棉铃虫细胞色素P450CYP6B7基因过量表达及调控机制研究

发布时间：2018-01-05 07:04

本文关键词：棉铃虫细胞色素P450CYP6B7基因过量表达及调控机制研究　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：棉铃虫已对多种杀虫剂产生不同程度的抗药性,对拟除虫菊酯类杀虫剂抗药性问题最为突出。细胞色素P450酶系(简称P450)介导的代谢能力增强是棉铃虫对拟除虫菊酯类杀虫剂产生抗药性的重要原因。国内外大量文献表明,多个和拟除虫菊酯类杀虫剂抗性相关的P450基因在抗性种群棉铃虫中过量表达。基因的过量表达主要由转录水平调控。本论文以相对敏感种群和田间抗性种群棉铃虫为研究对象,探究了细胞色素P450 CYP6B7基因与其他抗性相关P450基因的表达水平,通过基因组步移技术克隆了 CYP6B7基因的5'非翻译区(5'UTR)序列,并鉴定了和2-十三烷酮、氰戊菊酯诱导表达相关的顺式作用元件和响应区,并用转录组测序的方法从整体角度分析了棉铃虫对氰戊菊酯产生抗药性差异表达的代谢相关基因和转录因子,旨在探究棉铃虫CZP6B7基因过量表达的调控机制。主要研究结果如下:1、田间棉铃虫抗性水平及抗性相关P450基因表达水平测定从北京、河北、河南和山东等地田间采集多个棉铃虫种群,用点滴法测定其对常用杀虫剂的敏感性,与相对敏感种群相比,田间种群棉铃虫对氰戊菊酯具有较高水平抗性,抗性倍数在5.4-114.7倍之间,对高效氯氟氰菊酯、辛硫磷和灭多威抗药性水平较低,抗性倍数均小于10倍。增效试验表明PBO对氰戊菊酯增效倍数最高,增效倍数在91.5-4497.2倍之间,DEF和DEM的增效效果较弱。采用实时荧光定量PCR方法测定不同种群棉铃虫抗性相关P450基因相对表达水平,结果表明,田间抗性种群棉铃虫中CYP6B7、CYP332A1和CZP9A12存在高水平的过量表达。2、CYP6B7基因5'非翻译区序列的克隆和分析根据CZP6B7基因的编码区序列设计特异性引物,采用基因组步移技术克隆得到相对敏感种群HDS棉铃虫CYP6B7基因起始密码子(ATG)上游长度为1934bp的5'UTR序列,GenBank登录号为KY196470。在NCBI上比对结果显示,该序列与棉铃虫CYP6B2、CYP6B6、CYP6B7等P450基因的串联序列以及钠离子通道α亚基部分序列有很高的相似性。在线预测转录起始位点是一个腺嘌呤碱基,也预测到一些转录因子结合位点,包括蜕皮激素响应元件(EcRE)、芳香烃受体外源物质响应元件(XRE-AhR)、Oct-1、Dfd、ftz、Eve和ZEN等。在Beijing种群中克隆得到一段ATG前1395 bp的CYP6B7基因5'UTR序列。序列比对发现,HDS种群和Beijing种群棉铃虫CYP6B7基因5'UTR中存在较多碱基差异,其中HDS种群在732-791 bp之间有一处短片段缺失,Beijing种群在961-1013 bp之间有一处长片段缺失。3、2-十三烷酮对CYP6B7基因的诱导和顺式作用元件的鉴定植物次生物质2-十三烷酮对棉铃虫中肠CYP6B7基因的相对表达量有显著的诱导作用。将CYP6B7基因5'UTR的5个长度缺失片段与无启动子的pGL3-Basic载体分别连接,构建重组质粒 pGL3-(-1703/+232),pGL3-(-680/+232),pGL3-(-320/+232),pGL3-(-238/+232)和pGL3-(-72/+232),细胞转染和双荧光酶活性测定发现2-十三烷酮对重组质粒pGL3-(-320/+232)有显著的诱导,诱导倍数为3.56倍,对重组质粒pGL3-(-238/+232)无诱导。在-320和-238 bp之间进一步构建5'UTR长度缺失的重组质粒pGL3-(-292/+232)和pGL3-(-256/+232),发现2-十三烷酮对重组质粒pGL3-(-292/+232)有显著的诱导,诱导倍数为2.15倍,对重组质粒pGL3-(-256/+232)无诱导作用。将-292和-257 bp之间的序列分成M1、M2和M3,分别对其进行碱基突变,发现M1中的碱基突变对2-十三烷酮的诱导效果无显著影响,M2和M3中的碱基突变显著降低了 2-十三烷酮诱导后pGL3-(-320/+232)的荧光活性。确定介导2-十三烷酮过量表达的顺式作用元件位于M3,即CYP6B7基因5'UTR的-280 到-257 bp 之间。4、氰戊菊酯对CYP6B7基因的诱导和响应区的鉴定以含有0.1 mg/g氰戊菊酯的人工饲料饲喂棉铃虫,处理48 h后发现对幼虫中肠CYP6B7基因表达量有显著的诱导作用。对已构建的五个重组质粒的诱导试验表明,氰戊菊酯对重组质粒pGL3-(-320/+232)有显著的诱导作用,诱导倍数为1.91倍。对重组质粒pGL3-(-320/+232)、pGL3-(-292/+232)、pGL3-(-256/+232)和 pGL3-(-238/+232)进行进一步的诱导,发现氰戊菊酯对重组质粒pGL3-(-292/+232)有显著的诱导作用,诱导倍数为1.75倍,对重组质粒pGL3-(-256/+232)无显著诱导作用,但重组质粒pGL3-(-292/+232)的基础相对荧光活性和诱导相对荧光活性均显著低于重组质粒pGL3-(-320/+232),说明CYP6B7基因5'UTR中介导氰戊菊酯诱导表达的响应区位于-320到-257 bp之间。与重组质粒pGL3-(-292/+232)的基础相对荧光活性相比,2-十三烷酮、氰戊菊酯以及二者的联合诱导倍数分别为2.52、1.59和2.03倍。5、氰戊菊酯抗性和敏感种群棉铃虫转录组测序分析将Beijing种群在室内用氰戊菊酯多代汰选后得到BJR种群,其抗性倍数比HDS种群高至少1275倍。将这两个种群进行转录组测序分析,发现BJR种群中有6130个上调表达的unigene,选取33个和代谢相关的unigene进行实时荧光定量PCR验证,发现10个P450基因、1个酯酶基因、1个GST基因和5个UGT基因显著上调。对转录组测序得到的转录因子进行分析,发现BJR种群中有199个上调和158个下调的转录因子unigene,在上调的转录因子unigene中包括已被报道调节P450基因表达的转录因子Maf和Arh核转运蛋白,分别属于TF_bZIP和bHLH转录因子家族。
[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.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S433

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