二化螟田间种群酯酶CsCaE026的表达检测
发布时间：2019-06-25 15:21
【摘要】：二化螟是我国水稻上重要的钻蛀性害虫之一,可造成枯心和白穗等为害症状,进而导致严重的产量与经济损失。二化螟体内具有丰富的酯酶同工酶,并与其杀虫剂抗性密切相关。发掘二化螟酯酶基因资源,可以为害虫田间抗性监测与治理、新型高效“环境友好型”药剂创制与开发以及农药残留环境监测和控制提供基础。本论文就二化螟酯酶基因CsCaE026进行克隆和表达研究,获得以下主要结果： 1二化螟酯酶基因CsCaE026的克隆和序列分析 克隆获得二化螟酯酶基因CsCaE026的全长cDNA序列,其开放阅读框为1,671bp,编码557个氨基酸,预测分子量63.66kD,等电点为5.86。预测编码蛋白N-端前16个氨基酸为信号肽序列,且含有α/β-酯酶家族特征基序Gly-x-Ser-x-Gly和催化三聚体(Ser207、Glu339和His453)等保守结构域以及二硫键结合位点(Cys82和Cys103)等。CsCaE026基因属α-酯酶亚家族。 2二化螟酯酶基因CsCaE026的原核表达分析 构建获得CsCaE026基因重组表达载体,并在大肠杆菌Rosetta菌株中诱导表达。重组表达产物分子量为70kD,且形成包涵体,未能检测到酯酶活性。提取、纯化重组表达蛋白,并制备获得多克隆抗体。Western杂交结果证实,二化螟CsCaE026基因在大肠杆菌中成功表达。 3二化螟酯酶基因CsCaE026的时空表达特征分析 分别采用实时荧光定量PCR技术和蛋白Western杂交技术,分析二化螟CsCaE026基因在转录与翻译水平的时空表达分布。明确CsCaE026基因在二化螟幼虫的中肠中高水平特异性表达。 4二化螟硝酶基因CsCaE026的真核表达分析 构建获得CsCaE026基因重组杆状病毒,并在昆虫Sf9细胞中进行表达。重组表达产物分子量为90kD。Western杂交结果证实,二化螟CsCaE026基因在昆虫细胞中成表达。
[Abstract]:Chilo suppressalis (Chilo suppressalis) is one of the important borer pests in rice in China, which can cause bad symptoms such as withered heart and white panicle, and then lead to serious yield and economic losses. There are abundant esterase isozymes in Chilo suppressalis, which are closely related to their insecticide resistance. Exploring the esterase gene resources of Chilo suppressalis can provide a basis for the field resistance monitoring and control of pests, the creation and development of new and efficient "environmentally friendly" agents, and the environmental monitoring and control of pesticide residues. In this paper, the esterase gene CsCaE026 of Chilo suppressalis was cloned and expressed. The main results were as follows: (1) the full-length cDNA sequence of the esterase gene CsCaE026 of Chilo suppressalis was obtained by cloning and sequence analysis. The open reading frame was 1671 BP, encoding 557 amino acids, predicting molecular weight 63.66 KD and isoelectric point 5.86. It is predicted that the first 16 amino acids at the N-terminal of the coding protein are signal peptide sequences, and contain conserved domains such as 伪 / 尾-esterase family characteristic motifs Gly-x-Ser-x-Gly and catalytic trimers (Ser207,Glu339 and His453), as well as disulfide binding sites (Cys82 and Cys103). CsCaE026 gene belongs to 伪-esterase subfamily. (2) the recombinant expression vector of CsCaE026 gene was constructed by prokaryotic expression analysis of esterase gene CsCaE026 of Chilo suppressalis, and was induced to express in E. coli Rosetta strain. The molecular weight of the recombinant expression product was 70 KD, and the inclusion body was formed, and the esterase activity could not be detected. The recombinant expression protein was extracted and purified, and the polyclonal antibody was prepared. Western blot analysis confirmed that the CsCaE026 gene of Chilo suppressalis was successfully expressed in E. coli. 3The temporal and spatial expression characteristics of esterase gene CsCaE026 in Chilo suppressalis were analyzed by real-time fluorescence quantitative PCR and protein Western hybridization, respectively. The temporal and spatial expression and distribution of CsCaE026 gene in Chilo suppressalis at transcriptional and translation levels were analyzed. To determine the high level specific expression of CsCaE026 gene in the midgut of Chilo suppressalis larvae. (4) the recombinant baculovirus of CsCaE026 gene was constructed by eukaryotic expression analysis of nitrogenase gene CsCaE026 of Chilo suppressalis and expressed in insect Sf9 cells. The molecular weight of the recombinant expression product was 90kD.Western. The results showed that the CsCaE026 gene of Chilo suppressalis was expressed in insect cells.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433
本文编号：2505788
[Abstract]:Chilo suppressalis (Chilo suppressalis) is one of the important borer pests in rice in China, which can cause bad symptoms such as withered heart and white panicle, and then lead to serious yield and economic losses. There are abundant esterase isozymes in Chilo suppressalis, which are closely related to their insecticide resistance. Exploring the esterase gene resources of Chilo suppressalis can provide a basis for the field resistance monitoring and control of pests, the creation and development of new and efficient "environmentally friendly" agents, and the environmental monitoring and control of pesticide residues. In this paper, the esterase gene CsCaE026 of Chilo suppressalis was cloned and expressed. The main results were as follows: (1) the full-length cDNA sequence of the esterase gene CsCaE026 of Chilo suppressalis was obtained by cloning and sequence analysis. The open reading frame was 1671 BP, encoding 557 amino acids, predicting molecular weight 63.66 KD and isoelectric point 5.86. It is predicted that the first 16 amino acids at the N-terminal of the coding protein are signal peptide sequences, and contain conserved domains such as 伪 / 尾-esterase family characteristic motifs Gly-x-Ser-x-Gly and catalytic trimers (Ser207,Glu339 and His453), as well as disulfide binding sites (Cys82 and Cys103). CsCaE026 gene belongs to 伪-esterase subfamily. (2) the recombinant expression vector of CsCaE026 gene was constructed by prokaryotic expression analysis of esterase gene CsCaE026 of Chilo suppressalis, and was induced to express in E. coli Rosetta strain. The molecular weight of the recombinant expression product was 70 KD, and the inclusion body was formed, and the esterase activity could not be detected. The recombinant expression protein was extracted and purified, and the polyclonal antibody was prepared. Western blot analysis confirmed that the CsCaE026 gene of Chilo suppressalis was successfully expressed in E. coli. 3The temporal and spatial expression characteristics of esterase gene CsCaE026 in Chilo suppressalis were analyzed by real-time fluorescence quantitative PCR and protein Western hybridization, respectively. The temporal and spatial expression and distribution of CsCaE026 gene in Chilo suppressalis at transcriptional and translation levels were analyzed. To determine the high level specific expression of CsCaE026 gene in the midgut of Chilo suppressalis larvae. (4) the recombinant baculovirus of CsCaE026 gene was constructed by eukaryotic expression analysis of nitrogenase gene CsCaE026 of Chilo suppressalis and expressed in insect Sf9 cells. The molecular weight of the recombinant expression product was 90kD.Western. The results showed that the CsCaE026 gene of Chilo suppressalis was expressed in insect cells.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433
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