棉铃虫对转基因抗虫棉的抗性筛选、监测及抗性机理研究
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摘要
转基因抗虫棉的大面积种植,有效地控制了棉铃虫的暴发为害,获得了显著的社会经济效益,与此同时,其环境安全性问题尤其是抗性风险问题备受国内外关注。本文以室内连续筛选17代的棉铃虫抗性品系(AYBC、AYBT)和对照品系(AYCK)为基础,运用人工饲料添加抗虫棉种子粉的方法继续筛选至26代,并以此为试虫,研究了不同品系抗性适合度的差异和棉铃虫体内与抗性相关的13种酶活性变化,应用RAPD技术分析了种群的遗传分化程度;监测了2007~2009年黄河流域主产棉区棉铃虫田间种群的耐受性变化和抗性基因频率;并对2009年田间种群的遗传分化程度进行了RAPD分析。取得了如下的主要结果和结论:
1、室内连续筛选26代后,与AYCK品系相比,AYBC和AYBT品系的幼虫发育历期明显延长,蛹重、产卵量、卵孵化率均明显降低,蛹历期和雌虫百分率差异不显著;与AYBT品系相比,AYBC品系的幼虫发育历期明显延长,产卵量明显降低,蛹重、蛹历期、雌虫百分率和卵孵化率差异不显著。AYBC品系和AYBT品系对Cry1Ac毒素的抗性均呈波动上升趋势,抗性倍数分别上升到5.63和11.44。表明转双价基因抗虫棉对棉铃虫抗性的产生具有延缓作用。
2、对AYBC、AYBT和AYCK品系第26代棉铃虫幼虫中肠内的解毒酶、保护酶和蛋白酶进行活性测定。解毒酶测定结果表明,与AYCK品系相比,AYBC品系的GSTs和ACP活性明显降低,ANAE和ALP的活性明显升高;AYBT品系的AchE的活性显著降低,GSTs活性显著升高;其它酶变化不明显。与AYBT品系相比,AYBC品系的GSTs和ACP活性明显降低,其它酶类活性明显上升。保护酶测定结果表明,与AYCK品系相比,AYBC和AYBT品系的SOD、POD和CAT的活性均显著升高;与AYBT品系相比,AYBC品系3种酶的活性均显著降低。蛋白酶测定结果表明,与AYCK品系相比,AYBC和AYBT品系的胰凝乳蛋白酶和弱碱性类胰蛋白酶活性均明显降低;AYBT品系的强碱性类胰蛋白酶和APN活性均显著升高。与AYBT品系相比,AYBC品系的强碱性胰蛋白酶和弱碱性胰蛋白酶的活性降低。
3、利用改进的单雌系F1/F2代法,系统监测了2007~2009年安阳、武城和威县的棉铃虫田间种群对Cry1Ac的耐受性变化和抗性基因频率。结果表明,3个地区中武城种群对Cry1Ac的耐受水平最高,且呈下降趋势;安阳种群次之,且呈逐年上升趋势;威县种群最低,且年际间变化不显著。保守估计了安阳、武城和威县三个地区的抗性基因频率分别为0~1.03‰、0.95‰~1.03‰和0,表明抗性基因频率仍处于正常水平。
4、优化了棉铃虫RAPD-PCR反应体系,分析了第26代室内筛选的棉铃虫种群和2009年安阳、武城、威县田间种群的遗传分化程度。结果表明,室内种群AYBT、AYBC和AYCK聚类的遗传距离分别为0.161、0.177和0.213,三个品系最终在0.402处成聚,说明筛选26代后,AYBT、AYBC与AYCK出现了遗传分化。安阳、武城和威县田间种群聚类的遗传距离分别为0.524、0.437和0.446,三个地区种群最终在0.610处成聚,说明三个地区间的棉铃虫种群出现了明显的地域分化,地区间各自成聚。
The transgenic cotton expressing the Cry1Ac toxin shows an excellent effect on controlling the occurrence and damage of cotton bollworm. Meanwhile, the cultivation of Bt cotton also brought potential ecological and environmental security risk, in particular, the risk of resistance to transgenic cotton has been much attention at nation and abroad. The effects of transgenic cotton on cotton bollworm fitness, enzyme activities and genetic differentiation, at the same time, detection for the frequency of resistance to Cry1Ac in field were studied by lab feeding, chemistry analyzing, field monitoring from the view of biology, biochemistry, molecular biology. This paper based on cotton bollworm resistant strains (AYBC, AYBT) and the control strain (AYCK) which selected 17 generations by artificial feed mixed cotton seed powder, used the same method continued screening to 26th generation. And use it as test insects, the effects of transgenic cotton on cotton bollworm fitness, enzyme activities and genetic differentiation, as well as, monitoring for the frequency of resistance to Cry1Ac in the Yellow River Basin during 2007~2009 were studied by lab feeding, chemistry analyzing, field monitoring from the view of biology, biochemistry, molecular biology. The main results and conclusions as following:
1. After 26 generations of selection, compared with the susceptible strain, in the AYBC and AYBT strain, larval development period was delayed; pupal weight, fecundity and egg hatching rate were reduced. Compared with AYBT strain, larval development period was delayed; fecundity was reduced. After 26 generations of selection, resistance to Cry1Ac of the AYBC strain and AYBT strain increased to 5.63 fold and 11.44 fold, respectively, compared with that of the susceptive strain. The analysis of changes in resistance of these two strains revealed that the screening material in AYBC strain could effectively delay the evolution of resistance to Cry1Ac in bollworm.
2. We determined enzymes activities in larvae of H.armigera which from AYBC strain and AYBT strain. The results have demonstrated that: compared with the control, in AYBC strain, the activities of GSTs, ACP, chymotrypsin-like enzyme and weak alkaline trypsin-like enzyme were reduced; the activities ofα-naphthyl acetate esterase, ALP and three protective enzymes (SOD, CAT, and POD) were increased. In AYBT strain, the activities of AchE, chymotrypsin-like enzyme and weak alkaline trypsin-like enzyme were reduced; the activities of GST, SOD, POD, CAT, APN and active alkaline trypsin were increased. Compared with the AYBT strain, in AYBC strain, the activities of two trypsin-like enzymes were reduced; activity of chymotrypsin-like was not obvious change.
3.During 2007~2009, we used genetic method of isofemale lines F1/F2 to detect the frequency of Bt resistance genes in Helicoverpa armigera populations which collected from Anyang County, Weixian County and Wucheng County. The results show that the tolerance levels of Helicoverpa armigera to Cry1Ac was highest and tended to decline during 2008~2009 in Wucheng population, higher and tended to increase year by year in Anyang population, and have not significant change in Weixian population.
4. The optimal RAPD system for H.armigera was established. Based on this, the study used RAPD technique to analyze the genetic differentiation of populations from lab screening and field in 2009. The results show that, in population from lab screening, the genetic distance of clustering populations was 0.161 for AYBT strain, 0.177 for AYBC strain, and 0.213 for AYCK strain. Each population was clustered at first, and the genetic distance (0.402) of clustering for one species. The above result indicated that there was a significant genetic differentiation after 26 generations of selection. In field population, the genetic distance of clustering populations was 0.524 for Anyang population, 0.437 for Wucheng population, and 0.446 for Weixian population. Each population was clustered at first, and the genetic distance (0.610) of clustering for one species. The above result indicated that there was a clear geographical differentiation among the populations from three regions.
1、室内连续筛选26代后,与AYCK品系相比,AYBC和AYBT品系的幼虫发育历期明显延长,蛹重、产卵量、卵孵化率均明显降低,蛹历期和雌虫百分率差异不显著;与AYBT品系相比,AYBC品系的幼虫发育历期明显延长,产卵量明显降低,蛹重、蛹历期、雌虫百分率和卵孵化率差异不显著。AYBC品系和AYBT品系对Cry1Ac毒素的抗性均呈波动上升趋势,抗性倍数分别上升到5.63和11.44。表明转双价基因抗虫棉对棉铃虫抗性的产生具有延缓作用。
2、对AYBC、AYBT和AYCK品系第26代棉铃虫幼虫中肠内的解毒酶、保护酶和蛋白酶进行活性测定。解毒酶测定结果表明,与AYCK品系相比,AYBC品系的GSTs和ACP活性明显降低,ANAE和ALP的活性明显升高;AYBT品系的AchE的活性显著降低,GSTs活性显著升高;其它酶变化不明显。与AYBT品系相比,AYBC品系的GSTs和ACP活性明显降低,其它酶类活性明显上升。保护酶测定结果表明,与AYCK品系相比,AYBC和AYBT品系的SOD、POD和CAT的活性均显著升高;与AYBT品系相比,AYBC品系3种酶的活性均显著降低。蛋白酶测定结果表明,与AYCK品系相比,AYBC和AYBT品系的胰凝乳蛋白酶和弱碱性类胰蛋白酶活性均明显降低;AYBT品系的强碱性类胰蛋白酶和APN活性均显著升高。与AYBT品系相比,AYBC品系的强碱性胰蛋白酶和弱碱性胰蛋白酶的活性降低。
3、利用改进的单雌系F1/F2代法,系统监测了2007~2009年安阳、武城和威县的棉铃虫田间种群对Cry1Ac的耐受性变化和抗性基因频率。结果表明,3个地区中武城种群对Cry1Ac的耐受水平最高,且呈下降趋势;安阳种群次之,且呈逐年上升趋势;威县种群最低,且年际间变化不显著。保守估计了安阳、武城和威县三个地区的抗性基因频率分别为0~1.03‰、0.95‰~1.03‰和0,表明抗性基因频率仍处于正常水平。
4、优化了棉铃虫RAPD-PCR反应体系,分析了第26代室内筛选的棉铃虫种群和2009年安阳、武城、威县田间种群的遗传分化程度。结果表明,室内种群AYBT、AYBC和AYCK聚类的遗传距离分别为0.161、0.177和0.213,三个品系最终在0.402处成聚,说明筛选26代后,AYBT、AYBC与AYCK出现了遗传分化。安阳、武城和威县田间种群聚类的遗传距离分别为0.524、0.437和0.446,三个地区种群最终在0.610处成聚,说明三个地区间的棉铃虫种群出现了明显的地域分化,地区间各自成聚。
The transgenic cotton expressing the Cry1Ac toxin shows an excellent effect on controlling the occurrence and damage of cotton bollworm. Meanwhile, the cultivation of Bt cotton also brought potential ecological and environmental security risk, in particular, the risk of resistance to transgenic cotton has been much attention at nation and abroad. The effects of transgenic cotton on cotton bollworm fitness, enzyme activities and genetic differentiation, at the same time, detection for the frequency of resistance to Cry1Ac in field were studied by lab feeding, chemistry analyzing, field monitoring from the view of biology, biochemistry, molecular biology. This paper based on cotton bollworm resistant strains (AYBC, AYBT) and the control strain (AYCK) which selected 17 generations by artificial feed mixed cotton seed powder, used the same method continued screening to 26th generation. And use it as test insects, the effects of transgenic cotton on cotton bollworm fitness, enzyme activities and genetic differentiation, as well as, monitoring for the frequency of resistance to Cry1Ac in the Yellow River Basin during 2007~2009 were studied by lab feeding, chemistry analyzing, field monitoring from the view of biology, biochemistry, molecular biology. The main results and conclusions as following:
1. After 26 generations of selection, compared with the susceptible strain, in the AYBC and AYBT strain, larval development period was delayed; pupal weight, fecundity and egg hatching rate were reduced. Compared with AYBT strain, larval development period was delayed; fecundity was reduced. After 26 generations of selection, resistance to Cry1Ac of the AYBC strain and AYBT strain increased to 5.63 fold and 11.44 fold, respectively, compared with that of the susceptive strain. The analysis of changes in resistance of these two strains revealed that the screening material in AYBC strain could effectively delay the evolution of resistance to Cry1Ac in bollworm.
2. We determined enzymes activities in larvae of H.armigera which from AYBC strain and AYBT strain. The results have demonstrated that: compared with the control, in AYBC strain, the activities of GSTs, ACP, chymotrypsin-like enzyme and weak alkaline trypsin-like enzyme were reduced; the activities ofα-naphthyl acetate esterase, ALP and three protective enzymes (SOD, CAT, and POD) were increased. In AYBT strain, the activities of AchE, chymotrypsin-like enzyme and weak alkaline trypsin-like enzyme were reduced; the activities of GST, SOD, POD, CAT, APN and active alkaline trypsin were increased. Compared with the AYBT strain, in AYBC strain, the activities of two trypsin-like enzymes were reduced; activity of chymotrypsin-like was not obvious change.
3.During 2007~2009, we used genetic method of isofemale lines F1/F2 to detect the frequency of Bt resistance genes in Helicoverpa armigera populations which collected from Anyang County, Weixian County and Wucheng County. The results show that the tolerance levels of Helicoverpa armigera to Cry1Ac was highest and tended to decline during 2008~2009 in Wucheng population, higher and tended to increase year by year in Anyang population, and have not significant change in Weixian population.
4. The optimal RAPD system for H.armigera was established. Based on this, the study used RAPD technique to analyze the genetic differentiation of populations from lab screening and field in 2009. The results show that, in population from lab screening, the genetic distance of clustering populations was 0.161 for AYBT strain, 0.177 for AYBC strain, and 0.213 for AYCK strain. Each population was clustered at first, and the genetic distance (0.402) of clustering for one species. The above result indicated that there was a significant genetic differentiation after 26 generations of selection. In field population, the genetic distance of clustering populations was 0.524 for Anyang population, 0.437 for Wucheng population, and 0.446 for Weixian population. Each population was clustered at first, and the genetic distance (0.610) of clustering for one species. The above result indicated that there was a clear geographical differentiation among the populations from three regions.
引文
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