Os04g0178400

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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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   The CYP99A2 and CYP99A3 cDNAs are 1780 and 1750 bp in length with ORFs encoding 502 and 507 amino acid residues, respectively, and the CYP99A2 and CYP99A3 amino acid sequences are 83.9% identical, and no other P-450 ORFs with greater than 40% identity to these ORFs are present in the rice genome.The amino acid sequences encoded by CYP99A3 and CYP99A1 share 55.0%  identity.

Mutation

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  Because of the 87% identity at the nucleotide sequence level(83.9% identity at the amino acid sequence level) between CYP99A2 and CYP99A3, RNAi-mediated knockdown of either     CYP99A2 or CYP99A3 resulted in production of the double knockdown of the two genes.The RNAi knockdown of CYP99A2 consisted of a region of 108-bp 3’-UTR and a 217-bp 3’’-terminal region of the ORF, which is highly homologous to that of CYP99A3.The RNAi-mediated knockdown of CYP99A3 consisted of an 81-bp 3’-UTR and a 207-bp 3’-terminal region of the CYP99A3 ORF, resulting in production of the double knockdown lines.
 In the CYP99A2/CYP99A3 double-knockdown lines, momilactone biosynthesis was specifically suppressed, but the total amounts of phytocassanes in the double knockdown are similar to those in the control, so CYP99A2, CYP99A3 or both are involved in the biosynthetic steps between 9ˇH-pimara-7,15-diene and 3ˇ- hydroxy-9ˇH-pimara-7,15-dien-19,6ˇ-olide in the momilactone biosynthetic pathway.

Extension knowledge

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  In two independent OsDCL3a RNAi lines,Ithe transcript levels of CYP99A3 increased with decreasing accumulation of 24-nt siRNAs from MITEs(miniature inverted repeat transposable elements) in the 5’ or intron regions of the corresponding genes, indicating that an OsDCL3a deficiency and loss of 24-nt siRNAs causes the up-regulation of genes critical for diterpenoid biosynthesis, which may influence GA biogenesis and therefore reduce plant height.
  In rice, there is a 168-kb gene cluster on chromosome 4 that consists of a possible dehydrogenase gene (AK103462), P-450 genes (CYP99A2 and CYP99A3), and two diterpene cyclase genes (OsCyc1 and OsKS4) involved in momilactone biosynthesis . AK103462, CYP99A2, and CYP99A3, together with OsCyc1 and OsKS4, form a chitin oligosaccharide elicitor- and UV irradiation-inducible gene cluster on chromosome 4 and they are involved in phytoalexin biosynthesis.

Labs working on this gene

Please input related labs here. 1. Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA 2. Genome Resource Unit, Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602 3. Genome Informatics Department, Mitsubishi Space Software Co., Ltd. Takezono, Tsukuba, Ibaraki 305-0032 4. Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 5. College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China 6. Institute of Food Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China 7. Key Laboratory of Integrated Management of Crop Diseases and Pests (Nanjing Agricultural University), Ministry of Education, Nanjing 210095, China 8. Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 9. The Department of Bioresource Engineering, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan 10. The College of Agriculture, Ibaraki University, 3-21-1 Chuo, Ami, Ibaraki 300-0393, Japan 11. The Department of Life Science, Meiji University, Kawasaki, Kanagawa, 214-8571 Japan 12. The Food and Health Research and Development Laboratories, Meiji Seika Kaisha, Ltd., Sakado, Saitama 350-0289, Japan 13. Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 14. Bio Science Laboratories, Meiji Seika Kaisha, Ltd., 5-3-1 Chiyoda, Sakado, Saitama 350-0289 15. The Department of Life Sciences, Faculty of Agriculture, Meiji University, 1-1-1 Higashi-mita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan 16. The Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011 17. The Biotechnology Research Center, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 18. State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China 19. College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China 20. Food Crops Research Institute,Yunnan Academy of Agricultural Sciences, Kunming 650205, China 21. Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19711

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	Chromosome 4
Location	Chromosome 4:5324744..5326855
Sequence Coding Region	5324944..5325573,5325907..5326785
Expression	GEO Profiles:Os04g0178400
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>
External Link(s)	NCBI Gene:Os04g0178400, RefSeq:Os04g0178400