摘要
蛋白激酶c受体l(receptor for activated C kinase 1,Rackl)属于一类含有WD-40重复结构蛋白家族的亚家族。广泛分布于真核生物和原核生物中(Smith et al.,2000)。最初Rack1蛋白被确定为哺乳动物蛋白激酶C(PKC)的受体(Ron et al.,1994),现在认为Rackl蛋白作为支架蛋白在多个信号转导途径中起重要作用(chen et al.,2006)。棉铃虫[Helicoverpa armigera(Hübner)]是一种世界性农业害虫,危害棉花、玉米、小麦等多种农作,广泛分布于非洲、亚洲、澳大利亚、太平洋和欧洲等地区。人们对其进行了大量的遗传,分子水平的研究,但目前尚未发现对Rack1基因的研究。本实验对棉铃虫Rack1蛋白基因进行研究,为探明昆虫Rack1蛋白的保护作用机制提供理论依据。
主要研究结果如下:
(1)本实验利用RT-PCR和RACE的方法扩增获得了棉铃虫Rack1基因全序列,序列分析结果表明,该基因编码区长为957bp,编码319个氨基酸残基。5’端非编码区长为36bp,3’端非编码区长为112bp。同源性分析显示,棉铃虫Rack1的氨基酸序列具有高度的保守性,与其他昆虫相比高达85%以上。
(2)本实验利用荧光定量PCR方法,研究了Rack1基因在棉铃虫的不同发育时期的转录情况。结果表明,Rack1基因总是在蜕皮过程中表达量升高,这一结果暗示着Rack1基因可能参与棉铃虫的蜕皮过程。
(3)本实验利用注射激素和荧光定量PCR方法,研究了激素处理后Rack1基因在棉铃虫中的转录情况。结果表明,从处理3h到12h蜕皮激素均诱导Rack1的表达,从处理3h到12h保幼激素均显著抑制Rack1基因的表达,本结论再一次验证了蜕皮激素与保幼激素对Rack1基因的影响,暗示蜕皮激素与保有激素可能参与调控Rack1基因的表达。
(4)本实验利用饥饿处理和荧光定量PCR方法,研究了饥饿对Rack1基因在棉铃虫中的转录情况。结果表明在棉铃虫开始取食时对棉铃虫进行饥饿处理可以显著抑制Rack1的转录,从饥饿3 h开始一直持续到12 h,Rack1的转录明显被抑制。
Receptor for activated C kinase 1 (Rackl) belongs to a large family of WD 40 repeat proteins and is a highly conserved protein found in both eukaryote and prokaryote(Smith et al.,2000). At first,Rack1 was considered as a repceptor of protein kinase C(PKC)in mammals(Ron et al.,1994).But now, the role of RACK1 as a scaffold protein in a lot of signalling processes(chen et al.,2006). Cotton bollworm, Helicoverpa armigera(Hübner), is a world pest, cause huge damage to a lot of crops. In this experiment, the characterizations of receptor for activated C kinase 1 gene from Helicoverpa armigera (Hübner) were studied and these studies further provide foundation to understand the insect response to adversity, The main results are as follows:
1. Receptor for activated C kinase 1 (Rackl)cDNA was isolated by using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE).Nucleotide sequence analysis revealed that open reading frame (ORF) of Rack1 has 957 bp in size encoded 319 amino acid residues. 3’untranslated region (UTR) of Rack1 contains 112 nucleic acids and 5’UTR contain 36 nucleic acids.
2. The temporal expression of Rack1 transcript was studied by using Real-time PCR. The results showed that Rack1 was up-regulated in molting stage,It imply that Rack1 maybe concrol the molting stage.
3. The Rack1 expression analysis after hormones challange was studied by using Real- time PCR. The results showed that Rack1 was up-regulated by molting hormone (20e) and inhibited by juvenile hormone analogue and starvation.
4. The Rack1 expression analysis after hunger was studied by using Real- time PCR. The results showed that Rack1 was inhibited by hunger.
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