三个改造后的Bt抗虫基因在烟草中的杀虫活性研究

发布时间：2018-06-27 23:59

本文选题：棉铃虫 + cry2Ah2　；参考：《新疆农业大学》2016年硕士论文

【摘要】：棉铃虫(Helicoverpa armigera Hubner)属于鳞翅目夜蛾科,在世界各地广泛分布,是棉花生产最严重的害虫之一。20世纪80、90年代,棉铃虫在全世界主要产棉区大面积暴发,导致棉花严重减产,造成巨大的经济损失。经过20多年的研究和生产应用,转Bt基因抗虫棉取得了巨大的生态和经济效益。然而随着抗虫棉的大面积种植,在高生态选择压作用下,棉铃虫对抗虫基因产生抗性的风险也逐渐增加。通过对Bt蛋白的杀虫机理研究发现,转单一Bt基因棉花的大面积推广应用容易导致昆虫产生抗性。因此,通过发掘不同杀虫机理的Bt基因,创制含有不同杀虫机理复合抗虫棉花新材料,已经成为当前抗虫棉花基因工程的研究热点。转复合抗虫基因棉花,不仅可以提高杀虫效率,而且可有效地避免或延迟昆虫的抗性发生。cry2Ah2,cry2Ab4,vip3Aa11是从苏云金芽胞杆菌(Bacillus thuringiensis,Bt)中克隆到的3个新型Bt晶体蛋白杀虫基因。其中Cry2Ah2和Cry2Ab4是在孢子形成期产生的杀虫晶体蛋白(Insecticidal crystal proteins,ICPs),对鳞翅目、双翅目、鞘翅目等多种昆虫具有毒杀功能。Vip3Aa11是一种营养期杀虫蛋白(Vegetative insecticidal proteins,VIPs),对棉铃虫、小地老虎等一系列鳞翅目害虫均有较高的毒性作用。本研究依据棉花遗传密码子偏好性,对cry2Ah2,cry2Ab4,vip3Aa11基因进行了密码子优化改造,改造后的三个基因克隆到pGBI121植物表达载体,构建成pGBI-cry2Ah2(35S::cry2Ah2:Nos)、pGBI-cry2Ab4(35S::cry2Ab4:Nos)和pGBI-vip3Aa11(35S::vip3Aa11:Nos)单基因表达载体。农杆菌介导法转化烟草NC89(Nicotiana tabacum L.)后,分别获得30株转cry2Ah2基因阳性烟草植株,33株转cry2Ab4基因阳性烟草植株,40株转vip3Aa11基因阳性烟草植株。荧光定量PCR结果表明,三个抗虫基因的阳性转基因烟草植株均能稳定地高水平地表达外源抗虫基因,我们分别选取了表达水平较高的转基因植株进行Southern blot检测,三个基因已经整合到烟草基因组中,其中转cry2Ah2基因烟草进一步通过Western blot实验在蛋白水平验证了其表达。生物活性检测结果表明,用转cry2Ah2,cry2Ab4,vip3Aa11基因烟草叶片分别饲喂初孵棉铃虫幼虫,4天后幼虫校正死亡率分别在86.59%,86.51%和87.70%以上,均达到高抗水平,其中,转vip3Aa11植株抗虫效果优于转cry2Ah2和cry2Ab4植株,转cry2Ah2与cry2Ab4基因植株抗虫水平相当。这一结果表明cry2Ah2,cry2Ab4,vip3Aa11三个基因为现有抗虫棉的深化研究提供了新的抗性基因源。本研究证实了3个基因均可以在模式植物中高水平表达,并使转基因烟草具有高抗虫性,是防治棉铃虫等害虫的新型候选抗虫基因,可将其转入棉花等作物,用于培育新一代的转基因抗虫作物新品种。
[Abstract]:Helicoverpa armigera Hubner, a member of the family Lepidoptera, is widely distributed all over the world. Helicoverpa armigera Hubner is one of the most serious pests in cotton production. In the 1980s and 1990s, Helicoverpa armigera Hubner broke out in the major cotton producing areas of the world, resulting in serious reduction of cotton production. Causing enormous economic losses. After more than 20 years of research and application, transgenic BT transgenic cotton has achieved great ecological and economic benefits. However, with the extensive cultivation of cotton, the risk of resistance to cotton bollworm genes increased gradually under the action of high ecological selective pressure. By studying the insecticidal mechanism of BT protein, it was found that the extensive application of transgenic cotton with single BT gene could lead to insect resistance. Therefore, by exploring BT gene of different insecticidal mechanism and creating new material containing different insecticidal mechanism, it has become a hot spot in genetic engineering of insect-resistant cotton flower. Transgenic cotton with compound insect-resistant gene could not only improve the insecticidal efficiency, but also effectively avoid or delay the occurrence of insect resistance. Cry2Ah2H2Ab4vip3Aa11 is a new BT crystal protein insecticidal gene cloned from Bacillus thuringiensis Bt. Cry2Ah2 and Cry2Ab4 are insecticidal crystal proteinsICPs produced during spore formation. They are toxic to Lepidoptera, Diptera, Coleoptera and many other insects. Vip3Aa11 is a vegetative insecticidal proteinsVIPs. A series of Lepidoptera pests, such as small ground tiger, have high toxicity. Based on the preference of cotton genetic codon, the codon of cry2Ah2Ab4vip3Aa11 gene was optimized and cloned into pGBI121 plant expression vector pGBI121. The single gene expression vector pGBI-2Ah2 (35 S: cry2Ah2: Nos) pGBI-cry2Ab4 (35S: cry2Ab4: Nos) and pGBI-vip3Aa11 (35S: vivip3Aa11Nos) were constructed. Agrobacterium tumefaciens mediated transformation of tobacco NC89 (Nicotiana tabacum L.) After that, 30 cry2Ah2 gene positive tobacco plants, 33 cry2Ab4 gene positive tobacco plants and 40 vip3Aa11 gene positive tobacco plants were obtained. The results of fluorescence quantitative PCR showed that all the transgenic tobacco plants with three insect-resistant genes could express exogenous insect-resistant genes stably at a high level, and the transgenic plants with higher expression level were selected for Southern blot detection. The three genes had been integrated into the tobacco genome, and the transgenic cry2Ah2 gene was further verified by Western blot assay at the protein level. The results of bioactivity test showed that the corrected larval mortality was 86.59% and 87.70% respectively after the leaves of transgenic tobacco with cry2Ah2Ab4vip3Aa11 gene were fed with the newly hatched larvae of Helicoverpa armigera respectively for 4 days. The insect-resistant effect of vip3Aa11 transgenic plants was better than that of cry2Ah2 and cry2Ab4 plants, and the resistance level of transgenic cry2Ah2 and cry2Ab4 transgenic plants was the same. The results showed that the three bases of cry2Ah2 + cry2Ab4vip3Aa11 provided a new resistance gene source because of the further study of the existing insect-resistant cotton. This study confirmed that all three genes can be expressed at high level in model plants, and that transgenic tobacco has high insecticidal resistance. It is a new candidate insect-resistant gene for controlling cotton bollworm and other pests, which can be transferred to cotton and other crops. New varieties of transgenic insecticidal crops used to cultivate a new generation of transgenic insect-resistant crops.
【学位授予单位】：新疆农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S435.62

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