Takahashi Michiyoshi, Kikuchi Kyoko, Tomita Shuichiro, Imanishi Shigeo, Nakahara Yuichi, Kiuchi Makoto, Kamimura Manabu
Developmental Biology Department, National Institute of Agrobiological Sciences, 1-2, Owashi, Tsukuba, Ibaraki 305-8634, Japan.
Comp Biochem Physiol B Biochem Mol Biol. 2003 Jul;135(3):431-7. doi: 10.1016/s1096-4959(03)00094-0.
To analyze the molecular mechanisms underlying hormone-regulated gene expression during molt and metamorphosis, we developed a transient reporter gene assay system using the silkworm anterior silk gland. Reporter plasmids were delivered into dissected anterior silk glands by particle bombardment and bombarded glands transplanted into other larvae, to which hormones were then administered. When the green fluorescent protein gene, coupled with the constitutive cytoplasmic actin gene A3 promoter, was introduced into the anterior silk gland, strong green fluorescence was observed a few days later. Bombarded silk glands transplanted into other larvae showed the same morphological changes as intrinsic glands after 20-hydroxyecdysone (20E) alone or 20E plus juvenile hormone (JH) treatment, indicating that the transplanted gland received hormonal signals properly. When a 20E-responsive reporter construct containing four tandemly repeated pal-1 ecdysone response elements upstream from the luciferase gene was delivered into the gland, an approximately 50-fold increase in luciferase activity was detected 30 h after 20E injection. This induction was comparable to that in an ecdysteroid-responsive Bombyx cell line. This in vivo reporter assay system is thus a rapid, effective tool for analyzing gene expression regulated by 20E and probably by JH.
为了分析蜕皮和变态过程中激素调节基因表达的分子机制，我们利用家蚕前部丝腺开发了一种瞬时报告基因检测系统。通过粒子轰击将报告质粒导入解剖后的前部丝腺，然后将轰击后的腺体移植到其他幼虫体内，再对这些幼虫施加激素。当将与组成型细胞质肌动蛋白基因A3启动子相连的绿色荧光蛋白基因导入前部丝腺时，几天后观察到强烈的绿色荧光。移植到其他幼虫体内的轰击丝腺在单独使用20-羟基蜕皮酮（20E）或20E加保幼激素（JH）处理后，显示出与固有腺体相同的形态变化，这表明移植的腺体能够正确接收激素信号。当将一个在荧光素酶基因上游含有四个串联重复的pal-1蜕皮激素反应元件的20E反应性报告构建体导入腺体时，在注射20E后30小时检测到荧光素酶活性增加了约50倍。这种诱导与在蜕皮类固醇反应性家蚕细胞系中的诱导相当。因此，这种体内报告基因检测系统是分析由20E以及可能由JH调节的基因表达的一种快速、有效的工具。
