飛蝗幾丁質(zhì)脫乙�；富虻漠愒幢磉_(dá)及其在后腸中的功能研究
發(fā)布時(shí)間：2019-02-20 20:47
【摘要】：飛蝗是我國(guó)重要農(nóng)業(yè)害蟲,目前對(duì)其防治主要依靠化學(xué)農(nóng)藥,但不合理的化學(xué)防治不僅導(dǎo)致環(huán)境的污染,而且導(dǎo)致飛蝗抗藥性的產(chǎn)生和對(duì)非靶標(biāo)生物的危害。因此,探索新型、環(huán)境友好型飛蝗防治方法日趨重要。昆蟲的發(fā)育會(huì)發(fā)生周期性去除舊表皮的幾丁質(zhì)代謝過(guò)程。幾丁質(zhì)是昆蟲表皮、消化道及體內(nèi)組織所特有的組成成分。在幾丁質(zhì)降解過(guò)程中幾丁質(zhì)脫乙�；�(CDAs)起重要作用,可以催化幾丁質(zhì)中由β-1,4糖苷鍵連接的N-乙�；咸前返囊阴０坊撊ヒ阴；�,形成脫乙酰幾丁質(zhì)(殼聚糖)。因此,基于昆蟲幾丁質(zhì)降解途徑研發(fā)環(huán)境友好型殺蟲劑備受關(guān)注。本研究以飛蝗LmCDAs基因?yàn)檠芯繉?duì)象,采用真核表達(dá)系統(tǒng),通過(guò)體外表達(dá)目的基因、純化目的蛋白,并對(duì)其酶活力進(jìn)行檢測(cè),從而探索該蛋白基因在體外的脫乙酰功能;其次,本研究還對(duì)該基因進(jìn)行了原核表達(dá)和親和純化,并制備其特異性的多克隆抗體,為探索該基因在飛蝗后腸的功能研究提供了基礎(chǔ)材料;最后,基于課題組前期的研究基礎(chǔ),我們使用制備成功的特異性多克隆抗體探索LmCDA1和LmCDA2基因?qū)︼w蝗后腸發(fā)育的影響。該研究為探討基因在幾丁質(zhì)代謝過(guò)程中的功能特性提供了重要的基礎(chǔ)數(shù)據(jù),并為篩選殺蟲劑新靶標(biāo),研發(fā)環(huán)境友好型殺蟲劑以及以農(nóng)業(yè)害蟲飛蝗的幾丁質(zhì)作為底物的殼聚糖生產(chǎn)方法提供理論依據(jù)。本文主要從以下三個(gè)內(nèi)容進(jìn)行研究:一、飛蝗LmCDA1和LmCDA2的真核表達(dá)、親和純化及酶活性研究采用PCR法擴(kuò)增飛蝗LmCDA1、LmCDA2a和LmCDA2b基因,純化回收后瓊脂糖凝膠電泳檢測(cè)回收產(chǎn)物。選擇pFastBac?-Dual作為中間載體,將中間載體和回收產(chǎn)物雙酶切進(jìn)行重組質(zhì)粒的構(gòu)建。隨后進(jìn)行重組桿狀病毒的構(gòu)建。將構(gòu)建好的重組桿狀病毒轉(zhuǎn)染Sf9細(xì)胞,獲得重組LmCDAs蛋白,通過(guò)western blot和12%SDS-PAGE膠上電泳等方法驗(yàn)證、純化重組LmCDAs蛋白,并進(jìn)行初步的酶活力測(cè)定。結(jié)論:飛蝗幾丁質(zhì)脫乙酰基酶LmCDA1、LmCDA2a和LmCDA2b在體外均具有脫乙酰功能,并且統(tǒng)計(jì)分析結(jié)果顯示三者在酶活力上表現(xiàn)顯著性差異。二、飛蝗LmCDA1和LmCDA2的原核表達(dá)、純化及抗體制備與驗(yàn)證設(shè)計(jì)引物進(jìn)行目的基因的克隆和純化,選取pET32a作為中間載體構(gòu)建重組質(zhì)粒pET32a-LmCDA1和pET32a-LmCDA2,將構(gòu)建成功的重組質(zhì)粒轉(zhuǎn)入大腸桿菌中進(jìn)行目的蛋白的誘導(dǎo)表達(dá),采用Western blot技術(shù)對(duì)表達(dá)產(chǎn)物進(jìn)行檢測(cè);將成功表達(dá)的蛋白使用Ni-NTA親和層析柱進(jìn)行目的蛋白的純化,純化組分采用12%SDS-PAGE方法檢測(cè)其純化結(jié)果,然后測(cè)定純化后的蛋白濃度,將濃度達(dá)到10mg的純化蛋白送至生物公司進(jìn)行抗體制備。抗體合成后,使用Western blot技術(shù)進(jìn)行抗體的驗(yàn)證。結(jié)論:重組載體質(zhì)粒pET32a-LmCDA1和pET32a-Lm CDA2可在大腸桿菌中經(jīng)過(guò)100mM IPTG誘導(dǎo)劑,37℃誘導(dǎo)4h獲得目的蛋白的表達(dá),并且可借助大腸桿菌原核表達(dá)系統(tǒng)獲得大量目的蛋白以達(dá)到抗體制備所需的濃度和純度。三、LmCDA1和LmCDA2基因在飛蝗后腸中的組織定位和功能分析通過(guò)免疫組化技術(shù)檢測(cè)LmCDAs基因在飛蝗后腸中的組織定位。通過(guò)RNAi干擾與透射電鏡技術(shù)相結(jié)合的技術(shù)手段,研究LmCDAs基因?qū)︼w蝗后腸發(fā)育的影響。結(jié)論:免疫組化檢測(cè)結(jié)果顯示在五齡第8天飛蝗后腸中,LmCDA1和LmCDA2均定位在細(xì)胞質(zhì)中。對(duì)五齡第8天飛蝗后腸中的超微結(jié)構(gòu)進(jìn)行觀察,結(jié)果表明LmCDA1和LmCDA2基因均對(duì)飛蝗后腸幾丁質(zhì)片層結(jié)構(gòu)的排布起關(guān)鍵作用。
[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，
本文編號(hào)：2427250
[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，
本文編號(hào)：2427250
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