本文關(guān)鍵詞： 姜黃素 朱砂葉螨 數(shù)字基因表達(dá)譜 鈣調(diào)蛋白 磷脂酶A2　出處：《西南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：本實(shí)驗(yàn)室通過篩選一系列化合物發(fā)現(xiàn),源于中草藥植物姜黃的活性物質(zhì)姜黃素對(duì)朱砂葉螨表現(xiàn)出良好的殺螨活性,具有開發(fā)成綠色殺螨劑的潛力。因此明確姜黃素殺螨的分子機(jī)制,從而以姜黃素的作用靶標(biāo)為依據(jù),以姜黃素為模板,進(jìn)行分子優(yōu)化,將有助于新的綠色殺螨劑的研制。目前雖然在姜黃素的殺螨機(jī)理方面取得了一定的進(jìn)展,但是殺螨的分子機(jī)制仍不清楚。并且,朱砂葉螨基因信息以及姜黃素殺螨機(jī)制密切相關(guān)的應(yīng)答基因的缺乏一直是該領(lǐng)域的一大阻礙。本研究采用了數(shù)字基因表達(dá)譜升級(jí)版測(cè)序技術(shù)對(duì)朱砂葉螨響應(yīng)姜黃素脅迫的分子機(jī)制以及響應(yīng)基因進(jìn)行了研究,擴(kuò)展了參與姜黃素殺螨的基因信息資源,為進(jìn)一步全面闡述姜黃素的殺螨機(jī)理提供了思路,同時(shí)豐富了天然活性物質(zhì)殺蟲/螨機(jī)制的研究?jī)?nèi)容。主要的研究成果如下:1.明確了姜黃素對(duì)朱砂葉螨的LC50為2.642mg/m L,并表現(xiàn)出類似神經(jīng)毒劑的中毒癥狀姜黃素的生物活性測(cè)定結(jié)果表明,姜黃素對(duì)朱砂葉螨48 h的毒力回歸方程為y=-0.278+0.659x,致死中濃度LC50為2.642 mg/m L,95%的置信區(qū)間為1.680~6.082。玻片浸漬法和葉碟噴霧法處理朱砂葉螨后進(jìn)行癥狀觀察發(fā)現(xiàn),朱砂葉螨的中毒癥狀大致可分為:靜止期(0 h-4h);復(fù)蘇興奮期(4 h-48 h);麻痹昏迷期(48 h-72 h);死亡期(72 h-144 h)。這跟神經(jīng)毒劑的致毒癥狀有一定的相似性。并根據(jù)中毒癥狀明確了姜黃素作用的重要時(shí)間節(jié)點(diǎn)24 h和48 h,為下步測(cè)序?qū)嶒?yàn)提供理論依據(jù)。2.獲得并驗(yàn)證了數(shù)字基因表達(dá)譜測(cè)序結(jié)果,明確了姜黃素處理下朱砂葉螨響應(yīng)基因參與的主要生物學(xué)過程處理組和溶劑對(duì)照組測(cè)序分別得到53 419 438條和52 286 859條Reads。24 h和48 h的差異表達(dá)基因分別為15450條和15371條。滿足偏離概率值≥0.8并且log2Ratio≥1的顯著差異基因分別有111條和96條。此外,熒光定量證實(shí)了m RNA測(cè)序結(jié)果的準(zhǔn)確性,同時(shí)說明參與驗(yàn)證的15條基因也確實(shí)對(duì)姜黃素發(fā)生了響應(yīng)。GO功能顯著性富集分析明確了顯著差異基因參與的主要生物學(xué)過程包括生理過程、代謝過程、單生物過程、細(xì)胞凋亡、連接和催化、離子跨膜轉(zhuǎn)運(yùn)、神經(jīng)傳導(dǎo)等。KEGG通路富集分析羅列了富集程度排名前20的生化代謝途徑和信號(hào)轉(zhuǎn)導(dǎo)途徑,其中內(nèi)質(zhì)網(wǎng)蛋白質(zhì)加工和MAPK信號(hào)轉(zhuǎn)導(dǎo)通路是24 h和48 h均具有的最具代表性的生化途徑。另外與化合物分解代謝、信號(hào)傳導(dǎo)、細(xì)胞吞噬等作用有關(guān)的基因約占整個(gè)KEGG通路基因的64%。這些GO條目和KEGG條目里所包含的基因很可能與姜黃素殺螨機(jī)制密切相關(guān)。3.獲得23條與螨體代謝、信號(hào)傳導(dǎo)等相關(guān)的顯著差異基因,測(cè)定了谷胱甘肽-S-轉(zhuǎn)移酶活性,明確了螨體解毒代謝情況通過RPKM和Noiseq算法,篩選出兩處理時(shí)間段的顯著差異基因總條數(shù)為190條,共有基因?yàn)?7條。獲得了醫(yī)學(xué)上姜黃素的靶標(biāo)絲氨酸/蘇氨酸激酶以及與姜黃素的醫(yī)學(xué)靶標(biāo)相關(guān)的鳥苷酸激酶、硫氧還蛋白樣4A和Ras GTP酶。另外本研究還獲得了很多與鈣離子相關(guān)的酶,如磷脂酶C家族、磷脂酶A2、鈣調(diào)蛋白、鈣聯(lián)接蛋白和內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白1,其中磷脂酶A2和鈣調(diào)蛋白是重要的信號(hào)傳導(dǎo)類蛋白也是差異表達(dá)最明顯的基因,可以作為姜黃素殺螨作用的候選靶標(biāo)。同時(shí)獲得了GSTs基因,明確了谷胱甘肽-S-轉(zhuǎn)移酶隨姜黃素處理時(shí)間的變化情況,初步說明朱砂葉螨體內(nèi)姜黃素的減少并非是與谷胱甘肽硫轉(zhuǎn)移酶發(fā)生作用所致。4.克隆并分析了朱砂葉螨鈣調(diào)蛋白(calmodulin,Tc Ca M)和磷脂酶A2(phospholipase A2,Tc PLA2)基因的c DNA序列克隆獲得了Tc Ca M和Tc PLA2基因c DNA序列,分別命名為Tc Ca M、Tc PLA2,登錄號(hào)分別為:KY436226、KY436225。預(yù)測(cè)蛋白質(zhì)相對(duì)分子量分別為19.03KDa和12.04KDa,等電點(diǎn)分別為4.32和4.97。另外通過信號(hào)肽、蛋白跨膜結(jié)構(gòu)、糖基化位點(diǎn)和結(jié)構(gòu)域預(yù)測(cè)發(fā)現(xiàn),鈣調(diào)蛋白和磷脂酶A2均無信號(hào)肽和跨膜結(jié)構(gòu),均為非跨膜蛋白。鈣調(diào)蛋白具有四個(gè)EF-hand結(jié)構(gòu)域和兩個(gè)糖基化位點(diǎn),磷脂酶A2只有一個(gè)糖基化位點(diǎn),未發(fā)現(xiàn)結(jié)構(gòu)域。進(jìn)化樹結(jié)果顯示這兩條基因所處的分支分別與二斑葉螨最接近,該結(jié)果與傳統(tǒng)意義上的分類結(jié)果一致。5.朱砂葉螨Tc Ca M和Tc PLA2基因在不同螨態(tài)均有表達(dá),對(duì)姜黃素殺螨機(jī)制的研究具有重要價(jià)值Tc Ca M和Tc PLA2基因在朱砂葉螨不同螨態(tài)的表達(dá)表明:這兩個(gè)基因在朱砂葉螨的四個(gè)螨態(tài)中均有表達(dá),并且幼螨、若螨期的表達(dá)量要顯著高于卵期和成螨期。鈣調(diào)蛋白基因的表達(dá)由高到低依次為若螨幼螨成螨卵,除幼螨、若螨差異不顯著外,其余均呈顯著性差異。磷脂酶A2基因的表達(dá)量高低依次為幼螨若螨成螨卵,各螨態(tài)均成顯著性差異。Tc Ca M和Tc PLA2基因因其特殊的功能和在朱砂葉螨不同發(fā)育階段的廣泛存在使之成為維持螨體正常生命活動(dòng)的重要蛋白,因此它們的抑制和活化都將對(duì)螨體產(chǎn)生顯著影響,鈣調(diào)蛋白和磷脂酶A2在姜黃素的殺螨機(jī)制中具有很重要的研究?jī)r(jià)值。
[Abstract]:The laboratory by screening a series of compounds that originated from the herbal plant turmeric active substances of curcumin on Zhu Shaye mites showed good acaricidal activity, has developed into a green acaricide. The molecular mechanism of curcumin so clear potential acaricide targets, thus curcumin as the basis, with curcumin as a template for the molecular optimization, will help to develop new green acaricides. Although the acaricidal mechanism of curcumin has made some progress, but the molecular mechanism of acaricidal remains unclear. Moreover, the lack of response gene tetranychuscinnabarinus gene information and closely related to the killing mechanism of curcumin has been a major obstacles in this field. This study adopts digital gene expression profile sequencing technology upgrades to tetranychuscinnabarinus molecular response mechanism of curcumin and stress response genes were studied and extended In the genetic information resources curcumin acaricide, provides ideas for further comprehensive exposition of acaricidal mechanism of curcumin, and enrich the research content of natural insecticidal / mite mechanism. The main results are as follows: 1. the LC50 of curcumin to tetranychuscinnabarinus is 2.642mg/m L, and showed similar results for determination of nerve agent the symptoms of poisoning the bioactivities of curcumin showed that curcumin on toxicity regression equation of 48 h tetranychuscinnabarinus y=-0.278+0.659x, lethal concentration LC50 was 2.642 mg/m L, 95% confidence interval 1.680~6.082. slide impregnation method and leaf disc tetranychuscinnabarinus treatment spray method after symptoms were observed, symptoms of poisoning tetranychuscinnabarinus roughly can be divided into: stationary phase (0 h-4h); recovery of exciting period (4 h-48 h); paralysis coma period (48 h-72 h); death period (72 h-144 h). This is similar to the nerve agent poisoning symptoms According to the symptoms of poisoning. The important time node curcumin 24 h and 48 h, for the next step sequencing experiments provide a theoretical basis for.2. to obtain and verify the digital gene expression profile sequencing results, the response of genes involved in the biological process of the main treatment group and solvent control group were respectively 53419438 and 52286859 Reads.24 h h and 48 differentially expressed genes were 15450 and 15371, tetranychuscinnabarinus curcumin treatment. Meet the deviation probability value is more than 0.8 and 1 log2Ratio genes were respectively 111 and 96. In addition, fluorescence quantitative m confirmed the accuracy of the results of RNA sequencing, and 15 genes involved in verification does the curcumin occurred in response to the.GO function significant enrichment analysis clearly the main differences of genes involved in the biological processes including physiological process, metabolic process, biological single Process, apoptosis, connection and catalysis, ion transport across the membrane, nerve conduction and.KEGG pathway enrichment analysis lists the enrichment degree of the top 20 biochemical metabolic pathways and signal transduction pathways, including endoplasmic reticulum protein processing and signal transduction pathway of MAPK is 24 h and 48 h are some of the most representative biochemical pathways. In addition to the compound decomposition metabolism, signal transduction, cell phagocytosis gene effects about which contains the entire KEGG pathway genes 64%. these GO and KEGG entries in the gene may kill mite mechanism and curcumin is closely related to.3. was 23 and the mites metabolism genes were related to signal transduction, glutathione -S- transferase activity determination, clear the mites detoxification through the RPKM and Noiseq algorithms, selected genes differ significantly between the two treatment period the total number of 190, a total of 17 genes. The target serine / threonine kinase and medicine curcumin and curcumin medical target associated guanylate kinase, thioredoxin like 4A and Ras GTP enzyme. In addition this study also received a lot of calcium ions and related enzymes, such as phospholipase C family, phospholipase A2, calmodulin, calmegin and the endoplasmic reticulum stress related protein 1, including phospholipase A2 and calmodulin gene is the most obvious signal transduction proteins is an important differential expression, can be used as a candidate target of curcumin and miticides. At the same time GSTs gene has been obtained, the changes of glutathione -S- transferase with curcumin treatment time, preliminary description of cinnabar spider mite in curcumin reduction is not analyzed tetranychuscinnabarinus and Calmodulin effect caused by.4. cloning and glutathione-s transferase (calmodulin, Tc Ca M (phospholipase) and phospholipase A2 A2, Tc PLA2) C DNA cloned gene sequence was Tc Ca M and Tc PLA2 C gene DNA sequence, named Tc Ca M, Tc PLA2, the accession number was KY436226, the relative molecular weight of KY436225. and 19.03KDa for the prediction of protein 12.04KDa, isoelectric point was 4.32 and 4.97. respectively by signal protein peptide, transmembrane structure, glycosylation sites and domain prediction found that calmodulin and phospholipase A2 have no signal peptide and transmembrane structure are non transmembrane protein. Calmodulin has four EF-hand domains and two glycosylation sites, phospholipase A2 only a glycosylation site not found domain. The phylogenetic tree showed that the branch of the two genes were the most close to the two spotted spider mite, the classification results with the results of the traditional sense of the.5. Tc Ca M tetranychuscinnabarinus and Tc PLA2 gene in different states were mite killing mechanism of curcumin, the research This has important value of Tc M and Ca Tc expression of PLA2 gene in tetranychuscinnabarinus different mite state: the two genes in four states mite Tetranychus cinnabarinus were expressed, and the expression of larval mites, if mites period was significantly higher than the egg and adult period. The expression of calmodulin gene from high to low as if the young adult mites mite eggs, in addition to David mite nymphs, the difference was not significant, the rest showed a significant difference. The expression level of phospholipase A2 gene in young adult mites mites if the mite eggs, there were significant differences in.Tc state of Ca M and Tc PLA2 gene its special function and in tetranychuscinnabarinus different developmental stages exist widely and has become an important protein to maintain the normal life activities of the mites, so their inhibition and activation will have a significant impact on the mites, calmodulin and phospholipase A2 has very important value in the research on price acaricidal mechanism of curcumin.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S482.52

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王丹;章冰川;丁偉;張永強(qiáng);羅金香;;姜黃素對(duì)朱砂葉螨幾丁質(zhì)酶基因表達(dá)的影響[J];農(nóng)藥學(xué)學(xué)報(bào);2016年02期

2 鄭越;鄭慶偉;;果園常見螨類害螨的鑒別與防治技巧[J];農(nóng)藥市場(chǎng)信息;2015年29期

3 張龍濤;呂建建;高保全;劉萍;;三疣梭子蟹鈣調(diào)蛋白基因的克隆及在蛻皮中的功能分析[J];中國(guó)水產(chǎn)科學(xué);2015年06期

4 張夢(mèng)翔;施高翔;嚴(yán)園園;陸克喬;邵菁;汪天明;汪長(zhǎng)中;;姜黃素對(duì)5種非白念珠菌菌絲及生物膜形成的抑制作用[J];中草藥;2015年04期

5 高麗嬌;黃家興;吳杰;;小峰熊蜂蜂毒磷脂酶A2基因的克隆及表達(dá)分析[J];昆蟲學(xué)報(bào);2013年09期

6 羅金香;丁偉;張永強(qiáng);楊振國(guó);李陽;;姜黃素異VA唑和吡唑衍生物的合成及殺螨活性[J];農(nóng)藥學(xué)學(xué)報(bào);2013年04期

7 羅金香;丁偉;張永強(qiáng);楊振國(guó);李陽;丁麗娟;;雙去甲氧基姜黃素及其N-甲基吡唑衍生物對(duì)朱砂葉螨生物活性和多種酶活性的影響[J];中國(guó)農(nóng)業(yè)科學(xué);2013年13期

8 王華森;懷其勇;;姜黃素衍生物的合成及抑菌活性研究[J];天然產(chǎn)物研究與開發(fā);2013年02期

9 張全芳;李軍;范仲學(xué);楊連群;步迅;;高通量測(cè)序技術(shù)在農(nóng)業(yè)研究中的應(yīng)用[J];山東農(nóng)業(yè)科學(xué);2013年01期

10 于旭蓉;仇雪梅;常亞青;王秀利;劉洋;;長(zhǎng)牡蠣鈣調(diào)蛋白基因克隆及多態(tài)性分析[J];南方農(nóng)業(yè)學(xué)報(bào);2012年06期

相關(guān)碩士學(xué)位論文 前2條

1 李彩新;姜黃素對(duì)朱砂葉螨的致毒癥狀及體壁穿透效應(yīng)研究[D];西南大學(xué);2015年

2 劉剛;喜樹堿對(duì)小菜蛾的生物活性及作用機(jī)理研究[D];浙江林學(xué)院;2007年

，

本文編號(hào)：1441982