本文選題：粘蟲　切入點：胰蛋白酶　出處：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：隨著人們對食品安全以及綠色農(nóng)業(yè)的持續(xù)深入的關(guān)注,對安全使用農(nóng)藥問題的認(rèn)識也越來越深刻,因此,利用天然產(chǎn)物研究開發(fā)新型農(nóng)藥成了近年的熱點之一。前期研究發(fā)現(xiàn),杠柳新苷類化合物對東方粘蟲具有一定的殺蟲活性,并且可以上調(diào)昆蟲體內(nèi)胰蛋白酶的表達(dá),使其發(fā)生過分表達(dá)分泌的現(xiàn)象,超出蟲體自身的生命代謝水平,從而可能使其自身的器官和組織被該類酶水解破壞,最終導(dǎo)致昆蟲死亡。但是,目前的研究尚未明確該類化合物如何產(chǎn)生這種效應(yīng),對其殺蟲機理也未有明確的研究。1.東方粘蟲中腸胰蛋白酶基因啟動子的缺失克隆本研究利用生物信息學(xué)方法預(yù)測粘蟲中腸胰蛋白酶基因啟動子上游的序列中潛在的轉(zhuǎn)錄因子及其結(jié)合位點,根據(jù)初步預(yù)測的結(jié)果,設(shè)計出7段缺失引物,對靶標(biāo)啟動子進行分段PCR擴增,并將得到質(zhì)粒重組到熒光素酶報告基因載體,共同轉(zhuǎn)染至昆蟲Sf21細(xì)胞系中,瞬時表達(dá)檢測相對熒光活性,從而比較出各段啟動子活性。試驗結(jié)果表明靶標(biāo)基因啟動子區(qū)在-1003/-788和-274/-188間可能包含某些對胰蛋白酶基因轉(zhuǎn)錄調(diào)控起關(guān)鍵作用的轉(zhuǎn)錄因子結(jié)合位點,并且在-1003/-788之間可能存在轉(zhuǎn)錄激活因子,在-274/-188之間可能存在轉(zhuǎn)錄抑制因子。2.東方粘蟲中腸胰蛋白酶基因啟動子的突變克隆采用生物信息學(xué)預(yù)測方法,結(jié)合缺失片段啟動子活性強弱,參考各個轉(zhuǎn)錄因子的結(jié)構(gòu)與功能,預(yù)測出符合先前實驗結(jié)果的可能存在的6個可能潛在的轉(zhuǎn)錄因子Zeste、Ubx、Myod、Cf2、SMAD、STAT的結(jié)合位點,并將其可能的結(jié)合位點序列突變,設(shè)計出6對突變引物,利用PCR技術(shù)對啟動子序列進行突變克隆,構(gòu)建包含有突變啟動子片段質(zhì)粒的表達(dá)載體,轉(zhuǎn)染至Sf21細(xì)胞,瞬時表達(dá)后比較其各段啟動子活性,發(fā)現(xiàn)突變Myod和Cf2的結(jié)合位點序列后,啟動子活性與空白對照組有差異,從而推測這兩個轉(zhuǎn)錄因子可能存在于粘蟲體內(nèi),并對于粘蟲中腸胰蛋白酶基因的轉(zhuǎn)錄調(diào)控有重要作用。3.杠柳化合物對靶標(biāo)基因啟動子的影響本試驗為了探究杠柳新苷類化合物是否可以直接作用于靶標(biāo)啟動子,將供試藥物利用DMSO溶解配制成不同濃度梯度的藥劑,在由啟動子全長構(gòu)建的熒光素表達(dá)載體轉(zhuǎn)染的Sf21細(xì)胞中直接用不同濃度的藥劑孵育5 h,待表達(dá)完成后與空白對照組比較啟動子活性。結(jié)果表明,粘蟲中腸胰蛋白酶基因啟動子在杠柳新苷T低濃度處理下活性均有所降低,且隨著杠柳新苷T處理濃度的升高,啟動子活性降低,表明杠柳新苷T對胰蛋白酶基因啟動子活性有影響。本研究通過對粘蟲中腸的胰蛋白酶基因進行了分析與預(yù)測,與空白對照的啟動子活性比較,初步找到了靶標(biāo)啟動子基因中可能存在的轉(zhuǎn)錄因子結(jié)合位點,并利用杠柳新苷類化合物作用靶標(biāo)啟動子,結(jié)果表明,該類化合物對靶標(biāo)啟動子有一定影響,為殺蟲劑作用于昆蟲基因轉(zhuǎn)錄及表達(dá)水平的作用機制的研究奠定了理論基礎(chǔ)。
[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.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：TQ453;S433.4

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