本文關(guān)鍵詞：棉鈴蟲細胞色素P450CYP6B7基因過量表達及調(diào)控機制研究　出處：《中國農(nóng)業(yè)大學》2017年博士論文　論文類型：學位論文

  更多相關(guān)文章： 棉鈴蟲 細胞色素P450 CYP6B7 2-十三烷酮 氰戊菊酯 順式作用元件 調(diào)控機制

【摘要】：棉鈴蟲已對多種殺蟲劑產(chǎn)生不同程度的抗藥性,對擬除蟲菊酯類殺蟲劑抗藥性問題最為突出。細胞色素P450酶系(簡稱P450)介導(dǎo)的代謝能力增強是棉鈴蟲對擬除蟲菊酯類殺蟲劑產(chǎn)生抗藥性的重要原因。國內(nèi)外大量文獻表明,多個和擬除蟲菊酯類殺蟲劑抗性相關(guān)的P450基因在抗性種群棉鈴蟲中過量表達�；虻倪^量表達主要由轉(zhuǎn)錄水平調(diào)控。本論文以相對敏感種群和田間抗性種群棉鈴蟲為研究對象,探究了細胞色素P450 CYP6B7基因與其他抗性相關(guān)P450基因的表達水平,通過基因組步移技術(shù)克隆了 CYP6B7基因的5'非翻譯區(qū)(5'UTR)序列,并鑒定了和2-十三烷酮、氰戊菊酯誘導(dǎo)表達相關(guān)的順式作用元件和響應(yīng)區(qū),并用轉(zhuǎn)錄組測序的方法從整體角度分析了棉鈴蟲對氰戊菊酯產(chǎn)生抗藥性差異表達的代謝相關(guān)基因和轉(zhuǎn)錄因子,旨在探究棉鈴蟲CZP6B7基因過量表達的調(diào)控機制。主要研究結(jié)果如下:1、田間棉鈴蟲抗性水平及抗性相關(guān)P450基因表達水平測定從北京、河北、河南和山東等地田間采集多個棉鈴蟲種群,用點滴法測定其對常用殺蟲劑的敏感性,與相對敏感種群相比,田間種群棉鈴蟲對氰戊菊酯具有較高水平抗性,抗性倍數(shù)在5.4-114.7倍之間,對高效氯氟氰菊酯、辛硫磷和滅多威抗藥性水平較低,抗性倍數(shù)均小于10倍。增效試驗表明PBO對氰戊菊酯增效倍數(shù)最高,增效倍數(shù)在91.5-4497.2倍之間,DEF和DEM的增效效果較弱。采用實時熒光定量PCR方法測定不同種群棉鈴蟲抗性相關(guān)P450基因相對表達水平,結(jié)果表明,田間抗性種群棉鈴蟲中CYP6B7、CYP332A1和CZP9A12存在高水平的過量表達。2、CYP6B7基因5'非翻譯區(qū)序列的克隆和分析根據(jù)CZP6B7基因的編碼區(qū)序列設(shè)計特異性引物,采用基因組步移技術(shù)克隆得到相對敏感種群HDS棉鈴蟲CYP6B7基因起始密碼子(ATG)上游長度為1934bp的5'UTR序列,GenBank登錄號為KY196470。在NCBI上比對結(jié)果顯示,該序列與棉鈴蟲CYP6B2、CYP6B6、CYP6B7等P450基因的串聯(lián)序列以及鈉離子通道α亞基部分序列有很高的相似性。在線預(yù)測轉(zhuǎn)錄起始位點是一個腺嘌呤堿基,也預(yù)測到一些轉(zhuǎn)錄因子結(jié)合位點,包括蛻皮激素響應(yīng)元件(EcRE)、芳香烴受體外源物質(zhì)響應(yīng)元件(XRE-AhR)、Oct-1、Dfd、ftz、Eve和ZEN等。在Beijing種群中克隆得到一段ATG前1395 bp的CYP6B7基因5'UTR序列。序列比對發(fā)現(xiàn),HDS種群和Beijing種群棉鈴蟲CYP6B7基因5'UTR中存在較多堿基差異,其中HDS種群在732-791 bp之間有一處短片段缺失,Beijing種群在961-1013 bp之間有一處長片段缺失。3、2-十三烷酮對CYP6B7基因的誘導(dǎo)和順式作用元件的鑒定植物次生物質(zhì)2-十三烷酮對棉鈴蟲中腸CYP6B7基因的相對表達量有顯著的誘導(dǎo)作用。將CYP6B7基因5'UTR的5個長度缺失片段與無啟動子的pGL3-Basic載體分別連接,構(gòu)建重組質(zhì)粒 pGL3-(-1703/+232),pGL3-(-680/+232),pGL3-(-320/+232),pGL3-(-238/+232)和pGL3-(-72/+232),細胞轉(zhuǎn)染和雙熒光酶活性測定發(fā)現(xiàn)2-十三烷酮對重組質(zhì)粒pGL3-(-320/+232)有顯著的誘導(dǎo),誘導(dǎo)倍數(shù)為3.56倍,對重組質(zhì)粒pGL3-(-238/+232)無誘導(dǎo)。在-320和-238 bp之間進一步構(gòu)建5'UTR長度缺失的重組質(zhì)粒pGL3-(-292/+232)和pGL3-(-256/+232),發(fā)現(xiàn)2-十三烷酮對重組質(zhì)粒pGL3-(-292/+232)有顯著的誘導(dǎo),誘導(dǎo)倍數(shù)為2.15倍,對重組質(zhì)粒pGL3-(-256/+232)無誘導(dǎo)作用。將-292和-257 bp之間的序列分成M1、M2和M3,分別對其進行堿基突變,發(fā)現(xiàn)M1中的堿基突變對2-十三烷酮的誘導(dǎo)效果無顯著影響,M2和M3中的堿基突變顯著降低了 2-十三烷酮誘導(dǎo)后pGL3-(-320/+232)的熒光活性。確定介導(dǎo)2-十三烷酮過量表達的順式作用元件位于M3,即CYP6B7基因5'UTR的-280 到-257 bp 之間。4、氰戊菊酯對CYP6B7基因的誘導(dǎo)和響應(yīng)區(qū)的鑒定以含有0.1 mg/g氰戊菊酯的人工飼料飼喂棉鈴蟲,處理48 h后發(fā)現(xiàn)對幼蟲中腸CYP6B7基因表達量有顯著的誘導(dǎo)作用。對已構(gòu)建的五個重組質(zhì)粒的誘導(dǎo)試驗表明,氰戊菊酯對重組質(zhì)粒pGL3-(-320/+232)有顯著的誘導(dǎo)作用,誘導(dǎo)倍數(shù)為1.91倍。對重組質(zhì)粒pGL3-(-320/+232)、pGL3-(-292/+232)、pGL3-(-256/+232)和 pGL3-(-238/+232)進行進一步的誘導(dǎo),發(fā)現(xiàn)氰戊菊酯對重組質(zhì)粒pGL3-(-292/+232)有顯著的誘導(dǎo)作用,誘導(dǎo)倍數(shù)為1.75倍,對重組質(zhì)粒pGL3-(-256/+232)無顯著誘導(dǎo)作用,但重組質(zhì)粒pGL3-(-292/+232)的基礎(chǔ)相對熒光活性和誘導(dǎo)相對熒光活性均顯著低于重組質(zhì)粒pGL3-(-320/+232),說明CYP6B7基因5'UTR中介導(dǎo)氰戊菊酯誘導(dǎo)表達的響應(yīng)區(qū)位于-320到-257 bp之間。與重組質(zhì)粒pGL3-(-292/+232)的基礎(chǔ)相對熒光活性相比,2-十三烷酮、氰戊菊酯以及二者的聯(lián)合誘導(dǎo)倍數(shù)分別為2.52、1.59和2.03倍。5、氰戊菊酯抗性和敏感種群棉鈴蟲轉(zhuǎn)錄組測序分析將Beijing種群在室內(nèi)用氰戊菊酯多代汰選后得到BJR種群,其抗性倍數(shù)比HDS種群高至少1275倍。將這兩個種群進行轉(zhuǎn)錄組測序分析,發(fā)現(xiàn)BJR種群中有6130個上調(diào)表達的unigene,選取33個和代謝相關(guān)的unigene進行實時熒光定量PCR驗證,發(fā)現(xiàn)10個P450基因、1個酯酶基因、1個GST基因和5個UGT基因顯著上調(diào)。對轉(zhuǎn)錄組測序得到的轉(zhuǎn)錄因子進行分析,發(fā)現(xiàn)BJR種群中有199個上調(diào)和158個下調(diào)的轉(zhuǎn)錄因子unigene,在上調(diào)的轉(zhuǎn)錄因子unigene中包括已被報道調(diào)節(jié)P450基因表達的轉(zhuǎn)錄因子Maf和Arh核轉(zhuǎn)運蛋白,分別屬于TF_bZIP和bHLH轉(zhuǎn)錄因子家族。
[Abstract]:The cotton bollworm has become resistant to different kinds of pesticides, pyrethroid insecticide resistance is a prominent problem. Cytochrome P450 enzymes (P450) metabolism mediated enhancement is important for producing Cotton Bollworm Resistance to pyrethroid insecticides. A large number of domestic and foreign literature shows that overexpression of a pyrethroid insecticide resistance related gene P450 in the resistant population of cotton bollworm. The overexpression of mainly by transcriptional regulation. In this paper, the relative susceptible population and field populations of the Cotton Bollworm Resistance as the research object, to explore the expression of cytochrome P450 CYP6B7 gene and other resistance related gene P450 cloning of CYP6B7 gene, the 5'untranslated region (5'UTR) by genome shift step sequence, and identified thirteen 2- and alkanones, fenvalerate induced expression of CIS associated with Element and response area and method for transcriptome sequencing from the perspective of the overall analysis of cotton bollworm metabolism related genes and transcription factor expression differences of resistance to fenvalerate, to control the mechanism of overexpression of CZP6B7 gene of Helicoverpa armigera. The main results are as follows: 1, the level of determination from Beijing, Hebei express cotton field the level of insect resistance and resistance related gene P450, Henan and Shandong to gather a lot of field populations of H.armigera, determine the susceptibility to insecticides by drip method compared with the susceptible population, the field populations of the cotton bollworm has a high level of resistance to fenvalerate, the resistance ratio between 5.4-114.7 times, of lambdacyhalothrin cyhalothrin, Phoxim and Do-win resistance level is low, the resistance ratio was less than 10 times. The highest synergistic test showed that PBO of fenvalerate synergism, synergism in 91.5-4497.2 times Between DEF and DEM, weak synergistic effect. By using real-time fluorescence quantitative PCR method for determination of relative expression levels, different populations of Cotton Bollworm Resistance Related Gene P450 results show that CYP6B7 resistance in field populations of the cotton bollworm, CYP332A1 and CZP9A12 had high level of overexpression of.2, cloning and analysis of nucleotide sequence encoding region sequence according to the specific design primers of CZP6B7 gene, CYP6B7 gene 5'was cloned by genome walking technology relatively sensitive population of cotton bollworm HDS start codon of CYP6B7 gene (ATG) upstream of the length of 5'UTR sequence 1934bp, GenBank accession number KY196470. results displayed on the NCBI, with the sequence of CYP6B2 CYP6B6 CYP6B7, Helicoverpa armigera, P450 gene the tandem sequence and sodium channel alpha subunit sequences have high similarity. The online prediction of the transcription initiation site is an adenine base also predicted to turn Transcription factor binding sites, including the ecdysone response element (EcRE), aryl hydrocarbon receptor response element of exogenous substances (XRE-AhR), Oct-1, Dfd, FTZ, Eve and ZEN. In the Beijing population in the cloned CYP6B7 gene 5'UTR sequence of 1395 BP a ATG. Sequence alignment showed that more base differences between populations and HDS Beijing population of cotton bollworm CYP6B7 gene 5'UTR, which has a population of HDS short deletions in 732-791 BP, a director of Beijing population.3,2- deletion was thirteen pyrrolidone on CYP6B7 gene induced by cis acting elements of the identification of plant secondary metabolites 2- thirteen pyrrolidone midgut of the relative expression of CYP6B7 gene induced by effect on 961-1013 BP. The 5 CYP6B7 length deletion fragments of 5'UTR gene and promoter pGL3-Basic vector respectively, the recombinant plasmid pGL3- (-1703/+232), pGL3- (-680/+232), P GL3- (-320/+232), pGL3- (-238/+232) and pGL3- (-72/+232), cell transfection and dual luciferase activity assay showed that pGL3- 2- thirteen pyrrolidone (-320/+232) and the recombinant plasmid was induced by multiples of 3.56 times, the recombinant plasmid pGL3- (-238/+232) induced by recombinant plasmid pGL3-. No further construction of 5'UTR deletion length between -320 and -238 BP (-292/+232) and pGL3- (-256/+232), pGL3- 2- thirteen pyrrolidone (-292/+232) and the recombinant plasmid was induced by multiples of 2.15 times, the recombinant plasmid pGL3- (-256/+232) - induced effect. The sequence between -292 and -257 BP into M1, M2 and M3, of the mutations found in M1 mutation had no significant effect on inducing effect of 2- thirteen alkanones, M2 and M3 mutations significantly reduced 2- induced pGL3- thirteen pyrrolidone (-320/+232) fluorescence activity. To determine the 2- mediated thirteen pyrrolidone. The expression levels of cis element located between CYP6B7 M3, -280 -257 BP 5'UTR gene to.4, induced by fenvalerate on CYP6B7 gene and response zone identification of artificial feed of cotton bollworm to contain 0.1 mg/g of fenvalerate, processing capacity have significant effect on the induced expression of CYP6B7 gene was found in 48 larval midgut H. That induced test of five recombinant plasmids have been constructed, fenvalerate on recombinant plasmid pGL3- (-320/+232) were induced, induced by multiples of 1.91. The recombinant plasmid pGL3- (-320/+232), pGL3- (-292/+232), pGL3- (-256/+232) and pGL3- (-238/+232) were further induced that discovery of fenvalerate on recombinant plasmid pGL3- (-292/+232) were induced, induced by multiples of 1.75 times, the recombinant plasmid pGL3- (-256/+232) was induced, but the recombinant plasmid pGL3- (-292/+232) on the basis of relative fluorescence activity And the relative fluorescence activity were significantly lower than that induced by the recombinant plasmid pGL3- (-320/+232), that is located in the region of the CYP6B7 gene of 5'UTR mediated response to fenvalerate induced expression of -320 and pGL3- to -257 BP. The recombinant plasmid (-292/+232) based on the relative fluorescence activity compared to 2- thirteen fold induction combined with pyrrolidone, Fenvalerate and two were 2.52,1.59 and 2.03 times.5, fenvalerate resistant and sensitive populations of cotton bollworm transcriptome sequencing analysis of Beijing population by fenvalerate jigging after multi generation BJR population in the interior, the resistance ratio of HDS population up to 1275 times less. The two populations were analyzed transcriptome sequencing, found there are 6130 up-regulated expression of UniGene BJR in the population, and selected 33 metabolism related UniGene real-time quantitative PCR validation, found that 10 P450 genes, 1 esterase genes, 1 GST genes and 5 UGT genes were significantly up-regulated. The transcription factor transcriptome sequencing are analyzed, found 199 up-regulated and 158 down regulated transcription factor UniGene BJR in the population have been reported including transcription factor Maf and nuclear translocation of Arh protein to regulate P450 gene expression in transcription factor UniGene is upregulated in TF_bZIP and bHLH respectively, which belongs to the family of transcription factors.

【學位授予單位】：中國農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S433

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