【作者】 徐丽娜；

【导师】 何康来；

【作者基本信息】 中国农业科学院 ， 生物安全， 2010， 博士

【摘要】 转Bt基因玉米为亚洲玉米螟(Ostrinia furnacalis)的防治开辟了新的途径,然而大面积种植转相同或相似的单基因Bt玉米将不可避免的引发靶标害虫对Bt产生抗性。研发具有不同杀虫机理的新基因,明确害虫对Bt毒素产生抗性的分子机理,培育双/多价转Bt基因玉米,对今后制定和实施合理的抗性治理策略,解决或延缓害虫抗性产生,保证转Bt基因玉米这一高效的玉米螟综合治理新措施的可持续利用具有重要的意义。本文通过饲料混合法,测定了Cry1Ab、Cry1Ac、Cry1Ah、Cry1Fa和Cry1Ie蛋白对亚洲玉米螟Bt敏感品系(ACB-BtS)和Cry1Ab抗性品系(ACB-AbR)的毒力;通过SDS-PAGE和配体杂交研究了生物素标记的Cry1Ac蛋白在亚洲玉米螟幼虫中肠BBMV上的受体蛋白及其与Cry1Ab、Cry1Ah、Cry1Fa和Cry1Ie蛋白在BBMV上的竞争结合模式;双向凝胶电泳、配体杂交、质谱鉴定等方法分析了Cry1Ab、Cry1Ac、Cry1Ah和Cry1Ie蛋白与亚洲玉米螟幼虫BBMV的结合图谱及结合位点蛋白;利用RT-PCR和RACE方法克隆亚洲玉米螟幼虫中肠4个氨肽酶N基因,进而采用荧光定量PCR技术分析4个APN和1个已知的钙粘蛋白基因在ACB-BtS和ACB-AbR品系不同龄期幼虫中肠的表达量。供试的5种Bt毒素Cry1Ab、Cry1Ac、Cry1Ah、Cry1Fa和Cry1Ie蛋白对ACB-BtS品系初孵幼虫的毒力存在显著差异,LC50分别为0.75、2.37、0.07、2.19和1.87μg/g。ACB-AbR品系对Cry1Ab蛋白产生了39.7倍的抗性,与此同时其对Cry1Ac、Cry1Ah和Cry1Fa蛋白分别产生了36.9、131.7和6.2倍的抗性,而对Cry1Ie没有产生抗性,这说明以Cry1Ab蛋白汰选得到的ACB-AbR品系与没有接触过的Cry1Ah和Cry1Ac蛋白具有很高的交互抗性,与Cry1Fa的交互抗性较低,而与Cry1Ie没有交互抗性。亚洲玉米螟幼虫中肠BBMV的蛋白质分子量在10->250 kDa,Cry1Ac蛋白与其结合的6个条带的分子量分别约在70、120、160、200、260和270 kDa,其中120 kDa和200 kDa与亚洲玉米螟氨肽酶和钙粘蛋白的分子量大小相符。Cry1Ac蛋白与ACB-BtS幼虫中肠BBMV的结合可被过量的Cry1Ab或Cry1Ah蛋白完全竞争抑制,也可被Cry1Fa蛋白部分竞争抑制,但不能被Cry1Ie竞争抑制。这说明亚洲玉米螟幼虫中肠BBMV上Cry1Ac蛋白的结合位点都是Cry1Ab及Cry1Ah的共同结合位点,Cry1Ac与Cry1Fa蛋白既存在共同的结合位点,亦存在不同的结合位点,而Cry1Ac与Cry1Ie的结合位点不同。不同Bt蛋白在亚洲玉米螟幼虫中肠上结合位点的差异是导致Cry1Ab抗性亚洲玉米螟与其它Bt蛋白有/无交互抗性的主要原因。发现了V-ATPase亚基A和热激蛋白70为Bt蛋白的受体蛋白。抗性品系可结合Bt蛋白的量更多。成功克隆了亚洲玉米螟幼虫中肠4个APN基因,即Ofapn1、Ofapn2、Ofapn3和Ofapn4,其cDNA分别编码994、940、1014和951个氨基酸的蛋白质,预测蛋白质分子量分别为113.1、106.8、115.1和107.8 kDa。其推导的氨基酸序列中具有鳞翅目昆虫APN典型结构特征,即N-末端具有18-20个氨基酸的剪切信号肽,谷氨酸锌化氨肽酶保守结构GAMEN,锌结合位点HEXXHX18E,C-末端具有21-22个氨基酸的糖基磷脂酰肌醇(GPI)锚信号肽。这4个APN氨基酸序列分别与欧洲玉米螟相对应的4个ANP序列的同源性高达96%、97%、96%和96%。与敏感品系相比,ACB-AbR品系的4个APN蛋白序列分别有9、5、10、和12个氨基酸发生改变,且Ofapn1的潜在O-糖基化位点增加1位,Ofapn2在3′UTR区2828-2838位有11个核苷酸发生缺失,在2893-2901位有9个核苷酸插入。与ACB-BtS品系相比,4个APN和钙粘蛋白基因在ACB-AbR品系幼虫中肠的表达量随着幼虫的生长总体呈现逐渐上升的趋势并显著高于敏感品系,这说明ACB-AbR对Cry1Ab蛋白的抗性水平上升可能与其对毒素的降解作用以及细胞本身的修复作用增强有关。更多还原

【Abstract】 Despite the proven track record of biotech Bt maize to provide an new tool for easier and more consistent Ostrinia furnacalis control, it is known for their ability to develop resistance in target pest to Bt toxins, particularly when the same or similar Bt maize expressing single toxin protein is planed in area wide. It is necessary to discover novel Bt genes, to develop novel insect resistant Bt maize with double or multiple genes, as well as to understand the molecular mechanism for evolution of resistance to Bt toxins in target pest, which will play an important roll in sustainable use of Bt maize as a valuable and innovative O. furnacalis control tactic through a management plan to delay or avoid the risk of resistance.Susceptibility to Cry1Ab, Cry1Ac, Cry1Ah, Cry1Fa, and Cry1Ie proteins from Bt was determined for ACB-AbR and ACB-BtS strains of O. furnacalis, a Cry1Ab-selected strain and a Bt susceptible strain, through bioassay by exposing neonate larvae to semi-artificial diet incorporated with Bt proteins. Model Assays of competition binding to BBMV of O. furnacalis were performed with biotinylated Cry1Ac protein mixed with excessive unlabeled Cry1Ab, Cry1Ah, Cry1Fa, and Cry1Ie proteins, respectively. Binding receptors of Cry1Ac protein in BBMV of O. furnacalis were detected by using ligand blotting with biotainylated Cry1Ac protein. A proteomic approach was explored together with ligand blotting to identify Cry1Ab, Cry1Ac, Cry1Ah, and Cry1Ie binding proteins from ACB-BBMV. cDNAs of aminopeptidase N (APN) from both ACB-BtS and ACB-AbR strains were cloned and sequenced using RT-PCR and RACE technologies. The expressions of 4 APN genes and a known cadherin gene from O. furnacalis were detected by Real Time PCR.There were significant differences in susceptibility of ACB-BtS strain to Cry1Ab, Cry1Ac, Cry1Ah, Cry1Fa, and Cry1Ie proteins. Their LC50 were 0.75, 2.37, 0.07, 2.19, and 1.87μg/g. ACB-AbR strain developed significan levels of resistance to Cry1Ab (39.7-fold), Cry1Ac (36.9-fold), Cry1Ah (131.7-fold), and Cry1Fa (6.2). In contrast, the susceptibility to Cry1Ie protein was not increased in ACB-AbR strain. This results susgest that the highest level of cross-resistance was observed with Cry1Ah (131.7-fold), followed by Cry1Ac (36.9-fold). A low level of cross-resistance (6.2-fold) to Cry1F was also detected. In contrast, ACB-AbR was equally susceptible to Cry1Ie as the unselected control strain.There were six binding peptides were identified as Cry1Ac binding proteins with the molecular weight 70, 120, 160, 200, 260, and 270kDa, respectively, among which the 120 and 200 kDa bindings were similar to that of APN and cadherin’s molecular weights. The bindings of Cry1Ac protein to BBMV of O. funacalis were completely significantly inhibited by Cry1Ab and Cry1Ah proteins and partial inhibited by Cry1Fa protein. In contrast, no competitive binding was observed between Cry1Ie and Cry1Ac. These results indicated that Cry1Ac protein shares all receptors with Cry1Ab and/or Cry1Ah proteins in BBMV of O. furnacalis but partial with Cry1Fa protein. It was different in binding receptors between Cry1Ac and Cry1Ie proteins. Therefore, the variance of cross-resistance levels among 5 proteins was due to the difference in their bingding receptors on the BBMV. In addition, the V-type proton ATPase catalytic subunit A and heat shock 70 kDa proteins were identified as Cry toxins binding proteins in BBMV of O. furnacalis.Four APN isoforms, Ofapn1, Ofanp2, Ofapn3, and Ofapn4, were identified from O. furnacalis by cDNA cloning. The deduced amino acid sequences were included 994, 940, 1014, and 951 amino acid residues, respectively. The predicted molecular weights of Ofapn1, Ofapn2, Ofapn3 and Ofapn4 were 113.1, 106.8, 115.1, and 107.8kDa, respectively. Sequences analysis indicated that the deduced protein sequences are most similar to the aminopeptidases from Ostrinia nubilalis with 96%, 97%, 96%, and 96% sequence identity. Compared with APN sequences of ACB-BtS, there were 9, 5, 10, and 12 amino acid mutations in the deduced protein sequences of Ofapn1, Ofapn2, Ofapn3, and Ofapn4 in ACB-AbR, respectively. In addition, there was one more O-glycosylation site in Ofapn1r. At the non-translatable 3′-end region, a deletion of 11 nucleotides (positions 2828-2838) and an insert of 9 nucleotides (postion 2893-2901) occurred in the Ofapn2r. The expression of Ofapn1, Ofapn2, Ofapn3, and Ofapn4 mRNA were 2.03-fold, 1.61-fold, 2.69-fold, and 2.62-fold higher in ACB-AbR than in ACB-BtS. This suggest that the reduced susceptibility to Cry1Ab toxins in ACB-AbR strain may due to the increased ability of detoxification and repair effect of epithelial cell.更多还原