棉鈴蟲HaDFP2基因功能的研究
發(fā)布時間：2018-12-05 16:21
【摘要】：酚氧化酶原激活反應在昆蟲先天性免疫中起著重要的作用。該反應是個非常復雜的系統(tǒng),已知有多種分子參與調(diào)控這一反應。本研究組在進行棉鈴蟲Helicoverpa armigera)血細胞轉(zhuǎn)錄組測序發(fā)現(xiàn)了一個免疫刺激后上調(diào)表達的基因,命名為defense protein 2 (HaDFP2)。在本項研究中我們利用熒光定量PCR、Western blotting、蛋白體外功能試驗以及RNA干擾等方法研究了HaDFP2基因在棉鈴蟲先天性免疫中的具體功能,結(jié)果表明HaDFP2基因可能參與了棉鈴蟲酚氧化酶原激活反應的調(diào)控。具體的實驗結(jié)果如下：1.HaDFP2基因的克隆及序列分析：Ha-DFP2基因全長425bp,具有一個可以編碼123個氨基酸的完整開放閱讀框。預測該基因編碼的蛋白質(zhì)無信號肽序列,無跨膜區(qū),分子量為13.5 kDa,等電點為8.82。2.重組蛋白的原核表達純化及多克隆抗體的制備：利用pET32a載體在大腸桿菌中誘導表達和純化了HaDFP2重組蛋白,并且制備了多克隆抗體。3.組織特異性表達分析：利用Western blotting檢測了HaDFP2蛋白在血細胞、血漿、脂肪體、中腸和表皮等五種組織中的表達和分布情況,結(jié)果顯示只有血漿和血細胞中有HaDFP2蛋白的表達和分布。這一結(jié)果表明HaDFP2蛋白質(zhì)是一種分泌型蛋白,由血細胞表達后分泌到血淋巴中。4.免疫刺激后棉鈴蟲血淋巴中HaDFP2基因表達量的變化：熒光定量分析結(jié)果顯示注射大腸桿菌能夠顯著上調(diào)HaDFP2基因在棉鈴蟲血細胞中的表達,同時Western blotting分析顯示棉鈴蟲血細胞和血漿中的HaDFP2蛋白在大腸桿菌刺激后表達量上調(diào)。這些結(jié)果表明HaDFP2基因可能參與了調(diào)控棉鈴蟲的先天性免疫反應。5. HaDFP2重組蛋白體外功能試驗：(1)rHaDFP2能夠促進棉鈴蟲血漿中酚氧化酶原的激活,并且能促進凝膠珠的體內(nèi)黑化反應。(2) rHaDFP2能夠與大腸桿菌和金黃色葡萄球菌結(jié)合,并且能夠促進這兩種菌體外凝集。6.RNA干擾試驗：通過注射dsHaDFP2敲降棉鈴蟲血細胞中HaDFP2基因的表達后：血漿中酚氧化酶活力顯著降低,血淋巴清除大腸桿菌和金黃色葡萄球菌的能力也顯著降低。7. HaDFP2基因5’上游區(qū)的克隆及啟動子活性分析：共擴增出HaDFP2基因5’上游區(qū)序列3200bp；利用雙熒光素酶報告系統(tǒng)驗證顯示靠近3’端的1005bp序列具有啟動子活性,并且該啟動子活性能夠在大腸桿菌的刺激下進一步增強。但要找出啟動子的關鍵序列還有待進一步的研究。上述研究結(jié)果顯示,HaDFP2基因可能通過介導酚氧化酶原激活反應而在棉鈴蟲先天性免疫中起著重要的作用。實驗結(jié)果還表明HaDFP2基因可能是一種新的酚氧化酶原級聯(lián)通路調(diào)控因子,但其具體的作用機理還有待于進一步的研究。
[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.
【學位授予單位】：華中師范大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q78;S433
本文編號：2365278
[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.
【學位授予單位】：華中師范大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q78;S433
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相關碩士學位論文 前1條
1 宋彩霞;棉鈴蟲HaDFP2基因功能的研究[D];華中師范大學;2016年
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