转Bt双价基因甘蓝的抗虫性及遗传稳定性研究
发布时间：2019-05-17 16:33
【摘要】：结球甘蓝简称甘蓝,是十字花科芸薹属的一种重要蔬菜。近年来,甘蓝上的虫害日趋严重,尤其是鳞翅目的小菜蛾和菜青虫等危害最为严重。培育抗虫品种是防治害虫最理想的方法,但在甘蓝类蔬菜中缺乏抗虫种质资源,采用基因工程技术将外源抗虫基因转入甘蓝,可为育种提供有效的抗虫材料。 Bt crylIa8和cry1Ba3均是具有自主知识产权的新型杀虫蛋白基因,其编码蛋白对小菜蛾等鳞翅目害虫具有高毒力,且Cry1Ia8. Cry1Ba3与生产上常用的Cry1A类蛋白之间没有交互抗性。利用cry1Ia8和cry1Ba3进行转双价基因甘蓝的研制,不但可以提高甘蓝的抗虫性,还可以降低害虫快速产生抗性的风险。此外,在获得转抗虫基因甘蓝材料后,研究转基因甘蓝的遗传稳定性、抗虫性和安全性,可为甘蓝抗虫育种提供理论指导,加速转基因甘蓝在实践中的应用。 本研究采用农杆菌介导法将cry1Ia8和cry1Ba3基因同时导入甘蓝高代自交系,然后对转基因甘蓝的遗传稳定性、抗虫性和安全性等方面进行了研究,主要结果如下： 1.转cry1Ia8-cry1Ba3双价基因甘蓝纯系的获得 以重组质粒表达载体pCSIaBaN(含有cry1Ia8和cry1Ba3)转化甘蓝,共获得53株卡那霉素抗性植株,其中的33株在RNA和蛋白质水平均得到了表达；转双价基因植株在离体条件下对敏感小菜蛾和CrylAc抗性小菜蛾均具有很强的抗性；通过连续多代自交和筛选获得9个转基因甘蓝纯系,并利用其配制了杂交组合。 2.转crylIa8-cry1Ba3双价基因甘蓝的遗传稳定性分析 在获得T0、T1、T2、T3四个世代转cry1Ia8-crylBa3双价基因植株的基础上,对外源基因的遗传稳定性进行了研究：T0-1、T0-9、T0-13、T0-17等4个单拷贝转化植株外源目的基因的分离规律均符合单基因显性遗传；Cry1Ia8、Cry1Ba3蛋白含量在同一转化事件的不同世代间均没有显著性差异；外源基因的导入未对转基因甘蓝纯合株系及其杂交后代的株高、开展度、外叶数、球高、球宽、中心柱长和单球重等主要农艺性状产生影响。 3.转基因甘蓝的抗虫性评价 通过网室和田间抗虫性的鉴定、田间主要害虫种类与数量的调查、田间主要害虫离体生物活性的测定,表明转双价基因甘蓝对小菜蛾和菜青虫具有极强的抗性,而对甘蓝夜蛾和甜菜夜蛾没有明显抗性。 4.转基因甘蓝花粉和高浓度的Bt蛋白对蜜蜂幼虫存活率的影响 将转cry1Ia8-crylBa3双价基因甘蓝花粉、Cry1Ia8蛋白(3000ng/ml)及Cry1Ba3蛋白(3000ng/ml)直接饲喂意大利蜜蜂的工蜂幼虫,20天后,与取食对照甘蓝花粉、杂花粉相比,取食转基因甘蓝花粉和Bt蛋白的蜜蜂存活率均未受到负面影响。
[Abstract]:Cabbage, abbreviated as cabbage, is an important vegetable of the genus Cruciferae. In recent years, the pests on cabbage have become more and more serious, especially the Plutella xylostella and cabbage insects in Lepidoptera. Breeding insect-resistant varieties is the most ideal method to control pests, but there is a lack of insect-resistant germplasm resources in cabbage vegetables. Genetic engineering technology can be used to transfer exogenous insect-resistant genes into cabbage, which can provide effective insect-resistant materials for breeding. Both Bt crylIa8 and cry1Ba3 are novel insecticidal protein genes with independent intellectual property rights. Their encoded proteins are highly virulent to Lepidoptera pests such as Plutella xylostella, and Cry1Ia8. is highly toxic to Lepidoptera pests such as Plutella xylostella. There is no cross resistance between Cry1Ba3 and Cry1A proteins commonly used in production. The preparation of transgenic cabbage with bivalent gene by cry1Ia8 and cry1Ba3 can not only improve the insect resistance of cabbage, but also reduce the risk of rapid resistance of pests. In addition, after obtaining transgenic cabbage materials, the genetic stability, insect resistance and safety of transgenic cabbage can be studied, which can provide theoretical guidance for insect-resistant breeding of cabbage and accelerate the application of transgenic cabbage in practice. In this study, cry1Ia8 and cry1Ba3 genes were introduced into cabbage inbred lines at the same time by Agrobacterium tumefaciens, and then the genetic stability, insect resistance and safety of transgenic cabbage were studied. The main results are as follows: 1. Transgenic cabbage lines with cry1Ia8-cry1Ba3 bivalent gene were transformed into cabbage with recombinant plasmid expression vector pCSIaBaN (containing cry1Ia8 and cry1Ba3). A total of 53 kanamycin resistant plants were obtained, 33 of which were expressed at RNA and protein levels. The transgenic plants were highly resistant to sensitive Plutella xylostella and CrylAc resistant Plutella xylostella in vitro, and 9 transgenic cabbage lines were obtained by continuous inbreeding and screening, and the hybrid combinations were prepared by using them. 2. The genetic stability of transgenic cabbage with crylIa8-cry1Ba3 bivalent gene was studied on the basis of obtaining four generations of transgenic cry1Ia8-crylBa3 bivalent gene plants: T0, T1, T2, T3. The genetic stability of foreign genes was studied. The isolation of foreign target genes from four single copy transformed plants, such as T0 13 and T0 17, were in accordance with the dominant inheritance of single gene. There was no significant difference in Cry1Ia8,Cry1Ba3 protein content among different generations of the same transformation event. The introduction of foreign genes had no effect on the plant height, development degree, number of outer leaves, ball height, ball width, central column length and single bulb weight of transgenic cabbage homozygous lines and their hybrid progenies. 3. The evaluation of insect resistance of transgenic cabbage was carried out through the identification of insect resistance in net room and field, the investigation of the species and quantity of main pests in the field, and the determination of in vitro biological activity of the main pests in the field. The results showed that transgenic cabbage had strong resistance to Plutella xylostella and Plutella xylostella, but no obvious resistance to Plutella xylostella and Plutella xylostella. 4. Effects of transgenic cabbage pollen and high concentration of Bt protein on the survival rate of honeybee larvae were directly fed with cry1Ia8-crylBa3 bivalent cabbage pollen, Cry1Ia8 protein (3000ng/ml) and Cry1Ba3 protein (3000ng/ml). After 20 days, compared with the control cabbage pollen and miscellaneous pollen, the survival rate of bees fed on transgenic cabbage pollen and Bt protein was not negatively affected.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：S635.1
本文编号：2479245
[Abstract]:Cabbage, abbreviated as cabbage, is an important vegetable of the genus Cruciferae. In recent years, the pests on cabbage have become more and more serious, especially the Plutella xylostella and cabbage insects in Lepidoptera. Breeding insect-resistant varieties is the most ideal method to control pests, but there is a lack of insect-resistant germplasm resources in cabbage vegetables. Genetic engineering technology can be used to transfer exogenous insect-resistant genes into cabbage, which can provide effective insect-resistant materials for breeding. Both Bt crylIa8 and cry1Ba3 are novel insecticidal protein genes with independent intellectual property rights. Their encoded proteins are highly virulent to Lepidoptera pests such as Plutella xylostella, and Cry1Ia8. is highly toxic to Lepidoptera pests such as Plutella xylostella. There is no cross resistance between Cry1Ba3 and Cry1A proteins commonly used in production. The preparation of transgenic cabbage with bivalent gene by cry1Ia8 and cry1Ba3 can not only improve the insect resistance of cabbage, but also reduce the risk of rapid resistance of pests. In addition, after obtaining transgenic cabbage materials, the genetic stability, insect resistance and safety of transgenic cabbage can be studied, which can provide theoretical guidance for insect-resistant breeding of cabbage and accelerate the application of transgenic cabbage in practice. In this study, cry1Ia8 and cry1Ba3 genes were introduced into cabbage inbred lines at the same time by Agrobacterium tumefaciens, and then the genetic stability, insect resistance and safety of transgenic cabbage were studied. The main results are as follows: 1. Transgenic cabbage lines with cry1Ia8-cry1Ba3 bivalent gene were transformed into cabbage with recombinant plasmid expression vector pCSIaBaN (containing cry1Ia8 and cry1Ba3). A total of 53 kanamycin resistant plants were obtained, 33 of which were expressed at RNA and protein levels. The transgenic plants were highly resistant to sensitive Plutella xylostella and CrylAc resistant Plutella xylostella in vitro, and 9 transgenic cabbage lines were obtained by continuous inbreeding and screening, and the hybrid combinations were prepared by using them. 2. The genetic stability of transgenic cabbage with crylIa8-cry1Ba3 bivalent gene was studied on the basis of obtaining four generations of transgenic cry1Ia8-crylBa3 bivalent gene plants: T0, T1, T2, T3. The genetic stability of foreign genes was studied. The isolation of foreign target genes from four single copy transformed plants, such as T0 13 and T0 17, were in accordance with the dominant inheritance of single gene. There was no significant difference in Cry1Ia8,Cry1Ba3 protein content among different generations of the same transformation event. The introduction of foreign genes had no effect on the plant height, development degree, number of outer leaves, ball height, ball width, central column length and single bulb weight of transgenic cabbage homozygous lines and their hybrid progenies. 3. The evaluation of insect resistance of transgenic cabbage was carried out through the identification of insect resistance in net room and field, the investigation of the species and quantity of main pests in the field, and the determination of in vitro biological activity of the main pests in the field. The results showed that transgenic cabbage had strong resistance to Plutella xylostella and Plutella xylostella, but no obvious resistance to Plutella xylostella and Plutella xylostella. 4. Effects of transgenic cabbage pollen and high concentration of Bt protein on the survival rate of honeybee larvae were directly fed with cry1Ia8-crylBa3 bivalent cabbage pollen, Cry1Ia8 protein (3000ng/ml) and Cry1Ba3 protein (3000ng/ml). After 20 days, compared with the control cabbage pollen and miscellaneous pollen, the survival rate of bees fed on transgenic cabbage pollen and Bt protein was not negatively affected.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：S635.1
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