【作者】 龚佑静；

【导师】 吴益东；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2010， 博士

【摘要】 化学农药的广泛使用对农业发展和粮食安全起了重要作用,但是由于害虫抗药性的发展,防治抗药性害虫正变得越来越困难。苏云金芽孢杆菌Bacillus thuringiensis (Bt)由于其杀虫晶体蛋白(ICPs)具有高度专一性,对人畜和非靶标昆虫非常安全而被广泛用于防治小菜蛾等鳞翅目害虫。小菜蛾Plutella xvlostella是一种全球性分布的蔬菜害虫,早在1990年就有田间小菜蛾种群对Bt制剂产生抗性的报道,但迄今为止,小菜蛾对Bt抗性的分子机理还是没有研究清楚。为了研究我国小菜蛾对Cry1Ac的抗性机理,首先在室内筛选获得一个高抗品系(SZBT),并用F1筛选法检测了3个小菜蛾田间种群Cry1Ac抗性基因的频率；通过蛋白组学方法,对小菜蛾CryAc抗性和敏感品系幼虫中肠毒素结合蛋白进行分离和鉴定,发现一种Bt毒素受体—氨肽酶N蛋白(PxAPN2)在抗性品系中表达量明显低于敏感品系；然后,克隆了小菜蛾PxAPN2基因,并采用RNAi技术明确了PxAPN2基因在小菜蛾对Cry1Ac抗性产生中的作用。明确小菜蛾对Bt毒素Cry1Ac抗性的生化和分子机理对于小菜蛾Bt抗性治理具有重要意义。1.小菜蛾Cry1Ac抗性遗传互补与田间种群Cry1Ac抗性基因频率检测由于Bt制剂的大量使用,我国小菜蛾田间种群对Cry1Ac已产生了不同程度的抗性。由于小菜蛾对Cry1Ac抗性为不完全隐性遗传,检测抗性基因频率对于制定抗性治理措施尤为重要。通过室内筛选获得对Cry1Ac抗性达2000倍左右的SZBT品系,该品系对CrylAc的抗性表现为Ⅰ型(Mode 1)。抗性遗传互补试验表明,SZBT品系与来自美国的Cry1Ac-R抗性品系对Cry1Ac的抗性由相同遗传位点控制。将2008年采自广东广州、惠州及云南玉溪的田间小菜蛾分别于SZBT进行单对杂交,通过检测120个单对杂交后代(F1)在CrylAc区分剂量(2μg/mL)下的死亡率,测得这3个田间种群Cry1Ac抗性基因频率分别为0.375、0.472和0.156。广州、惠州和玉溪田间种群对Cry1Ac的抗性分别为73、117和13倍,抗性程度与其抗性基因频率相一致。2.小菜蛾中肠Cry1Ac结合蛋白的分离与鉴定用生物素标记的CrylAc分别与敏感品系ROTH、抗性品系SZBT和对照品系SZ小菜蛾4龄幼虫中肠BBMV(用传统的SDS-PAGE分离)进行配基结合试验,发现Cry1Ac结合蛋白有10条主带。小菜蛾SZBT抗性品系110 kDa结合蛋白表达量低于敏感品系,而其他结合蛋白表达量差异不明显。据此推测,110 kDa的Cry1Ac结合蛋白可能为Cry1Ac的功能受体,其表达量下降可能与SZBT品系对Cry1Ac抗性相关。为了进一步分离和鉴定Cry1Ac结合蛋白,用2-D电泳和配基结合方法对小菜蛾抗性品系(SZBT)和敏感品系(ROTH)中肠BBMV中Cry1Ac结合蛋白进行分离,并通过肽质量指纹图谱(peptide mass fingerprinting, PMF)对主要结合蛋白进行鉴定。鉴定的小菜蛾Cry1Ac结合蛋白包括氨肽酶N(PxAPN2)、储存蛋白(storage protein)、肌动蛋白(actin)、硫酸酯酶(glucosinolate sulfatase)、V-ATPase亚基B (vacuolar ATPase B subunit)等。小菜蛾SZBT抗性品系中PxAPN2表达量明显低于敏感品系的相应结合蛋白,该PxAPN2(分子量为110 kDa)可能即为1-D电泳所发现的在抗性品系中表达量降低的110 kDa结合蛋白。氨肽酶N是鳞翅目昆虫中肠BBMV上Cry1Ac毒素的重要受体,小菜蛾PxAPN2蛋白的表达量降低可能与抗性品系Cry1Ac抗性相关。3.小菜蛾PxAPN2基因沉默后对Cry1Ac敏感性的影响采用RT-PCR技术分别克隆了抗性(SZBT)和敏感(ROTH)品系中编码110kDaCry1Ac结合蛋白的氨肽酶N基因(PxAPN2)。抗性和敏感品系PxAPN2基因间有多个位点的氨基酸发生替换。用实时定量PCR比较PxAPN2基因在小菜蛾SZBT和ROTH品系四龄幼虫中肠中的mRNA表达量,发现SZBT抗性品系PxAPN2 mRNA表达量下降为ROTH敏感品系的33.9%。为了证明小菜蛾PxAPN2在CryAc抗性形成中的作用,采用RNAi技术使PxAPN2基因mRNA表达下调,研究PxAPN2下调能否使小菜蛾敏感品系对Cry1Ac获得抗性。通过喂食体外合成的PxAPN2 dsRNA干扰小菜蛾ROTH敏感品系PxAPN2基因的表达,结果表明喂食PxAPN2 dsRNA最高可以使PxAPN2基因mRNA表达量下降53%,而另一种氨肽酶N基因(APNA) mRNA表达量则没有明显变化。小菜蛾PxAPN2基因被沉默后使ROTH敏感品系幼虫对Cry1Ac产生4-14倍抗性。上述结果表明,小菜蛾PxAPN2是Cry1Ac的功能受体,其表达量降低导致小菜蛾对CryAc产生抗性。本研究运用蛋白组学、配基结合、基因克隆和基因沉默等技术,系统研究了小菜蛾对Bt毒素Cry1Ac抗性的生化和分子机理,证明了PxAPN2为小菜蛾幼虫中肠中Cry1Ac的功能受体,该受体mRNA和蛋白表达量下降能够导致小菜蛾对Cry1Ac产生高水平抗性。由于该基因位点与Cry1Ac抗性基因并不位于同一连锁群,PxAPN2 mRNA表达下调可能与反式作用因子有关,PxAPN2蛋白表达量下降也可能与转录后调控有关,确切原因还有待进一步研究。更多还原

【Abstract】 The widespread use of chemical pesticides has played a very important role in agriculture and food security. Due to the development of pesticide resistance, pest prevention and control is becoming more and more difficult. Bacillus thuringiensis (Bt) based its crystal protein (ICPs) are highly friendly for humans and non-target insects and are widely used control diamondback moth and other Lepidoptera. Diamondback moth (Plutella xylostella) is a worldwide pest of cruciferous vegetables. As early as 1990, cases of resistance to Bt formulations were documented in open-field populations of diamondback moth. But, the precise mechanism of diamondback moth resistance to Bt toxin Cry1Ac is poorly understood. A Cry1Ac-resistant strain (SZBT) was selected in the laboratory in order to investigate resistance mechanisms. Resistance allele frequency of three field populations was detected with an F1 screen (single pair crossing between field-derived insects and SZBT insects). Using proteomic analysis, Cry1Ac binding proteins in midgut membranes from both CrylAc-resistant and susceptible strains of P. xylostella were compared and identified. An aminopeptidase N (PxAPN2) with reduced expression in the resistant strain was identified. PxAPN2 was demonstrated as a functional receptor of Cry1Ac by using RNAi technique. Understanding of biochemical and molecular mechanisms of CrylAc resistance is crucial for designing rational resistance management strategy in P. xylostella.1. Genetic complementation test for CrylAc resistance and detection of CrylAc resistance allele frequency in field populations of P. xylostellaBacillus thuringiensis (Bt) formulations have been widely used in China against the diamondback moth, P. xylostella, and different levels of resistance to Cry1Ac have been evolved in field populations of P. xylostella. Because Cry1Ac resistance is generally incompletely recessive in P. xylostella, detection of resistance allele frequency is very important for resistance management. A resistant strain (SZBT) selected under laboratory developed about 2000-fold resistance to Cry1Ac. Cry1Ac resistance in the SZBT strain was characterized as "Mole 1" resistance. Allelic complementation tests showed that Cry1Ac resistance in the SZBT strain and the Cry1Ac-R strain (originated from Florida of the USA) share a common locus. An F1 screen of 120 single-pair families between the SZBT strain and three field populations collected in 2008 was carried out. Based on this approach, the estimated frequencies of CrylAc resistance alleles were 0.156 in the Yuxi population from Yunnan province, and 0.375 and 0.472 respectively in the Guangzhou and Huizhou populations from Guangdong province. Cry1Ac resistance levels in Guangzhou. Huizhou and Yuxi populations were 73-,117- and 13-fold respectively, which is correlated with the resistance frequencies detected by thescreen.2. Idientification of CrylAc binding proteins in midgut membranes from P. xylostellaMidgut membranes of 4th instar larvae from the susceptible strain ROTH, the resistant strain SZBT and the reference strain SZ were separated by the traditional SDS-PAGE and blotted with biotin-labeled Cry1A toxins (Cry1Aa, Cry1Ab and Cry1Ac). About ten major Cry1Ac-binding bands were revealed in the ligand blotting. Pattern and darkness of the toxin-binding protein bands were similar between the resistant SZBT and the susceptible ROTH strains, except a 110 kDa protein band was significantly lighter in the resistant SZBT strain than in the ROTH strain. It is suggested that reduced expression of this 110 kDa Cry1Ac-binding protein may be associated with Cry1Ac resistance in the SZBT strain.Two-dimensional electrophoresis (2-DE) combined with ligand blotting assay were employed to separate and identify Cry1Ac-bingding proteins from both the resistant SZBT and the susceptible ROTH strains, and peptidase mass fingerprints were generated for several spots identified as Cry1Ac- binding proteins. The results showed that, in addition to the reported receptor aminopeptidase (PxAPN2), the storage protein, actin, glucosinolate sulfatase, V-ATPase subunit B were also identified as novel binding proteins in P. xylostella. As found in the 1-DE experiments, an 110 kDa Cry1Ac-binding protein was significantly reduced in the resistant SZBT strain. This 110 kDa protein was identified as an aminopeptidase N (PxAPN2). Aminopeptidase N is an important class of Bt toxin receptors in lepidopteran insects. Lower expression of PxAPN2 may relate to Cry1Ac resistance in the SZBT strain.3. Effect of RNAi-mediated gene silencing of PxAPN2 on Cry1Ac toxicity to the susceptible P. xylostella PxAPN2 gene encoding the 110 kDa toxin binding protein was cloned from both SZBT and ROTH strains using RT-PCR. Amino acid substitutions at several sites of PxAPN2 were present in both strains. Using quantitative real-time PCR, mRNA expression levels of PxAPN2 were compared between the resistant and the susceptible strains, and it showed that expression of PxAPN2 in the fourth instar larval midgut from the resistant SZBT strain was reduced to 33.9% of the susceptible ROTH strain.In order to verify the role of PxAPN2 on Cry1Ac resistance, PxAPN2 was down-regulated through RNAi to see whether it can make the susceptible strain obtain CrylAc resistance. Expression of PxAPN2 was reduced by as high as 53% when fed with PxAPN2 dsRNA. The third instar larvae from the ROTH fed with dsRNA of PxAPN2 obtained 4- to 14-fold resistance to Cry1Ac compare with the control larvae. This demonstrated that PxAPN2 is a functional receptor of Cry1Ac, and lower expression of PxAPN2 can result in resistance to Cry1Ac in P. xylostella.In the present study, biochemical and molecular mechanisms of resistance to CrylAc in the SZBT strain were extensively characterized through a combination of proteomic analysis, ligand blotting, gene cloning and RNA interference. PxAPN2 was confirmed as a functional receptor for CrylAc, and down-regulation of this receptor is associated with a high level resistance to CrylAc in the SZBT strain. PxAPN2 locus does not locate in the same linkage group as the CrylAc resistance gene. Therefore, lower mRNA expression of PxAPN2 may relate to changes of cis-acting factors, and lower expression of PxAPN2 protein may also be caused by post-transcriptional regulation in the resistant SZBT strain of P. xvlostella.更多还原