Sieglaff Douglas H, Duncan Kelli Adams, Brown Mark R
Department of Entomology, University of Georgia, Athens, GA 30602, USA.
Insect Biochem Mol Biol. 2005 May;35(5):471-90. doi: 10.1016/j.ibmb.2005.01.011.
A blood meal induces the ovaries of female Aedes aegypti mosquitoes to produce ecdysteroid hormones that regulate many processes required for egg maturation. Various proteins involved in the intracellular transport and biosynthesis of ecdysteroid precursors have been identified by analysis of Drosophila melanogaster mutants and by biochemical and molecular techniques in other insects. To begin examining these processes in mosquito ovaries, complete cDNAs were cloned for putative orthologs of diazepam-binding inhibitor (DBI), StAR-related lipid transfer domain containing protein (Start1), aldo/keto reductase (A/KR), adrenodoxin reductase (AR), and the cytochrome P450 enzymes, CYP302a1 (22-hydroxylase), CYP315a1 (2-hydroxylase) and CYP314a1 (20-hydroxylase). As shown by RT-PCR, transcripts for all seven genes were present in ovaries and other tissues both before and following a blood meal. Expression of these genes likely supports the low level of ecdysteroids produced in vitro (7-10 pg /tissue/6 h) by tissues other than ovaries. Ovaries from females not blood fed and up to 6 h post blood meal (PBM) also produced low amounts of ecdysteroids in vitro, but by 18 and 30 h PBM, ecdysteroid production was greatly increased (75-106 pg/ovary pair/6h) and thereafter (48 and 72 h PBM) returned to low levels. As determined by real-time PCR analysis, gene transcript abundance for AedaeCYP302 and AedaeCYP315a1 was significantly greater (9 and 12 fold, respectively) in ovaries during peak ecdysteroid production relative to that in ovaries from females not blood fed or 2 h PBM. AedaeStart1, AedaeA/KR and AedaeAR also had high transcript levels in ovaries during peak ecdysteroid production, and AedaeDBI transcripts had the greatest increase at 48 h PBM. In contrast, gene transcript abundance of AedaeCYP314a1 decreased PBM. This study shows for the first time that transcription of a few key genes for proteins involved in ecdysteroid biosynthesis is positively correlated with the rise in ecdysteroid production by ovaries of a female insect.
血液摄入会促使埃及伊蚊雌蚊的卵巢产生蜕皮甾体激素，这些激素调节着卵子成熟所需的许多过程。通过对黑腹果蝇突变体的分析以及在其他昆虫中运用生化和分子技术，已鉴定出多种参与蜕皮甾体前体的细胞内运输和生物合成的蛋白质。为了开始研究蚊子卵巢中的这些过程，我们克隆了与地西泮结合抑制剂（DBI）、含StAR相关脂质转移结构域蛋白（Start1）、醛糖/酮还原酶（A/KR）、肾上腺皮质铁氧化还原蛋白还原酶（AR）以及细胞色素P450酶CYP302a1（22-羟化酶）、CYP315a1（2-羟化酶）和CYP314a1（20-羟化酶）的假定直系同源物的完整cDNA。如逆转录聚合酶链反应（RT-PCR）所示，在血液摄入前后，所有这七个基因的转录本在卵巢和其他组织中均有存在。这些基因的表达可能支持卵巢以外的组织在体外产生的低水平蜕皮甾体（7-10 pg/组织/6小时）。未吸血雌蚊以及吸血后长达6小时（PBM）的雌蚊卵巢在体外也产生少量蜕皮甾体，但在吸血后18小时和30小时，蜕皮甾体的产生大幅增加（75-106 pg/卵巢对/6小时），此后（吸血后48小时和72小时）又恢复到低水平。通过实时定量PCR分析确定，相对于未吸血雌蚊或吸血后2小时的雌蚊卵巢，在蜕皮甾体产生高峰期，埃及伊蚊CYP302和埃及伊蚊CYP315a1的基因转录本丰度显著更高（分别为9倍和12倍）。埃及伊蚊Start1、埃及伊蚊A/KR和埃及伊蚊AR在蜕皮甾体产生高峰期的卵巢中也具有较高的转录水平，并且埃及伊蚊DBI转录本在吸血后48小时增加幅度最大。相比之下，埃及伊蚊CYP314a1的基因转录本丰度在吸血后降低。这项研究首次表明，参与蜕皮甾体生物合成的几种关键蛋白质基因的转录与雌虫卵巢中蜕皮甾体产生的增加呈正相关。
