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棉铃虫HaDFP2基因功能的研究

发布时间:2018-12-05 16:21
【摘要】:酚氧化酶原激活反应在昆虫先天性免疫中起着重要的作用。该反应是个非常复杂的系统,已知有多种分子参与调控这一反应。本研究组在进行棉铃虫Helicoverpa armigera)血细胞转录组测序发现了一个免疫刺激后上调表达的基因,命名为defense protein 2 (HaDFP2)。在本项研究中我们利用荧光定量PCR、Western blotting、蛋白体外功能试验以及RNA干扰等方法研究了HaDFP2基因在棉铃虫先天性免疫中的具体功能,结果表明HaDFP2基因可能参与了棉铃虫酚氧化酶原激活反应的调控。具体的实验结果如下:1.HaDFP2基因的克隆及序列分析:Ha-DFP2基因全长425bp,具有一个可以编码123个氨基酸的完整开放阅读框。预测该基因编码的蛋白质无信号肽序列,无跨膜区,分子量为13.5 kDa,等电点为8.82。2.重组蛋白的原核表达纯化及多克隆抗体的制备:利用pET32a载体在大肠杆菌中诱导表达和纯化了HaDFP2重组蛋白,并且制备了多克隆抗体。3.组织特异性表达分析:利用Western blotting检测了HaDFP2蛋白在血细胞、血浆、脂肪体、中肠和表皮等五种组织中的表达和分布情况,结果显示只有血浆和血细胞中有HaDFP2蛋白的表达和分布。这一结果表明HaDFP2蛋白质是一种分泌型蛋白,由血细胞表达后分泌到血淋巴中。4.免疫刺激后棉铃虫血淋巴中HaDFP2基因表达量的变化:荧光定量分析结果显示注射大肠杆菌能够显著上调HaDFP2基因在棉铃虫血细胞中的表达,同时Western blotting分析显示棉铃虫血细胞和血浆中的HaDFP2蛋白在大肠杆菌刺激后表达量上调。这些结果表明HaDFP2基因可能参与了调控棉铃虫的先天性免疫反应。5. HaDFP2重组蛋白体外功能试验:(1)rHaDFP2能够促进棉铃虫血浆中酚氧化酶原的激活,并且能促进凝胶珠的体内黑化反应。(2) rHaDFP2能够与大肠杆菌和金黄色葡萄球菌结合,并且能够促进这两种菌体外凝集。6.RNA干扰试验:通过注射dsHaDFP2敲降棉铃虫血细胞中HaDFP2基因的表达后:血浆中酚氧化酶活力显著降低,血淋巴清除大肠杆菌和金黄色葡萄球菌的能力也显著降低。7. HaDFP2基因5’上游区的克隆及启动子活性分析:共扩增出HaDFP2基因5’上游区序列3200bp;利用双荧光素酶报告系统验证显示靠近3’端的1005bp序列具有启动子活性,并且该启动子活性能够在大肠杆菌的刺激下进一步增强。但要找出启动子的关键序列还有待进一步的研究。上述研究结果显示,HaDFP2基因可能通过介导酚氧化酶原激活反应而在棉铃虫先天性免疫中起着重要的作用。实验结果还表明HaDFP2基因可能是一种新的酚氧化酶原级联通路调控因子,但其具体的作用机理还有待于进一步的研究。
[Abstract]:Phenoloxidase activation plays an important role in innate immunity of insects. This reaction is a very complex system, and many molecules are known to be involved in regulating the reaction. A gene named defense protein 2 (HaDFP2), which was up-regulated after immunostimulation, was identified by our team by sequencing the Helicoverpa armigera) hematopoietic transcriptome of Helicoverpa armigera (Helicoverpa armigera). In this study, we studied the specific function of HaDFP2 gene in congenital immunity of Helicoverpa armigera by fluorescence quantitative PCR,Western blotting, protein function test in vitro and RNA interference. The results suggest that HaDFP2 gene may be involved in the regulation of phenoloxidase activation in Helicoverpa armigera. The results are as follows: cloning and sequence analysis of 1.HaDFP2 gene: Ha-DFP2 gene is 425 BP in length and has a complete open reading frame which can encode 123 amino acids. The protein encoded by this gene was predicted to have no signal peptide sequence, no transmembrane region, and a molecular weight of 13.5 kDa, isoelectric point of 8.82.2. Prokaryotic expression and purification of recombinant protein and preparation of polyclonal antibody: recombinant HaDFP2 protein was induced and purified by pET32a vector in Escherichia coli, and polyclonal antibody was prepared. Tissue specific expression: Western blotting was used to detect the expression and distribution of HaDFP2 protein in blood cells, plasma, fat body, midgut and epidermis. The results showed that only HaDFP2 protein was expressed and distributed in plasma and blood cells. These results suggest that HaDFP2 protein is a secretory protein expressed by blood cells and secreted into hemolymph. 4. Changes of HaDFP2 gene expression in hemolymph of Helicoverpa armigera after immunostimulation: the results of fluorescence quantitative analysis showed that E. coli injection could significantly up-regulate the expression of HaDFP2 gene in Helicoverpa armigera hemocytes. At the same time, Western blotting analysis showed that the expression of HaDFP2 protein in blood cells and plasma of Helicoverpa armigera was up-regulated after stimulation by Escherichia coli. These results suggest that HaDFP2 gene may be involved in regulating the innate immune response of Helicoverpa armigera. In vitro functional test of HaDFP2 recombinant protein: (1) rHaDFP2 could promote the activation of phenoloxidase in plasma of Helicoverpa armigera and the melanization reaction of gel beads in vivo. (2) rHaDFP2 could bind to Escherichia coli and Staphylococcus aureus. 6.RNA interference test: the expression of HaDFP2 gene in blood cells of Helicoverpa armigera was reduced by injection of dsHaDFP2: the activity of phenoloxidase in plasma decreased significantly. The ability of hemolymph to clear Escherichia coli and Staphylococcus aureus was also significantly decreased. Cloning and promoter activity analysis of 5'upstream region of HaDFP2 gene: the sequence of 5'upstream region of HaDFP2 gene was amplified from 3200bp; Double luciferase reporting system was used to verify that the 1005bp sequence near the 3 'end had promoter activity, and the promoter activity could be further enhanced under the stimulation of Escherichia coli. However, further research is needed to find the key sequence of the promoter. These results suggest that the HaDFP2 gene may play an important role in the innate immunity of Helicoverpa armigera by mediating the activation of phenoloxidase. The results also indicated that HaDFP2 gene may be a new regulation factor of prophenol oxidase cascade pathway, but its specific mechanism needs to be further studied.
【学位授予单位】:华中师范大学
【学位级别】:硕士
【学位授予年份】:2016
【分类号】:Q78;S433

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1 宋彩霞;棉铃虫HaDFP2基因功能的研究[D];华中师范大学;2016年



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