本文選題：棉鈴蟲 + 絲氨酸蛋白酶抑制劑基因��； 參考：《河北大學》2015年碩士論文

【摘要】：絲氨酸蛋白酶抑制劑是一類最大的、分布最為廣泛的蛋白酶抑制劑超家族,它通過抑制絲氨酸蛋白酶活性來集中控制許多重要的蛋白水解級聯(lián)反應,影響蛋白質(zhì)的代謝,在生物體中執(zhí)行多種生物學功能。在昆蟲中,絲氨酸蛋白酶抑制劑主要通過調(diào)節(jié)黑化反應和Toll路徑來參與先天免疫反應。在植物中,絲氨酸蛋白酶抑制劑能夠阻礙昆蟲的生長和發(fā)育,是抵制害蟲和病原體侵染的重要防御蛋白,因其抗蟲譜廣和作用機制獨特,成為一種新型抗蟲蛋白,也成為了植物抗蟲基因工程中研究和應用越來越廣泛的靶標蛋白。獲得棉鈴蟲絲氨酸蛋白酶抑制劑基因是探究其結構、功能及作用機制的基礎。本研究從棉鈴蟲中克隆出幾個全新的絲氨酸蛋白酶抑制劑基因,利用生物信息學技術、實時定量PCR技術和RNA干涉技術對該基因的基本特征、在棉鈴蟲中的分布及功能進行了初步探究,研究成果如下:(1)通過與已報道的家蠶serpin基因同源比對,利用RT-PCR技術成功克隆到三條與家蠶serpin相似性高的棉鈴蟲絲氨酸蛋白酶抑制劑基因分別命名為:Haspi-5、Haspi-6和Haspi-10。利用生物信息學技術對Haspi-5、Haspi-6和Haspi-10基因c DNA及其編碼氨基酸序列進行了分析,構建了棉鈴蟲與其它昆蟲serpin氨基酸序列的分子系統(tǒng)進化樹。(2)利用基因重組技術成功構建了原核表達載體p ET17b-Haspi-5、p ET17b-Haspi-6和p ET17b-Haspi-10,轉(zhuǎn)入大腸桿菌(Escherichia coli)BL21(DE3)中,經(jīng)0.8 mmol/L的IPTG,30℃誘導5 h。原核表達結果表明,Haspi-5和Haspi-6基因均高效表達出融合蛋白,SDS-PAGE顯示該蛋白主要以包涵體形式存在,表達條帶分子量大小與預測相符,而p ET17b-Haspi-10并沒有表達出融合蛋白。(3)采用實時定量PCR技術對Haspi-5、Haspi-6和Haspi-10在棉鈴蟲五齡幼蟲各組織部位的轉(zhuǎn)錄水平進行了檢測,結果表明三個基因在棉鈴蟲各組織部位均有轉(zhuǎn)錄,但在不同組織部位處的表達水平差異顯著。Haspi-5基因在表皮中的表達水平最高;Haspi-6基因在頭部的轉(zhuǎn)錄水平遠遠高于其他組織部位;Haspi-10基因在前腸中表達水平最高,中腸的轉(zhuǎn)錄水平也相比其他組織較高。(4)成功構建了干涉載體p L4440-Haspi-5和p L4440-Haspi-6,通過活菌飼喂的方式對棉鈴蟲二齡幼蟲Haspi-5和Haspi-6基因進行RNA干涉。結果表明干涉后棉鈴蟲致死率有所提高,實時定量PCR檢測顯示,經(jīng)RNA干涉處理后,Haspi-5基因在棉鈴蟲的轉(zhuǎn)錄水平明顯下降,約降低了6.5倍,而Haspi-6基因轉(zhuǎn)錄水平略有下降但并不明顯,約降低了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.
【學位授予單位】：河北大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：Q78;S433

【參考文獻】

相關期刊論文 前10條

1 王彥云;何漸鳴;李國勝;王明慧;沈衛(wèi)德;許雅香;;家蠶絲氨酸蛋白酶抑制劑基因Bmserpin-6的特異性表達分析[J];蠶業(yè)科學;2013年02期

2 李國勝;王彥云;王明慧;徐開遵;陳玉華;沈衛(wèi)德;許雅香;;家蠶絲氨酸蛋白酶抑制劑5的多克隆抗體制備及組織表達分析[J];蠶業(yè)科學;2013年02期

3 王路;陸濤;王海勇;王振;;第二代丙型肝炎病毒NS3/4A絲氨酸蛋白酶抑制劑研究進展[J];國際藥學研究雜志;2013年01期

4 周劍,尹麗紅,王琛柱;注射親水性硅珠對棉鈴蟲血漿酚氧化酶活性的影響[J];昆蟲學報;2002年06期

5 劉襯麗;王東;李兵;管京敏;俞燕芳;g晗，

本文編號：2047594