甜菜夜蛾几丁质脱乙酰酶1（CDA1）的鉴定与分析

发布时间：2018-05-26 23:09

本文选题：甜菜夜蛾 + 几丁质脱乙酰酶　；参考：《河北农业大学》2015年硕士论文

【摘要】：甜菜夜蛾(Spodoptera exigua Hübner),属鳞翅目夜蛾科,是一种世界性分布的害虫。其取食范围非常广泛,对多种粮食作物、蔬菜、经济作物和油料作物等造成严重危害。昆虫几丁质脱乙酰酶(Chitin deacetylase,CDA)是几丁质的修饰酶之一,可水解几丁质中的乙酰胺基,将几丁质降解成脱乙酰几丁质,是昆虫几丁质代谢中的一种关键酶,在昆虫的多种生理活动中发挥重要作用。本研究以甜菜夜蛾S.exigua 5龄幼虫中肠为材料提取总RNA,利用RT-PCR及RACE技术,扩增得到甜菜夜蛾几丁质脱乙酰酶1(chitin deacetylase 1)基因的c DNA全长序列,Gen Bank登录号为KJ621414。该基因全长1659bp,包括一个1554bp的开放阅读框,编码518个氨基酸,N-端具有23个氨基酸的信号肽序列,预测蛋白(Se CDA1)分子量为61.5k Da,等电点为4.83;NCBI BLAST分析结果表明,Se CDA1含有低密度脂蛋白结合区(LDLa)、几丁质结合区(Ch BD)和脱乙酰基酶催化区(CDA),属于GroupⅠ类CDA蛋白。氨基酸序列分析发现,该序列分别含有3个N-联糖基化位点和5个O-联糖基化位点。secda1基因能够在原核细胞中表达61.5k Da目的蛋白,免疫家兔获得Se CDA1蛋白的特异性抗体。secda1基因在毕赤酵母细胞中表达80k Da的目的蛋白。构建重组杆状病毒表达载体p Fast Bac-secda1,Western blot分析表明secda1基因在昆虫细胞BTI-Tn-5B1-4(High Five)中成功表达80k Da蛋白。脱乙酰基酶活性测定结果显示昆虫细胞表达的Se CDA1蛋白及毕赤酵母表达的Se CDA1蛋白均具有脱乙酰基酶活性,分别为1.88U/m L及1.35U/m L。利用Se CDA1特异性抗体对Se CDA1进行免疫组织定位显示,Se CDA1蛋白在卵、幼虫和蛹期中均有表达。q PCR结果显示secda1基因在卵、幼虫和蛹期中均有表达,而在卵中表达量最高,在5龄幼虫头、中肠、马氏管、脂肪体中也有表达,且在体壁中表达水平最高,推测Se CDA1可能参与卵的孵化及幼虫蜕皮过程。为进一步验证Se CDA1蛋白功能,利用RNA干扰(RNAi)技术将secda1 536bp保守序列克隆至p GEM-T载体,成功构建了RNAi重组质粒,大量提取纯化重组质粒,经线性化后合成ds RNA-secda1,为下一步功能研究提供了材料。
[Abstract]:Spodoptera exigua H 眉 bneridae, belonging to the family Lepidoptera, is a worldwide pest. Its feeding range is very extensive, which causes serious harm to many kinds of food crops, vegetables, cash crops and oil crops. Chitin deacetylase (Chitin deacetylase CDA) is one of the modification enzymes of chitin, which can hydrolyze acetamide group in chitin and deacetylate into deacetylated chitin, which is a key enzyme in the metabolism of insect chitin. It plays an important role in many physiological activities of insects. In this study, total RNAs were extracted from the midgut of 5th instar larvae of Spodoptera exigua (S.exigua). By using RT-PCR and RACE techniques, the full-length sequence of c DNA 1(chitin deacetylase 1) gene of Spodoptera exigua was amplified and its accession number was KJ621414. The gene is 1659bp in length and contains an open reading frame of 1554bp encoding 518 amino acids with a signal peptide sequence of 23 amino acids. The molecular weight of predicted protein se CDA1 was 61.5kDa.The isoelectric point was 4.83NCBI BLAST. The results showed that se CDA1 contained low density lipoprotein binding region (LDLaN), chitin binding region (CHBDD) and deacetylase catalyzed region (CDAN), which belonged to Group class I CDA protein. Amino acid sequence analysis showed that the sequence contained three N-glycosylation sites and five O-glycosylation sites. Secda1 gene was able to express 61.5 kDa target protein in prokaryotic cells. The specific antibody of se CDA1 protein. Secda1 gene was expressed in Pichia pastoris cells by immunizing rabbits with 80 kDa target protein. Western blot analysis of recombinant baculovirus expression vector p Fast Bac-secda1 showed that the secda1 gene was successfully expressed in insect cell line BTI-Tn-5B1-4(High. The results of deacetylase activity test showed that se CDA1 protein expressed by insect cells and se CDA1 protein expressed by Pichia pastoris had deacetylase activity, which were 1.88U/m L and 1.35U/m L, respectively. Immunohistochemical localization of se CDA1 with se CDA1 specific antibody showed that se CDA1 protein was expressed in eggs, and in larval and pupa stage. Q PCR showed that secda1 gene was expressed in eggs, larvae and pupal stages, but the highest expression level was found in eggs. The expression of se CDA1 was also found in the head, midgut, Markov tube and adipose body of the 5th instar larva, and the highest expression level was found in the body wall. It was suggested that se CDA1 might be involved in the hatching of eggs and the molting process of larvae. In order to further verify the function of se CDA1 protein, the conserved sequence of secda1 536bp was cloned into p GEM-T vector by RNA interference RNAi technique. The recombinant plasmid of RNAi was successfully constructed, and a large number of purified recombinant plasmids were extracted and purified. After linearization, DS RNA-secda1 was synthesized, which provided materials for further functional research.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.4

【共引文献】