【作者】 宋小玲；

【导师】 强胜；

【作者基本信息】 南京农业大学 ， 植物学， 2003， 博士

【摘要】 以时间关系序列、空间关系序列和种群数量关系为原则，选择抗除草剂转基因作物的伴生杂草、亲缘关系较远的和亲缘关系较近的近缘种为研究对象，从抗性基因漂移的条件，特别是首要条件亲和性为出发点，采用生殖生物学方法结合人工杂交和田间混合种植的试验结果，探讨了抗除草剂转基因作物的抗性基因向不同亲缘关系近缘种，特别针对早期不亲和的近缘种漂移的检测方法和技术。以抗除草剂转基因水稻(Oryza sativa)和油菜(Brassica napus)与其不同亲缘关系近缘种为材料进行了抗性基因漂移的风险评价。为抗除草剂转基因作物抗性基因漂移的安全性评价提供了快速检测技术和方法。首次提出了针对亲和性不同近缘种的分层次评价抗性基因漂移的措施。主要内容如下： 1．抗除草剂转基因水稻的基因漂移 1．1 从稗类植物(Echinochloa spp．)的开花生物学特性评价其与抗除草剂转基因水稻的基因交流可能性。首次全面报道了稗类植物的开花生物学特性，结果表明稗类植物的每日开花节律为80％左右的颖花在7：00之前开，7：00-8：30为15-20％，8：30之后零星开花，10：30尚未见开花。稗类植物和栽培水稻存在花时不遇现象，因而在田间稗草和水稻的基因交流可能性因花时不遇而降低。无芒稗花粉活力在活体条件下下降较慢，3h后仍有17.31％的花粉有活力。说明无芒稗花粉在水稻开花时仍有部分保持活力，因而存在潜在的基因漂移可能性。 1．2 采用光学显微镜观察，首次证明了抗除草剂转基因水稻和无芒稗的不亲和性发生在柱头，表现在转基因水稻花粉不能在无芒稗柱头上正常萌发生长更不能穿过无芒稗柱头，而且该柱头不亲和不能用花粉蒙导的方法打破。结合人工杂交和田间混合种植没有杂交种产生，证明抗除草剂转基因水稻的抗性基因漂移到无芒稗的可能性较小。本方法为花粉早期萌发阶段就不亲和的物种间的基因漂移提供了科学快速检测的方法。通过植物的亲和性评价抗除草剂转基因作物基因漂移的安全性研究 1 .3采用光学显微镜观察结合人工杂交试验结果研究了药用野生稻(口，口‘咖cilolis)和转ba)’基因水稻花粉杂交的基因漂移.结果表明，供试水稻花粉在药用野生稻柱头上的萌发生长与药用野生稻自花授粉花粉的萌发生长有一定差异，表现在穿过柱头的花粉粒百分率及内容物释放和正在凝缩、释放的花粉粒百分率较少。杂交后结实率为O，表明转基因水稻和药用野生稻杂交不亲和.但转基因水稻花粉能在药用野生稻柱头上正常萌发生长，并能释放内容物，增加了水平基因转移的风险.在本实验条件下转基因水稻和药用野生稻没有发生成功的基因交流.显然用普通光学显微镜不适合于观察发生在柱头之后的不亲和过程。 1 .4采用荧光显微镜观察了杭性水稻花粉在杂草稻(口尽翻sativa)柱头上的萌发、花粉管在花柱中的生长以及进入胚囊的情况，结合杂交结实率，评价了杭性基因向杂草稻漂移的可能性.结果表明转基因水稻花粉在供试5种杂草稻柱头上都能正常萌发生长，花粉管能顺利穿过花柱进入子房和胚囊，和杂草稻自花授粉没有明显差异.人工杂交后不同生态型杂草稻和2种转基因水稻的杂交结实率不同，江苏招稻和安徽塘稻与2种水稻的结实率类似，与Y0003的结实率都较低，不超过20%，而与99一t的结实率较高，达60%之多，马来西亚1号和2种转基因水稻的杂交结实率都比较低，都不超过20%，而马来西亚2号和3号与2种水稻的杂交结实率都高于50%，最高达82“、。从杂交结实结果来看，虽然不同抗性水稻品种向不同杂草稻漂移的可能性大小存在一定差异，但2种转基因水稻的基因都能以高频率向杂草稻漂移，风险性大。利用荧光显微镜可以观察和检测花粉管能否进入子房。.抗除草剂转基因油菜的抗性基因漂移 2 .1采用荧光显微镜观察了3种类型栽培油菜(B~ica spp.)花粉在两地采集的野芥菜(B.jl，ncea var.罗叹‘11115)柱头上的萌发生长情况，结合杂交后的结实率，探讨了3种类型油菜和野芥菜杂交的亲和性.结果表明，甘蓝型油菜和芥菜型油菜与野芥菜的亲和性都非常高，亲和性指数达ro.o以上，而白菜型油菜和野芥菜的亲和性非常低，亲和指数小于0.2。子一代的适合度研究结果表明，莽菜型油莱和野芥菜杂交一代的适合度没有降低，而甘蓝型及白菜型和野芥菜杂交一代的适合度明显下降，表现在花粉活力降低，结实率极低。上述结果表明，白菜型油菜和野莽菜的基因转移可能性最小，甘蓝型居中、而芥菜型极易和野芥菜发生基因转移.摘要 2.2通过人工去雄授粉和田间混合种植，研究了杭草甘磷和草丁麟转基因油莱向野芥菜基因漂移。结果表明两种转基因油菜向野芥菜基因漂移可能性类似，杂交后结实率都很高，达13粒/角果，和母本自交结实率没有明显差异，田间混合种植能产生杂交种;杂交一代的适合度研究结果表明，杂交一代的适合度明显下降，表现在花粉活力降低，结实率极低.杂交一代和父母本回交结果表明，以杂交一代为父本，野芥菜为母本，结实率虽低，为2.29一2.45粒/角果，但仍能产生少量种子，以杂交一代为母本，野芥菜和杭性油菜为父本结?更多还原

【Abstract】 The concominant weeds and relatives with different level (near and far comparatively) of relatedness from transgenic herbicide-resistant crops were selected as experimental materials based on the principle of time relation, space relation and quantity relation of those related population. By means of reproductive biology plus hand-pollination and open-pollination, the assessment methods and techniques of gene flow from transgenic herbicide-resistant crops to different relatives were studied based on the sexual compatibility which is the first important condition of gene flow. Especially the fast test method for these species which incompatibility occurred at early stage of pollen germination was provided by this experiment. Meanwhile the possibilities of gene flow from transgenic herbicide-resistant rice (Oryza sativd) and oilseed rape (Brassica napus)io their relative species were assessed. The fast test techniques and methods for assessment on the gene flow of transgenic herbicide-resistant crops were provided. And the assessment measure of different level to different relatives was put forward firstly.1. Gene flow of transgenic herbicide-resistant rice1.1 The biology of anthesis of Echinochloa spp. was studied and reported firstly. The daily anthesis rhythm was 77-80% before 7:00, 15-20 % during 7:00-8:30, sporadic after 8:30, and no anthesis was obseved after 10:30. This showed the flowering time didn’t overlapped between Echinochloa spp. and rice due to 9:00-10:30’s rice anthesis, which the possibility of gene exchange may be low. The pollen vitality of E. crusgalli var. mitis collecting directly from spikes decreased slowly. At 3h after anthesis, the pollen vitality was still 17.31%. This showed the pollen ofE. crusgalli var. mitis could keep vitality until rice was blooming. The possibility of potential gene flow could still exist between transgenic herbicide-resistant rice and E. crusgalli var. mitis .1.2 The fact that the sexual incompatibility between transgenic rice and Echinochloa crusgalli var. mitis occurred at the stigma was observed with light-microscope firstly bymeans of reproductive biology. The sexual incompatibility lie in the fact that pollen grains of transgenic rice on the stigmas of barnyard grass couldn’t germinate nor grow normally after cross, and couldn’t penetrate the stigmas of barnyard grass. Moreover the sexual incompatibility couldn’t be broken under the mentor pollen inducement. Furthermore the fact that emasculated barnyard grass pollinated with the rice pollen grains couldn’t seed and produce no hybrid in field supported the incompatibility between them. Therefore, the possibility of gene flow between them was relatively low. The fast test method for these species which incompatibility occurred at early stage of pollen germination was developed from this experiment.1.3 The gene flow of pollen cross between wild rice (O. offidnalis ) and two varieties of transgenic rice with bar-gene was studied by means of reproductive biology. The results showed that the pollen grains of two varieties of transgenic rice could germinate on the stigmas of wild rice and their pollen tubes could grow and penetrate stigmas of wild rice normally, but the percent of pollen grains penetrated wild rice stigmas or condensed and released pollen inclusions after cross was lower than that of self-pollination. The fact that the emasculated wild rice pollinated with transgenic rice pollen grains couldn’t seed showed they were sexual incompatibility. However the probability of horizontal gene transfer could be increased because the pollen grains of rice could germinate and grow on the stigmas of wild rice and released the inclusions. Under this experiment condition, gene flow between wild rice and transgenic rice with bar-gene didn’t occur successfully. The optical microscope was not suitable for the observation of the sexual incompatibility after stigma.1.4 The possibility of gene flow between transgenic rice and weedy rice(0. saliva) was assessed by the results of the germina更多还原