【作者】 刘琳莉；

【导师】 强胜；

【作者基本信息】 南京农业大学 ， 植物学， 2004， 硕士

【摘要】 转基因植物田间释放带来的主要问题之一，就是抗性基因通过基因流转移到野生植株，从而给农田生态环境造成潜在的危害，所以在释放前对其潜在的基因漂移做出确切的评估是很必要的。自然条件下种与种之间是否存在亲和性是发生基因交流的关键因素，本文即通过生殖生物学研究，以种间亲和性作为评价标准，用转基因水稻与不同亲缘关系的杂草稻、药用野生稻和稗为实验材料，研究了判断不亲和性发生的各个阶段的方法，为评价其能否发生基因漂移提供技术支持，为最终建立安全性评价体系中个体及种群水平的评价系统提供依据。主要内容如下： 杂交亲和性的障碍可能在生殖生长过程中的许多阶段起作用，即花粉管不能萌发和不能进入柱头，不能到达子房，不能座果或种子发育不良，杂种死亡、不育或破损。寻找简单、快速检测其不亲和阶段的方法，特别是杂交早期的不亲和性观察研究，为风险评估提供有力支持。这也是目前转基因环境安全性评价研究和技术手段方面较为薄弱的环节。 杂交不亲和的第一个阶段，即花粉管能否萌发和到达柱头，用普通显微镜观察即可得到较为满意的结果。转基因水稻花粉授在稗草柱头，普通显微镜观察，其花粉萌发后花粉管扭曲变形不能进入柱头，因而不可能杂交，发生基因漂移的可能性极小。 第二个阶段即花粉管能否进入子房，因花粉管生长在花柱组织内而难以观察，用特异的荧光染料使花粉管染色，在荧光显微镜下呈明亮的黄绿色，而花柱和子房组织因自发弱荧光呈很暗的颜色，反差明显极易区分，可以确定花粉管在花柱中的位置，对于判断不亲和性的第二阶段十分方便。用该方法观察转基因水稻花粉授于药用野生稻柱头，花粉萌发较好并进入柱头，花粉管到达花柱基部时顶端膨大并停止生长，这说明转基因水稻的遗传物质可进入药用野生稻花柱，虽不能到达子房，但也有可能发生基因交换，所以可判断其发生基因漂移的可能性较小。 第三个阶段，即不能座果或种子发育不良、杂种死亡，主要观察胚囊发育。 用石蜡切片法，将植物子房用石蜡包埋后，切成8um左右薄片，再通过染色，显微镜观察胚囊内部结构，其结果准确、清晰、精细。转基因水稻花粉授于杂草稻柱头，用荧光显微镜技术观察发现花粉管可正常进入胚囊，但其结实率较低，用石蜡切片法观察发现其受精过程也较正常，但后期胚和胚乳发育不协调导致败育，所以种间亲和性评价抗除草剂转荃因作物安全性的技术研究以可判断其发生基因漂移的可能性较大。 但石蜡切片法操作过程相当繁冗，并且根据观察连续切片来建立立体概念也需要耐。心与经验。 整体透明是将子房整体处理透明后直接观察胚发育的方法，操作简便又富于立体感，具备原位地形学观察和指定性观察两个优点，但只能做粗略观察、判断胚胎发育的阶段。用该方法观察转基因水稻自交胚胎发育，只有球形胚阶段可清楚看到，可能因胚较小时不易染色和其它组织反差小而难以观察，胚较大时淀粉积累较多染色深遮住了胚。 另外，以上人工授粉及其后观察方法无法区分自身花粉和外来花粉，只能在确保去雄率达到100%时，才能做出有效判断，寻找特异标记的染料或先对父本花粉活体标记的方法，将可省去去雄这一步骤，且与母本自身花粉混合授粉更接近自然条件。 采用32P示踪法，对转基因水稻花粉进行标记，用自显影法可直接看到标记花粉管到达位置。该方法标记精确，谨慎操作也可避免人身伤害和环境污染。用该方法标记转基因水稻花粉授于稗草和药用野生稻柱头，自显影结果显示同位素没有进入稗草花柱，而药用野生稻柱头到花柱基部都有显像，这同前面用普通显微镜和荧光显微镜观察结果一致. 本论文还试验了多种染料及不同引入方法对花粉的标记，结果不理想，其原因还有待进一步研究，但实验过程为以后继续研究提供思路和借鉴. 通常研究基因漂移均以野生近缘种为母本，因漂移也可能由转基因植物获得野生近缘种的特异基因而自然存活，本文以转基因植物为母本进行了反向杂交的研究。用稗草、药用野生稻和几种杂草稻为父本，转基因水稻为母本进行杂交，其中授转稗草和药用野生稻花粉的结实率为O，授几种杂草稻的结实率与其以转基因水稻为父本杂草稻为母本的杂交结实率也有差异.对以转基因抗除草剂植物为母本野生亲缘种为父本的杂交子代，因母本携带杭性基因不能用喷除草剂或检验转基因启动子的方法来检验，本论文以子代种子的特异蛋白凝胶电泳法判断是否为杂交种.用种子清蛋白和球蛋白凝胶电泳法判断马来西亚杂草稻和安徽杂草稻花粉授于转基因水稻柱头结实是否为杂交种.清蛋白和球蛋白均有二十多条带，且父母本和子代谱带有差异，说明其子代是杂交种。关键词:转基因水稻;安全胜评价;技术方法;基因漂移;亲和性更多还原

【Abstract】 The main problem to release transgenic plant in the field is the target-gene flow to wild plant and causes potential danger to farmland ecological environment. So it is essential to make a precise assessment about gene flow before transgenic plant field release.Sexual compatibility between species in natural conditions plays a key role in occurrence of gene flow. The methods of assessment of occurrence stage of sexual incompatibility between species were developed on the basis of sexual compatibility between species by means of reproductive biology. The objectives of the studies are to develop assessment techniques of possibility of gene flow through observing sexual compatibility at the early stage using trans-genic rice and weedy rice, medical wild rice (Oryza officinalis) and Echinochloa spp. as experimental materials, and ultimately to integrate those techniques into safety assessment system of transgenic crops at individual and population level. The main content is given as follows:Hybridization incompatibility may occur at many stages in reproduction. It includes pollens can’t germinated and pollen tubes can’t enter the stigma or reach the ovary, the fruit or the seed can’t develop, hybrid seeds die, sterile or damage. Looking for the simple, quick methods to observe incompatibility stage, especially at very early stage, can offer strong support for the assessment. This is also the weak link on environmental safety assessment research and technique at present comparatively.The first stage, whether pollen can germinate and pollen tube enter the stigma, could be clearly observed under ordinary microscope. Pollen tubes of the transgenic rice pollinated on the stigma of barnyard grass (Echinochloa spp.) were so twisted and out of shape that can’t enter the stigma. Therefore they couldn’t hybrid and the possibility of gene flow should be extremely low.It is difficult to observe the second stage of incompatibility, namely pollen tube can’t enter the ovary with the above ordinary microscope, because the pollen tube is growing inside the style tissue. Pollen tube stained with the peculiar fluorescence dyestuffs could present bright yellowish green in style, and ovary be observed under the fluorescence microscope, while the style and ovary of spontaneous weak fluorescence are distinguished with very dark color comparing the stained pollen tubes. The above method for judge thesecond stage was convenient. When the transgenic rice pollen pollinated on the medical wild rice stigma was observed with this method, it was found that the pollen could germinated and entered the stigma normally, but the tube expanded and stopped growth when reaching the style base. This proved that hereditary materials of the transgenic rice could reach the medical wild rice style, so the possibility of gene exchange might increase. From above, it was concluded that possibility of gene flow from transgenic rice to the medical wild rice was relatively low but higher than to barnyard grass.Third stage, namely fruit or seed can’t develop, hybrid seed die, could be judged by observing the embryo sac development mainly. After cutting ovary buried with paraffin into 8 microns slices and dyed, the structure of the embryo sac including in the slices was observed under ordinary microscope accurately, clearly, and meticulously. Transgenic rice pollen on weedy rice’s stigma was observed with the above technology. The results showed that transgenic rice pollen could enter the embryo sac of weedy rice normally, but seed rate were low. It was found that embryo and endosperm development was incongruity although fertilization could occur. From above it was concluded that the possibility of gene flow was relatively high.But operation course of paraffin quite was great superfluous, and setting up three-dimensional concept according to plan slices observation needed patience and experience.The whole clearing method was to observe embryo directly after dealing with wholly ovary by transparent, which had advantages of easy and simple handling and three-dimensional eff更多还原