【摘要】：二化螟是我國(guó)水稻上重要的鉆蛀性害蟲之一,可造成枯心和白穗等為害癥狀,進(jìn)而導(dǎo)致嚴(yán)重的產(chǎn)量與經(jīng)濟(jì)損失。二化螟體內(nèi)具有豐富的酯酶同工酶,并與其殺蟲劑抗性密切相關(guān)。發(fā)掘二化螟酯酶基因資源,可以為害蟲田間抗性監(jiān)測(cè)與治理、新型高效“環(huán)境友好型”藥劑創(chuàng)制與開發(fā)以及農(nóng)藥殘留環(huán)境監(jiān)測(cè)和控制提供基礎(chǔ)。本論文就二化螟酯酶基因CsCaE026進(jìn)行克隆和表達(dá)研究,獲得以下主要結(jié)果： 1二化螟酯酶基因CsCaE026的克隆和序列分析 克隆獲得二化螟酯酶基因CsCaE026的全長(zhǎng)cDNA序列,其開放閱讀框?yàn)?,671bp,編碼557個(gè)氨基酸,預(yù)測(cè)分子量63.66kD,等電點(diǎn)為5.86。預(yù)測(cè)編碼蛋白N-端前16個(gè)氨基酸為信號(hào)肽序列,且含有α/β-酯酶家族特征基序Gly-x-Ser-x-Gly和催化三聚體(Ser207、Glu339和His453)等保守結(jié)構(gòu)域以及二硫鍵結(jié)合位點(diǎn)(Cys82和Cys103)等。CsCaE026基因?qū)佴?酯酶亞家族。 2二化螟酯酶基因CsCaE026的原核表達(dá)分析 構(gòu)建獲得CsCaE026基因重組表達(dá)載體,并在大腸桿菌Rosetta菌株中誘導(dǎo)表達(dá)。重組表達(dá)產(chǎn)物分子量為70kD,且形成包涵體,未能檢測(cè)到酯酶活性。提取、純化重組表達(dá)蛋白,并制備獲得多克隆抗體。Western雜交結(jié)果證實(shí),二化螟CsCaE026基因在大腸桿菌中成功表達(dá)。 3二化螟酯酶基因CsCaE026的時(shí)空表達(dá)特征分析 分別采用實(shí)時(shí)熒光定量PCR技術(shù)和蛋白Western雜交技術(shù),分析二化螟CsCaE026基因在轉(zhuǎn)錄與翻譯水平的時(shí)空表達(dá)分布。明確CsCaE026基因在二化螟幼蟲的中腸中高水平特異性表達(dá)。 4二化螟硝酶基因CsCaE026的真核表達(dá)分析 構(gòu)建獲得CsCaE026基因重組桿狀病毒,并在昆蟲Sf9細(xì)胞中進(jìn)行表達(dá)。重組表達(dá)產(chǎn)物分子量為90kD。Western雜交結(jié)果證實(shí),二化螟CsCaE026基因在昆蟲細(xì)胞中成表達(dá)。
[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 偽 / 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 偽-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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S433

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本文編號(hào)：2505788