杠柳新苷类化合物对粘虫中肠胰蛋白酶基因启动子活性的影响

发布时间：2018-08-02 20:58

【摘要】：前期研究发现,杀虫活性化合物杠柳新苷P和T可显著上调粘虫Mythimna separata(Walker)(Lepidoptera:Noctuidae)中肠胰蛋白酶基因的表达,引起昆虫体内胰蛋白酶活性升高,从而破坏昆虫的中肠细胞,引起昆虫死亡。为了阐明杠柳新苷活性化合物上调胰蛋白酶基因表达的作用机理,本研究通过基因克隆获得了粘虫中肠胰蛋白酶基因5′端侧翼启动子序列,并对其进行序列分析和启动子功能验证,为进一步探索昆虫胰蛋白酶活性调控机制奠定基础。主要研究结果如下:1.粘虫中肠胰蛋白酶基因启动子的克隆本研究根据粘虫中肠胰蛋白酶基因编码区5′端外显子序列采用染色体步移法设计两轮步移用的特异性引物,并通过构建粘虫基因组步移的文库成功克隆了胰蛋白酶基因5′端1 863 bp侧翼序列。经NCBI比对和启动子在线分析软件NNPP v.2.2预测,此序列与家蚕转座子TRAS4基因同源性达到73%,且含有TATA框、CAAT框等启动子核心序列和GATA、STAT、C/EBP等转录调控元件。2.粘虫中肠胰蛋白酶基因启动子功能分析将胰蛋白酶基因启动子片段定向插入到萤火虫荧光素酶报告基因载体中,通过转染草地贪夜蛾Spodoptera frugiperda(J.E.Smith)(Lepidoptera:Noctuidae)Sf21细胞系,瞬时表达后用双荧光素酶报告基因检测系统分析该启动子活性。结果表明,相对于空载体pGL3-Basic,所构建的重组载体p(-1 673/+25)具有明显的启动子活性,可启动报告基因表达,说明所插入片段确为粘虫中肠胰蛋白酶基因启动子。3.杠柳新苷类化合物对胰蛋白酶基因启动子活性的影响在胰蛋白酶基因启动子-荧光素酶报告基因载体瞬时转染至昆虫细胞后,用不同浓度的杠柳新苷类化合物处理细胞,同时设置不加药剂处理的转染细胞为空白对照,孵育5 h并检测报告基因表达水平。结果表明,与对照相比,不同浓度的药剂处理对报告基因表达水平没有显著影响,说明这些化合物对胰蛋白酶基因启动子活性也无显著影响。本研究克隆了粘虫中肠胰蛋白酶基因侧翼启动子序列并分析其结构特征和可能的转录因子结合位点,通过瞬时表达分析法对其功能进行了分析,并进一步证明胰蛋白酶基因启动子可能不是杠柳新苷类活性化合物的直接作用位点,从而为在转录因子水平研究杠柳新苷活性化合物的激活机理奠定基础。
[Abstract]:The previous studies showed that the insecticidal compounds P and T could significantly upregulate the expression of trypsin gene in the midgut of Mythimna separata (Walker) (Lepidoptera:Noctuidae, thus causing the increase of trypsin activity in insects, thus destroying the midgut cells of insects and causing insect death. In order to elucidate the mechanism of the up-regulation of trypsin gene expression by the active compounds, the 5 'flanking promoter sequence of the midgut trypsin gene was obtained by gene cloning. The sequence analysis and promoter function verification were carried out, which laid a foundation for further exploring the regulation mechanism of insect trypsin activity. The main results are as follows: 1. Cloning of Promoter of Myxomycetes midgut trypsin Gene; based on the 5 'exon sequence of trypsin gene coding region in Myxomycetes, a specific primer was designed by chromosome step method for two rounds of trypsin gene translocation. The 5'end 1863 BP flanking sequence of trypsin gene was cloned successfully by constructing the genomic walking library. NCBI alignment and promoter on-line analysis software NNPP v.2.2 predicted that the sequence had 73 homology with TRAS4 gene of Bombyx mori transposon, and contained promoter core sequences such as TATA frame and CAAT-CAAT-box, and transcriptional regulatory element .2. The promoter of trypsin gene was inserted into the luciferase reporter gene vector of firefly, and transfected into Sf21 cell line of Spodoptera frugiperda (J.E.Smith (Lepidoptera:Noctuidae). The promoter activity was analyzed by double luciferase reporter gene detection system after transient expression. The results showed that compared with the empty vector pGL3-Basic, the recombinant vector p (1.673 / 25) had obvious promoter activity and could initiate the expression of the reporter gene, which indicated that the inserted fragment was the promoter of the intestinal trypsin gene of Myxomycetes armyworm. The effect of Acetylenein on the activity of trypsin Gene Promoter after transient transfection of trypsin Gene Promoter-luciferase report Gene Vector into insect cells, the cells were treated with different concentrations of Salicylate glycosides. At the same time, untreated transfected cells were set as blank control, incubated for 5 h and detected the expression level of reporter gene. The results showed that the expression of reporter gene was not significantly affected by different concentrations of medicament treatment, which indicated that these compounds had no significant effect on the promoter activity of trypsin gene. In this study, the flanking promoter sequence of the midgut trypsin gene was cloned and its structural characteristics and possible transcription factor binding sites were analyzed, and its function was analyzed by transient expression analysis. It is further proved that the promoter of trypsin gene may not be the direct action site of the active compounds of the triamoside, thus laying a foundation for the study of the activation mechanism of the active compounds at the level of transcription factor.
【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】：2016
【分类号】：S433.4

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