东方粘虫中肠胰蛋白酶基因上游启动子核心序列的克隆及功能验证

发布时间：2018-03-23 13:55

本文选题：粘虫　切入点：胰蛋白酶　出处：《西北农林科技大学》2017年硕士论文

【摘要】：随着人们对食品安全以及绿色农业的持续深入的关注,对安全使用农药问题的认识也越来越深刻,因此,利用天然产物研究开发新型农药成了近年的热点之一。前期研究发现,杠柳新苷类化合物对东方粘虫具有一定的杀虫活性,并且可以上调昆虫体内胰蛋白酶的表达,使其发生过分表达分泌的现象,超出虫体自身的生命代谢水平,从而可能使其自身的器官和组织被该类酶水解破坏,最终导致昆虫死亡。但是,目前的研究尚未明确该类化合物如何产生这种效应,对其杀虫机理也未有明确的研究。1.东方粘虫中肠胰蛋白酶基因启动子的缺失克隆本研究利用生物信息学方法预测粘虫中肠胰蛋白酶基因启动子上游的序列中潜在的转录因子及其结合位点,根据初步预测的结果,设计出7段缺失引物,对靶标启动子进行分段PCR扩增,并将得到质粒重组到荧光素酶报告基因载体,共同转染至昆虫Sf21细胞系中,瞬时表达检测相对荧光活性,从而比较出各段启动子活性。试验结果表明靶标基因启动子区在-1003/-788和-274/-188间可能包含某些对胰蛋白酶基因转录调控起关键作用的转录因子结合位点,并且在-1003/-788之间可能存在转录激活因子,在-274/-188之间可能存在转录抑制因子。2.东方粘虫中肠胰蛋白酶基因启动子的突变克隆采用生物信息学预测方法,结合缺失片段启动子活性强弱,参考各个转录因子的结构与功能,预测出符合先前实验结果的可能存在的6个可能潜在的转录因子Zeste、Ubx、Myod、Cf2、SMAD、STAT的结合位点,并将其可能的结合位点序列突变,设计出6对突变引物,利用PCR技术对启动子序列进行突变克隆,构建包含有突变启动子片段质粒的表达载体,转染至Sf21细胞,瞬时表达后比较其各段启动子活性,发现突变Myod和Cf2的结合位点序列后,启动子活性与空白对照组有差异,从而推测这两个转录因子可能存在于粘虫体内,并对于粘虫中肠胰蛋白酶基因的转录调控有重要作用。3.杠柳化合物对靶标基因启动子的影响本试验为了探究杠柳新苷类化合物是否可以直接作用于靶标启动子,将供试药物利用DMSO溶解配制成不同浓度梯度的药剂,在由启动子全长构建的荧光素表达载体转染的Sf21细胞中直接用不同浓度的药剂孵育5 h,待表达完成后与空白对照组比较启动子活性。结果表明,粘虫中肠胰蛋白酶基因启动子在杠柳新苷T低浓度处理下活性均有所降低,且随着杠柳新苷T处理浓度的升高,启动子活性降低,表明杠柳新苷T对胰蛋白酶基因启动子活性有影响。本研究通过对粘虫中肠的胰蛋白酶基因进行了分析与预测,与空白对照的启动子活性比较,初步找到了靶标启动子基因中可能存在的转录因子结合位点,并利用杠柳新苷类化合物作用靶标启动子,结果表明,该类化合物对靶标启动子有一定影响,为杀虫剂作用于昆虫基因转录及表达水平的作用机制的研究奠定了理论基础。
[Abstract]:Along with the people to the food security and green agriculture continued in-depth attention, awareness of the safe use of pesticides has become increasingly profound, therefore, the use of natural products research and development of new pesticide has become one of the hot spots in recent years. The preliminary study found that Periplocoside compounds have certain insecticidal activity of Oriental armyworm, and can up regulate the expression of insect trypsin, the overexpression of secretion phenomenon beyond the metabolic level of insect life itself, which may make its own organs and tissues by the enzymatic hydrolysis of damage, eventually lead to the death of insects. However, the current study is not yet clear how these compounds produce this effect, the lack of study on the insecticidal mechanism also.1. Oriental midgut trypsin gene has no clear cloning of the promoter of this study using bioinformatic prediction methodology of midgut trypsin Start the gene transcription factor binding sites and protease sequences upstream of the potential, according to preliminary forecast results, designed 7 deletion primers for the target promoter fragments were amplified by PCR, and will get the recombinant plasmid into the luciferase reporter gene vector, CO transfection to Sf21 insect cell lines, transient expression of relative fluorescence detection to compare the activity of each promoter activity. Test results showed that the target gene promoter region may contain certain plays a key role in trypsin gene transcription factor binding sites in -1003/-788 and -274/-188, and there may be a transcription factor between -1003/-788, there may be a transcriptional repressor.2. Oriental midgut trypsin gene mutations in promoter cloning using bioinformatics prediction method in -274/-188, combined with the deletion and promoter activity is strong Weak, the structure and function of reference to various transcription factors, to predict the 6 potential transcription factor Zeste, are consistent with the previous experimental results may be Ubx, Myod, Cf2, SMAD, STAT binding site, and the possible binding site sequence mutations, designed 6 pairs of primers for promoter mutations. The sequence of mutant clones using PCR technology to construct expression vector containing mutant promoter fragment of plasmid and transfected into Sf21 cells. After comparing the transient expression of each promoter activity, found that the binding site sequence mutation of Myod and Cf2, the promoter activity and blank group, which suggested that these two transcription factors may in vivo and in transcriptional regulation of armyworm, midgut trypsin gene has significant effect on the promoter of.3. sepium compound target gene in this experiment in order to explore Periplocoside compounds can be straight Can be directly used for the target promoter will be tested using DMSO prepared drug dissolved agent of different concentration gradient vector, transfected Sf21 cells with different concentrations of drugs were directly bred 5 expression in H promoter was constructed by fluorescein, to be expressed after the completion and the blank control group were compared. The results showed that the promoter activity, midgut trypsin gene promoter in Periplocoside T lower concentration activity decreased, and with increased Periplocoside T concentration, reduced promoter activity, showed that Periplocoside T of trypsin gene promoter activity were studied. This study analyzed and predicted by trypsin gene in the midgut, compared with the control of the promoter activity, preliminarily found start possible binding sites of transcription factor gene in the target, and the use of Periplocoside compounds and targets The results showed that these compounds had certain effects on target promoters, and laid a theoretical foundation for the research on the mechanism of insecticides acting on insect gene transcription and expression level.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ453;S433.4

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