本文選題：亞洲玉米螟 + 多巴脫羧酶��； 參考：《揚州大學》2017年碩士論文

【摘要】：為研究亞洲玉米螟Ostrinia furnacalis(Guenee)幼蟲體內一種與昆蟲黑化相關的多巴脫羧酶(dopa decarboxylase,DDC)的分子功能,本文利用RACE技術從亞洲玉米螟幼蟲體內克隆得到DDC的cDNA序列,對DDC基因序列進行了生物信息學分析,對DDC原核表達表達條件進行優(yōu)化,采用Western blot對表達的目標蛋白進行了鑒定,對玉米螟5齡幼蟲進行亞致死濃度蟲酰肼飼喂處理以及methoprene點滴處理,通過Real time PCR檢測了蟲酰肼和methoprene處理前后DDC基因mRNA水平上的表達變化情況,主要研究結果如下:(1)采用RT-PCR及RACE技術從亞洲玉米螟幼蟲中獲得了 DDC基因全長cDNA序列。該基因全長1688 bp(GenBank 登錄號:KT428965),包含一個1431 bp的開放閱讀框(ORF),一個202 bp的5'非編碼區(qū)(5'UTR)和一個55 bp的3'非編碼區(qū)(3'UTR)。開放閱讀框從第203個核苷酸開始,終止于第1633個核苷酸,起始密碼子ATG,終始密碼子TGA,由其推導的氨基酸序列長為476個氨基酸。Of-DDC的計算分子量約為53.8 kDa,估測等電點pI為5.95。生物信息學分析表明:DDC蛋白無跨膜結構域,N端不含有信號肽,有1個糖基化位點,位于32位,含有36個磷酸化位點,均勻分布于整個多肽鏈中。BlastP分析結果表明:(Of-DDC的氨基酸序列與甜菜夜蛾Spodoptera exigua、豆天蛾Clanis bilinata、水稻粘蟲Mythimna separata、天蠶Antheraea ynaamai、大紅斑蝶Danus plexippus、冬尺蠖蛾 Operophtera brumata、斜紋夜蛾Spodoptera ltura等昆蟲的DDC基因高度同源。(2)采用pET-28a表達系統(tǒng)對DDC基因編碼的蛋白進行了原核表達,結果顯示,Of-DDC重組蛋白在不同的IPTG誘導之后在上清和沉淀中均有表達,重組表達蛋白質的分子量大小與預測結果一致,約為53 kDa。pET-28a-Of-DDC在16℃、1.2 mmol/LIPTG誘導;28℃、1.2 mmol/L IPTG 誘導;和 37℃、1.0 mmol/L 和 1.2 mmol/L IPTG 誘導條件下,均可在碎菌上清中有明顯的蛋白表達。Western blot鑒定結果表明可實現(xiàn)DDC蛋白的體外可溶表達。(3)通過Real time PCR分別檢測了亞致死濃度蟲酰肼和methoprene處理后亞洲玉米螟5齡幼蟲不同時間(0h、12h、24h、36h、48h、60h、72h)后DDC基因mRNA水平上的表達變化情況。結果表明:隨著時間的延長,對照組Of-DDC基因表達水平不斷上升,處理48h后,對照組Of-DDC基因表達水平開始下降。與對照組相比,蟲酰肼LC30處理后,Of-DDC基因在轉錄水平的表達在處理后0-24 h與對照組無明顯差異,在處理后36 h水平稍高于對照組,但處理后48h至72hOf-DDC的基因表達明顯被抑制(p0.05)。與對照組相比methoprene LC30處理后,初期(0-24h)Of-DDC基因表達水平呈現(xiàn)上升趨勢;在處理后72 h Of-DDC基因表達水平達到峰值,為對照組表達量的3.38倍。
[Abstract]:In order to study the molecular function of a dopa decarboxylase (DDC) associated with insect melanization in the larva of Asian corn borer (Ostrinia furnacalis Gueneee), the cDNA sequence of DDC was cloned from the larva of Asian corn borer by RACE technique. The DDC gene sequence was analyzed by bioinformatics, and the prokaryotic expression conditions of DDC were optimized. The target protein was identified by Western blot. The 5th instar larvae of corn borer were treated with fenhydrazide and methoprene. The expression of DDC gene mRNA was detected by Real time PCR before and after treatment with methoprene and propoxyhydrazide. The main results were as follows: (1) RT-PCR and RACE techniques were used to obtain the full-length cDNA sequence of DDC gene from Asian corn borer larvae. The total length of the gene is 1688 bp(GenBank accession number: KT428965, which contains an open reading frame of 1431 BP, a 202bp 5'non-coding region and a 55bp 3'noncoding region. The open reading frame starts from the 203rd nucleotide, terminates at 163nucleotide, starts at the beginning codon ATG, and ends with TGA. The deduced amino acid sequence is 476 amino acids. The calculated molecular weight of Of-DDC is about 53.8 kDa. the estimated isoelectric point Pi is 5.95. Bioinformatics analysis showed that there was no signal peptide in the N terminal of WDDC protein, and there was one glycosylation site, located at 32 position, containing 36 phosphorylation sites. The amino acid sequence of Of-DDC in the whole polypeptide chain showed that the amino acid sequences of Spodoptera exigua, Clanis bilinata, Mythimna separata, Antheraea ynaamai, Danus plexippus, Operophtera brumata, Spodoptera ltura et al. DDC gene was highly homologous. (2) pET-28a expression system was used to express the protein encoded by DDC gene in prokaryotic expression. The results showed that the recombinant Of-DDC protein was expressed in supernatant and precipitate after different IPTG induction. The molecular weight of the recombinant protein was consistent with the predicted results, and the molecular weight of the recombinant protein was about 53 kDa.pET-28a-Of-DDC induced at 28 鈩，

本文編號：1966472