飞蝗几丁质脱乙酰基酶基因的异源表达及其在后肠中的功能研究

发布时间：2019-02-20 20:47

【摘要】：飞蝗是我国重要农业害虫,目前对其防治主要依靠化学农药,但不合理的化学防治不仅导致环境的污染,而且导致飞蝗抗药性的产生和对非靶标生物的危害。因此,探索新型、环境友好型飞蝗防治方法日趋重要。昆虫的发育会发生周期性去除旧表皮的几丁质代谢过程。几丁质是昆虫表皮、消化道及体内组织所特有的组成成分。在几丁质降解过程中几丁质脱乙酰基酶(CDAs)起重要作用,可以催化几丁质中由β-1,4糖苷键连接的N-乙酰基葡糖胺的乙酰胺基脱去乙酰基,形成脱乙酰几丁质(壳聚糖)。因此,基于昆虫几丁质降解途径研发环境友好型杀虫剂备受关注。本研究以飞蝗LmCDAs基因为研究对象,采用真核表达系统,通过体外表达目的基因、纯化目的蛋白,并对其酶活力进行检测,从而探索该蛋白基因在体外的脱乙酰功能;其次,本研究还对该基因进行了原核表达和亲和纯化,并制备其特异性的多克隆抗体,为探索该基因在飞蝗后肠的功能研究提供了基础材料;最后,基于课题组前期的研究基础,我们使用制备成功的特异性多克隆抗体探索LmCDA1和LmCDA2基因对飞蝗后肠发育的影响。该研究为探讨基因在几丁质代谢过程中的功能特性提供了重要的基础数据,并为筛选杀虫剂新靶标,研发环境友好型杀虫剂以及以农业害虫飞蝗的几丁质作为底物的壳聚糖生产方法提供理论依据。本文主要从以下三个内容进行研究:一、飞蝗LmCDA1和LmCDA2的真核表达、亲和纯化及酶活性研究采用PCR法扩增飞蝗LmCDA1、LmCDA2a和LmCDA2b基因,纯化回收后琼脂糖凝胶电泳检测回收产物。选择pFastBac?-Dual作为中间载体,将中间载体和回收产物双酶切进行重组质粒的构建。随后进行重组杆状病毒的构建。将构建好的重组杆状病毒转染Sf9细胞,获得重组LmCDAs蛋白,通过western blot和12%SDS-PAGE胶上电泳等方法验证、纯化重组LmCDAs蛋白,并进行初步的酶活力测定。结论:飞蝗几丁质脱乙酰基酶LmCDA1、LmCDA2a和LmCDA2b在体外均具有脱乙酰功能,并且统计分析结果显示三者在酶活力上表现显著性差异。二、飞蝗LmCDA1和LmCDA2的原核表达、纯化及抗体制备与验证设计引物进行目的基因的克隆和纯化,选取pET32a作为中间载体构建重组质粒pET32a-LmCDA1和pET32a-LmCDA2,将构建成功的重组质粒转入大肠杆菌中进行目的蛋白的诱导表达,采用Western blot技术对表达产物进行检测;将成功表达的蛋白使用Ni-NTA亲和层析柱进行目的蛋白的纯化,纯化组分采用12%SDS-PAGE方法检测其纯化结果,然后测定纯化后的蛋白浓度,将浓度达到10mg的纯化蛋白送至生物公司进行抗体制备。抗体合成后,使用Western blot技术进行抗体的验证。结论:重组载体质粒pET32a-LmCDA1和pET32a-Lm CDA2可在大肠杆菌中经过100mM IPTG诱导剂,37℃诱导4h获得目的蛋白的表达,并且可借助大肠杆菌原核表达系统获得大量目的蛋白以达到抗体制备所需的浓度和纯度。三、LmCDA1和LmCDA2基因在飞蝗后肠中的组织定位和功能分析通过免疫组化技术检测LmCDAs基因在飞蝗后肠中的组织定位。通过RNAi干扰与透射电镜技术相结合的技术手段,研究LmCDAs基因对飞蝗后肠发育的影响。结论:免疫组化检测结果显示在五龄第8天飞蝗后肠中,LmCDA1和LmCDA2均定位在细胞质中。对五龄第8天飞蝗后肠中的超微结构进行观察,结果表明LmCDA1和LmCDA2基因均对飞蝗后肠几丁质片层结构的排布起关键作用。
[Abstract]:The locust is an important agricultural pest in China, and its prevention and control mainly depends on the chemical pesticide, but the unreasonable chemical control not only leads to the pollution of the environment, but also leads to the production of the drug resistance of the locust and the harm to the non-target organism. Therefore, it is becoming more and more important to explore new and environment-friendly locust control methods. the development of the insect can periodically remove the chitin metabolism process of the old cuticle. Chitin is an essential component of the insect's epidermis, digestive tract and in-vivo tissue. In the process of chitin degradation, the chitin deacetaminidase (CDAs) plays an important role, and can be used for catalyzing the deethanolinyl of N-ethanone-based glucamine which is linked by the HCO3-1 and the 4-sugar linkage in the chitin to form the deethanone chitin (chitosan). Therefore, the development of an environment-friendly insecticide based on the insect chitin degradation pathway is of great concern. By using the LmCDAs gene as a research object, a true nuclear expression system is adopted, and the target protein is purified through the in vitro expression target gene, and the enzyme activity of the target protein is detected, so that the deethanizing function of the protein gene in the body is explored; and secondly, The research also carried out the prokaryotic expression and affinity purification of the gene, and prepared the specific polyclonal antibody, and provided a basic material for exploring the functional research of the gene after the migratory locust; and finally, based on the research basis of the early stage of the research group, We investigated the effects of the LmCDA1 and LmCDA2 genes on the intestinal development of the migratory locust by using the successful specific polyclonal antibody. In order to study the functional characteristics of the gene in the process of chitin metabolism, this paper provides a theoretical basis for the selection of new targets for pesticides, the development of environment-friendly insecticides, and the chitosan production method with the chitin as a substrate. The expression of LmCDA1, LmCDA2a and LmCDA2b was amplified by PCR, and the recovered products were detected by agarose gel electrophoresis. pFastBac?-Dual was used as the intermediate vector to construct the recombinant plasmid by double-enzyme digestion of the intermediate vector and the recovery product. and then the construction of the recombinant baculovirus is carried out. The constructed recombinant baculovirus was transfected into Sf9 cells to obtain the recombinant LmCDAs protein, and the recombinant LmCDAs protein was purified by Western blot and 12% SDS-PAGE gel electrophoresis. The recombinant LmCDAs protein was purified and the initial enzyme activity was determined. Conclusion: LmCDA1, LmCDA2a and LmCDA2b of the chitin deethanone of the migratory locust have the function of deethylation in vitro, and the results of the statistical analysis show that there is a significant difference in the activity of the enzyme. 2, cloning and purifying the prokaryotic expression, purification and antibody preparation and verification design primers of the LmCDA1 and LmCDA2 of the migratory locust, and selecting the pET32a as an intermediate vector to construct the recombinant plasmid pET32a-LmCDA1 and the pET32a-LmCDA2, and transferring the constructed recombinant plasmid into the Escherichia coli for inducing expression of the target protein, the expression product is detected by Western blot; the protein is purified by using a Ni-NTA affinity chromatography column; the purified component is used for detecting the purification result by adopting a 12% SDS-PAGE method, and then the purified protein concentration is measured, The purified protein with a concentration of 10 mg was sent to a biological company for antibody preparation. After the antibody was synthesized, the antibody was verified by Western blot. Conclusion: The recombinant vector pET 32a-LmCDA1 and pET32a-Lm CDA2 can be induced by 100 mM IPTG in E. coli, and the expression of the target protein can be obtained at 37 鈩，

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