【作者】 孙伟；

【导师】 何林；

【作者基本信息】 西南大学 ， 农药学， 2010， 硕士

【摘要】 朱砂叶螨(Tetranychus cinnabarinus)是一种为害严重而义难以防治的重要农业害螨,在我国分布广泛,对包括棉花、多种蔬菜在内的许多植物都有危害。该螨世代周期短,繁殖力强,近亲交配率高,受药机会多,很容易产生抗药性。酯酶作为一种水解酶,能够水解羧酸酯键和磷酸酯键,代谢多种含有酯键的杀虫剂,是害虫产生抗性的主要因素之一本研究在前人已经克隆得到朱砂叶螨酯酶基因TCE1，TCE2的基础上,建立了适于定量检测朱砂叶螨mRNA表达的real-time PCR方法,并用该方法检测了酯酶基因TCE1和TCE2的mRNA分别在朱砂叶螨不同发育时期(卵、幼螨、若螨和成螨)、不同品系(阿维菌素、甲氰菊酯、氧化乐果抗性品系和敏感品系)以及药剂(阿维菌素、甲氰菊酯、氧化乐果)诱导前后的表达差异,从基因水平探讨了朱砂叶螨酯酶基因的表达变化与其抗药性形成之间的内在联系。此外,还成功构建了酯酶TCE1, TCE2基因的重组原核表达载体pET43a-TCE1, pET43a-TCE2,且在E.coli Transetta中成功进行了蛋白表达。本研究获得的主要成果如下：1.利用RT-PCR技术,从朱砂叶螨体内获得了七条候选内参基因的扩增片段,并提交到GenBank,取得相应的登录号分别为RPS18 (FJ608659):262bp,5.8SrRNA (FJ526334):470bp, GAPDH (FJ526335):293bp, RPL13a (FJ608662):242bp, TBP (FJ608661):365bp, SDHA (FJ608660):594bp,α-TUB (FJ526336):962bp。采用实时荧光定量PCR方法并使用geNorm和NormFinder两个软件共同筛选最适合朱砂叶螨的内参基因。内参基因筛选结果为：5.8s rRNA,α-TUB, RPS18, GAPDH, RPL13a,β-actin表达稳定度的平均值M在不同品系中依次为0.381,0.449,0.352,0.460,0.445,0.643,表达稳定度由高到低排序依次为RPS18> 5.8srRNA> RPL13a> 0.449>GAPDH>β-actin; M值在不同螨态中依次为0.762,0.555,0.537,0.855,0.574,0.768,表达稳定度由高到低排序依次为RPS18>α-TUB> RPL13a> 5.8srRNA>β-actin> GAPDH。再通过NormFinder软件计算各内参基因的稳定度值,最终确定了在不同品系朱砂叶螨中以5.8srRNA和RPS18两个看家基因作为内参基因最理想,在不同螨态中以RPS18,α-TUB两个看家基因作为内参基因最理想,并据此建立了基于SYBR Green I染料技术的适于朱砂叶螨mRNA表达定量检测的real-time PCR方法,即实时荧光定量PCR (qrtPCR)。2.采用建立的qrtPCR疗法检测了酯酶基因TCE1和TCE2的mRNA在朱砂叶螨敏感品系的卵、幼螨、若螨和成螨四个螨态的相对表达。检测结果表明朱砂叶螨TCE2基因的表达量在各螨态中均显著高于TCE1基因(幼螨除外),而两条酯酶基因(TCE1和TCE2) mRNA的表达量均在成螨体内最高。3.相对于敏感品系,TCE1基因在朱砂叶螨抗性品系(阿维菌素、甲氰菊酯和氧化乐果抗性品系)中的相对表达量为1.06-1.14倍,无显著性差异；TCE2基因的相对表达量为1.39-2.47倍,均显著高于在敏感品系中的表达。4.分别用亚致死剂量(LC30)的阿维菌素、氧化乐果和甲氰菊酯对朱砂叶螨敏感品系进行诱导,测定诱导前后酯酶基因TCE1和TCE2 mRNA的相对表达量,结果表明,经药剂处理后,酯酶基因TCE1的表达量在处理后4h显著下降,而后又上升,但其表达量在测定时间内(处理后0-20h)一直没有超过未施药的对照。酯酶基因TCE2的表达量随时间延长呈现先升高再降低的变化趋势,且与对照相比差异显著。TCE2基因的相对表达量在受到三种药剂诱导后的最高表达量分别是对照的1.64-,2.92-和2.24倍。以上研究表明朱砂叶螨酯酶基因TCE2扩增是其对杀螨剂产生抗性的重要原因之一。5.成功构建了酯酶TCE1,TCE2基因的重组原核表达载体pET43a-TCE1, pET43a-TCE2,转化入E.coli Transetta进行原核表达,分别经IPTG诱导,SDS-PAGE和Western Blot分析并检测融合蛋白,结果表明所构建的载体可以在宿主菌中稳定、正确的表达,为进一步研究这两条酯酶的性质和功能奠定了基础。更多还原

【Abstract】 The carmine spider mite, Tetranychus cinnabarinus (Boisduval), one of the most important pests on cotton and most vegetables, is widely distributed in China. Because of high fecundity and short generation time, the mite can rapid formation of a certain number of stocks in a short period of time, develop resistance easily and thus it is difficult to prevent and control this mite.As a large class of enzymes that metabolize a variety of pesticides, esterases play important roles in priority hydrolysis of water-soluble short-chain esters of the ester. The research of esterase is very active in foreign and relative behindhand in domestic.This study was based on the esterase genes TCE1 and TCE2 that had been cloned from carmine spider mite, and a real-time PCR method based on SYBR Green I dye was developed. The mRNA expression differences of the two genes in different developmental period (egg, protonymph, nymphae and adults), different strains (abamectin-resistant, AbR; fenpropathrin-resistant, FeR; omethoate-resistant, OmR and susceptive strains, S) and these three pesticides induced from carmine spider mite were also detected using real-time PCR technology, respectively. TCE1 and TCE2 genes were inserted into pET-43.1a(+) of prokaryotic expression vector, and recombinant expression vector was obtained, named pET43a-TCE1 and pET43a-TCE2. The recombinant expression vector could express fusion proteins in E. coli. The key results were as follows.1. Seven candidate genes has been cloned from carmine spider mite using RT-PCR technology, and submitted them to the GenBank, GenBank number and length were RPS18 (FJ608659):262bp, 5.8SrRNA (FJ526334):470bp, GAPDH (FJ526335):293bp, RPL13a (FJ608662):242bp, TBP (FJ608661):365bp, SDHA (FJ608660):594bp, a-TUB (FJ526336):962bp, respectively. The qrtPCR primers were designed and the stability of gene expression was assessed using two different analysis programs, geNorm and NormFinder. In this study, the ranking of gene expression stability value (M) of 5.8s rRNA,α-TUB, RPS18, GAPDH, RPL13a and P-actin were 0.381,0.449,0.352, 0.460,0.445 and 0.643, and the ranking of gene expression stability were RPS18> 5.8srRNA> RPL13a> 0.449>GAPDH>P-actin in different strains, the ranking of gene expression stability value (M) of this six reference genes were 0.762,0.555,0.537,0.855,0.574 and 0.768, the ranking of gene expression stability were RPS18>a-TUB> RPL13a> 5.8srRNA>P-actin> GAPDH in different life stages. The geNorm program was subsequently used to calculate the optimal number of reference genes was two. To further validate our findings regarding the most optimal reference gene to normalize transcript expression data, we assessed our data set with NormFinder, Based on these data, the NormFinder program validated the findings with the geNorm algorithm, in which the most stable single gene was RPS18, and the best combination of the reference genes was RPS18 and 5.8SrRNA in different strains, RPS18 and a-TUB in different life stages of carmine spider mite, respectively.2. The mRNA expression levels of TCE1 and TCE2 genes were detected from egg, protonymph, nymph and adults of T. cinnabarinus through the method of qrtPCR technology with RPS18 and a-TUB as reference genes. The results showed that the mRNA expression level of TCE2 gene was higher than that of TCE1 gene, only by contrary in protonymph. The mRNA expression levels of two esterase genes in adults of T. cinnabarinus were significantly higher than those from other instars.3. The mRNA relative expression level of the TCE1 and TCE2 genes were also detected from T. cinnabarinus AbR, FeR, OmR and S. Compared with S, TCE1 gene mRNA expression of the three resistant strains were 1.06～1.07 fold, and 1.386～2.473, respectively for mRNA expression of the TCE2 gene. The results showed that the mRNA expression levels of TCE2 genes of the three resistant strains compared with that of S, and the difference was significant, but there was no obvious difference of the mRNA expression levels of TCE1 genes among the four strains.4. After induced by abamectin, fenpropathrin and omethoate, compared with susceptive strain that not induced with pesticide, the mRNA expression level of TCE1 gene from T. cinnabarinus decreased at 4h and then increased, but not preponderate over the control. The expression levels of TCE2 gene increased gradually at the beginning,12 to 16h reached the maximum after being treated with insecticides, then decreased. The highest expression level of TCE2 was 1.64-,2.92-and 2.24-fold of the control when treated with omethoate, abamectin and fenpropathrin, respectively, and the difference was significant, illustrate that TCE2 related to pesticide resistance closely.5. TCE1 and TCE2 genes were inserted into pET-43.1a(+) of prokaryotic expression vector, and recombinant expression vector was obtained, named pET43a-TCE1 and pET43a-TCE2. The recombinant expression vectors could express fusion proteins of recombinant TCE1 and TCE2 in E. coli Transetta. Specific strip appeared on gel in SDS-PAGE and on PVDF-Membran in Western blotting. It supported important theoretical and practical significance for further study.更多还原