【作者】 陈庆园；

【导师】 沈晋良；

【作者基本信息】 南京农业大学 ， 农药学， 2005， 硕士

【摘要】 采用Bt棉叶片喂饲法对棉铃虫抗性品系（YCR）进行抗性筛选和保持,室内筛选YCR从第61代到72代。利用饲料感染法测定棉铃虫抗性品系的抗性倍数,结果表明:经室内12代筛选,YCR对21%MVP Ⅱ粉剂的抗性倍数稳定在2000倍左右。棉铃虫抗性品系得到保持和进一步的纯化,为研究棉铃虫对转Bt基因棉的抗性分子遗传机理提供了保证。 采用Bt棉叶片喂饲法和含Cry1Ac18.75μg/ml的饲料感染法,通过抗性、敏感亲本及其正、反交试验检测,建立了棉铃虫对转Bt基因棉抗性个体（rr）的检测标准。喂饲抗性品系（YCR）和敏感品系（HZS）亲本及正交F₁（YCR♀×HZS♂）、反交F₁′（YCR♂×HZS♀）后代的初孵幼虫。处理5天后的结果表明:喂饲Bt棉叶5天,F₁和F₁′存活幼虫的发育龄期为1龄到2龄初期,体重在0.1-0.7mg/头;HZS只有2头存活幼虫,龄期为1龄,体重均为0.1mg;YCR存活幼虫的80.9%发育龄期为2龄中期到3龄,体重≥0.8mg/头。喂饲含Cry1Ac 18.75μg/mL的饲料5天,F₁、F₁、HZS的存活幼虫体重均没有达到0.8mg/头,YCR存活幼虫体重≥0.8mg/头的个数（40头）占总处理虫数（50头）的80.0%,YCR、F₁、F₁′和HZS死亡率分别为（8.0±13.0）%、（66.0±11.4）%、（66.0±20.7）%和（88.0±10.6）%,YCR与F₁、F₁、HZS的死亡率差异极显著（p<0.01）.棉铃虫对转Bt基因棉的抗性个体（rr）检测标准为发育龄期达2龄中期,体重≥0.8mg/头。上述结果通过回交试验得到进一步验证。 在室内通过用Bt棉选育棉铃虫抗性品系的基础上,采用单雌系F₁代检测法检测了2004年河北邱县棉铃虫对Bt棉的抗性等位基因频率。结果表明:检测的131个单雌系中,有32个单雌系的存活幼虫达到抗性检测标准（即龄期达到2龄中期,体重≥0.8mg/头）,其河北邱县大田棉铃虫种群的抗性等位基因频率E（q）及方差Var（q）分别为12.4×10^-2和8.17×10^-4,95%的置信区间为（0.1526～0.0954）。 采用等位基因特异性PCR扩增技术对32个单雌系的达标幼虫进行检测。结果表明:9个单雌系为阳性,即河北邱县Bt棉棉铃虫大田种群中携带钙粘素cDNA大片断缺失的抗性等位基因频率E（q）及方差Var（q）,分别为3.76×10^-2和2.70×10^-4,95%的置信区间为（0.0054～0.0698）。更多还原

【Abstract】 Using a Bt cotton leaf-feeding method, selection and maintenance of cotton bollworm resistance strain (YCR) were conducted. YCR was selected from 61st to 72nd generation. Resistance ratio of YCR was measured with diet infection method. The results showed that, resistant ratio of YCR to 21% MVP II WP (only containing Cry1Ac active component) was stable -2000-fold during 12episodes of selection with Bt cotton. Levels of resistance was maintained and enhanced. It would provide a guarantee for the research work about molecular mechanism of cotton bollworm resistance to transgenic Bt cotton.Using a Bt cotton leaf-feeding and toxin-impregnated Cry1Ac 18.75μg/ml diet method, the resistance criterion of cotton bollworm to transgenic Bt cotton was established through experiment of reisistant, susceptible parent and reciprocal cross. After 5 days, the results showed that, survival larvae of F1 and F1’ developed to 1st~ initial-2nd instar and weighted 0.1mg~0.7mg/larvae. There were only two survival larvae of HZS that developed to 1st instar and weighted 0.1 mg. The 80.9% of surviving larvae of YCR developed to mid-2nd ~ 3rd instar and weighted≥0.8mg/larvae. The bioassay was carried out for five days with toxin-impregnated Cry1Ac 18.75μg/mL diet. The results showed that F1, F1’, and HZS developed to 1st~ initial-2nd instar and weighted 0.1~0.5mg. 80% of surviving larvae of YCR developed to mid-2nd~3rd instar and weighted≥0.8mgAarvae in treated total number (50 larvae). The mean mortality of YCR, F1, F1’ and HZS were, respectively, (8.0±13.0)%, (66.0±11.4)%, (66.0 ±20.7)% and (88.0±10.6)%, which of YCR was significantly different to which of F1, F1’ and HZS (P<0.01). Resistance larvae criterion of cotton bollworm to transgenic Bt cotton was that developmental stage and weight of survival larvae were mid-2nd instar and ≥ 0.8mg/larvae, respectively. The results were verified by backcross inheritance experiment.On the basis of resistant strain selected by Bt cotton, Applying isofemale lines F1 method detected the resistance alleles frequency of cotton bollworm to transgenic Bt cottonin Qiu County, Hebei Province, in 2004. The results showed that, there were survival larvae developed the resistance criterion (developmental stage mid-2nd instar and weight ≥ 0.8mg/larvae ) in 32 isofemale lines with detecting 131 isofemale lines. The E(q) and Var(q) of resistance alleles in field cotton bollworm resistance to transgenic Bt cotton were respectively 12.4 × 10-2 and 4.46 × 10-4,95% confidence interval was 0.1526-0.0954.Using the PASA (PCR amplification of specific alleles) method, the results showed that 9 isofemale lines were positive in 32 isofemale lines of F1 method. The E(q) and Var(q) of resistance alleles of cDNA lesion in cadherins in field cotton bollworm resistance to transgenic Bt cotton were respectively 3.76×10-2 and 2.70× 10-4, 95% confidence interval was 0.0054-0.0698.更多还原