转Bt基因抗虫玉米田间试验与遗传稳定性分析
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摘要
近20年来,人们采用基因工程手段成功培育了一批抗虫、抗病、耐除草剂和高产优质的农作物新种质,取得了令人瞩目的成就。而一个转基因的植株获得后,通常还要对其后代进行一系列基因纯合鉴定、遗传稳定性的研究、农艺和经济性状的筛选、产业化过程中的种性保持等工作,所有这些过程都需要对目的基因进行跟踪调查,需要对大量的转基因后代进行筛选、鉴定。因此在转基因作物研究继续深入的同时,科学家们越来越重视转基因与常规育种技术的结合,并加强了对转基因作物后代性状的大田遗传表达与变异研究试验,为利用转基因技术进行作物种质创新,改良现有作物品种,最终实现作物转基因植株从实验室走向大田生产奠定坚实的基础。而基因转入宿主后的有效表达及其稳定遗传,则成为转基因植物实用化中的关键问题。
国内关于玉米转Bt基因及Bt玉米遗传稳定性有一些报道,但多数是在转基因株系的早期世代,以转化植株的性能和基因功能的分析以及对国外Bt玉米的利用效果评价为目的,以国内具有自主知识产权的基因转化获得的Bt玉米材料为基础的鉴定工作不多,尤其是在转Bt基因抗虫玉米高代自交系的遗传稳定性的研究还未见报道。
本研究对采用花粉管通道法转cry1Ah基因抗虫玉米高代自交系(及其后代)和采用基因枪法获得的转Bt cry1Ah和cry1Ie双价基因抗虫玉米植株,通过田间及室内玉米螟抗性鉴定,利用PCR、RT-PCR、Southern blot、Western blot、ELISA等各种分子检测手段对外源基因的遗传稳定性和遗传规律等进行了研究分析,获得的主要研究结果如下：
1.通过PCR检测确认了阳性转化植株,用Southern杂交进一步证实了Bt基因已整合到受体植物基因组,用RT-PCR验证了Bt基因在植物细胞中的转录,用Western杂交证实了Bt基因在植物细胞中的表达。遗传分析表明,Bt基因在遗传传递过程中符合孟德尔遗传规律,是单位点显性方式遗传。外源基因cry1Ah、cry1Ie和2mG2-epsps三个外源基因在转基因玉米基因组中完全连锁,表现为共分离,就像单个基因位点一样一起传递到下一代而没有分离。
2.Cry1Ah在同一转基因事件的不同植株间表达量有所不同,但经统计分析差异不显著；在不同转基因事件间的表达量存在显著差异；同一植株同一发育时期不同器官Cry1Ah的表达量不同,苞叶和叶片外源基因蛋白表达量比较高,花粉最低,各组织部位外源基因蛋白表达量顺序：苞叶>叶>茎>雄穗>雄穗柄>雌穗尖>籽粒>花丝>花粉。Bt杀虫蛋白在玉米中的表达具有时空特异性,不同生育时期,CryAh在叶片和茎中的表达量不同,呈动态下降趋势。
3.不同的转基因事件与不同常规玉米自交系进行杂交,杂交后代的外源基因表达量不同,这与亲本外源基因表达量高低及另一亲本的基因型有很大关系。要想得到高表达量的杂交后代,则需要选择外源基因表达量高的转基因自交系做亲本；通过玉米螟抗性鉴定和外源基因表达量的测定,表明外源Bt基因通过雌配子的传递频率高于通过雄配子的传递频率,因此尽量选择抗性亲本正向方式进行杂交,这样获得表达量高的杂交后代的几率会高一些。
4.无论是转cry1Ah单价基因抗虫玉米自交系及其杂交、回交后代,还是转cry1Ah和cry1Ie双价基因抗虫玉米经连续四代的田间、室内玉米螟抗性检测,结果对亚洲玉米螟均表现出很好的抗性,并且这种抗性可以逐代稳定遗传,说明外源Bt基因的导入对防治亚洲玉米螟的危害是有效的。通过ELISA对Cry1Ah蛋白表达量的检测,其变化规律与虫测结果基本一致,说明田间、室内鉴定与分子鉴定相结合能更准确地说明Bt杀虫蛋白对亚洲玉米螟的抗性强弱,进行相关分析,结果表明抗虫玉米Cry1Ah蛋白表达量与雄穗柄倒折率和室内生测7天幼虫死亡率具有显著的相关性,与食叶级别、百株隧道长度具有极显著的相关性。说明玉米中Bt蛋白含量的高低是转基因玉米对玉米螟产生抗性强弱的内因。
5.转cry1Ah和cry1Ie单、双价基因抗虫玉米及其后代与受体对照比较,在穗行粒数及秃尖长上有一定的差异,差异显著,秃尖变短和穗行粒数增加表现为产量的提高,而在株高、穗位高、穗长、穗粗、穗行数及千粒重上差异不大,经检验差异不显著。
6.转cry1Ah基因抗虫玉米与受体对照比较,除了在栽培地试验时产量比受体自交系高外,在栽培地、荒地条件下,其出苗率、长势、株型、生育期、株高等方面均无显著差异；在荒地条件下,玉米出苗率均很低,撒播处理的出苗率还不到5%；杂草的覆盖度高于玉米的覆盖度,存在显著或极显著差异,说明玉米的生存竞争能力要远远小于杂草；种子发芽试验显示,转cry1Ah基因抗虫玉米后代种子发芽活力方面与对照也无显著差异。
7.确定了“两高—阳—优良”(即抗虫性高、外源基因蛋白表达量高、PCR检测为阳性、农艺性状优良)的转基因抗虫玉米筛选原则。依据这个筛选原则从转cry1Ah单价基因抗虫玉米中筛选出5个抗性好、农艺性状优良的纯合转化事件,分别为P66、P33、P25、P26和P14。从转cry1Ah和cry1Ie双价基因抗虫玉米中也筛选出1个纯合转化事件(S8)。
In the last two decades, remarkable achievements has been made on successful cultivation of a large number of genetic engineering crops, including those with resistance to insect, disease, herbicide and those with high yield and quality. However, after obtaining one transgenic plant, much work had to do, for their offspring, on pure line identification, genetic stability analysis, agronomic and economic traits selection and germplasm conservation in commercialization, all the above work was fuscous on tracking survey to the interesting gene and selection/identification of large number of progenies. Therefore, at the time on development of genetically modified crops, more and more attention paid to combination of transgenic and conventional breeding technology, and genetic expression and variation test of GM crops offspring in the field also strengthen. As a result, crop innovation and improvement by genetic engineering would make it possible to enable transgenic plants to commercialization, and effective expression and genetic stability were the key issues on the process.
There were some national reports on Bt-transgenic maize and their genetic stability analysis, but they were concerned about performance of transformed plants and gene function, as well as gene usage evaluation of foreign Bt-transgenic maize, mainly in the early generations. Besides, only a few articles has been reported on identification of Bt maize harboring genes with intellectual property, and it was notice that there was few story about genetic stability analysis of inbred line high generation of insect-resistant Bt maize.
The applied plant materials were high generations of inbred insect-resistant maize harboring cry1Ah transformed by Pollen tube pathway and insect-resistant maize harboring cry1Ah and cry1Ie by gene gun. Indoor and field resistance identification on corn borer, plus a series of molecular detection including PCR、RT-PCR、Southern blot、Western blot and ELISA has been served to genetic stability and pattern analysis. Results were as follows:
1. Positive plants were confirmed by PCR. Bt gene was proved by Southern blot that it was integrated into plant genome, further it was transcripted and expressed in plant cells detected by RT-PCR, Western blot respectively. Genetic analysis showed that Bt gene was single-locus dominant inheritance, conforming to Mendelian genetic pattern. Three foreign genes, cry1Ah, cry1Ie and 2mG2-epsps, was complete linkage and coseparation in transgenic maize genome, namely they were transferred to offspring like single gene locus.
2. Cry1Ah expression varied among plants from the same transformed event, but statistical analysis indicated that the difference was not significant, while there was significant difference among different transformed events. And Cry1Ah expression varied among different tissues from the same stage of one plant, higher in the bract and leaf, lowest in the pollen (bract>leaf>stalk>tassel>tassel handle>ear tip>seed>filament>pollen). Bt toxic protein expression possessed temporal and special specificity, Cry1Ah expression varied in different stage of leaf and stalk, taking on dynamic downtrend.
3. Foreign gene expression varied in progenies from different transformed events hybridized with different conventional maize, largely related to foreign gene expression of one parent and gene type of the other. Therefore, it required transgenic inbred lines with high foreign gene expression to be parent. Resistance analysis on corn borer and foreign expression detection showed that transmission frequency of mega gamete was higher than andro gamete, so that choosing resistant parent to crossbred would make it possible to obtain high expression progenies.
4. Indoor and field resistance analysis on corn borer demonstrated that, not only maize harboring cry1Ah and their progenies, but also transgenic maize harboring cry1Ah and cry1Ie were highly resistant to corn borer, besides the very resistance was able to transfer from generation to generation. Therefore it was effective to control Asia corn borer by means of transforming foreign Bt genes. Variety pattern of Cry1Ah protein expression detected through ELISA was on the whole consistent with resistance analysis, indicated that combination of indoor/field resistance analysis and molecular detection would illuminate resistance on corn borer more precisely, and the results showed that Cry1Ah protein expression was significant relative to Fracture rate of tassel handle and indoor larva death ratio in 7 days, and extremely significant relative to leaf-feeding level and tunnel length per 100 plants. So it made clear that Bt protein content was the internal cause of resistance to corn borer.
5. Comparing conventional maize and transgenic maize harboring single cry1Ah or harboring cry1Ah and cry1Ie and their progenies, statistical analysis indicated that there was significant difference in kernals per row and bare tip length, yet indistinct difference in plant height, height, width, line number of ear and g/1000 seeds. Shorter bald tip and increasing in grains per ear indicated the possible product raising.
6. Comparing conventional maize and transgenic maize harboring single cry1Ah, both in experimental field and wilderness, there was indistinct difference in geminating rate, growing vigor, plant type, bearing period and plant height, except for yield. In wilderness, maize geminating rate was only 5%, and weeds coverage was higher than that of maize (significant difference or extremely significant difference), showing that survival competitiveness of maize was much weaker. Germinating test showed that there was indistinct difference between conventional maize and progenies of transgenic maize harboring cry1Ah.
7. Five pure lines of transgenic maize harboring cry1Ah and 1 transgenic maize harboring cry1Ah and cry1Ie have been selected.
国内关于玉米转Bt基因及Bt玉米遗传稳定性有一些报道,但多数是在转基因株系的早期世代,以转化植株的性能和基因功能的分析以及对国外Bt玉米的利用效果评价为目的,以国内具有自主知识产权的基因转化获得的Bt玉米材料为基础的鉴定工作不多,尤其是在转Bt基因抗虫玉米高代自交系的遗传稳定性的研究还未见报道。
本研究对采用花粉管通道法转cry1Ah基因抗虫玉米高代自交系(及其后代)和采用基因枪法获得的转Bt cry1Ah和cry1Ie双价基因抗虫玉米植株,通过田间及室内玉米螟抗性鉴定,利用PCR、RT-PCR、Southern blot、Western blot、ELISA等各种分子检测手段对外源基因的遗传稳定性和遗传规律等进行了研究分析,获得的主要研究结果如下：
1.通过PCR检测确认了阳性转化植株,用Southern杂交进一步证实了Bt基因已整合到受体植物基因组,用RT-PCR验证了Bt基因在植物细胞中的转录,用Western杂交证实了Bt基因在植物细胞中的表达。遗传分析表明,Bt基因在遗传传递过程中符合孟德尔遗传规律,是单位点显性方式遗传。外源基因cry1Ah、cry1Ie和2mG2-epsps三个外源基因在转基因玉米基因组中完全连锁,表现为共分离,就像单个基因位点一样一起传递到下一代而没有分离。
2.Cry1Ah在同一转基因事件的不同植株间表达量有所不同,但经统计分析差异不显著；在不同转基因事件间的表达量存在显著差异；同一植株同一发育时期不同器官Cry1Ah的表达量不同,苞叶和叶片外源基因蛋白表达量比较高,花粉最低,各组织部位外源基因蛋白表达量顺序：苞叶>叶>茎>雄穗>雄穗柄>雌穗尖>籽粒>花丝>花粉。Bt杀虫蛋白在玉米中的表达具有时空特异性,不同生育时期,CryAh在叶片和茎中的表达量不同,呈动态下降趋势。
3.不同的转基因事件与不同常规玉米自交系进行杂交,杂交后代的外源基因表达量不同,这与亲本外源基因表达量高低及另一亲本的基因型有很大关系。要想得到高表达量的杂交后代,则需要选择外源基因表达量高的转基因自交系做亲本；通过玉米螟抗性鉴定和外源基因表达量的测定,表明外源Bt基因通过雌配子的传递频率高于通过雄配子的传递频率,因此尽量选择抗性亲本正向方式进行杂交,这样获得表达量高的杂交后代的几率会高一些。
4.无论是转cry1Ah单价基因抗虫玉米自交系及其杂交、回交后代,还是转cry1Ah和cry1Ie双价基因抗虫玉米经连续四代的田间、室内玉米螟抗性检测,结果对亚洲玉米螟均表现出很好的抗性,并且这种抗性可以逐代稳定遗传,说明外源Bt基因的导入对防治亚洲玉米螟的危害是有效的。通过ELISA对Cry1Ah蛋白表达量的检测,其变化规律与虫测结果基本一致,说明田间、室内鉴定与分子鉴定相结合能更准确地说明Bt杀虫蛋白对亚洲玉米螟的抗性强弱,进行相关分析,结果表明抗虫玉米Cry1Ah蛋白表达量与雄穗柄倒折率和室内生测7天幼虫死亡率具有显著的相关性,与食叶级别、百株隧道长度具有极显著的相关性。说明玉米中Bt蛋白含量的高低是转基因玉米对玉米螟产生抗性强弱的内因。
5.转cry1Ah和cry1Ie单、双价基因抗虫玉米及其后代与受体对照比较,在穗行粒数及秃尖长上有一定的差异,差异显著,秃尖变短和穗行粒数增加表现为产量的提高,而在株高、穗位高、穗长、穗粗、穗行数及千粒重上差异不大,经检验差异不显著。
6.转cry1Ah基因抗虫玉米与受体对照比较,除了在栽培地试验时产量比受体自交系高外,在栽培地、荒地条件下,其出苗率、长势、株型、生育期、株高等方面均无显著差异；在荒地条件下,玉米出苗率均很低,撒播处理的出苗率还不到5%；杂草的覆盖度高于玉米的覆盖度,存在显著或极显著差异,说明玉米的生存竞争能力要远远小于杂草；种子发芽试验显示,转cry1Ah基因抗虫玉米后代种子发芽活力方面与对照也无显著差异。
7.确定了“两高—阳—优良”(即抗虫性高、外源基因蛋白表达量高、PCR检测为阳性、农艺性状优良)的转基因抗虫玉米筛选原则。依据这个筛选原则从转cry1Ah单价基因抗虫玉米中筛选出5个抗性好、农艺性状优良的纯合转化事件,分别为P66、P33、P25、P26和P14。从转cry1Ah和cry1Ie双价基因抗虫玉米中也筛选出1个纯合转化事件(S8)。
In the last two decades, remarkable achievements has been made on successful cultivation of a large number of genetic engineering crops, including those with resistance to insect, disease, herbicide and those with high yield and quality. However, after obtaining one transgenic plant, much work had to do, for their offspring, on pure line identification, genetic stability analysis, agronomic and economic traits selection and germplasm conservation in commercialization, all the above work was fuscous on tracking survey to the interesting gene and selection/identification of large number of progenies. Therefore, at the time on development of genetically modified crops, more and more attention paid to combination of transgenic and conventional breeding technology, and genetic expression and variation test of GM crops offspring in the field also strengthen. As a result, crop innovation and improvement by genetic engineering would make it possible to enable transgenic plants to commercialization, and effective expression and genetic stability were the key issues on the process.
There were some national reports on Bt-transgenic maize and their genetic stability analysis, but they were concerned about performance of transformed plants and gene function, as well as gene usage evaluation of foreign Bt-transgenic maize, mainly in the early generations. Besides, only a few articles has been reported on identification of Bt maize harboring genes with intellectual property, and it was notice that there was few story about genetic stability analysis of inbred line high generation of insect-resistant Bt maize.
The applied plant materials were high generations of inbred insect-resistant maize harboring cry1Ah transformed by Pollen tube pathway and insect-resistant maize harboring cry1Ah and cry1Ie by gene gun. Indoor and field resistance identification on corn borer, plus a series of molecular detection including PCR、RT-PCR、Southern blot、Western blot and ELISA has been served to genetic stability and pattern analysis. Results were as follows:
1. Positive plants were confirmed by PCR. Bt gene was proved by Southern blot that it was integrated into plant genome, further it was transcripted and expressed in plant cells detected by RT-PCR, Western blot respectively. Genetic analysis showed that Bt gene was single-locus dominant inheritance, conforming to Mendelian genetic pattern. Three foreign genes, cry1Ah, cry1Ie and 2mG2-epsps, was complete linkage and coseparation in transgenic maize genome, namely they were transferred to offspring like single gene locus.
2. Cry1Ah expression varied among plants from the same transformed event, but statistical analysis indicated that the difference was not significant, while there was significant difference among different transformed events. And Cry1Ah expression varied among different tissues from the same stage of one plant, higher in the bract and leaf, lowest in the pollen (bract>leaf>stalk>tassel>tassel handle>ear tip>seed>filament>pollen). Bt toxic protein expression possessed temporal and special specificity, Cry1Ah expression varied in different stage of leaf and stalk, taking on dynamic downtrend.
3. Foreign gene expression varied in progenies from different transformed events hybridized with different conventional maize, largely related to foreign gene expression of one parent and gene type of the other. Therefore, it required transgenic inbred lines with high foreign gene expression to be parent. Resistance analysis on corn borer and foreign expression detection showed that transmission frequency of mega gamete was higher than andro gamete, so that choosing resistant parent to crossbred would make it possible to obtain high expression progenies.
4. Indoor and field resistance analysis on corn borer demonstrated that, not only maize harboring cry1Ah and their progenies, but also transgenic maize harboring cry1Ah and cry1Ie were highly resistant to corn borer, besides the very resistance was able to transfer from generation to generation. Therefore it was effective to control Asia corn borer by means of transforming foreign Bt genes. Variety pattern of Cry1Ah protein expression detected through ELISA was on the whole consistent with resistance analysis, indicated that combination of indoor/field resistance analysis and molecular detection would illuminate resistance on corn borer more precisely, and the results showed that Cry1Ah protein expression was significant relative to Fracture rate of tassel handle and indoor larva death ratio in 7 days, and extremely significant relative to leaf-feeding level and tunnel length per 100 plants. So it made clear that Bt protein content was the internal cause of resistance to corn borer.
5. Comparing conventional maize and transgenic maize harboring single cry1Ah or harboring cry1Ah and cry1Ie and their progenies, statistical analysis indicated that there was significant difference in kernals per row and bare tip length, yet indistinct difference in plant height, height, width, line number of ear and g/1000 seeds. Shorter bald tip and increasing in grains per ear indicated the possible product raising.
6. Comparing conventional maize and transgenic maize harboring single cry1Ah, both in experimental field and wilderness, there was indistinct difference in geminating rate, growing vigor, plant type, bearing period and plant height, except for yield. In wilderness, maize geminating rate was only 5%, and weeds coverage was higher than that of maize (significant difference or extremely significant difference), showing that survival competitiveness of maize was much weaker. Germinating test showed that there was indistinct difference between conventional maize and progenies of transgenic maize harboring cry1Ah.
7. Five pure lines of transgenic maize harboring cry1Ah and 1 transgenic maize harboring cry1Ah and cry1Ie have been selected.
引文
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