棉铃虫Serpin基因的克

发布时间：2018-06-21 06:30

本文选题：棉铃虫 + 丝氨酸蛋白酶抑制剂基因　；参考：《河北大学》2015年硕士论文

【摘要】：丝氨酸蛋白酶抑制剂是一类最大的、分布最为广泛的蛋白酶抑制剂超家族,它通过抑制丝氨酸蛋白酶活性来集中控制许多重要的蛋白水解级联反应,影响蛋白质的代谢,在生物体中执行多种生物学功能。在昆虫中,丝氨酸蛋白酶抑制剂主要通过调节黑化反应和Toll路径来参与先天免疫反应。在植物中,丝氨酸蛋白酶抑制剂能够阻碍昆虫的生长和发育,是抵制害虫和病原体侵染的重要防御蛋白,因其抗虫谱广和作用机制独特,成为一种新型抗虫蛋白,也成为了植物抗虫基因工程中研究和应用越来越广泛的靶标蛋白。获得棉铃虫丝氨酸蛋白酶抑制剂基因是探究其结构、功能及作用机制的基础。本研究从棉铃虫中克隆出几个全新的丝氨酸蛋白酶抑制剂基因,利用生物信息学技术、实时定量PCR技术和RNA干涉技术对该基因的基本特征、在棉铃虫中的分布及功能进行了初步探究,研究成果如下:(1)通过与已报道的家蚕serpin基因同源比对,利用RT-PCR技术成功克隆到三条与家蚕serpin相似性高的棉铃虫丝氨酸蛋白酶抑制剂基因分别命名为:Haspi-5、Haspi-6和Haspi-10。利用生物信息学技术对Haspi-5、Haspi-6和Haspi-10基因c DNA及其编码氨基酸序列进行了分析,构建了棉铃虫与其它昆虫serpin氨基酸序列的分子系统进化树。(2)利用基因重组技术成功构建了原核表达载体p ET17b-Haspi-5、p ET17b-Haspi-6和p ET17b-Haspi-10,转入大肠杆菌(Escherichia coli)BL21(DE3)中,经0.8 mmol/L的IPTG,30℃诱导5 h。原核表达结果表明,Haspi-5和Haspi-6基因均高效表达出融合蛋白,SDS-PAGE显示该蛋白主要以包涵体形式存在,表达条带分子量大小与预测相符,而p ET17b-Haspi-10并没有表达出融合蛋白。(3)采用实时定量PCR技术对Haspi-5、Haspi-6和Haspi-10在棉铃虫五龄幼虫各组织部位的转录水平进行了检测,结果表明三个基因在棉铃虫各组织部位均有转录,但在不同组织部位处的表达水平差异显著。Haspi-5基因在表皮中的表达水平最高;Haspi-6基因在头部的转录水平远远高于其他组织部位;Haspi-10基因在前肠中表达水平最高,中肠的转录水平也相比其他组织较高。(4)成功构建了干涉载体p L4440-Haspi-5和p L4440-Haspi-6,通过活菌饲喂的方式对棉铃虫二龄幼虫Haspi-5和Haspi-6基因进行RNA干涉。结果表明干涉后棉铃虫致死率有所提高,实时定量PCR检测显示,经RNA干涉处理后,Haspi-5基因在棉铃虫的转录水平明显下降,约降低了6.5倍,而Haspi-6基因转录水平略有下降但并不明显,约降低了1.2倍。
[Abstract]:Serine protease inhibitors are the largest and most widely distributed superfamily of protease inhibitors, which control many important proteolytic cascades by inhibiting the activity of serine proteases and affect the metabolism of proteins. Perform a variety of biological functions in an organism. In insects, serine protease inhibitors play an important role in innate immune response by regulating the melanization and Toll pathways. In plants, serine protease inhibitors can hinder the growth and development of insects and are important defense proteins against pests and pathogens. Because of their wide spectrum and unique mechanism, serine protease inhibitors have become a new type of insect resistance protein. It has also become a widely used target protein in plant insect resistance gene engineering. Obtaining the serine protease inhibitor gene of Helicoverpa armigera is the basis for exploring its structure, function and mechanism. In this study, several novel serine protease inhibitor genes were cloned from Helicoverpa armigera. The basic characteristics of the gene were analyzed by bioinformatics, real-time quantitative PCR and RNA interference. The distribution and function of Helicoverpa armigera were preliminarily studied. The results are as follows: 1) by homologous alignment with the reported serpin gene of Bombyx mori (Bombyx mori), Three serine protease inhibitor genes of Helicoverpa armigera with high similarity to silkworm serpin were cloned by RT-PCR and named as: Haspi-5 Haspi-6 and Haspi-10, respectively. Using bioinformatics technique, the cDNA of Haspi-6 and Haspi-10 genes and their encoding amino acid sequences were analyzed. The molecular phylogenetic tree of serpin amino acid sequence of Helicoverpa armigera and other insects was constructed. The prokaryotic expression vectors pET17b-Haspi-6 and pET17b-Haspi-6 were successfully constructed by gene recombination technique. The results of prokaryotic expression showed that both the Haspi-5 and Haspi-6 genes were highly expressed, and SDS-PAGE showed that the fusion protein mainly existed in the form of inclusion body, and the molecular weight of the expressed band was in accordance with the predicted molecular weight. However, pET17b-Haspi-10 did not express the fusion protein. The transcription level of Haspi-6 and Haspi-10 in the tissues of the fifth instar larvae of Helicoverpa armigera was detected by real-time quantitative PCR. The results showed that the three genes were transcribed in all tissues of Helicoverpa armigera. But the expression level of Haspi-5 gene in epidermis was the highest in the epidermis. The transcription level of Haspi-6 gene in the head was much higher than that in the foregut of other tissues. The interference vectors pL4440-Haspi-5 and pL4440-Haspi-6 were constructed successfully. The RNA interference of Haspi-5 and Haspi-6 genes of the second instar larvae of Helicoverpa armigera was carried out by feeding live bacteria. The results showed that the fatality rate of Helicoverpa armigera increased after interference. Real-time quantitative PCR analysis showed that the transcription level of Happi-5 gene in Helicoverpa armigera was significantly decreased after RNA interference, about 6.5 times lower than that of Haspi-6 gene, but the transcription level of Haspi-6 gene was slightly decreased but not obvious. About 1.2 times lower.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q78;S433

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