【作者】 韩超；

【导师】 翟保平； 陈法军； 韩兰芝；

【作者基本信息】 南京农业大学 ， 生态学， 2011， 硕士

【摘要】 二化螟Chilo supperssalis (Walker)和大螟Sesamia inferens (Walker)均为我国水稻生产上的重要害虫。随着水稻耕作制度的变更、害虫抗药性的产生和气候变暖等因素,二化螟和大螟种群的发生呈逐年上升趋势。转Bt基因抗虫水稻的成功研制为螟虫的防治提供了新策略。然而,Bt水稻所引发的生态安全问题是制约其商业化生产的重要因子。Bt杀虫晶体蛋白的种类不同,其杀虫谱也不同。Bt Cry晶体蛋白对二化螟具有较高的毒力效果,但对具有相近生态位的大螟毒力效果显著下降。田间试验也发现,转Bt Cry基因水稻对二化螟的控制效果显著高于大螟,可能引起转基因稻田害虫发生地位的变化。基于此,本文以二化螟、大螟和产业化前景的转Bt基因水稻为研究对象,系统开展了大螟人工饲养技术研究；Bt水稻对二化螟和大螟存活和营养利用的影响及二化螟和大螟对不同Bt毒蛋白的敏感基线研究。该研究结果可望明确二化螟和大螟对Bt水稻的耐受性差异,为害虫的抗性风险治理及Bt水稻的可持续应用和健康发展提供科学依据。主要研究结果如下：1.利用大豆粉、玉米粉、麦胚和鲜茭白等成分研制了大螟的半合成人工饲料,在人工饲料的基础上发展了大螟的人工饲养,即初孵幼虫-2日龄幼虫在茭白上饲养,3日龄-化蛹在人工饲料上饲养。利用该方法连续饲养大螟3代,幼虫的发育历期、蛹重、幼虫存活率、化蛹率、羽化率、卵孵化率和单雌产卵量等生活史参数与在天然饲料茭白上饲养的大螟相比,二者没有任何显著差异。而且利用该方法饲养,成本低、省工省力,能显著减少病原菌的感染。2.测定了Bt水稻(TT51、Cry2Aa)对鳞翅目害虫二化螟和大螟存活及营养利用影响。离体茎测定结果表明,Bt水稻TT51和Cry2Aa对二化螟和大螟均有较好的抗虫效果,6天后初孵幼虫死亡率几乎达100%；从幼虫取食第2d和第4d的校正死亡率来看,大螟在两转基因水稻的校正死亡率显著低于二化螟,表现出较高的耐受性。营养利用指标测定结果表明,大螟在不同生育期的两种转Bt基因水稻上的校正相对增长率(CRGR)明显高于二化螟或与二化螟差异不显著,但其校正相对取食量(CRCR)和校正近似消化率(CAD)低于二化螟或与二化螟差异不显著.这说明大螟在降低相对增长率,减少相对取食量的同时,提高了近似消化率,对转Bt水稻比二化螟具有更强的适应能力3.采用毒饲料饲喂法建立了二化螟和大螟对不同Bt毒蛋白的敏感基线。生测结果表明,大螟对不同Bt毒蛋白的LC50值显著高于二化螟,对不同Bt毒素二者LC50的相对比值分别为20.21 (Cry1Ac)、39.13 (Cry1Ab)、3.2(Cry1Ah)、1.85(Cry1Ca)和1.57(Cry2Aa)。与二化螟相比,大螟对不同Bt毒蛋白表现了较强的耐受性。大螟对Bt毒蛋白的敏感基线测定结果表明,不同地理种群的大螟初孵幼虫对Cry1Ac和Cry1Ab毒蛋白的LC50值的变化范围分别为138.09-1521.76mg(AI)/L和17.25-173.08mg(AI)/L。敏感种群和耐受种群对Cq1Ac和Cry1Ab毒蛋白敏感性的相对比值分别为11倍和10倍。不同地理种群大螟对两种Bt毒蛋白的LC50值存在显著的正相关关系,说明同一地理种群的大螟对Cry1Ac和Cry1Ab的耐受性是一致的,且对Cry1Ac的耐受性显著高于CrylAb。更多还原

【Abstract】 The striped stem borer, Chilo supperssalis Walker(SSB) and the pink stem borer, Sesamia inferens Walker (PSB) are the important rice pests widely distributed in all rice growing regions in China. In recent years, SSB and PSB populations have increased gradually, because of changes in rice cultivation, use of new hybrid varieties and global climate and the occurrence of pest resistance to chemical insecticides. The development of transgenic rice lines expressing Bacillus thuringiensis (Bt) insecticidal protein provide the new strategy for the control of SSB and PSB. However, the ecological safety is the main factor for transgenic insect-resistant rice not being approved to be commercial application. Generally speaking, protein types of Bt insecticidal crystals are different, so are their insecticides spectrums. The Cry protein has showed high virulent effect on SSB, but low toxicity on PSB with similar niche to SSB. A preliminary field test also showed that the insecticidal Cry protein expressed in transgenic rice has effective control on SSB, but its efficacy on SSB decresed, which might change the component proportion of major pests in transgenic rice field. Therefore, researches on artifical rearing technique of PSB, the effect of Bt rice on the survival and nutritional utilization of SSB and PSB, baseline susceptibility of SSB and PSB to different Bt toxins were conducted. The main results were as follows:1. An artificial diet for PSB was formulated with soybean powder, corn powder, wheat germ powder and fresh water bamboo as major components. Rearing technique for mass and successive production of PSB was developed using fresh water bamboo (natural food source) for young larvae (neonates-2rd instar) and the artificial diet for older larvae (3rd instar and beyond). The performance of PSB reared on this fresh water bamboo/artificial diet regimen was evaluated over 3 consecutive generations by comparison with individuals fed on fresh water bamboo only for the whole generation. The fitness traits of larval durations, pupal weight, larval survival rate, pupation rate, eclosion rate and number of eggs laid per female were similar between these two diet regimens. Additionally, the rearing technique with fresh water bamboo/artificial diet combination could lower the cost by saving considerable amount of labor and time and significantly reduce the rate of insecticidal pathogen contaminations. The formulated diet and rearing technique were suitable for the mass and successive production of PSB.2. Effects of two transgenic Bt rice lines, TT51 and Cry2A, and their untransformed parental cultivar, Minghui63, at four different growing stages on the survival and nutritional utilization of SSB and PSB were conducted. The results indicated that both transgenic rice lines showed higher resistance to SSB than that to PSB at four different stages. The corrected mortalities of SSB feeding on both transgenic lines for 2 day and 4 day are significantly higher than that of PSB at different stages, respectively. Indices, Corrected relative growth rate, corrected relative consumption rate and corrected approximate digestibility, for larval consumption and digestibility indicated that both transgenic lines showed significant inhibition on the larval growth and development of SSB and PSB. CRGRs for SSB feeding on TT51 and Cry2Aa at different stages were lower than or similar to that for PSB on both lines, While CRCRs and CADs for SSB were higher than or similar to that for PSB.3. Baseline susceptibilities of SSB and PSB to different Bt toxins were established. The results of bioassay showed that the LC50 values of PSB to Bt toxins were very higher than that of SSB to Bt toxins. The ratio values of PSB and SSB LC50 values were 20.21 (CrylAc),39.13 (Cry1Ab),3.2 (Cry1Ah),1.85 (Cry1Ca) and 1.57 (Cry2Aa), which indicated that PSB showed higher tolerant to Bt toxin compared with SSB. Geographic variability in susceptibility of field-collected PSB to CrylAc and CrylAb was also studied using a diet incorporation method. Colonies of PSB were established from 6 different geographic areas of China. Among the different populations, the LC50 of CrylAc ranged from 138.09 mg (A.I.)/L to 1521.76 mg (A.I.)/L, and the LC50 of CrylAb ranged from 17.25 mg (A.I.)/L to 173.08 mg (A.I.)/L. The intra-specific variations in Cry1Ac and CrylAb susceptibility were 11- and 10-fold, respectively. Within a given population, the susceptibility to CrylAc and CrylAb were positively correlated and CrylAb was more susceptive to PSB than CrylAc. The results provided much needed groundwork for establishing susceptibility baseline and monitoring Bt tolerance of PSB.更多还原