抗Bt水稻的二化螟品系筛选和机制研究

Target insects have the potential to evolve resistance to Bt crops and threat the long-term application. Striped stem borer (Chilo suppressalis) is one of the target insects of Bt rice. It is less susceptive to Bt toxin and has the potential to evolve resistant population under continuous selection pressure. We have obtained tolerant populations of striped stem borer to Bt proteins through resistance selection in room, using larvae from Bt rice plant in the field as original individuals. In this study, we keep feeding these tolerant populations using Bt poisonous food in room and Bt rice plants in netting field, survey the change of susceptivity and livability on Bt rice, for the objective of obtaining striped stem borer strains resistant to Bt rice. Then, backcross population is founded using resistant strain for analyzing genetic characteristic of the resistance. Molecular markers are also explored to originally map the resistance locus. According to the results of mapping, we measure the activity of midgut protease in the strains with broad-spectrum resistance. The strains resistant to Cry1Ac are likely to have variation in midgut receptors, so we clone their cadherin protein and aminopeptidase N genes through PCR and RACE amplification. At last, the sequences make sure whether the resistance is caused by mutation on midgut receptor. This study has important significance for evaluating the risk of resistance and guiding the insect resistance management.

目标昆虫有对Bt 作物进化出抗性的风险，影响其可持续应用。二化螟是Bt 水稻防治的目标昆虫之一，对毒蛋白的敏感性较低，在持续的选择压下有形成抗性群体的潜力。我们以田间Bt 水稻植株上收集的二化螟幼虫为起始个体，室内用Bt 毒饲料对后代进行抗性筛选，获得了对蛋白敏感性降低的群体。本项目用室内毒饲料结合网室Bt 水稻对这些群体继续饲养，监测其敏感性和在Bt 水稻上存活率的变化，筛选对Bt 水稻产生抗性的二化螟品系。然后，利用抗性品系构建回交群体，分析抗性的遗传特性，并开发分子标记对单位点遗传的抗性基因进行初步定位。根据定位的结果，测定广谱抗性品系的中肠蛋白酶活性；对可能是蛋白结合受体变异的Cry1Ac 抗性品系，通过PCR 扩增结合RACE 技术克隆其钙粘蛋白和氨肽酶N 基因并测序，确定抗性是否由它们的突变引起。该研究对于评估二化螟对Bt 水稻进化出抗性的风险，指导昆虫抗性治理具有重要意义。

二化螟是Bt水稻防治的目标昆虫之一，对Bt蛋白的敏感性较低，有产生抗性的潜力，影响Bt水稻可持续应用。我们以田间Bt水稻植株上收集的二化螟幼虫为起始个体，在室内用Bt饲料进行抗性筛选，获得了对Cry1Ac、Cry1C和Cry2A敏感性分别降低的品系。本项目用室内Bt饲料和网室Bt水稻对这些品系继续进行筛选，得到了2个Cry1Ac抗性品系、2个Cry1C抗性品系和1个Cry2A抗性品系，抗性倍数分别达到6.29、4.54和5.27，都能够在Bt水稻植株上完成生活史。对每种Bt蛋白分别选择抗性倍数最高的品系，发现Cry1Ac抗性品系的总蛋白酶和强碱性蛋白酶活性显著增强，Cry2A抗性品系的类胰凝乳蛋白酶活性显著增强。3个抗性品系分别与敏感品系正反交，对F1代分析发现抗性的显性度分别为0.34、-0.58和0.25，表现为常染色体上的不完全显性或不完全隐性遗传。Cry1Ac和Cry2A抗性品系对另外两种Bt蛋白表现出明显的交叉抗性，Cry1C抗性品系没有表现出交叉抗性。当用不含Bt蛋白的饲料喂养时，3个抗性品系都存在不同程度的适合度代价，主要表现为幼虫历期和世代历期延长，幼虫存活率降低。随着筛选代数的增加，抗性品系的适合度代价变大。转录组分析发现Cry1Ac和Cry2A抗性品系中肠蛋白酶基因的表达出现了明显的上调，因此推测它们对Bt水稻的抗性是由中肠蛋白酶活性增强引起。Cry1C抗性品系中肠蛋白酶基因和几种主要的Bt蛋白中肠受体基因的表达水平没有出现明显的变化，其抗性应该是由其它的机制引起。本项目研究结果表明在持续的选择压下，二化螟具有对Bt水稻进化出抗性的潜力，涉及到的抗性机制复杂多样。但是由于受到抗性遗传特性和适合度代价的制约，二化螟难以在田间Bt水稻植株上形成稳定的抗性群体，产生抗性的风险较小。这些结论可以为二化螟的抗性治理提供依据，有助于Bt水稻的可持续利用。