL IVARLLYYFDWSLPDGMQPGDIDMELVVGATARRKNHLQLVASPYKPISMQS</aaseq> |
| Gene Sequence |
<dnaseqindica>1283..1912#71..949#aagctaaactgccaagtaattaaacctaatcaatcatcaagccatatatacatcctctatctatcgatcgatgatggagataaactcagaagccaccgtcaccctcgtctccgtcgtcaccctgcccattcttctcgcgttgctgacccgcaagtcgtcgtcaaagaagagacggcctccggggccatggaacctccccttggtcggcggcctcctccacctcctccggtcgcaaccgcaggtcgcactccgcgacctcgccggcaagtacggcccggtgatgttcctgcggacggggcaggtggacaccgtcgtgatctcctcgccggcggcggcgcaggaggtgctccgcgacaaggacgtcaccttcgcgtcgcggccgagcctcctcgtctcggagatcttctgctacggcaacctcgacatcggcttcgcgccctacggcgcctactggcggatgctgcgcaagctctgcacggtggagctcctcagcaccaagatggtgcggcagctcgcgccgatcagggacggcgagacgctggcccttgtcaggaacatcgaggccgccgccggcggcaagaagccgttcaccctcgccacgctgctcatctcgtgcacgaacacgttcaccgcgaaggcggcgttcgggcaggcgtgcggcggcgagctccaggagcagttcttgacggctctggacgaagcgctcaagttcagcaatgggttctgcttcggcgacctcttcccgtcgctgcggttcatcgacgccatgaccgggctgaggagccgcctggagcgactgcgcttgcagctggacaccgtcttcgacaagatcgtcgctcagtgcgagtcgaatccgggtgattcccttgtcaacgtcctgctcaggatcaaggaccagggcgagcttgacttcccctttagctcaacacacgtcaaggcaattatactggtaataatttcatttatttactgctgaaaaatatatactccctccatcataaaatataagcatttttagaatagtgacaagtcaagcatttataactttgactattaatagcaaaaaaataaaaaagaccaatcgtgttactagatttatcactaaacaaaatattataacatgcaactctttttatttaaaacatcttacttttataaaaattattggtcaaagtagtatctcaaagaccgtgtcagcgtctaaaaatgcttatattttgggacggagggagtatatgtttcttcaatcttcattcttgctcatgtagttggtttcattcaggatatgttcacgggagggacagagacgacatcatctaccaccgagtggctcatgtcggagttgatgaggaacccagaggtgatggccaaggtgcaggctgaggtgcgaggagtattcgacaacaagagcccacaagaccacgagggcctgcttgaaaacctatcatacatgaagttggtgatcaaggagaccctgaggctgaatccggtgttgccgctgctactcccccatttatgtcgggagacctgcgaaatcggagggtatgagattgtggagggcaccagagtgttgatcaattcgtgggcgatggcgaggagccccgagtattgggacgatgcggagaagttcataccagagaggtttgaggatggcacagcggacttcaaaggctcccgttttgagtacttgccatttgggaccgggagaaggaggtgccctggtgatatatttgcaatggccacgctggaactcatcgtggcgcgccttctatactacttcgactggagcctcccggatgggatgcagccaggtgatatcgacatggagctcgtcgttggtgcaacggcgaggagaaagaatcatctccagctagtggcatcaccatataagccgatttccatgcaaagttgatggatgtgtgtgactgtgtgtcggtgtacttgcggttcgcttacgtgcttgcatacctcttaataagttttgtttgtcctggggaaatggacaaacaaattaataatactagttgataccccgtgctttgccgtgggatgtgtggatgaatgcattttatatatgatataaatgatagatgtacaagtttaaataatgtg</dnaseqindica> |
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