Kinjoh Terunori, Kaneko Yu, Itoyama Kyo, Mita Kazuei, Hiruma Kiyoshi, Shinoda Tetsuro
Faculty of Agriculture and Life Sciences, Hirosaki University, Hirosaki 036-8561, Japan.
Insect Biochem Mol Biol. 2007 Aug;37(8):808-18. doi: 10.1016/j.ibmb.2007.03.008. Epub 2007 Mar 19.
We have isolated the cDNAs of all enzymes involved in the mevalonate pathway portion of the juvenile hormone (JH) biosynthetic pathway in Bombyx mori, i.e., those responsible for the formation of farnesyl diphosphate from acetyl-CoA. There is a single gene encoding each enzyme of this pathway, with the exception of farnesyl diphosphate synthase (FPPS), for which we identified three homologs. All but two of these enzymes are expressed almost exclusively in the corpora allata (CA), as indicated by quantitative RT-PCR analyses. Phosphomevalonate kinase (MevPK) was expressed in many tissues, including the CA. In day 2 4th instars, FPPS1 expression was detected primarily in the Malpighian tubules, but expression of the structurally related FPPS2 and FPPS3 occurred mainly in the CA. Since FPPS3 transcripts were 55 times less abundant than those of FPPS2, the latter is expected to play a major role in JH biosynthesis at this stage. Studies on the developmental expression of these enzymes in the CA showed that the levels of all transcripts were high during the 4th instar larvae, a stage at which in vitro JH biosynthesis was high. However, the transcripts of all the mevalonate enzymes declined to low levels and JH acid O-methyltransferase (JHAMT) transcript disappeared by day 3 when CA ceased JH production after the final larval molt. The CA did not synthesize JH during the pupal stage, coincident with the limited expression of mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate kinase and isopentenyl diphosphate isomerase, and the inactivation of the JHAMT gene. Only female CA produced JH in the adult stage, a feature associated with the re-expression of JHAMT in female but little in male adult CA. Altogether, our results point to a relationship between JH biosynthesis and expression of most JH biosynthetic enzymes in the CA.
我们已经分离出了家蚕保幼激素（JH）生物合成途径中甲羟戊酸途径部分所涉及的所有酶的cDNA，即那些负责从乙酰辅酶A形成法呢基二磷酸的酶。该途径的每种酶都由一个单一基因编码，但法呢基二磷酸合酶（FPPS）除外，我们鉴定出了它的三个同源物。定量逆转录聚合酶链反应（RT-PCR）分析表明，除两种酶外，其他所有酶几乎都仅在咽侧体（CA）中表达。磷酸甲羟戊酸激酶（MevPK）在包括CA在内的许多组织中都有表达。在4龄第2天，FPPS1主要在马氏管中被检测到表达，但结构相关的FPPS2和FPPS3主要在CA中表达。由于FPPS3转录本的丰度比FPPS2少55倍，预计后者在这个阶段的JH生物合成中起主要作用。对这些酶在CA中的发育表达研究表明，在4龄幼虫阶段，所有转录本水平都很高，这一阶段体外JH生物合成水平也很高。然而，在最后一次幼虫蜕皮后，当CA停止产生JH时，所有甲羟戊酸酶的转录本在第3天下降到低水平，并且保幼激素酸O-甲基转移酶（JHAMT）转录本消失。蛹期CA不合成JH，这与甲羟戊酸激酶、磷酸甲羟戊酸激酶、二磷酸甲羟戊酸激酶和异戊烯基二磷酸异构酶的有限表达以及JHAMT基因的失活相一致。只有雌性CA在成虫阶段产生JH，这一特征与雌性成虫CA中JHAMT的重新表达有关，而在雄性成虫CA中则很少。总之，我们的结果表明了JH生物合成与CA中大多数JH生物合成酶表达之间的关系。
