本文關(guān)鍵詞： 小菜蛾 Cry1Ac蛋白 鈣粘蛋白 氨肽酶氮 堿性磷酸酶 增效特性　出處：《湖南農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：昆蟲中腸鈣粘蛋白(Cadherin)、氨肽酶氮(Aminopeptidase N)及堿性磷酸酶(Alkaline phosphatase)是蘇云金芽胞桿菌(Bacillus thuringiensis, Bt)殺蟲晶體蛋白受體中的三個(gè)。它們能夠促進(jìn)毒素單體的寡聚化,形成毒素寡聚體后誘導(dǎo)毒素分子的空間構(gòu)象發(fā)生變化,介導(dǎo)毒素寡聚體與各受體之間的特異性結(jié)合。已有報(bào)道棉鈴蟲(Helicoverpa armigera)和煙草天蛾(Manduca sexta)等的鈣粘蛋白、氨肽酶氮及堿性磷酸酶的毒素結(jié)合區(qū)片段等能增強(qiáng)或抑制殺蟲晶體蛋白Cryl對(duì)靶標(biāo)昆蟲的活性。本文根據(jù)已報(bào)道的昆蟲毒素結(jié)合區(qū)鈣粘蛋白、氨肽酶氮及堿性磷酸酶片段對(duì)CrylAc有增效或抑制作用,以3齡小菜蛾幼蟲為研究對(duì)象,選取其鈣粘蛋白、氨肽酶氮及堿性磷酸酶片段相同功能區(qū)的五個(gè)受體片段,將其進(jìn)行克隆。通過pGEX-6P-1載體,在大腸桿菌中超量表達(dá)了五個(gè)功能區(qū)片段：鈣粘蛋白片段PxCAD-1,該片段全長(zhǎng)735bp,共編碼245個(gè)氨基酸：鈣粘蛋白片段PxCAD-2,該片段全長(zhǎng)627bp,共編碼209個(gè)氨基酸；鈣粘蛋白片段PxCAD-3,該片段全長(zhǎng)510bp的,共編碼170個(gè)氨基酸克隆：氨肽酶氮片段PxAPN1,該片段全長(zhǎng)192bp,共編碼64個(gè)氨基酸；堿性磷酸酶片段PxALPO,該片段全長(zhǎng)967bp,共編碼322個(gè)氨基酸。使用致死中濃度劑量的CrylAc及中高濃度的PxCADa、PxAPN1、PxALP1、PxCADb與PxCADc片段對(duì)小菜蛾幼蟲進(jìn)行體外復(fù)配生測(cè),1.87μg/mL的Cry1Ac可引起小菜蛾三齡幼蟲46.7%的死亡率,當(dāng)加入終濃度為551.25 μg/mL的GST-PxCADb時(shí),其死亡率為45%；當(dāng)加入終濃度為204.67 μg/mL的GST-PxCADc時(shí),其死亡率為48.9%,當(dāng)加入終濃度為187.33 μg/mL的GST-PxAPN1時(shí),其死亡率為45.6%；而對(duì)照組加入終濃度為300.27μg/mL的pGEX-6p-l時(shí),小菜蛾三齡幼蟲的死亡率為45.6%,處理(GST-PxCADb、GST-PxCADc及GST-PxAPN1a)與對(duì)照(CrylAc及pGEX-6p-1)之間并無顯著差異,表明較高濃度的PxCADb、PxCADc及PxAPN1均不能增強(qiáng)CrylAc蛋白的殺蟲活性。但是當(dāng)加入終濃度為207.58 μg/mL的GST-PxCADa時(shí),其死亡率為85.6%；當(dāng)加入終濃度為290.25 μg/mL的GST-PxALP1時(shí),其死亡率為70.0%,而對(duì)照組加入終濃度為300.27μg/mL的pGEX-6p-1時(shí),其死亡率為45.6%,處理(GST-PxCADa及GST-PxALPl)與對(duì)照(CrylAc及pGEX-6p-1)之間有顯著差異,表明中高濃度的PxCADa和PxALP1能顯著增強(qiáng)CrylAc蛋白的殺蟲活性。結(jié)果表明,PxCADb、PxCADc和PxAPN1a多肽均不能增強(qiáng)或抑制CrylAc蛋白的殺蟲活性。PxCADa和PxALP1片段可以增強(qiáng)CrylAc蛋白的殺蟲活性,且效果顯著。研究結(jié)果將為篩選有效的小菜蛾增效片段及小菜蛾毒素結(jié)合區(qū)多肽在高效殺蟲Bt工程菌和轉(zhuǎn)基因植物中的應(yīng)用提供理論依據(jù),對(duì)于揭示Bt殺蟲蛋白的毒理機(jī)制和害蟲對(duì)Bt殺蟲蛋白的抗性機(jī)制具有重要意義。
[Abstract]:The insect midgut cadherin, aminopeptidase N and alkaline phosphatase Alkaline phosphatase are three of the crystal protein receptors of Bacillus thuringiensis (BT), which can promote the oligomerization of toxin monomers. The formation of toxin oligomers induces changes in the spatial conformation of toxin molecules and mediates the specific binding between toxin oligomers and receptors. Calmodulin such as Helicoverpa armigera and Manduca sexta) have been reported. Aminopeptidase nitrogen and alkaline phosphatase toxin binding region fragments can enhance or inhibit the activity of insecticidal crystal protein Cryl to target insects. Aminopeptidase nitrogen and alkaline phosphatase fragments had synergistic or inhibitory effects on CrylAc. Five receptor fragments with the same functional regions of cadherin, aminopeptidase nitrogen and alkaline phosphatase were selected from the 3rd instar diamondback moth larvae. By using pGEX-6P-1 vector, five functional regions were overexpressed in E. coli: cadherin fragment PxCAD-1, the total length of the fragment was 735 BP, encoding 245 amino acids and cadherin fragment PxCAD-2, the total length of the fragment was 627 BP, encoding 209 amino acids. Calmodulin fragment PxCAD-3, a 510bp fragment, encodes 170 amino acid clones: aminopeptidase nitrogen fragment PxAPN1, which encodes 64 amino acids. Alkaline phosphatase fragment (PxALPO), a 967bpfull length fragment encoding 322 amino acids, was used to detect the third instar of Plutella xylostella by using median lethal dose of CrylAc and moderate and high concentration of PxCADa1, PxALP1PxALP1PxCADb and PxCADc fragments. In vitro, the Cry1Ac of Plutella xylostella larvae was determined by 1.87 渭 g / mL Cry1Ac. The death rate of 46.7% larva, The mortality rate was 45 when the final concentration of GST-PxCADb was 551.25 渭 g / mL, when the final concentration of GST-PxCADc was 204.67 渭 g / mL, the mortality was 48.9, when the final concentration of GST-PxAPN1 was 187.33 渭 g / mL, the mortality rate was 45.6, while the control group added 300.27 渭 g / mL pGEX-6p-l. The mortality of the third instar larvae of Plutella xylostella was 45.6. There was no significant difference between the treatment of GST-PxCADc and GST-PxAPN1a and that of the control, which indicated that the higher concentration of PxCADbPxCADc and PxAPN1 could not enhance the insecticidal activity of CrylAc protein, but when the final concentration of GST-PxCADa was 207.58 渭 g / mL, PxCADbPxCADc and PxAPN1 could not enhance the insecticidal activity of CrylAc protein. The mortality rate was 85.6, when the final concentration of GST-PxALP1 was 290.25 渭 g / mL, the mortality rate was 70.0 and 70.0, while in the control group it was 45.6 when the final concentration of pGEX-6p-1 was 300.27 渭 g / mL. There was a significant difference between the treatment of GST-PxCADa and GST-PxALPland the control group (CrylAc and pGEX-6p-1). The results showed that PxCADbPxCADc and PxAPN1a peptides could not enhance or inhibit the insecticidal activity of CrylAc protein. PxCADa and PxALP1 fragments could enhance the insecticidal activity of CrylAc protein. The results will provide theoretical basis for the screening of effective diamondback moth synergistic fragments and the application of polypeptides of diamondback moth toxin binding region in the efficient insecticidal BT engineering bacteria and transgenic plants. It is of great significance to reveal the toxicological mechanism of BT insecticidal protein and the resistance mechanism of pests to BT insecticidal protein.
【學(xué)位授予單位】：湖南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S433.4

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