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Molecular Biological Assessment of Soil Environment Using Earthworm (Annelida, Olagoch aeta)

使用蚯蚓（Annelida、Olagoch aeta）对土壤环境进行分子生物学评估

基本信息

批准号：
12480158
负责人：
GAMOU Shinobu
金额：
$ 5.76万
依托单位：
Kyorin University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

Earthworms are very sensitive to their circumstance, and thus, a possible indicator for soil environment. The field investigations of earthworm were carried out in Okutama forestry region and in our campus for 2 years. Anatomical analysis of Pheretimoid earthworms revealed more then 90% of them possess incomplete male gonads, whereas all possess complete female gonad. It is not yet clear whether the anomaly of male gonad come from environmental contaminations or the species characteristics of Pheretimoids.In order to assess soil environment in molecular biological technique, we first established the clean cultivation of Eisenia fetida, which is a world-widely distributed Lumricide. E. fetida can grow in a sawdust media with rice bran in our laboratory, and reproduce once a month. E. fetida shows the significant growth retardation in synthetic estrogen DES containing-growth media, suggesting the common mechanism for steroid sex hormones reception despite of no apparent endocrine organ. Their genetic information is extremely scarce, but it is worthy to investigate the molecular mechanism of gonad differentiation. We are carrying out the molecular cloning of homolog(s) of DMRT, which is isolated from nematode C. elegans, fruit fly D. melanogaster and also mammals as male gonad-determining gene.Enchytraeus japonensis reproduces in a unique asexual manner, in which the mature E. japonensis divided into 〜10 fragments and each fragment grow into mature worms within 10 days. However, under starved and low-density culture, gonad differentiation into hermaphrodites and sexual reproduction takes place. 17-b-estradiol or DES induces neither gonad differentiation nor sexual reproduction. However, 17-b-estradiol induces a dose-dependent delay of growth, while DES exhibits toxic effect. To further investigate these reproduction and regeneration process, immunological staining procedures were established using anti-phosphotyrosine and anti-BrdU antibodies.

蚯蚓对它们的环境非常敏感，因此可能是土壤环境的指示器。在冈山林区和我校进行了为期2年的蚯蚓田间调查。对螺形蚯蚓的解剖分析表明，90%以上的蚯蚓具有不完全的雄性性腺，而所有的蚯蚓都具有完整的雌性性腺。目前尚不清楚雄性性腺的异常是由环境污染引起的，还是由环纹夜蛾的种类特征引起的。为了利用分子生物学技术评价土壤环境，我们首先建立了世界上分布广泛的杀线虫--赤子爱胜虫的清洁栽培。在本实验室的木屑和米皮基质中可以生长，每个月繁殖一次。E.Fetida在含有合成雌激素的培养液中表现出明显的生长迟缓，这表明在没有明显的内分泌器官的情况下，类固醇性激素的接收是共同的机制。它们的遗传信息极其稀缺，但性腺分化的分子机制值得研究。我们正在进行Dmrt同源基因(S)的分子克隆，Dmrt是从线虫、果蝇和哺乳动物中分离出来的雄性性腺决定基因。日本线虫以独特的无性繁殖方式繁殖，成熟的日本线虫分为10个片段，每个片段在10天内发育成成虫。然而，在饥饿和低密度培养下，性腺分化为两性和有性繁殖。17-b-雌二醇或DES既不能诱导性腺分化，也不能诱导有性繁殖。然而，17-b-雌二醇会引起剂量依赖性的生长延迟，而DES则表现出毒性效应。为了进一步研究这些繁殖和再生过程，建立了使用抗磷酸酪氨酸和抗BrdU抗体的免疫染色程序。