Gene cloning and nucleotide sequence analysis of eclosion hormone of the silkworm, Bombyx mori

家蚕羽化激素基因克隆及核苷酸序列分析

基本信息

批准号：
62560117
负责人：
NAGASAWA Hiromichi
金额：
$ 1.22万
依托单位：
Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

Eclosion hormone (EH), an insect neuropeptide, triggers behavior of larval, pupal and adult ecdyses. We have purified EH to a homogeneous state from pupal and adult heads of the silkworm, Bombyx mori, and proposed its amino-terminal 61 amino acid residues. However, we could not establish the complete amino acid sequence of EH because of the limitted amount of the purified peptide. In order to estimate the whole amino acid sequence of EH we tried to isolate a genomic DNA encoding for EH.A DNA fraction was prepared from whole bodies of 3rd instar larvae of B. mori. It was digested with Sau3AI and the resulting DNA fragments (9-23 kb) were inserted into EMBL3 phage to construct a genomic library. Three unique oligonucleotide probes (54mer, 33mer and 36mer) and mix probe (17mer) were synthesized according to the amino acid sequence. Screening of 1.2 x 10^5 clones with ^<32>P-labled probes permitted us to find out only one clone, which hybridized with 54mer, 33mer and 17mer probes. A XbaI-EcoRI fragment (380 bp) of this clone, which hybridized with all the three probes, was sequenced by the dideoxy method. A portion of the translated sequence matches exactly with the known peptide sequence, ALa(5)-LYs(61). The DNA sequence of this portion is followed by the sequence of CTT(Leu)-TAA(stop codon). On the other hand, the sequence encoding for Ser(1)-Ile(4) could not be found in this fragment and in addition, there was a sequence similar to the consensus sequence of the intron acceptor just upstream of Ala(5), suggesting that there is an intron between Ile(4) and Ala(5). Thus, EH was estimated to be a 62-residue peptide with a Leu residue at the carboxyl-terminus. We are now searching for the sequence encoding for the signal peptide and EH(1-4).

羽化激素 (EH) 是一种昆虫神经肽，可引发幼虫、蛹和成虫的蜕皮行为。我们从家蚕（Bombyx mori）的蛹和成虫头部中将EH纯化至均质状态，并提出其氨基末端61个氨基酸残基。然而，由于纯化肽的数量有限，我们无法确定EH的完整氨基酸序列。为了估计EH的整个氨基酸序列，我们试图分离编码EH的基因组DNA。从家蚕3龄幼虫的整个身体中制备DNA片段。用 Sau3AI 对其进行消化，并将所得 DNA 片段 (9-23 kb) 插入 EMBL3 噬菌体中以构建基因组文库。根据氨基酸序列合成了三种独特的寡核苷酸探针（54mer、33mer和36mer）和混合探针（17mer）。用^ 32 P标记的探针筛选1.2×10^5个克隆使我们仅找出一个与54mer、33mer和17mer探针杂交的克隆。该克隆的 XbaI-EcoRI 片段（380 bp）与所有三个探针杂交，通过双脱氧方法进行测序。翻译序列的一部分与已知的肽序列 ALa(5)-LYs(61) 完全匹配。该部分的DNA序列后面是CTT(Leu)-TAA(终止密码子)的序列。另一方面，在该片段中找不到编码Ser(1)-Ile(4)的序列，此外，在Ala(5)上游存在与内含子受体共有序列相似的序列，表明 Ile(4) 和 Ala(5) 之间存在一个内含子。因此，EH 被估计为 62 个残基的肽，在羧基末端有一个 Leu 残基。我们现在正在寻找编码信号肽和 EH(1-4) 的序列。