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PHYSIOLOGICAL AND BIOCHEMICAL STUDIES ON EMBRYONIC DEVELOPMENT OF SILKWORMS

蚕胚胎发育的生理生化研究

基本信息

批准号：
06304014
负责人：
TAKEI Ryuzo
金额：
$ 7.3万
依托单位：
SHINSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

项目摘要

Vitellins (Vns) were purified form the eggs of the two wild silkworm, Antheraea pernyi (Ap) and Antheraea yamamai (Ay) by Butyl-cellulofine chromatography, DE-52 cellulose column chromatography and hydroxyapatite column chromatography. Native molecular weight of ApVg is approximately 470000 as same as ApVn, while the molecular weights of AyVg and AyVn are 465000 and 450000, respectively. The amounts of Antheraea vitellogenin in hemolymph increased remarkably after spinning and kept constant concentration until the commencement of vitellogenesis. In vitro translation of RNA from fat body and immunoprecipitation of translation products using anti-Vn antiserum showed that the synthesis of Vgs occurred in the fat bodies after spinning but reduced remarkably after pupation.In an initial effort to understand the molecular mechanism of how-low temperature induces sorbitol dehydrogenase gene expression in diapause eggs of the silkworm, the sorbitol dehydrogenase gene was isolated from a Bombyx genomic library using a cDNA encoding the Bombyx homologue of mammalian sorbitol dehydrogenase as a probe. The gene extended for about 10 kb, consisting of eight exons and seven introns. AATTAA,instead of AATAAA,was localized in the upstream region of the polyadenylation site. Although a single copy of thie gene was present per haploid genome, 1.2kb and 1.1kb transcripts were found from yolk cells in diapause eggs and from larval fat body cells, respectively. The two major transcription initiation sites corresponding to both transcripts were localized at 355 and 226 base pairs upstream from the translation start site, indicating an alternative use of promoter. The 5'-upstream region of the gene contained a consensus sequence, TGA (A/T) AA (A/G/T), that has been found in insect genes expressed mainly in larval and pupal fat bodies.

用丁基纤维素层析、DE-52纤维素柱层析和羟基磷灰石柱层析从柞蚕卵和天蚕卵中分离纯化卵黄蛋白（Vns）。ApVg的天然分子量约为470000，与ApVn相同，而AyVg和AyVn的分子量分别为465000和450000。蚕血淋巴中卵黄原蛋白的含量在吐丝后显著增加，并在卵黄发生前保持恒定浓度。脂肪体RNA的体外翻译和翻译产物的抗Vn抗血清免疫沉淀实验表明，家蚕吐丝后脂肪体中有Vgs的合成，但化蛹后Vgs的合成显著减少。使用编码哺乳动物山梨糖醇脱氢酶的pGEX同源物的cDNA作为探针，从pGEX基因组文库中分离山梨糖醇脱氢酶基因。该基因长约10 kb，由8个外显子和7个内含子组成。AATTAA，而不是AATAAA，定位在上游区域的多聚腺苷酸化位点。虽然该基因在每个单倍体基因组中只有一个拷贝，但在滞育卵的卵黄细胞和幼虫的脂肪体细胞中分别发现了1.2kb和1.1kb的转录本。两个主要的转录起始位点分别位于翻译起始位点上游的355和226个碱基对处，这表明启动子的另一种用途。该基因的5 '-上游区域含有一个共有序列TGA（A/T）AA（A/G/T），该序列已在主要在幼虫和蛹脂肪体中表达的昆虫基因中发现。