棉铃虫对转Bt基因棉的抗性等位基因频率及其监测方法研究
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摘要
棉铃虫(Helicoverpa armigera(Hübner))是危害棉花的一种重要世界性害虫，在我国由于大量使用化学农药防治，棉铃虫已对常用的几类杀虫剂均产生抗性，1998年，河北省首先大面积种植转Bt基因棉，由于转Bt基因棉在整个生长季都表达Bt杀虫蛋白，因此与Bt生物制剂相比，棉铃虫对Bt棉存在更易产生抗性的风险。鉴于室内用Bt棉筛选的棉铃虫抗性品系的抗性基因遗传特性为不完全隐性(孟凤霞博士论文)，无法用常规方法监测Bt棉大田棉铃虫种群的早期抗性，因此采用单雌系F_2代法，从2000年至2002年，连续三年监测了从1998年开始大面积种植转Bt基因棉的河北省威县大田棉铃虫对对新棉33~B的抗性等位基因频率的变化，并初步建立了可快速、简便检测抗性等位基因频率的单雌系F_1代法，以便为抗性治理和延长Bt棉的应用寿命提供科学依据。
1999年，本实验室何丹军用单雌系F_2代法检测到种植Bt棉一年地区(河北邱县)棉铃虫种群对转Bt基因棉的起始抗性基因频率为0.58％，2000年，我们再次应用单雌系F_2代法监测到大面积种植转Bt基因棉三年地区的河北省(威县和成安)大田棉铃虫对对新棉33~B的抗性等位基因频率已上升到3.3％。继续用F_2代法检测河北威县棉铃虫种群对新棉33~B的抗性等位基因频率，2001年为6.9％，2002年二代幼虫、三代卵、三代幼虫这三个种群分别为6.9％、7.8％和9.4％。监测结果表明随着棉花种植年数和田间棉铃虫取食Bt棉代次的的增加，河北棉铃虫种群对Bt棉的抗性等位基因频率在快速上升。
初步建立用于检测田间棉铃虫Bt棉抗性等位基因频率的单雌系F1代法，采集田间幼虫或卵，饲养至羽化后与室内筛选的对转Bt基因棉有极高抗性水平的纯合的抗性品系单对交配，每对棉铃虫的所有F_1代幼虫作为一个单雌系用新棉33~B检测其中有无抗性个体，抗性等位基因频率估计方法参照Andow and Alstad(2000)描述的Bayesian统计方法。用此法其监测到2002年威县Bt棉田棉铃虫对新棉33~B的抗性等位基因频率分别为8.8％(二代幼虫)、8.9％(三代卵)、9.3％(三代幼虫)，经统计分析，与用单雌系F_2代法的检测频率无显著差异，表明单雌系F_1代法检测棉铃虫对转Bt基因棉的抗性等位基因频率的灵敏度与F_2代相同，该法可用于检测田间棉铃虫对Bt棉的低水平抗性等位基因频率，且比单雌系F_2代法快速、经济。
威县棉铃虫对MVPⅡ的抗性水平测定结果表明：参照孟凤霞测定的敏感基线，威县棉铃虫各种群对有效成分为Cry1Ac毒素的21％MVPⅡ可湿性粉剂的抗性
棉铃虫对转Bt基因棉的抗性等位基因频率及其监测方法研究
倍数分别为7．2倍（2001年三代幼虫）、7．1倍（2002年二代幼虫）、6．9倍（2002
年三代卵L10．9倍（2o02年三代幼虫），2O02年对各种群对20删w11胶悬剂抗
性分别为15．3倍（2002年二代幼虫）、N．5倍（2m2年三代卵）和28．O倍（2002
年三代幼虫），表明随着棉花种植年数和田间棉铃虫取食Bt棉代次的的增加，威
县棉铃虫对Bt棉的杭性水平迅速上升，这一结果与应用单雌系F；代法和F；代法
检测出田间抗性等位基因频率上升的趋势相一致。
根据对河北省威县棉铃虫的抗性监测的结果，我们认为，目前河北省采取的
类似高剂量／庇护区的抗性治理对策对延缓棉铃虫对Bt棉的抗性发展存在不足，
有必要及时调整策略，实施合理的抗性治理措施，否则将失去进行预防性抗性治
理的最后机会；对转 B t基因棉种植二至三年以上的地方，应立即开展抗性监测
工作，尽早设计和实施抗性治理对策。
Cotton bollworm (Helicoverpa armigera (Hubner)) was an important worldwide pest that damaged cotton. In China, cotton bollworm had already developed resistance to several kinds of insectisides because of the slather using of chemical pestiside. Since 1998, genetically engineered cotton ( Bt cotton ) that produced an insecticidal protein originally found in the biocontrol agent, Bacillus thuringiensis (Bt), has been planted millions hectares every year in China. Because these toxins were produced by the plants from seedling stage to harvest, there would be intense selection for the resistant genotypes of the pest as the acreage planted to this transgenic cotton increase. Therefore, widespread adoption of Bt cotton increase the risk that pest would evolve resistance than that of the Bt biocontrol agents. Doctor Fengxia Meng had studied that the inheritance trait of resistance in laboratory selected cotton bollworm strain was incompletely recessive, so there were no proper general methods to monitor the early resistance in cotton bollworm population to Bt cotton . From 2000 to 2002, we used F_(2) method of isofemale lines (F2 screen) to monitor the change of resistance alleles frequency in the field population of cotton bollworm to transgenic Bt cotton, NuCOTN 33" in Heibei Province where Bt cotton had planted since 1998 . Furthermore, we established an F1 method of isofemale lines to detect quickly and conveniently the frequency of resistance alleles to transgenic cotton in field cotton bollworm population. The goals of our research were to offer scientific evidences for resistance management and to prolong the application of Bt cotton.
In 1999, He et al used the F2 method to detect the frequency of resistance alleles to Bt toxin from transgenic Bt cotton, NuCOTN 33" , in cotton bollworm. The results showed that the resistance alleles frequency in the natural population of cotton bollworm reached to >0. 58%, in Qiu County,
Hebei Province .where transgenic Bt cotton had been planted for only one year. In 2000, we monitored the resistance allele frequency to transgenic Bt cotton, NuCOTN 33" in the field population of cotton bollworm with F2 method, in Wei Xian county and Chengan county, Hebei Province, where transgenic Bt cotton had been planted for three years. And the frequency of resistance allele of the bollworm population had increase to 3.3%. We continued to monitor the frequency of resistance alleles in cotton bollworm to Bt cotton, NuCOTN 33" by F2method, in Wei County, Hebei Province. In 2001, the frequency increased to 6. 9% (in larvae of the third generation); in 2002,the frequency was 6. 9 % (in larvae of the second generation), 7.8% (in eggs of the third generation) and 9.4% (in larvae of the third generation) in field. We could conclude that the frequency of resistance was rising quickly with the time of cotton planted increasingo
The F1 method of isofemale lines was established to detect the frequency of resistance alleles to transgenic cotton in field cotton bollworm population. Virgin females or males that developed from larvae and eggs collected in the cotton field were individually mated to virgin males or females from laboratory resistance strain, respectively. The genotypes of the offspring of all the F1 progeny of each isofemale lines were determined in a Bt cotton assay, the expected frequency and confidence intervals of resistance alleles were calculated using Bayesian statistics described by Andow and Alstad (2000). The results of the monitoring showed that the frequency of resistance alleles to transgenic Bt cotton in the field populations were 8. 8% (in larvae of field second generation), 8. 9 % (in eggs of field third generation) and 9.3% (in larvae of field third generation) in Wei County in 2002, which had no distinguish difference with the research result of the F. method by statistical analysis. The data suggested that the F, method had the same sensibility as that of the F2 method when they were applied to detect (monitor) the rare resistance allele, and the F1 method was mo
1999年，本实验室何丹军用单雌系F_2代法检测到种植Bt棉一年地区(河北邱县)棉铃虫种群对转Bt基因棉的起始抗性基因频率为0.58％，2000年，我们再次应用单雌系F_2代法监测到大面积种植转Bt基因棉三年地区的河北省(威县和成安)大田棉铃虫对对新棉33~B的抗性等位基因频率已上升到3.3％。继续用F_2代法检测河北威县棉铃虫种群对新棉33~B的抗性等位基因频率，2001年为6.9％，2002年二代幼虫、三代卵、三代幼虫这三个种群分别为6.9％、7.8％和9.4％。监测结果表明随着棉花种植年数和田间棉铃虫取食Bt棉代次的的增加，河北棉铃虫种群对Bt棉的抗性等位基因频率在快速上升。
初步建立用于检测田间棉铃虫Bt棉抗性等位基因频率的单雌系F1代法，采集田间幼虫或卵，饲养至羽化后与室内筛选的对转Bt基因棉有极高抗性水平的纯合的抗性品系单对交配，每对棉铃虫的所有F_1代幼虫作为一个单雌系用新棉33~B检测其中有无抗性个体，抗性等位基因频率估计方法参照Andow and Alstad(2000)描述的Bayesian统计方法。用此法其监测到2002年威县Bt棉田棉铃虫对新棉33~B的抗性等位基因频率分别为8.8％(二代幼虫)、8.9％(三代卵)、9.3％(三代幼虫)，经统计分析，与用单雌系F_2代法的检测频率无显著差异，表明单雌系F_1代法检测棉铃虫对转Bt基因棉的抗性等位基因频率的灵敏度与F_2代相同，该法可用于检测田间棉铃虫对Bt棉的低水平抗性等位基因频率，且比单雌系F_2代法快速、经济。
威县棉铃虫对MVPⅡ的抗性水平测定结果表明：参照孟凤霞测定的敏感基线，威县棉铃虫各种群对有效成分为Cry1Ac毒素的21％MVPⅡ可湿性粉剂的抗性
棉铃虫对转Bt基因棉的抗性等位基因频率及其监测方法研究
倍数分别为7．2倍（2001年三代幼虫）、7．1倍（2002年二代幼虫）、6．9倍（2002
年三代卵L10．9倍（2o02年三代幼虫），2O02年对各种群对20删w11胶悬剂抗
性分别为15．3倍（2002年二代幼虫）、N．5倍（2m2年三代卵）和28．O倍（2002
年三代幼虫），表明随着棉花种植年数和田间棉铃虫取食Bt棉代次的的增加，威
县棉铃虫对Bt棉的杭性水平迅速上升，这一结果与应用单雌系F；代法和F；代法
检测出田间抗性等位基因频率上升的趋势相一致。
根据对河北省威县棉铃虫的抗性监测的结果，我们认为，目前河北省采取的
类似高剂量／庇护区的抗性治理对策对延缓棉铃虫对Bt棉的抗性发展存在不足，
有必要及时调整策略，实施合理的抗性治理措施，否则将失去进行预防性抗性治
理的最后机会；对转 B t基因棉种植二至三年以上的地方，应立即开展抗性监测
工作，尽早设计和实施抗性治理对策。
Cotton bollworm (Helicoverpa armigera (Hubner)) was an important worldwide pest that damaged cotton. In China, cotton bollworm had already developed resistance to several kinds of insectisides because of the slather using of chemical pestiside. Since 1998, genetically engineered cotton ( Bt cotton ) that produced an insecticidal protein originally found in the biocontrol agent, Bacillus thuringiensis (Bt), has been planted millions hectares every year in China. Because these toxins were produced by the plants from seedling stage to harvest, there would be intense selection for the resistant genotypes of the pest as the acreage planted to this transgenic cotton increase. Therefore, widespread adoption of Bt cotton increase the risk that pest would evolve resistance than that of the Bt biocontrol agents. Doctor Fengxia Meng had studied that the inheritance trait of resistance in laboratory selected cotton bollworm strain was incompletely recessive, so there were no proper general methods to monitor the early resistance in cotton bollworm population to Bt cotton . From 2000 to 2002, we used F_(2) method of isofemale lines (F2 screen) to monitor the change of resistance alleles frequency in the field population of cotton bollworm to transgenic Bt cotton, NuCOTN 33" in Heibei Province where Bt cotton had planted since 1998 . Furthermore, we established an F1 method of isofemale lines to detect quickly and conveniently the frequency of resistance alleles to transgenic cotton in field cotton bollworm population. The goals of our research were to offer scientific evidences for resistance management and to prolong the application of Bt cotton.
In 1999, He et al used the F2 method to detect the frequency of resistance alleles to Bt toxin from transgenic Bt cotton, NuCOTN 33" , in cotton bollworm. The results showed that the resistance alleles frequency in the natural population of cotton bollworm reached to >0. 58%, in Qiu County,
Hebei Province .where transgenic Bt cotton had been planted for only one year. In 2000, we monitored the resistance allele frequency to transgenic Bt cotton, NuCOTN 33" in the field population of cotton bollworm with F2 method, in Wei Xian county and Chengan county, Hebei Province, where transgenic Bt cotton had been planted for three years. And the frequency of resistance allele of the bollworm population had increase to 3.3%. We continued to monitor the frequency of resistance alleles in cotton bollworm to Bt cotton, NuCOTN 33" by F2method, in Wei County, Hebei Province. In 2001, the frequency increased to 6. 9% (in larvae of the third generation); in 2002,the frequency was 6. 9 % (in larvae of the second generation), 7.8% (in eggs of the third generation) and 9.4% (in larvae of the third generation) in field. We could conclude that the frequency of resistance was rising quickly with the time of cotton planted increasingo
The F1 method of isofemale lines was established to detect the frequency of resistance alleles to transgenic cotton in field cotton bollworm population. Virgin females or males that developed from larvae and eggs collected in the cotton field were individually mated to virgin males or females from laboratory resistance strain, respectively. The genotypes of the offspring of all the F1 progeny of each isofemale lines were determined in a Bt cotton assay, the expected frequency and confidence intervals of resistance alleles were calculated using Bayesian statistics described by Andow and Alstad (2000). The results of the monitoring showed that the frequency of resistance alleles to transgenic Bt cotton in the field populations were 8. 8% (in larvae of field second generation), 8. 9 % (in eggs of field third generation) and 9.3% (in larvae of field third generation) in Wei County in 2002, which had no distinguish difference with the research result of the F. method by statistical analysis. The data suggested that the F, method had the same sensibility as that of the F2 method when they were applied to detect (monitor) the rare resistance allele, and the F1 method was mo
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