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  The 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][2][7]


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   Rice plants (O. sativa L. ssp. Nipponbare) were cultivated in growth chambers under 12 h light (28℃) and 12 h dark (24℃) 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.[1]     
  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).[1]

CYP99A3 gene-specific primers (forward: 5'-GAGCCTCCTCGTCTCG-3') GA; reverse: 5'-AATTGCCTTGACGTGTGTTGA-3').[5]


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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.[5]


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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.

knowledge Extension

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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.[8] 
  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.[3][5][6]

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


Please input cited references here. 1. Qiang Wang, Matthew L. Hillwig and Reuben J. Peters* (2011)CYP99A3: functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice. The Plant Journal 65: 87–95

2. Yisheng Wu, Matthew L. Hillwig, Qiang Wang, Reuben J. Peters* (2011) Parsing a multifunctional biosynthetic gene cluster from rice: Biochemical characterization of CYP71Z6 & 7. FEBS Letters 585:3446–3451

3. Yutaka Sato1, Nobukazu Namiki2, Hinako Takehisa1, Kaori Kamatsuki2, Hiroshi Minami2, Hiroshi Ikawa2, Hajime Ohyanagi2, Kazuhiko Sugimoto3, Jun-Ichi Itoh4, Baltazar A. Antonio1,* and Yoshiaki Nagamura1,*(2013) RiceFREND: a platform for retrieving coexpressed gene networks in rice. Nucleic Acids Research,Vol. 41:D1214–D1221

4. Wenqi Li a,b,c,1, Min Shaoa,c,1, Jie Yangb, Weigong Zhongb, Kazunori Okadad, Hisakazu Yamaned, Guoliang Qiana,c, Fengquan Liua,c,(2013) Oscyp71Z2 involves diterpenoid phytoalexin biosynthesis that contributes tobacterial blight resistance in rice. Plant Science, 207 :98– 107

5. Kazuhiro Shimura‡, Atsushi Okada‡, Kazunori Okada‡, Yusuke Jikumaru‡, Kwang-Wook Ko‡,Tomonobu Toyomasu§, Takeshi Sassa§,Morifumi Hasegawa, Osamu Kodama, Naoto Shibuya,Jinichiro Koga**, Hideaki Nojiri‡, and Hisakazu Yamane‡1 (2007)Identification of a Biosynthetic Gene Cluster in Rice for Momilactones*. The Journal Of Biological Chemistry VOL. 282, NO. 47, pp. 34013–34018.

6. Atsushi Okada‡, Kazunori Okada‡1, Koji Miyamoto‡2, Jinichiro Koga§, Naoto Shibuya¶, Hideaki Nojiri‡, and Hisakazu Yamane‡ (2009) OsTGAP1, a bZIP Transcription Factor, Coordinately Regulates the Inductive Production of DiterpenoidPhytoalexins in Rice. The Journal Of Biological Chemistry VOL. 284, NO. 39, pp. 26510–26518.

7. Qiang Wang‡, Matthew L. Hillwig‡, Kazunori Okada§, Kohei Yamazaki§, Yisheng Wu‡, Sivakumar Swaminathan‡, Hisakazu Yamane§, and Reuben J. Peters‡1(2012)Characterization of CYP76M5–8 Indicates Metabolic Plasticity within a Plant Biosynthetic Gene Cluster*. The Journal Of Biological Chemistry VOL. 287, NO. 9, pp. 6159–6168.

8. Liya Weia,b,1, Lianfeng Gua,1, Xianwei Songa,1, Xiekui Cuia,b, Zhike Lua, Ming Zhoua, Lulu Wanga, Fengyi Huc, Jixian Zhaid,e, Blake C. Meyersd,e, and Xiaofeng Cao. (2014) Dicer-like 3 produces transposable element-associated 24-nt siRNAs that control agricultural traits in rice. PNAS vol. 111 no. 10 :3877–3882

Structured Information