Molecular cloning and characterization of cDNA for insect biogenic peptide, growth‐blocking peptide
Molecular cloning and characterization of cDNA for insect biogenic peptide, growth‐blocking peptide
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昆虫生物肽、生长阻断肽 cDNA 的分子克隆和表征
DOI：
10.1016/0014-5793(95)01273-7
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发表时间：
1995
期刊：
影响因子：
3.5
通讯作者：
S. Tomino
中科院分区：
文献类型：
--
作者：
Y. Hayakawa;A. Ohnishi;A. Yamanaka;S. Izumi;S. Tomino
生长阻滞肽（Growth Blocking Peptide，GBP）是一种昆虫生物源肽，可阻止幼虫到蛹的变态。描述了编码GBP的cDNA。使用衍生自GBP肽序列的混合寡核苷酸通过聚合酶链式反应（PCR）产生扩增的DNA。根据扩增的DNA序列，设计了一个41bp的寡核苷酸序列，用于筛选粘虫Pseudaletia separatalararachum寄生蜂Cotesia kariyai的cDNA文库。克隆的GBP cDNA全长为809 bp。一个429个碱基对的开放阅读框架编码一个143个氨基酸残基的前原肽，其中GBP位于C末端区域，其他三个肽包括一个推定的信号肽和适当的内切蛋白水解切割位点。北方印迹分析表明，脂肪体中存在800碱基的mRNA转录本，脑和神经索中存在2.5碱基的mRNA转录本，这表明GBP基因的转录可能以组织依赖性方式调节。从脑神经索的cDNA库中分离GBP cDNA片段支持了这一解释。GBP mRNA在被寄生和未被寄生的末龄幼虫中持续表达，并且两种幼虫之间的mRNA水平没有差异，因此表明寄生可能影响翻译或翻译后水平以升高血浆GBP浓度。
Growth‐blocking peptide (GBP) is an insect biogenic peptide that prevents the onset of metamorphosis from larva to pupa. A cDNA coding for GBP is described. Mixed oligonucleotides derived from a GBP peptide sequence were used to generate amplified DNA by the polymerase chain reaction (PCR). Based on the sequence of the amplified DNA, a 41 bases oligonucleotide was designed for screening a cDNA library which was constructed from the armywormPseudaletia separatalarvae parasitized with the parasitic waspCotesia kariyai. The cloned cDNA for GBP was 809 base pairs in length. An open reading frame of 429 base pairs encodes a pre‐pro‐peptide of 143 amino acid residues in which GBP is localized at the C‐terminal region, and other three peptides including a putative signal peptide and appropriate processing sites for endoproteolytic cleavage precede the GBP sequence. Northern blot analyses demonstrate the presence of a 800‐base mRNA transcript in fat body and 2.5‐kilobase transcript in brain and nerve cord, suggesting the possibility that the transcription of GBP gene is regulated in a tissue‐dependent manner. This interpretation was supported by isolating a GBP cDNA fragment from cDNA pool of brain‐nerve cords. GBP mRNA is constantly expressed in both parasitized and non‐parasitized last instar larvae and there is no difference in the levels of the mRNA between both larvae, thus indicating that parasitism may effect on translational or posttranslational level to elevate plasma GBP concentration.