Expressional and functional analysis of CYP15A1, a juvenile hormone epoxidase, in the red flour beetle Tribolium castaneum.
Juvenile hormone (JH) is synthesized and secreted by the corpora allata. In the final two steps of JH biosynthesis, farnesoic acid (FA) is converted to JH through methylation by JH acid O-methyltransferase (JHAMT) and epoxidation by the cytochrome P450 enzyme CYP15. In the present study, we identified a homolog of CYP15 from the red flour beetle Tribolium castaneum (TcCYP15A1), and analyzed its expression as well as its role in JH biosynthesis. Quantitative RT-PCR analysis showed that the level of TcCYP15A1 mRNA was high in the embryonic stage as well as in the middle of the final larval instar. In the embryonic stage, the transcript level of TcCYP15A1 started to increase 30h after egg laying (AEL), peaked 54-60h AEL, and was followed by an increase of TcJHAMT mRNA, suggesting that JH biosynthesis started at this time point. TcCYP15A1 mRNA was present, but not exclusively so in the larval corpora allata. The recombinant TcCYP15A1 protein epoxidized both FA and methyl farnesoate (MF) in highly stereo-specific manners. These results confirmed that TcCYP15A1 is involved in JH biosynthesis. The RNAi-mediated knockdown of TcCYP15A1 in the pre-final larval instar did not result in precocious metamorphosis to pupa, indicating that MF may exhibit JH-like activity in order to maintain the larval status. The double knockdown of TcJHAMT and TcCYP15A1 resulted in pupae and adults with shorter wings, suggesting that the precursors of JH, JH acid and MF, may be essential for wing expansion.
Graduate School of Bio-Agricultural Sciences, Nagoya University, Furocho, Chikusa, Nagoya 464-8601, Japan. Electronic address: c_mina@agr.nagoya-u.ac.jp.
Chieka
C
Minakuchi
Graduate School of Bio-Agricultural Sciences, Nagoya University, Furocho, Chikusa, Nagoya 464-8601, Japan.
Fumika
F
Ishii
Graduate School of Bio-Agricultural Sciences, Nagoya University, Furocho, Chikusa, Nagoya 464-8601, Japan.
Yumiko
Y
Washidu
National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba 305-8634, Japan.
Akio
A
Ichikawa
Graduate School of Bio-Agricultural Sciences, Nagoya University, Furocho, Chikusa, Nagoya 464-8601, Japan.
Toshiharu
T
Tanaka
Graduate School of Bio-Agricultural Sciences, Nagoya University, Furocho, Chikusa, Nagoya 464-8601, Japan.
Ken
K
Miura
National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba 305-8634, Japan.
Tetsuro
T
Shinoda
D000818:Animals; D003335:Corpora Allata / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D007605:Juvenile Hormones / Q000378:metabolism; D008018:Life Cycle Stages; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D014231:Tribolium / Q000196:embryology* / Q000201:enzymology* / Q000235:genetics
D019476:Insect Proteins; D007605:Juvenile Hormones; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C023111:epoxidase
CYP15; Epoxidation; Juvenile hormone; Tribolium castaneum