小菜蛾的五个受体片段对Cry1Ac蛋白的增效特性研究

发布时间：2018-02-25 02:30

本文关键词： 小菜蛾 Cry1Ac蛋白钙粘蛋白氨肽酶氮碱性磷酸酶增效特性　出处：《湖南农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：昆虫中肠钙粘蛋白(Cadherin)、氨肽酶氮(Aminopeptidase N)及碱性磷酸酶(Alkaline phosphatase)是苏云金芽胞杆菌(Bacillus thuringiensis, Bt)杀虫晶体蛋白受体中的三个。它们能够促进毒素单体的寡聚化,形成毒素寡聚体后诱导毒素分子的空间构象发生变化,介导毒素寡聚体与各受体之间的特异性结合。已有报道棉铃虫(Helicoverpa armigera)和烟草天蛾(Manduca sexta)等的钙粘蛋白、氨肽酶氮及碱性磷酸酶的毒素结合区片段等能增强或抑制杀虫晶体蛋白Cryl对靶标昆虫的活性。本文根据已报道的昆虫毒素结合区钙粘蛋白、氨肽酶氮及碱性磷酸酶片段对CrylAc有增效或抑制作用,以3龄小菜蛾幼虫为研究对象,选取其钙粘蛋白、氨肽酶氮及碱性磷酸酶片段相同功能区的五个受体片段,将其进行克隆。通过pGEX-6P-1载体,在大肠杆菌中超量表达了五个功能区片段：钙粘蛋白片段PxCAD-1,该片段全长735bp,共编码245个氨基酸：钙粘蛋白片段PxCAD-2,该片段全长627bp,共编码209个氨基酸；钙粘蛋白片段PxCAD-3,该片段全长510bp的,共编码170个氨基酸克隆：氨肽酶氮片段PxAPN1,该片段全长192bp,共编码64个氨基酸；碱性磷酸酶片段PxALPO,该片段全长967bp,共编码322个氨基酸。使用致死中浓度剂量的CrylAc及中高浓度的PxCADa、PxAPN1、PxALP1、PxCADb与PxCADc片段对小菜蛾幼虫进行体外复配生测,1.87μg/mL的Cry1Ac可引起小菜蛾三龄幼虫46.7%的死亡率,当加入终浓度为551.25 μg/mL的GST-PxCADb时,其死亡率为45%；当加入终浓度为204.67 μg/mL的GST-PxCADc时,其死亡率为48.9%,当加入终浓度为187.33 μg/mL的GST-PxAPN1时,其死亡率为45.6%；而对照组加入终浓度为300.27μg/mL的pGEX-6p-l时,小菜蛾三龄幼虫的死亡率为45.6%,处理(GST-PxCADb、GST-PxCADc及GST-PxAPN1a)与对照(CrylAc及pGEX-6p-1)之间并无显著差异,表明较高浓度的PxCADb、PxCADc及PxAPN1均不能增强CrylAc蛋白的杀虫活性。但是当加入终浓度为207.58 μg/mL的GST-PxCADa时,其死亡率为85.6%；当加入终浓度为290.25 μg/mL的GST-PxALP1时,其死亡率为70.0%,而对照组加入终浓度为300.27μg/mL的pGEX-6p-1时,其死亡率为45.6%,处理(GST-PxCADa及GST-PxALPl)与对照(CrylAc及pGEX-6p-1)之间有显著差异,表明中高浓度的PxCADa和PxALP1能显著增强CrylAc蛋白的杀虫活性。结果表明,PxCADb、PxCADc和PxAPN1a多肽均不能增强或抑制CrylAc蛋白的杀虫活性。PxCADa和PxALP1片段可以增强CrylAc蛋白的杀虫活性,且效果显著。研究结果将为筛选有效的小菜蛾增效片段及小菜蛾毒素结合区多肽在高效杀虫Bt工程菌和转基因植物中的应用提供理论依据,对于揭示Bt杀虫蛋白的毒理机制和害虫对Bt杀虫蛋白的抗性机制具有重要意义。
[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.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.4

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