亚致死浓度虫酰肼及Methoprene对亚洲玉米螟幼虫多巴脱羧酶基因表达的影响

发布时间：2018-06-02 00:37

  本文选题：亚洲玉米螟 + 多巴脱羧酶　； 参考：《扬州大学》2017年硕士论文

【摘要】：为研究亚洲玉米螟Ostrinia furnacalis(Guenee)幼虫体内一种与昆虫黑化相关的多巴脱羧酶(dopa decarboxylase,DDC)的分子功能,本文利用RACE技术从亚洲玉米螟幼虫体内克隆得到DDC的cDNA序列,对DDC基因序列进行了生物信息学分析,对DDC原核表达表达条件进行优化,采用Western blot对表达的目标蛋白进行了鉴定,对玉米螟5龄幼虫进行亚致死浓度虫酰肼饲喂处理以及methoprene点滴处理,通过Real time PCR检测了虫酰肼和methoprene处理前后DDC基因mRNA水平上的表达变化情况,主要研究结果如下:(1)采用RT-PCR及RACE技术从亚洲玉米螟幼虫中获得了 DDC基因全长cDNA序列。该基因全长1688 bp(GenBank 登录号:KT428965),包含一个1431 bp的开放阅读框(ORF),一个202 bp的5'非编码区(5'UTR)和一个55 bp的3'非编码区(3'UTR)。开放阅读框从第203个核苷酸开始,终止于第1633个核苷酸,起始密码子ATG,终始密码子TGA,由其推导的氨基酸序列长为476个氨基酸。Of-DDC的计算分子量约为53.8 kDa,估测等电点pI为5.95。生物信息学分析表明:DDC蛋白无跨膜结构域,N端不含有信号肽,有1个糖基化位点,位于32位,含有36个磷酸化位点,均匀分布于整个多肽链中。BlastP分析结果表明:(Of-DDC的氨基酸序列与甜菜夜蛾Spodoptera exigua、豆天蛾Clanis bilinata、水稻粘虫Mythimna separata、天蚕Antheraea ynaamai、大红斑蝶Danus plexippus、冬尺蠖蛾 Operophtera brumata、斜纹夜蛾Spodoptera ltura等昆虫的DDC基因高度同源。(2)采用pET-28a表达系统对DDC基因编码的蛋白进行了原核表达,结果显示,Of-DDC重组蛋白在不同的IPTG诱导之后在上清和沉淀中均有表达,重组表达蛋白质的分子量大小与预测结果一致,约为53 kDa。pET-28a-Of-DDC在16℃、1.2 mmol/LIPTG诱导;28℃、1.2 mmol/L IPTG 诱导;和 37℃、1.0 mmol/L 和 1.2 mmol/L IPTG 诱导条件下,均可在碎菌上清中有明显的蛋白表达。Western blot鉴定结果表明可实现DDC蛋白的体外可溶表达。(3)通过Real time PCR分别检测了亚致死浓度虫酰肼和methoprene处理后亚洲玉米螟5龄幼虫不同时间(0h、12h、24h、36h、48h、60h、72h)后DDC基因mRNA水平上的表达变化情况。结果表明:随着时间的延长,对照组Of-DDC基因表达水平不断上升,处理48h后,对照组Of-DDC基因表达水平开始下降。与对照组相比,虫酰肼LC30处理后,Of-DDC基因在转录水平的表达在处理后0-24 h与对照组无明显差异,在处理后36 h水平稍高于对照组,但处理后48h至72hOf-DDC的基因表达明显被抑制(p0.05)。与对照组相比methoprene LC30处理后,初期(0-24h)Of-DDC基因表达水平呈现上升趋势;在处理后72 h Of-DDC基因表达水平达到峰值,为对照组表达量的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，

本文编号：1966472