甘蓝夜蛾β-N-乙酰葡萄糖胺糖苷酶cDNA的克隆及功能研究
发布时间：2018-12-13 17:35
【摘要】：β-N-乙酰葡萄糖胺糖苷酶是降解昆虫体内几丁质的关键酶之一,具有调控昆虫发育、变态和生殖等功能,是一种重要的糖苷酶。几丁质被分解时,首先几丁质酶降解几丁质成小分子的寡聚物,之后被β-N-乙酰葡萄糖胺糖苷酶降解为单分子的N-乙酰氨基葡萄糖。本研究克隆甘蓝夜蛾β-N-乙酰葡萄糖胺糖苷酶全长c DNA序列,对基因推导的氨基酸序列进行分析,构建了进化树,并进一步在大肠杆菌中成功表达了该序列的蛋白,进行纯化和复性,获得了有活性的目的蛋白,为β-N-乙酰葡萄糖胺糖苷酶蛋白今后的研究奠定了基础。针对基因的时空表达进行研究,同时,体外注射蜕皮激素和ds RNA对β-N-乙酰葡萄糖胺糖苷酶基因进行功能的研究。本试验扩增得到甘蓝夜蛾β-N-乙酰葡萄糖胺糖苷酶基因的全长c DNA序列,命名为Mb NAG(Gen Bank登录号:KP730442)。该基因含有2445 bp,包括一个开放读码框大小为1782bp,编码一个多肽含有594个氨基酸,其相对分子量大小约为67.8 k Da,等电点为5.11。该序列含有G20家族保守活性位点HMGGDEVSERCW,判定该基因属于GH20家族中的一员。推导得到的β-N-乙酰葡萄糖胺糖苷酶基因含有三个N-位糖基化位点。与甘蓝夜蛾与八字地老虎和小地老虎的一致性达到85%和84%,综上所述,本试验克隆的基因是甘蓝夜蛾的一条新的β-N-乙酰葡萄糖胺糖苷酶基因。原核表达成功表达外源蛋白,分子量与预测融合蛋白分子量相符,表达的蛋白为包涵体蛋白,蛋白经过充分洗涤、纯化、复性获得纯的目的蛋白,蛋白经SDS-PAGE和Western blotting检测结果表明该蛋白为目的蛋白。复性及活性检测结果表明,随着p H的升高,重组蛋白活性先升高后降低,p H=7时活性最高为39.33 U/m L;随着温度的升高,蛋白的活性先升高后降低,在温度为34℃时活性最高为31.33 U/m L。利用荧光定量PCR技术检测基因表达量的变化,结果表明,该基因在甘蓝夜蛾的不同发育阶段和不同组织中都有mRNA水平的特异性表达,1-6龄虫体基因的表达量逐渐升高,预蛹期表达量最大,蛹期又降低。在围食膜中该基因的表达最高,是前肠的54倍。Real-time PCR分析激素处理后Mb NAG基因表达量的变化。结果表明,浓度为2μg/μl,基因的表达变化不明显,与对照趋势相同;浓度为6μg/μl和18μg/μl时基因表达量在48 h达到最大,分别是对照的3.92和6.56倍;表明该基因的表达受蜕皮激素影响,且浓度为18μg/μl时变化明显。RNAi结果表明,注射48 h后,处理组虫体中Mb NAG基因表达量下降60%;注射72 h后,处理组下降到67%。同时,试验组虫体出现不能正常蜕皮、羽化和死亡现象,到完全羽化为成虫死亡率达到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，
本文编号：2376941
[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，
本文编号：2376941
本文链接：https://www.wllwen.com/kejilunwen/nykj/2376941.html
