转Bt双价基因甘蓝的抗虫性及遗传稳定性研究
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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。
Cabbage is an important cruciferous vegetable. The cultivation of cabbage is severely challenged by infestation of Lepidopteran pests, including diamondback moth (DBM), Pieris rapae, and etc.. The use of insect-resistant cultivars is the most effective method of controlling the pests. Despite extensive screening, high levels of natural resistance to lepidopteran pests have not been found in cabbage germplasms. Therefore, introducing foreign insect-resistance genes into cabbages through genetic engineering may provide effective germplasm resources for breeding.
Both crylla8and cry1Ba3were cloned from the Bt strain. Bioassays showed that Cry1Ia8and Cry1Ba3toxins were highly resistant to DBM and predominant Lepidopteran pests, and there were no cross resistance with the CrylAc. Pyramiding cry1la8and cry1Ba3into cabbage can not only improve the insect resistance of cabage but also dealy the resistant evolution of pests. In addition, the research on the genetic stability, insect-resistance, and risk assessement of transgenic cabbages will make it possible to enable transgenic plants to commercialization.
In the study, cry1la8and cry1Ba3were pyramided into cabbage inbred line by Agrobacterium tumefaciens-mediated transformation. Then the genetic stability, insect-resistance, and risk assessement of transgenic cabbages were assessed. The main results were as follows.
1. Obtaining of homozygous transgenic cabbage line expressing both cry1Ia8and cry1Ba3.
cry1Ia8and cry1Ba3were introduced simultaneously into cabbage inbred line by Agrobacterium tumefaciens-mediated transformation and53kanamycin-resistant plantlets were obtained. There are33Southern blot positive plants. Molecular analyses confirmed that the33transfomed plants were also expressed in RNA and protein level. Bioassays showed that the transgenic plants could effectively control both susceptible and Cry1Ac-resistant DBM.9homozygous transgenic lines were obtained by means of self-pollination and molecular-assisted selection. Then Bt hybrid cabbage were recoveryed by pollinating with other cabbage inbred lines.
2. Genetic stability analysis of transgenes in Cryla8-Cry1Ba3cabbage.
To plants were selfed and advanced to the third generation. Selfed progeny were harvested from each individual plant for the analysis of genetic stability. Analysis of transgene activities in four sinlge-copy lineages T0-1, T0-9, To-13, T0-17provided evidence that inheritance of the introduced gene was in Mendelian fasion. ELISA analyses indicated that content of Bt toxin was not significantly difference in four successive generations of lineage T0-1. Furthermore, data from field trials indicated that there were no significant differences in most agronomic traits between transgenic plants and the control plants.
3. Insect-resistance evaluation of Cryla8-Cry1Ba3cabbage.
The results of insect-resistance evaluations in greenhouse and field domonstated that the homozygous transgenic lines were highly resistant against DBM and Pieris rapae as compared to the control while susceptible to Mamestra brassicae and Spodoptera exigua.
4. Influences of transgenic cabbage pollen or Bt toxins on survival rates of bees.
The bees were fed with the pollens of transgenic cabbages, Crylla8toxin (3000ng/ml), Cry1Ba3toxin (3000ng/ml), mixed pollens, and untransformed cabbages pollens, respectively.20days later, there were no significant differences in mortalities of bees among the5treatments.
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。
Cabbage is an important cruciferous vegetable. The cultivation of cabbage is severely challenged by infestation of Lepidopteran pests, including diamondback moth (DBM), Pieris rapae, and etc.. The use of insect-resistant cultivars is the most effective method of controlling the pests. Despite extensive screening, high levels of natural resistance to lepidopteran pests have not been found in cabbage germplasms. Therefore, introducing foreign insect-resistance genes into cabbages through genetic engineering may provide effective germplasm resources for breeding.
Both crylla8and cry1Ba3were cloned from the Bt strain. Bioassays showed that Cry1Ia8and Cry1Ba3toxins were highly resistant to DBM and predominant Lepidopteran pests, and there were no cross resistance with the CrylAc. Pyramiding cry1la8and cry1Ba3into cabbage can not only improve the insect resistance of cabage but also dealy the resistant evolution of pests. In addition, the research on the genetic stability, insect-resistance, and risk assessement of transgenic cabbages will make it possible to enable transgenic plants to commercialization.
In the study, cry1la8and cry1Ba3were pyramided into cabbage inbred line by Agrobacterium tumefaciens-mediated transformation. Then the genetic stability, insect-resistance, and risk assessement of transgenic cabbages were assessed. The main results were as follows.
1. Obtaining of homozygous transgenic cabbage line expressing both cry1Ia8and cry1Ba3.
cry1Ia8and cry1Ba3were introduced simultaneously into cabbage inbred line by Agrobacterium tumefaciens-mediated transformation and53kanamycin-resistant plantlets were obtained. There are33Southern blot positive plants. Molecular analyses confirmed that the33transfomed plants were also expressed in RNA and protein level. Bioassays showed that the transgenic plants could effectively control both susceptible and Cry1Ac-resistant DBM.9homozygous transgenic lines were obtained by means of self-pollination and molecular-assisted selection. Then Bt hybrid cabbage were recoveryed by pollinating with other cabbage inbred lines.
2. Genetic stability analysis of transgenes in Cryla8-Cry1Ba3cabbage.
To plants were selfed and advanced to the third generation. Selfed progeny were harvested from each individual plant for the analysis of genetic stability. Analysis of transgene activities in four sinlge-copy lineages T0-1, T0-9, To-13, T0-17provided evidence that inheritance of the introduced gene was in Mendelian fasion. ELISA analyses indicated that content of Bt toxin was not significantly difference in four successive generations of lineage T0-1. Furthermore, data from field trials indicated that there were no significant differences in most agronomic traits between transgenic plants and the control plants.
3. Insect-resistance evaluation of Cryla8-Cry1Ba3cabbage.
The results of insect-resistance evaluations in greenhouse and field domonstated that the homozygous transgenic lines were highly resistant against DBM and Pieris rapae as compared to the control while susceptible to Mamestra brassicae and Spodoptera exigua.
4. Influences of transgenic cabbage pollen or Bt toxins on survival rates of bees.
The bees were fed with the pollens of transgenic cabbages, Crylla8toxin (3000ng/ml), Cry1Ba3toxin (3000ng/ml), mixed pollens, and untransformed cabbages pollens, respectively.20days later, there were no significant differences in mortalities of bees among the5treatments.
引文
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