【摘要】：前期研究發(fā)現(xiàn),殺蟲活性化合物杠柳新苷P和T可顯著上調(diào)粘蟲Mythimna separata(Walker)(Lepidoptera:Noctuidae)中腸胰蛋白酶基因的表達(dá),引起昆蟲體內(nèi)胰蛋白酶活性升高,從而破壞昆蟲的中腸細(xì)胞,引起昆蟲死亡。為了闡明杠柳新苷活性化合物上調(diào)胰蛋白酶基因表達(dá)的作用機(jī)理,本研究通過基因克隆獲得了粘蟲中腸胰蛋白酶基因5′端側(cè)翼啟動(dòng)子序列,并對(duì)其進(jìn)行序列分析和啟動(dòng)子功能驗(yàn)證,為進(jìn)一步探索昆蟲胰蛋白酶活性調(diào)控機(jī)制奠定基礎(chǔ)。主要研究結(jié)果如下:1.粘蟲中腸胰蛋白酶基因啟動(dòng)子的克隆本研究根據(jù)粘蟲中腸胰蛋白酶基因編碼區(qū)5′端外顯子序列采用染色體步移法設(shè)計(jì)兩輪步移用的特異性引物,并通過構(gòu)建粘蟲基因組步移的文庫成功克隆了胰蛋白酶基因5′端1 863 bp側(cè)翼序列。經(jīng)NCBI比對(duì)和啟動(dòng)子在線分析軟件NNPP v.2.2預(yù)測(cè),此序列與家蠶轉(zhuǎn)座子TRAS4基因同源性達(dá)到73%,且含有TATA框、CAAT框等啟動(dòng)子核心序列和GATA、STAT、C/EBP等轉(zhuǎn)錄調(diào)控元件。2.粘蟲中腸胰蛋白酶基因啟動(dòng)子功能分析將胰蛋白酶基因啟動(dòng)子片段定向插入到螢火蟲熒光素酶報(bào)告基因載體中,通過轉(zhuǎn)染草地貪夜蛾Spodoptera frugiperda(J.E.Smith)(Lepidoptera:Noctuidae)Sf21細(xì)胞系,瞬時(shí)表達(dá)后用雙熒光素酶報(bào)告基因檢測(cè)系統(tǒng)分析該啟動(dòng)子活性。結(jié)果表明,相對(duì)于空載體pGL3-Basic,所構(gòu)建的重組載體p(-1 673/+25)具有明顯的啟動(dòng)子活性,可啟動(dòng)報(bào)告基因表達(dá),說明所插入片段確為粘蟲中腸胰蛋白酶基因啟動(dòng)子。3.杠柳新苷類化合物對(duì)胰蛋白酶基因啟動(dòng)子活性的影響在胰蛋白酶基因啟動(dòng)子-熒光素酶報(bào)告基因載體瞬時(shí)轉(zhuǎn)染至昆蟲細(xì)胞后,用不同濃度的杠柳新苷類化合物處理細(xì)胞,同時(shí)設(shè)置不加藥劑處理的轉(zhuǎn)染細(xì)胞為空白對(duì)照,孵育5 h并檢測(cè)報(bào)告基因表達(dá)水平。結(jié)果表明,與對(duì)照相比,不同濃度的藥劑處理對(duì)報(bào)告基因表達(dá)水平?jīng)]有顯著影響,說明這些化合物對(duì)胰蛋白酶基因啟動(dòng)子活性也無顯著影響。本研究克隆了粘蟲中腸胰蛋白酶基因側(cè)翼啟動(dòng)子序列并分析其結(jié)構(gòu)特征和可能的轉(zhuǎn)錄因子結(jié)合位點(diǎn),通過瞬時(shí)表達(dá)分析法對(duì)其功能進(jìn)行了分析,并進(jìn)一步證明胰蛋白酶基因啟動(dòng)子可能不是杠柳新苷類活性化合物的直接作用位點(diǎn),從而為在轉(zhuǎn)錄因子水平研究杠柳新苷活性化合物的激活機(jī)理奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S433.4

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