【摘要】：β-N-乙酰葡萄糖胺糖苷酶是降解昆蟲體內(nèi)幾丁質(zhì)的關(guān)鍵酶之一,具有調(diào)控昆蟲發(fā)育、變態(tài)和生殖等功能,是一種重要的糖苷酶。幾丁質(zhì)被分解時,首先幾丁質(zhì)酶降解幾丁質(zhì)成小分子的寡聚物,之后被β-N-乙酰葡萄糖胺糖苷酶降解為單分子的N-乙酰氨基葡萄糖。本研究克隆甘藍夜蛾β-N-乙酰葡萄糖胺糖苷酶全長c DNA序列,對基因推導(dǎo)的氨基酸序列進行分析,構(gòu)建了進化樹,并進一步在大腸桿菌中成功表達了該序列的蛋白,進行純化和復(fù)性,獲得了有活性的目的蛋白,為β-N-乙酰葡萄糖胺糖苷酶蛋白今后的研究奠定了基礎(chǔ)。針對基因的時空表達進行研究,同時,體外注射蛻皮激素和ds RNA對β-N-乙酰葡萄糖胺糖苷酶基因進行功能的研究。本試驗擴增得到甘藍夜蛾β-N-乙酰葡萄糖胺糖苷酶基因的全長c DNA序列,命名為Mb NAG(Gen Bank登錄號:KP730442)。該基因含有2445 bp,包括一個開放讀碼框大小為1782bp,編碼一個多肽含有594個氨基酸,其相對分子量大小約為67.8 k Da,等電點為5.11。該序列含有G20家族保守活性位點HMGGDEVSERCW,判定該基因?qū)儆贕H20家族中的一員。推導(dǎo)得到的β-N-乙酰葡萄糖胺糖苷酶基因含有三個N-位糖基化位點。與甘藍夜蛾與八字地老虎和小地老虎的一致性達到85%和84%,綜上所述,本試驗克隆的基因是甘藍夜蛾的一條新的β-N-乙酰葡萄糖胺糖苷酶基因。原核表達成功表達外源蛋白,分子量與預(yù)測融合蛋白分子量相符,表達的蛋白為包涵體蛋白,蛋白經(jīng)過充分洗滌、純化、復(fù)性獲得純的目的蛋白,蛋白經(jīng)SDS-PAGE和Western blotting檢測結(jié)果表明該蛋白為目的蛋白。復(fù)性及活性檢測結(jié)果表明,隨著p H的升高,重組蛋白活性先升高后降低,p H=7時活性最高為39.33 U/m L;隨著溫度的升高,蛋白的活性先升高后降低,在溫度為34℃時活性最高為31.33 U/m L。利用熒光定量PCR技術(shù)檢測基因表達量的變化,結(jié)果表明,該基因在甘藍夜蛾的不同發(fā)育階段和不同組織中都有mRNA水平的特異性表達,1-6齡蟲體基因的表達量逐漸升高,預(yù)蛹期表達量最大,蛹期又降低。在圍食膜中該基因的表達最高,是前腸的54倍。Real-time PCR分析激素處理后Mb NAG基因表達量的變化。結(jié)果表明,濃度為2μg/μl,基因的表達變化不明顯,與對照趨勢相同;濃度為6μg/μl和18μg/μl時基因表達量在48 h達到最大,分別是對照的3.92和6.56倍;表明該基因的表達受蛻皮激素影響,且濃度為18μg/μl時變化明顯。RNAi結(jié)果表明,注射48 h后,處理組蟲體中Mb NAG基因表達量下降60%;注射72 h后,處理組下降到67%。同時,試驗組蟲體出現(xiàn)不能正常蛻皮、羽化和死亡現(xiàn)象,到完全羽化為成蟲死亡率達到52%。
[Abstract]:尾 -N-acetylglucosaminidase is one of the key enzymes for the degradation of chitinase in insects. It has the functions of regulating insect development, metamorphosis and reproduction, and is an important glucosidase. When chitin is decomposed, first chitinase degrades chitinase into a small molecule oligomer, then 尾 -N-acetylglucosaminidase degrades to monolayer N-acetylglucosamine glucosamine. In this study, the full-length c DNA sequence of Spodoptera exigua 尾 -N-acetylglucosaminidase was cloned, the deduced amino acid sequence was analyzed, the evolutionary tree was constructed, and the protein was successfully expressed in Escherichia coli. After purification and renaturation, the active target protein was obtained, which laid a foundation for the future study of 尾 -N-acetylglucosaminidase protein. At the same time, the function of 尾 -N-acetylglucosaminidase gene was studied by injection of ecdysone and ds RNA in vitro. In this experiment, the full-length c DNA sequence of 尾 -N-acetylglucosaminidase gene was amplified and named Mb NAG (Gen Bank accession number: KP730442). The gene contains 2445 bp, including an open reading frame size of 1782 BP, a polypeptide containing 594 amino acids, and a relative molecular weight of about 67.8 k Da, isoelectric point (5.11). The sequence contains HMGGDEVSERCW, a conserved active locus of the G20 family, which identified the gene as a member of the GH20 family. The 尾-N-acetyl glucosaminidase gene contains three N-site glycosylation sites. The concordance was 85% and 84% with Spodoptera spp. It was concluded that the gene cloned in this study was a new 尾 -N-acetylglucosaminidase gene of Spodoptera brassica. The prokaryotic expression of the foreign protein was successful. The molecular weight of the fusion protein was consistent with that of the predicted fusion protein. The expressed protein was an inclusion body protein. The protein was washed, purified, and renatured to obtain a pure target protein. The results of SDS-PAGE and Western blotting showed that the protein was the target protein. The results of renaturation and activity detection showed that with the increase of pH, the activity of recombinant protein first increased and then decreased, the highest activity of pH = 7 was 39.33 U / mL; With the increase of temperature, the activity of protein first increased and then decreased, and the highest activity was 31.33 U / mL at 34 鈩，

本文編號：2376941