【作者】 李艳梅；

【导师】 赵奎军；

【作者基本信息】 东北农业大学 ， 作物遗传育种， 2009， 博士

【摘要】 转基因棉花的大规模种植对控制鳞翅目害虫的为害发挥了重要作用,但害虫长期处于转基因作物Bt毒素的高压选择下,害虫对Bt作物的抗性问题将不容忽视。昆虫对Bt的抗性是一种遗传性状,其抗性基因就位于昆虫体内,参与体内各种代谢过程。目前,还没有一致的结论阐明昆虫对Bt毒素的抗性机制,其抗性机制是复杂和多样的。害虫抗性的遗传机制研究不仅有助于了解抗性本质和特征,而且是制定抗性治理策略的基础,是研究抗性的一个重要工具。无论是昆虫抗性适合度研究,还是昆虫抗性机理研究,试验材料都是非常重要的。通常研究害虫抗性都是通过比较抗、感品系之间的差异,所以尽可能缩小二者之间的差异,可以更大概率的找到抗性差异,这对研究害虫抗性,尤其是研究害虫抗性基因的DNA分子遗传标记是至关重要的。近等基因系(NIL)是一组遗传背景相同或相近,只在个别染色体区段上存在差异的品系,如果一对近等基因系在目标性状上表现差异,那么,凡是能在这对近等基因系间揭示多态性的分子标记,就可能位于目标基因的附近。因此,利用近等基因系材料,可以寻找与目标基因紧密连锁的分子标记。通过10多年的室内筛选,本研究室已经获得了对Bt具有较高抗性的棉铃虫种群,正在进一步研究其抗性机制。用大约一年半的时间,我们建立了棉铃虫抗性近等基因系。经过一系列回交和抗性再筛选,我们得到一个与敏感品系整个基因组具有至少93%的同源性、又具有较高抗性倍数的抗性近等基因系。以棉铃虫室内Bt抗性品系、敏感品系为试验材料,我们研究了棉铃虫对Bt毒素抗性的分子遗传机理。体重抑制法测定结果表明,用含区分剂量1.5μg/mL CrylAc毒料饲喂棉铃虫回交后代(BC)5d后,存活虫数:死亡虫数的比例为1:1,证实该虫对Bt毒素CrylAc的抗性为常染色体上不完全隐性遗传。利用AFLP分子标记技术,研究棉铃虫对Bt毒素CrylAc抗性的分子遗传机理,结果发现抗性基因BtR与AFLP标记EaaMctc2连锁,位于第4连锁群上EaaMctc2与EcaMcac1两个多态性标记之间,遗传距离分别为12.2cM±6cM和13.1cM±6cM。对标记EaaMctc2回收、克隆、测序,仅出现对于301bp处的单一条带。序列分析结果表明,该片段与GenBank中已知序列同源性较低。昆虫对Bt产生抗性后,在没有选择压力时,有些昆虫的抗性表现出不同程度的衰退,而有些则趋于稳定。棉铃虫抗性稳定性研究结果表明,在没有Bt选择压下,即用未加入Bt毒素的正常人工饲料饲养棉铃虫抗性品系五代,抗性倍数呈现快速下降的趋势,从3626.67倍下降到1033.33倍,下降了2593.34倍,抗性极不稳定。更多还原

【Abstract】 The wide spread growth of Bacillus thuringiensis cotton has played an important role in Lepidopteran pests control.But because pests have been selected under the pressure of high-dose Bt toxin which produced by Bt cotton for a long time, thereby the resistance evolution of pests to Bt cotton should not be ignored. The resistance of insect pests is a genetic character; its resistance genes are in the body of insects and they participate in various kinds of metabolic processes. At present, there are no unanimous conclusions that can elucidate the molecular mechanism of Bt resistance, because it is so complex and variable. The genetic analysis of insect resistance can not only help to understand the resistance character and mechanism but also creat the basic to design the resistance management strategy.It is an important tool to investigate insect resistance. The experimental materials are very critical to research either on fitness costs or mechanisms of resistance. To study insect resistance always need to compare the differences between resistant lines and their susceptible parental lines, so it is very important to reduce the differences between them as soon as possible, which can ensure to find the resistant differences with more probability.This is important for studying on the resistance of insect, especially for researching on the DNA molecular genetic markers on resistance. The near-isogenic lines are the strains that have the same or close genetic backgrounds; they just have differences in one or two chromosome section（s）. All of the markers that can reveal the polymorphisms between a pair of near-isogenic lines may locate in the vicinity of the goal traits, if there are differences in goal traits between them. Therefore, using the near-isogenic lines materials, we can find the molecular markers which tightly linked to the goal genes.By selection resistance for more than ten years in our laboratory, we have obtained a Bt resistant cotton bollworm （Helicoverpa armigera Hübner） strain with high level resistance to Bt Cry1Ac.Now the further resistance mechanism are being researched. A near-isogenic line of cotton bollworm was gotted using serial crossing and reseletion of the resistant allele in almost 1.5 years.This resistant strain （NIL₄） has at least 93% genetically homogeneity with the susceptible strain, and also has high resistance to Cry1Ac.In this dissertation, the molecular genetic mechanism of resistance to Bt in H.armigera were studied using the resistant （BtR） and susceptible （96s） strain as the materials. The determination results of weight inhibition method showed that the ratio between the survival and the dead in the backcross population （BC） of H. armigera was 1:1 after feeding diet with 1.5μg/mL Cry1Ac, a determining dose between resistant colony and heterozygote colony for 5d.This approved that the resistance genetic model was incompletely recessive autosomal heredity.AFLP method was applied to map the resistant gene marker in H. armigera to Bt toxin Cry1Ac on the basis of previous studies. The results showed that BtR gene flanked with two AFLP markers, EaaMctc2 and EcaMcac1, on the fourth linkage group in H. armigera, with 12.2cM±6cM and 13.1cM±6cM linkage distance. The AFLP marker EaaMctc2 was collected, cloned, and sequenced, and a band with 301bp length was obtained. The BLAST result showed that the sequence had low homology with other sequences in GenBank.After insects have evolved resistance, if there has no selection pressure, the resistance level will decline in some insect, whereas the resistance in other insects shows stable. We tested the resistance stability of H.armigera to Bt, the results showed that the resistance ratio declined rapidly in the absence of exposure of Cry1Ac 5 generations, the resistance ratio dropped to 1033.33-fold from 3626.67-fold.The resistance of H.armigera to Bt was not extremely stable.更多还原