【作者】 Bashir Lubna；

【导师】 崔金杰；

【作者基本信息】 中国农业科学院 ， 农业昆虫与害虫防治， 2014， 博士

【摘要】 从1997年,中国开始种植转Bt基因抗虫棉,2013年成为第二大转Bt基因抗虫棉种植国,种植面积达到420万公顷。转Bt基因抗虫棉的种植,有效地控制了棉铃虫的为害,但长期种植抗虫棉也带来棉铃虫对Bt毒蛋白产生抗性的风险。本研究利用生态学、昆虫毒理学和分子生物技术,对中棉所41、中棉所79和Bollgard Ⅱ等三种类型转基因抗虫棉的抗虫性及外源杀虫蛋白的表达量进行研究,并于2012-2013年对黄河流域棉区棉铃虫的抗性发展动态进行系统监测,同时对棉铃虫抗性相关基因钙粘蛋白的变异情况了进行了研究。主要研究结果如下：1.2012-2013年,对中棉所41、中棉所79和Bollgard Ⅱ等三种类型抗虫棉的抗虫性进行研究。三种类型转基因棉花均具有较好的抗虫性,但品种间和生育时期间的抗虫性存在差异。在苗期,三种类型抗虫棉抗虫效果都高于其他时期,中棉所41的抗虫效果最好,在2012年和2013年棉铃虫死亡率分别为87.98%和75.48%；在蕾期,Bollgard Ⅱ的抗虫效果最好,在2012年和2013年棉铃虫死亡率分别为49.56%和54.47%;在铃期,2012年Bollgard Ⅱ的抗虫效果最好,棉铃虫死亡率为39.51%,而2013年,中棉所41的抗虫效果最好,棉铃虫死亡率为47.68%。棉铃虫低龄幼虫对转基因棉花叶片敏感,取食转基因棉花叶片后,未死亡的棉铃虫低龄幼虫取食和消化均受到抑制,取食5天后,Bollgard Ⅱ叶片上的棉铃虫平均体重最大。以上结果说明,抗虫棉的抗虫效果存在发育时期、年度和品种间差异,这些差异能直接影响田间棉铃虫种群的抗性发展。2.转Bt基因抗虫棉主要通过表达Bt杀虫蛋白来抵御铃虫,Bt蛋白的表达量与抗虫性密切相关。为了研究中棉所41、中棉所79和Bollgard Ⅱ在大田生长季中Bt蛋白表达量变化情况,于2012-2013年对大田种植的三种棉花不同生长阶段Bt蛋白进行定量检测。在整个生长季,不同类型的转基因棉花中的Cry1Ac蛋白的表达量存在显著地差异(P<0.05)。CrylAc蛋白在棉花叶片中的表达量显著高于蕾和铃中,蛋白表达量随棉花的生长逐渐降低,这与它们的抗虫性变化趋势一致。在Bollgard Ⅱ中,整个生长季Cry2Ab蛋白的表达量显著高于CyrlAc蛋白;Cry2Ab蛋白在蕾和铃中的表达量显著高于叶片。Bollgard Ⅱ中Cry2Ab蛋白的表达量和CrylAc蛋白的表达量没有明显的相关性,试验数据表明在整个棉花生长季Cry2Ab蛋白的高表达没有减少CrylAc蛋白的表达量。以上结果说明外源杀虫蛋白的表达量与棉花的抗虫效果呈正相关,通过检测棉花外源杀虫蛋白的含量,可以推断出该品种的抗虫效果。3.2012-2013连续两年对中棉所41、中棉所79和Bollgard Ⅱ等3种类型转基因棉花和广泛应用的常规棉品种中棉所49的纤维品质进行了检测。结果显示这3种转基因棉花质量性状与中棉所49相比,除纤维强度和马克隆值外,没有明显的差异。所以种植这3种转基因抗虫棉不会影响棉花的纤维品质。外源基因对棉花纤维品质是否具有影响还需进一步研究。4.为了实时监测黄河流域棉区在长期种植转Bt基因抗虫棉后,田间棉铃虫种群的抗性发展动态,本研究对黄河流域棉区田间棉铃虫种群的抗性基因频率进行测定。在2010-2013年,分别从河南、河北、山东棉田采集棉铃虫成虫。使用含有1.Oμg/ml Cry1Ac蛋白的人工饲料,在室内对单雌家系F1/F2代抗性水平进行测定。2010年共对河南、山东、河北的58,43和12个家系成功进行生物测定；2011年共对河南、山东、河北的48,25和26个家系成功进行生物测定；2012年共对河南、山东、河北的96,36和17个家系成功进行生物测定；2013年共对河南、山东、河北的342,146和40个家系成功进行生物测定。总体而言,黄河流域棉区棉铃虫抗性仍处于较低的水平,2010—2013年三省抗性基因频率无明显的差异(2010:χ2=0.0001<χ20.052=5.99;2011:χ2=0.0001<χ20.052=5.99;2012:χ2=0.0001<χ20.052=5.99;2013:χ2=0.0001<χ20.052=5.99)。2010-2013年,棉铃虫F1代平均相对发育级别没有明显的上升。但为防止棉铃虫对Bt抗性升高,有必要种植转复合Bt基因抗虫棉花并结合其他防治策略来防治棉铃虫。5.本研究对采自于田间的棉铃虫抗性基因缺失突变情况进行了研究,以发展从分子水平监测棉铃虫抗性的方法。本研究期望筛选出田间抗性个体,但在2012-2013年没有检测到抗性个体。这一结果也符合田间棉铃虫种群抗性基因频率较低的监测结果。对4类在Bt饲料上发育级别不同的棉铃虫家系钙粘蛋白基因的全长序列进行克隆测序,钙粘蛋白全长5190bp,编码1730个aa,所有4类棉铃虫钙粘蛋白基因与NCBI注册的钙粘蛋白长度一致,没有发生缺失的情况。序列比对结果显示,4类棉铃虫钙粘蛋白氨基酸序列间存在突变位点。更多还原

【Abstract】 In China transgenic Bt(Bacillus thuringiensis) cotton has been broadly planted since1997, which makes the China second largest Bt cotton growing country with planting area of4.2million hectares in2013. For the control of H. armigera (Hubner)(Lepidoptera:Nocruidae) the planting of transgenic Bt cotton has been extensively successful and competent tool. Although, the cultivation of Bt crops on large-scale may be the source of evolution in pest resistance to Bt toxin. Therefore, the main object of this study was biosafety of transgenic Bt cotton after continuous cultivation of Bt cotton in Yellow River cotton growing region of China during2012-2013and appropriate understanding the evolution of resistance of H. armigera to Bt cotton. The present scientifically studies helps to understand the efficacy of different transgenic Bt cotton, expression level of Bt insecticidal proteins and their effect on the survival of H. armigera. Also in this study, we estimate the frequency of Cryl Ac resistance genes in H. armigera populations with logical monitoring and find variation in cadherin gene related with resistance in H. armigera. As well as this study will help to improve the biosafety Bt cotton crops. The main conclusions of the study were as follows:1. During three growing stages of Bt cotton, larval mortality of H. armigera were calculated on cotton leaf tissue of three transgenic Bt cotton varieties after five days of trail in2012and2013. The result indicates that transgenic Bt cotton are resistant to H. armigera during early stage of cotton but as plant grow up the mortality is declining with cotton crop age. The highest mortality%was recorded during seedling stage and less mortality%during boll stage of CCRI41and Bollgard Ⅱ but in CCR179the less mortality%was recorded in bud stage during2012-2013. During seedling stage, the higher mortality%was recorded in CCRI4187.97%and75.48%in2012-2013respectively. During bud stage, the maximum mortality%was recorded in Bollgard Ⅱ49.56%and54.47%in2012-2013, in boll stage; the highest mortality%was recorded39.51%in Bollgard Ⅱ in2012and highest mortality was recorded in CCRI4147.68%in2013. The data showed that bollworms were more susceptible to Bollgard Ⅱ and CCRI41than CCRI79. The average weight of each insect also calculated after five days of feeding on three transgenic Bt cotton varieties. The result indicates that Bt toxins which are present in the transgenic Bt cotton reduced the growth and development of the H. armigera larvae. Among all transgenic Bt cotton varieties the highest average of weight was recorded in Bollgard Ⅱ. These results suggested that differences in mortality%between developmental stages of Bt cotton in two years can directly affect the development of resistance in populations of H. armigera.2. B. thuringiensis produced insecticidal protein CrylAc which is an important natural biological agent for the control of H. armigera considered as one of the most important economic insect pests in many parts of the world. To quantify the expression level of CrylAc and Cry2Ab during different growth stages of cotton plant, three Bt cotton varieties CCRI41(Bt+CpTI), CCRI79(CrylAc) and Bollgard II (CrylAc+Cry2Ab) were planted in2012and2013. For the quantification of the CrylAc and Cry2Ab content Envirologix Qualiplate kit for CrylAc and Cry2Ab were used. The Result showed some clear significant differences in the amount of CrylAc protein present in various plant parts of transgenic Bt cotton throughout the growing season (P<0.05). The expression levels of CrylAc proteins in the leaves of transgenic Bt cotton was significantly higher than buds and bolls but gradually decreasing as plant grew. Result showed that in Bt cotton CCRI41(5/+CpTI) which is toxic to H. armigera the CrylAc and CpTI proteins together have synergistic effect which enhances the level of Cry lAc Protein. In2012, measure the level of Cry2Ab, result showed that in Bollgard II the Cry2Ab present at much higher level throughout the season in the plant compared with CrylAc. Data showed that at the same time in fruiting bodies the expression level of Cry2Ab is higher but the expression level of Cry2Ab was lower in leaves, in Bollgard II expression level of the two Cry proteins found to be different from one another. However, data proved that in Bollgard II both proteins were present throughout the season and the addition of Cry2Ab had no deleterious effect on levels of CrylAc. These results recommend that expression levels of insecticidal proteins are linked with the cotton growth.3. During2012and2013, the cotton fiber quality characters were tested to recognize the effect of different transgenic Bt cotton on the lint. Result shows that the fiber quality characters of transgenic cotton varieties were not significantly affected when compared with conventional non Bt variety. But in2013, fiber strength and micronaire are significantly affected. Finally it is concluded that there was no significant effect of transgenic Bt cotton on the fiber quality of cotton during2012and2013. To know the effect of transgenic Bt cotton on fiber qualities further research work will be carry on in future.4. For the biosafety and continuous cultivation of Bt cotton in Yellow River region of China, the most important to gain a timely understanding the evolution of resistance of H. armigera to Bt toxin and estimate the frequency of alleles conferring resistance to CrylAc toxin in field populations of H. armigera. Adult female moths of H. armigera were collected from Henan Province, Shandong Province and Hebei Province in2010-2013. The female moths trapped in the field used for screening through bioassay test of F1and F2generations. The females moths reared on a diet containing1.0μg/ml CrylAc to estimate the frequency of resistance alleles. In2010, totals58,43and12isofemale lines tested for the F1generation bioassay, total48,25and26isofemale lines for the F1generation bioassay in2011, total of96,36and17isofemale lines for the F1generation in2012, and342,146and40isofemale were screened out during2013from Henan, Shandong and Hebei Provinces respectively. Yellow River region is the largest growing region of cotton in China, result shows that resistance gene frequency was still very low, fluctuating and it did not increase significantly from2010to2013in Henan Province, Shandong Province and Hebei Province(2010:χ2=0.0001<χ200.052=5.99;2011:χ2=0.0001<χ20.053=5.99;2012:=0.0001<χ20.0052=5.99;2013:χ2=0.0001<χ20.0052=5.99).Result shows that in F, tests the relative average development rating (RADR) of H. armigera larvae had no substantial increase in Cry1Ac tolerance during the four years period. In H. armigera to keep away from further increases in Bt resistance frequency, it is necessary to introduce Bt cotton expressing several Bt toxins and put together this technology with other strategy for management of H. armigera.5. To identify the CrylAc binding protein in H. armigera, characterized the deletion mutation of field collected strain; which significantly monitor the changes in the frequency of resistance gene. We tried to identify the mutant in field populations of H. armigera, but no mutant was found in moths collected in2012-2013. This result is constant with the existing low gene frequency of Bt resistance in field population of H. armigera. The results shows sequencing of four types of H. armigera, the full length of H. armigera were5190bp that encoding1730Amino Acid proteins of the same length with bollworm cadherin sequence in NCBI registered bollworm cadherin (CAD accession AF519180.2). Sequences are of the same length and all sequences have no deletion mutations. Sequence alignments showed that amino acid sequences of four groups were different.更多还原