转基因植物中筛选标记基因的利用及消除 Using of Selective Marker Gene in Transgenic Plants an
标记基因技术在转基因作物育种中的应用研究
标记基因技术在转基因作物育种中的应用研究随着人口的增长和城市化进程的加速，粮食安全问题越来越受到关注。
为了满足日益增长的粮食需求，转基因技术成为农业领域的热点话题。
而标记基因技术作为转基因技术中的一个重要组成部分，在转基因作物育种中的应用研究也越来越受到重视。
一、标记基因技术的概念和原理标记基因技术是一种在基因工程领域中常用的技术。
其主要原理是针对目标基因，通过特定的分子标记对其进行标记和检测，从而降低转基因育种的繁琐程度。
目前常用的分子标记有DNA序列标记、酶标记和抗体标记等。
二、标记基因技术在转基因作物育种中的应用1. 筛选适合杂交的亲本标记基因技术可以识别携带特定基因的个体，从而筛选出适合杂交的亲本。
这可以大大缩短育种周期，提高转基因作物的育种效率。
2. 评价和选择优良转基因品种标记基因技术可以快速、准确地评价和选择优良的转基因品种。
通过对大量基因型信息的分析，可以对转基因品种的质量和性状进行评估，为农业生产提供更好的品种资源。
3. 植物基因组研究标记基因技术可以帮助植物基因组研究的开展。
通过对基因组DNA序列的标记和分析，可以研究植物基因组的结构和功能，以及植物遗传学的各种规律。
三、标记基因技术在转基因作物育种中的前景标记基因技术在转基因作物育种中的应用已经取得了一定的成果，但其前景仍然非常广阔。
随着先进科技的不断发展，标记基因技术在作物遗传育种和演化基因组学等领域的应用必将继续扩大。
同时，在经济和实用性等方面的需求不断提高的情况下，标记基因技术将在转基因作物育种中发挥更为重要的作用。
总之，标记基因技术作为转基因技术中的重要一环，对于转基因作物育种有着极其重要的意义。
它的应用将加快转基因作物育种领域的发展，促进粮食安全和农业可持续发展。
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标记基因筛选原理
标记基因筛选原理标记基因筛选是一种常用的分子生物学技术，它通过引入特定的标记基因来筛选和鉴定转基因生物。
标记基因通常与目标基因共同转入宿主细胞，然后利用标记基因的特定性质对转基因生物进行筛选和鉴定。
本文将介绍标记基因筛选的原理及其在生物技术领域中的应用。
标记基因筛选的原理主要包括标记基因的选择、转基因生物的构建和筛选方法。
首先，标记基因的选择是标记基因筛选的关键。
常用的标记基因包括抗生素抗性基因和草除剂抗性基因等。
这些标记基因在转入宿主细胞后，能够使细胞对特定抗生素或草除剂产生抗性，从而实现对转基因生物的筛选。
其次，转基因生物的构建是标记基因筛选的基础。
通过基因工程技术，将目标基因和标记基因共同导入宿主细胞，并确保它们在细胞中能够稳定表达。
最后，筛选方法是标记基因筛选的关键环节。
常用的筛选方法包括对转基因植物进行抗生素或草除剂处理，对转基因动物进行PCR或Southern blotting等分子生物学方法。
标记基因筛选在生物技术领域中有着广泛的应用。
首先，它在转基因作物的培育中起着关键作用。
通过引入抗生素或草除剂抗性基因，可以实现对转基因作物的筛选和鉴定，从而加快转基因作物的培育进程。
其次，标记基因筛选也在基因治疗和基因编辑领域中得到广泛应用。
通过引入特定的标记基因，可以实现对基因治疗和基因编辑技术的筛选和鉴定，从而提高基因治疗和基因编辑的效率和准确性。
总之，标记基因筛选是一种重要的分子生物学技术，它通过引入特定的标记基因来实现对转基因生物的筛选和鉴定。
标记基因筛选的原理包括标记基因的选择、转基因生物的构建和筛选方法。
它在转基因作物的培育、基因治疗和基因编辑等领域有着广泛的应用前景。
随着生物技术的不断发展，标记基因筛选技术将进一步完善和应用，为生物技术领域的发展提供更多的可能性和机遇。
转基因移植技术真题单选题100道及答案
转基因移植技术真题单选题100道及答案1. 转基因技术是指将（）导入生物体基因组中，以改变其遗传特性。
A. 外源基因B. 内源基因C. 蛋白质D. 核酸答案：A2. 下列哪项不是转基因技术的应用领域？A. 农业B. 医学C. 环境保护D. 历史学答案：D3. 转基因植物中常用的基因载体是（）A. 质粒B. 病毒C. 细菌D. 真菌答案：A4. 转基因动物的制备方法不包括（）A. 显微注射法B. 胚胎干细胞法C. 核移植法D. 化学合成法答案：D5. 以下哪种酶常用于切割DNA 以获取目的基因？A. 解旋酶B. 限制酶C. DNA 聚合酶D. RNA 聚合酶答案：B6. 转基因食品的安全性评估不包括（）A. 营养学评价B. 毒理学评价C. 社会学评价D. 致敏性评价答案：C7. 转基因技术中，将目的基因导入受体细胞的过程称为（）A. 转化B. 转染C. 感染D. 转导答案：A8. 下列哪种生物不能作为转基因的受体？A. 细菌B. 病毒C. 植物细胞D. 动物细胞答案：B9. 用于鉴定转基因植株的常用方法是（）A. PCR 技术B. 电泳技术C. 层析技术D. 离心技术答案：A10. 转基因植物可能带来的环境风险不包括（）A. 基因污染B. 生物多样性减少C. 土壤肥力增加D. 产生超级杂草答案：C11. 以下关于转基因技术的描述，正确的是（）A. 只能在同种生物间进行基因转移B. 可以随意改变生物的性状C. 遵循自然界的遗传规律D. 不需要对受体细胞进行筛选答案：C12. 转基因动物在医学研究中的应用不包括（）A. 疾病模型的建立B. 药物筛选C. 器官移植D. 考古研究答案：D13. 目的基因在受体细胞中的表达水平可以通过（）来检测。
A. 荧光定量PCRB. 显微镜观察C. 化学分析D. 肉眼观察答案：A14. 下列哪项不是转基因技术面临的伦理问题？A. 对人类健康的潜在影响B. 对生态平衡的破坏C. 对传统文化的冲击D. 对宗教信仰的违背答案：C15. 转基因技术在农业上的优势不包括（）A. 提高农作物产量B. 增加农作物抗病虫害能力C. 减少农药使用D. 降低农产品营养价值答案：D16. 以下哪种生物的基因组最适合作为转基因的模板？A. 细菌B. 真菌C. 植物D. 动物答案：A17. 转基因植物的安全性评价主要依据（）A. 实验数据B. 专家意见C. 公众舆论D. 政府决策答案：A18. 转基因技术中，用于连接目的基因和载体的酶是（）A. 限制酶B. DNA 连接酶C. 解旋酶D. RNA 聚合酶答案：B19. 下列哪种方法不能用于去除转基因植物中的筛选标记基因？A. 共转化法B. 位点特异性重组系统C. 随机突变D. 杂交育种答案：C20. 转基因食品标识的目的是（）A. 保障消费者知情权B. 限制转基因食品销售C. 增加食品生产成本D. 区分转基因和非转基因食品答案：A21. 以下关于转基因动物的描述，错误的是（）A. 可以用于生产药用蛋白B. 培育过程简单快捷C. 可能存在伦理争议D. 有助于研究基因功能答案：B22. 转基因技术中常用的报告基因有（）A. 绿色荧光蛋白基因B. 胰岛素基因C. 生长激素基因D. 干扰素基因答案：A23. 下列哪项不是转基因植物可能带来的生态风险？A. 改变土壤微生物群落B. 影响非靶标生物C. 促进生态系统稳定D. 与野生近缘种杂交答案：C24. 目的基因导入植物细胞后，整合到基因组的位置是（）A. 随机的B. 固定的C. 可预测的D. 由载体决定的答案：A25. 转基因技术在环境保护中的应用不包括（）A. 生物修复B. 减少温室气体排放C. 制造新型污染物D. 开发新能源答案：C26. 以下哪种方法可以检测转基因食品中的外源基因？A. 蛋白质印迹法B. 气相色谱法C. 高效液相色谱法D. 红外光谱法答案：A27. 转基因动物的培育过程中，受体细胞通常是（）A. 受精卵细胞B. 体细胞C. 造血干细胞D. 神经细胞答案：A28. 下列哪项不是转基因技术在工业中的应用？A. 生产生物材料B. 提高工业发酵效率C. 制造传统手工艺品D. 开发新型酶制剂答案：C29. 转基因技术引发的知识产权问题主要涉及（）A. 基因序列的专利保护B. 植物品种的保护C. 动物品种的保护D. 以上都是答案：D30. 以下关于转基因植物的抗虫性，说法错误的是（）A. 可以减少农药使用B. 可能导致害虫产生抗性C. 对所有害虫都有效D. 不会影响生态平衡答案：C31. 目的基因在受体细胞中的稳定遗传需要（）A. 整合到染色体上B. 存在于细胞质中C. 独立复制D. 随机分布答案：A32. 转基因技术在农业生产中的应用面临的挑战不包括（）A. 公众接受度低B. 技术成本高C. 基因漂移风险D. 农产品价格下降答案：D33. 下列哪种作物最常进行转基因改良？A. 小麦B. 水稻C. 玉米D. 高粱答案：C34. 转基因植物的筛选标记基因通常（）A. 有利于植物生长B. 对植物生长无影响C. 会影响植物品质D. 会增加植物的抗性答案：C35. 以下关于转基因动物的食品安全问题，说法正确的是（）A. 与传统动物食品完全相同B. 存在潜在风险，需要严格评估C. 一定不安全D. 不需要关注答案：B36. 转基因技术中，目的基因的获取方法不包括（）A. 化学合成法B. 从基因文库中筛选C. 随机合成D. PCR 扩增答案：C37. 下列哪项不是转基因技术在医学领域的应用前景？A. 基因治疗B. 器官再生C. 美容整形D. 疫苗生产38. 转基因植物的推广需要经过（）A. 严格的审批程序B. 简单的登记手续C. 无需审批D. 消费者投票决定答案：A39. 目的基因导入受体细胞后，可能出现的情况不包括（）A. 不表达B. 低表达C. 高表达D. 立即死亡答案：D40. 以下关于转基因技术的社会争议，主要集中在（）A. 安全性B. 伦理道德C. 经济效益D. 以上都是答案：D41. 转基因技术在畜牧业中的应用不包括（）A. 改良畜禽品种B. 提高饲料利用率C. 生产皮革制品D. 保护野生动物答案：D42. 下列哪种基因常用于提高转基因植物的抗逆性？A. 抗冻基因B. 甜味基因C. 香味基因D. 彩色基因答案：A43. 转基因食品的检测技术不断发展，其目的是（）A. 提高检测精度B. 降低检测成本C. 简化检测流程D. 以上都是答案：D44. 以下关于转基因植物的知识产权保护，说法正确的是（）A. 不受保护B. 与传统植物相同C. 有专门的法律法规D. 完全由企业自主决定答案：C45. 转基因技术在水产养殖中的应用优势不包括（）A. 提高养殖产量B. 改善水产品品质C. 减少养殖水域污染D. 增加水产品种类答案：D46. 目的基因与载体连接时，黏性末端的形成依靠（）A. 限制酶B. DNA 连接酶C. 核酸内切酶D. 核酸外切酶答案：A47. 下列哪项不是转基因技术在农业可持续发展中的作用？A. 节约水资源B. 增加土壤侵蚀C. 提高肥料利用率D. 减少化学物质排放答案：B48. 转基因植物的商业化种植需要考虑（）A. 市场需求B. 种植成本C. 政策法规D. 以上都是答案：D49. 以下关于转基因动物的伦理问题，说法错误的是（）A. 可能涉及动物福利B. 不存在伦理问题C. 引发道德争议D. 需要规范和监管答案：B50. 转基因技术在生物制药中的应用不包括（）A. 生产疫苗B. 合成抗生素C. 提取中药成分D. 制造抗体51. 下列哪种方法可以提高转基因植物的表达效率？A. 优化启动子B. 增加筛选标记基因C. 减少目的基因拷贝数D. 降低转化效率答案：A52. 转基因技术引发的国际贸易争端主要涉及（）A. 技术壁垒B. 知识产权C. 产品质量D. 以上都是答案：D53. 以下关于转基因植物的监管，说法正确的是（）A. 各国监管政策相同B. 监管力度逐渐减弱C. 不断完善和加强D. 无需监管答案：C54. 转基因动物的繁殖过程中，目的基因的遗传遵循（）A. 孟德尔遗传定律B. 连锁遗传定律C. 自由组合定律D. 细胞质遗传定律答案：A55. 目的基因导入受体细胞前需要进行（）A. 测序分析B. 活性鉴定C. 蛋白表达D. 以上都是答案：D56. 转基因技术在农业领域的应用对农民的影响不包括（）A. 增加收入B. 提高劳动强度C. 降低生产成本D. 面临市场风险答案：B57. 下列哪种作物不是通过转基因技术获得的？A. 抗虫棉B. 太空椒C. 黄金大米D. 转基因大豆答案：B58. 转基因植物的安全性评价实验不包括（）A. 急性毒性实验B. 慢性毒性实验C. 致癌实验D. 考古实验答案：D59. 以下关于转基因技术的发展趋势，说法错误的是（）A. 更加精准B. 更加安全C. 应用范围缩小D. 与其他技术融合答案：C60. 转基因动物的生产过程中，需要对（）进行筛选。
使用基因工程技术进行植物转基因的关键步骤
使用基因工程技术进行植物转基因的关键步骤基因工程技术在植物领域的应用越来越广泛，其中最重要的应用之一就是植物转基因。
通过植物转基因，科学家们能够改变植物的基因组，使其获得更好的抗病性、耐旱性、抗虫性以及提高产量等特性。
下面将介绍植物转基因的关键步骤。
1. 目标基因的挑选和克隆植物转基因的第一步是选择需要改变的目标基因。
根据需求，科学家们可以选择增强某种抗性、改善某种品质或增加植物的营养价值等。
一旦确定目标基因，就需要在染色体上将其克隆出来，以便后续的基因转移。
2. 基因载体的构建转基因技术中，基因载体是一个不可或缺的工具。
基因载体是一个DNA分子，用于将目标基因转移到植物细胞中。
一般来说，研究人员会选择合适的质粒作为基因载体，并将目标基因插入到其中。
此外，基因载体还可以含有选择标记基因，用于筛选转基因植株。
3. 基因传递技术选择适当的基因传递技术是植物转基因的关键步骤之一。
常用的基因传递技术有农杆菌介导的转化、生物质粒介导的转化和基因枪转化等。
农杆菌介导的转化是最常用的方法之一，它利用一种土壤中的细菌农杆菌，通过插入一段目标基因的DNA序列到其载体上，然后转移到植物组织中。
而生物质粒介导的转化则是将目标基因导入含有质粒的金属微粒，通过炮弹或其他装置将其射入植物的细胞中。
4. 选择标记基因筛选转基因植株为了筛选出转基因植株，科学家会在基因载体中加入选择标记基因。
选择标记基因常常与目标基因一起转移。
通过选择合适的标记基因，可以利用生物学、生化或者抗生素抑制等方法筛选出含有目标基因的转基因植株。
5. 转基因植株的再生和培养一旦得到转基因植株，接下来的步骤是将其再生和培养。
科学家们将含有目标基因的转基因组织继续培养，通过选择合适的培养基和生长条件，促进其生长和分化。
在培养过程中，经过筛选得到的转基因植株会不断繁殖，直到形成一批具备目标基因特征的转基因植株。
最后，进行多代观察和评估，确保目标基因在转基因植株中稳定遗传。
tk基因作为筛选标记基因的原理
tk基因作为筛选标记基因的原理TK基因，全称双重抗性标记基因（twin-killer gene），是一种常用的筛选标记基因。
它是一种用于标记转基因植物的方法，可以通过它的独特特性来筛选出带有特定基因的真正转基因植物，从而实现基因在植物中的稳定表达。
TK基因的原理是利用一对相互作用的基因组成，分别是克隆选择基因（Selectable Marker Gene）与草酸苯甲酯酶（TK）。
克隆选择基因通常是一种对外界环境具有抗性的基因，例如抗生素抗性基因或除草剂抗性基因等。
而草酸苯甲酯酶（TK）是一种外源酶，能够将草酸苯甲酯（TK基因座上的杀手物质）转化为香豆素和对甲酚。
在筛选转基因植株的过程中，首先将带有TK基因的转基因体细胞与未转基因细胞进行杂交杂交，形成杂合体。
然后在培养基中加入草酸苯甲酯，草酸苯甲酯会被杂合体细胞中的草酸苯甲酯酶转化为香豆素和对甲酚，从而在细胞中产生杀手效应。
由于草酸苯甲酯对未转基因细胞没有杀伤效应，只有杂合体中带有TK基因的转基因细胞可以存活下来。
在培养基中存活的转基因细胞可以再次进行分化培养，形成新的转基因植株。
通过这种方式，可以筛选出带有TK基因的真正转基因植株。
TK基因标记方法具有一些优点。
首先，转基因植株筛选过程简单，只需加入草酸苯甲酯即可，无需复杂的培养条件和处理过程。
其次，筛选效率高，只有带有TK基因的转基因细胞才能存活下来，可以确保筛选出真正的转基因植株。
此外，TK基因在转基因植物中稳定表达，不会发生基因沉默等现象。
然而，TK基因标记方法也存在一些问题。
首先，筛选过程中使用的草酸苯甲酯对植株细胞有毒性作用，可能对植株的生长和发育产生不利影响。
其次，TK基因可能会对植株自身代谢过程产生干扰，影响植株正常生长。
此外，由于TK基因是通过转化杂交体细胞的方式进行筛选，可能会出现杂合体细胞的混合性质，导致筛选结果的不确定性。
总之，TK基因作为筛选标记基因具有一些优点和问题。
在实际应用中，需要根据具体情况权衡利弊，选择合适的筛选方法。
植物转基因Transgenic Plants
1Transgenic PlantsAn Historical PerspectiveLuis Herrera-Estrella, June Simpson, and Miguel Martínez-TrujilloSummaryThe development of technologies that allow the introduction and functional expres-sion of foreign genes in plant cells has extended in less than two decades to the pro-duction of transgenic plants with improved insect and disease resistance, seeds and fruits with enhanced nutritional qualities, and plants that are better adapted to adverse environmental conditions. Vaccines against serious human diseases and other impor-tant products have also been developed using transgenic plants. Many more agronomic and quality traits are currently being engineered in both academic and industrial labo-ratories, which are limited only by our poor knowledge of plant gene function. The emergence of new functional genomic strategies for the identification and character-ization of genes promises to provide a wealth of information with an enormous poten-tial to enhance traditional plant breeding and to genetically engineer plants for specific purposes. This chapter describes some of the highlights in the development of these technologies and some of the major achievements in production and commercializa-tion of transgenic crops. We also discuss some of the biosafety issues related to release of this novel class of plants into the environment.Key Words:Biosafety regulations; disease and pest resistance; genetic engineering;metabolic engineering; plant protection; transgenic plants.1. IntroductionTo date, the world population stands at more than 6 billion people, and it is expected to reach 9 billion by the year 2050. Food production will need to increase at the same rate or more to satisfy the needs of such an enormous number of people. Plants, the first link in the food chain, obtain energy from sunlight and transform it into compounds that directly or indirectly provide the From:Methods in Molecular Biology, vol. 286: Transgenic Plants: Methods and ProtocolsEdited by: L. Peña © Humana Press Inc., Totowa, NJ3food necessary for the survival of other living organisms. Traditionally, plants have been improved through selection during many crop cycles, and to date this has produced high yielding varieties, especially in the case of hybrids, which formed the basis of the green revolution. One challenge that faces tradi-tional agriculture is the fact that normally, only individuals of the same species can be crossbred. If natural resistance to a specific insect or fungus does not exist, then traditional breeders cannot create resistance or introgress this trait. Therefore, it is necessary to search for alternative sources of genes in other species of plants, microbes, or fungi. The genes harbored in other species can now be transferred to different plant species with the appropriate regulatory sequences so as to add a new trait or modify an existing one. Plant genetic engineering has become possible as a result of the work of many researchers during the last two decades.Twenty years ago, results of the first experiments describing the successful transfer and expression of foreign genes in plant cells were published. Since then, transgenic plants have become an essential tool for studying plant biol-ogy and for the development of novel plant varieties that have been cultivated extensively in some regions of the world. Transgenic technology has had a profound impact on the rapid development of plant biology in the past 15 yr by providing the means of producing gene-tagged populations, cell markers to study plant development, and the technology to study gene function. In terms of agricultural production, the impact of transgenic technology has not achieved its full potential because of the controversies that this new technology has gen-erated and the strict regulatory systems that have been adopted by many coun-tries. This chapter briefly discusses some of the highlights that led to the development of transgenic plants; also reviews some of the tools that transgenic technology has provided to study plant biology together with several of the major plant improvements achieved using this technology.2. Plant Transformation MethodsDifferent methods have been developed to introduce foreign genes into plants. A common feature is that the transforming DNA has to bypass differ-ent membrane barriers; it first has to enter the plant cell by penetrating the plant cell wall and the plasma membrane and then must reach the nucleus and integrate into the resident chromosomes. For the majority of species gene transfer is carried out using explants competent of regeneration to obtain complete, fertile plants. This implies the development of a tissue culture tech-nology that frequently becomes an art. Although gene transfer technology has become routine in working with several plant species, in others the limit-ing step is not the transformation itself but rather the lack of efficient regen-eration protocols.The most widely used and successful transformation methods are the Agrobacterium tumefaciens-mediated DNA transfer and direct transfer through particle bombardment.2.1. The Agrobacterium SystemIn 1907, Smith and Townsend demonstrated that the Gram-negative soil bac-terium Agrobacterium tumefaciens, a member of the eubacterial family Rhizobiaceae, is the organism responsible for the elicitation of crown gall tumors in plants; formation of these tumors occurs as a result of bacterial infec-tion, usually at wound sites, on many dicotyledonous and some monocotyledon-ous plants (1). This discovery had no major repercussion until Armin Braun demonstrated that tumor cells are transformed and that the uncontrolled prolif-eration of the tumor cells was not dependent on the continuous presence of Agrobacterium, implying the existence of a transformation-inducing principle (2). In 1974, Ivo Zaenen, Jeff Schell and Marc Van Montagu (3) at the Univer-sity of Ghent, Belgium, identified a megaplasmid that was present only in the virulent strains of Agrobacterium and absent in the avirulent ones, and named it Ti plasmid for tumor-inducing plasmid. Three years later, Eugene Nester, Milton Gordon, and Mary-Dell Chilton (4), at the University of Washington, demon-strated that only some genes of the Ti plasmid were transferred to the chromo-somes of the plant cell and were responsible for inducing tumors. The DNA segment transferred to plant cells was named T-DNA and is delimited by left and right borders, which are 25-basepair imperfect, direct repeats. Researchers reasoned that any piece of DNA between these borders could be transferred into the plant cell and randomly integrated into the genome of the plant. Taking into account this consideration, research teams at the University of Ghent, the Monsanto Company, and the University of Washington at St. Louis, Missouri, inserted heterologous genes with the appropriate regulatory regions into the T-DNA region and showed that foreign genes became integrated and functionally expressed in plant cells. Later, disarmed Ti plasmids, which con-tain a T-DNA lacking genes involved in tumor formation, were used to produce the first transgenic plants (5–7).In the years since these early experiments using Nicotiana tabacum and Petunia hybrida, the Agrobacterium system has been used to transform a large range of dicotyledonous plant species. Although initially the transfor-mation of cereals was considered impossible, a few years later it was shown that cereals such as maize and rice could also be transformed. More recently fungi have also been transformed using this system (8). A large number of plant species has been transformed with this method (9).The Agrobacterium system has several advantages over other transforma-tion methods and it is considered as the first option to transform plants. Theseadvantages include the following aspects: (a) In a significant percentage of the transformation events, a single copy of the T-DNA is integrated into the chromosomes of the transformed cell (10). (b) Numerous vector systems are now available containing the T-DNA borders and various reporter and select-able marker genes, allowing researchers to choose the most appropriate com-bination to insert heterologous genes. (c) It is possible to transfer large fragments of DNA, including bacterial artificial chromosomes (11). (d) Trans-formation in planta, without the necessity of tissue culture, is possible in some species such as Arabidopsis thaliana and Medicago trunculata (12). For a more detailed description of the Agrobacterium-mediated gene transfer sys-tem,see Chapter 2.2.2. The Biolistic MethodThe biolistic method was developed as a necessity to transform plant spe-cies originally recalcitrant to transformation by the Agrobacterium system including the economically important cereals. This method consists of the delivery of microprojectiles, usually of tungsten or gold, coated with DNA and propelled into the target cells by acceleration. The acceleration can be provided by an explosion of gunpowder or a discharge of high-pressure gases such as helium or CO2(13,14). Molecular analysis of plants transformed biolistically in general reveals a complex pattern of transgene, indicating the integration of multiple copies of the bombarded DNA. However, it has been demonstrated that in most cases, these multiple copies are arranged as a single locus and segregate in a Mendelian pattern (15). As with Agrobacterium, a great number of diverse plant species have been transformed by the biolistic method(9). Some advantages of the biolistic method are the following: (a) A wide variety of types of explants can be used to undergo bombardment and obtain fertile plants. (b) There is no need for specialized transformation vec-tors. (c) This is the only reliable method for chloroplast transformation. More detail information of the biolistic method is provided in Chapter 4.2.3. Other Transformation MethodsThe direct transfer of DNA to protoplasts using polyethylene glycol (PEG), calcium phosphate, or electroporation has been shown to be possible in vari-ous of plants including maize (see Chapters 5 and 8 and ref.16). Low repro-ducibility and the regeneration of plants were the main problems because these methods are often specific for certain cultivars. The microinjection technique employs immobilized cells into which the DNA is internalized individually. However, the tedious manipulation, need for sophisticated equip-ment, and difficulty of regeneration of plants have not permitted its wider utili-zation.3. The Use of Transgenic Plants to Study Gene Expression and FunctionTransgenic plants have been used extensively to study gene expression and function. For this purpose, plants are transformed with chimeric gene constructs in which a reporter gene is under the control of the regulatory sequences of the gene to be analyzed.Several reporter genes are commonly used in plants, including G-glucu-ronidase, luciferase, and genes involved in anthocyanin biosynthesis (see Chapter 14 and ref.17). More recently, the gene for the green fluorescent pro-tein (GFP) has become an important in vivo reporter in plants. When expressed in plant cells and illuminated with blue light, GFP produces a stable bright green fluorescence that is easily monitored nondestructively (see Chapter 15 and ref.18). Thus, it can be used as a means to visualize the fate of trans-formed cells over time and rapidly test the influence of various factors on gene expression.These new generations of reporter genes are easily monitored for expres-sion, and allow rapid determination of sequences important in regulating the temporal, spatial, and environmental expression of a gene in great detail. Reporter genes have been instrumental in the analysis of gene expression under a myriad of environmental stimuli, including light, wounding, tem-perature, growth hormones, and so forth and in different plant tissues. These studies are leading to the unraveling of the complex interactions involved in the responses of plants to these stimuli.Reducing or increasing the expression of the target gene by sense and antisense or cosuppression strategies can be used to study gene function. Analy-sis of the phenotype or changes in mRNA or metabolite profiles can provide valuable information to determine gene function. Plant transformation is now also widely used as a tool for insertional mutagenesis, either directly by the T-DNA or by the mobilization of transposons into species in which these ele-ments have not been characterized. This strategy produces a collection of indi-viduals containing transposon or T-DNA insertions throughout the genome. These insertion mutants can then be systematically screened for interesting phenotypes and the affected genes identified and isolated with relative ease. This strategy has been carried out successfully in several plant species to date, including Arabidopsis, tomato, and rice (19–21).4. Production of Transgenic Plants With ImportantApplications in Agriculture, Industry, and MedicineWith the development of plant transformation methods, knowledge of the structure and function of certain genes, and the desire to resolve some of the classic problems in traditional agriculture, the race to obtain better plants by genetic engineering began with satisfactory results. Initial strategies consid-ered the introduction of single genes into plants of interest; now, however, strategies involving multiple genes from a single metabolic pathway can be used. The main strategies used to produce improved transgenic plants with commercial or agricultural applications are mentioned in the following sub-headings.4.1. Nutrients and Quality of Seeds and FruitsPostharvest losses are one of the main constraints in preservation and com-mercialization of agricultural products. In the case of fruits, it is essential to conserve quality properties during transport and storage to ensure successful marketing. The main problem is fruit softening resulting from the ripening pro-cess. Using plant antisense technology it has been possible to delay ripening by decreasing the expression of genes important in this process, such as those involved in cell wall degradation or the biosynthesis of ethylene (22,23). To date, tomatoes have been modified for slower ripening and higher solid con-tent and commercialized by three different companies (24). This strategy has an enormous potential for tropical fruit such as mango and papaya, grown in many developing countries. In such countries, the lucrative export market can-not be exploited because the fruits ripen rapidly and there is a lack of appropri-ate storage conditions and efficient transport systems to enable them to reach the end consumer (25).The major sources of proteins for a large portion of the human population are cereal grains and legume seeds. However, a characteristic of these seeds is a deficiency in lysine in cereals and cysteine and methionine in legumes. One obvious solution to this problem would be the consumption of both kinds of seeds in adequate proportions; however, in the case of human nutrition there are cultural traditions and economical factors that prevent this. An alternative is to change the seed protein composition of certain crops. Efforts in this direction include the production of methionine-rich proteins in transgenic tobacco (26) and canola seeds, which results in an increase of up to 33% in methionine (27); the expression of a sunflower seed albumin gene in lupins (Lupinus angustifolius) causing the methionine content to double (28); or a synthetic gene that encodes a protein with 43% lysine content in tobacco seeds (29). A 100-fold increase in free lysine in soybean and canola was possible by modifying the regulatory properties of enzymes involved in synthesis of this essential amino acid(30). Potato, the most important vegetable food crop, was transformed with a gene from amaranth that encodes a seed-specific nonallergenic protein (AmA1), with a balanced amino acid composition that promises to improve the nutritional value of this food source (31). Recently, the Indian government has authorized cultivation of these transgenic potatoes to help alleviate the serious malnutrition problems in that country.Vitamin A deficiency is a very important nutritional problem in many coun-tries, especially in Asia, where 124 million children suffer from blindness caused by deficiency of this vitamin. As a potential solution to this problem, a strategy has been developed to produce vitamin A in rice. Rice does not normally produce vitamin A, but the genes encoding the three enzymes for G-carotene (pro-vitamin A) biosynthesis that are absent in rice were specifically expressed in the endosperm of transgenic rice seeds (32). Because rice is an important dietary component in Asia, consumption of the transgenic so-called “golden rice” could help alleviate vitamin A deficiency in this region.Plant oils have many uses in both food and industrial applications, there-fore, the manipulation of fatty acid composition and content is one of the areas of greatest interest in metabolic engineering. One of the goals in manipulating fatty acid composition is to produce healthier vegetable oils. Most vegetable oil used for food applications is partially or fully hydroge-nated to produce semisolid spreads, a process that results in the production of trans-isomers of unsaturated fatty acids that are normally not present in plants and have been associated with coronary heart disease. Increasing the content of stearic acid in soybean, cotton and Brassica oilseeds using antisense, cosuppression, and RNA interference to down-regulate endogenous stearyl-ACP synthase has allowed the production of semisolid margarine without the need for hydrogenation (33). An oxidatively stable liquid oil low in saturated fatty acids has also been produced in soybeans by suppression of the oleoyl desaturase. This oil has been produced commercially and is extremely stable for high-temperature frying applications (33). In the future, it will be neces-sary to increase the production of oil, including novel types of oils, to satisfy the demand not only for edible oils but also for industrial oils, considering that the nonrenewable fossil oils will be depleted in the future, and vegetable oils are a renewable resource that can be harvested at a rate of several million tons annually.The phytoene synthase from the bacterium Erwinia uredovora has been overexpressed in tomato (Lycopersicon esculentum). Fruit-specific expression was achieved by using the tomato polygalacturonase promoter and the levels of phytoene, lycopene, G-carotene, and lutein levels were increased 2.4-, 1.8-, and 2.2-fold, respectively (34). These changes in flux coefficients have revealed a shift in the regulatory step of carotenogenesis, which has important implica-tions for future metabolic engineering strategies. In addition, a high consump-tion of tomatoes in the human diet could help to reduce the onset of chronic diseases such as coronary heart diseases and certain cancers.Although coffee has no nutritive function in the human diet, it does have an important social and psychological role as it accompanies various social events and is part of the daily habits of many individuals. The consumptionof caffeine, however, can adversely affect sensitive individuals by producing insomnia and increasing blood pressure, among other effects. With this in mind, in plants of Coffea canephora the gene encoding theobromine syn-thase, an enzyme involved in the synthesis of caffeine, was inhibited using RNA interference (35). The caffeine content of these plants was reduced by up to 70%, indicating that it would be possible to produce naturally “decaffeinated” coffee seeds. This strategy is now being applied to Coffea arabiga, which accounts for roughly 70% of the world coffee market.4.2. Insect and Virus ResistanceThe damage to crops caused by insects, viruses, and other pathogens repre-sents one of the most important problems in agricultural production. There-fore, major efforts both in public institutions and the private sector have focused on developing pest- and disease-resistant transgenic plants that have resulted in some of the most successful genetically engineered plant products.The development of insect-resistant transgenic plants was based on the knowledge of insecticidal proteins. Bacillus thuringiensis (Bt) is a soil micro-organism that produces proteins called I-endotoxins during sporulation. When I-endotoxins are ingested by insects, they bind to midgut epithelial cells, caus-ing their osmotic lysis (36). Many Bt strains express different I-endotoxins, each with their own spectrum of activity, against different types of insects (37). For example, the Cry1A and Cry1C proteins are specific to lepidopteran larvae such as those of the E uropean corn borer (Ostrinia nubilalis), whereas the Cry3A protein is toxic to coleopteran larvae such as those of the Colorado potato beetle (Leptinotarsa decemlineata)(38).I-Endotoxin genes have been manipulated for expression in plants by generation of truncated versions of the genes, decreasing their GC content and changing codons that are seldom used in plants (36). Transgenic plants expressing I-endotoxins from several strains of Bacillus thuringiensis(known as Bt lines), have to date been generated and effectively used for insect control in various species, including tobacco (39), tomato(40), cotton (41), potato (42), maize (43), canola (44), soybean (45), and rice (46).Bt maize plants provide excellent protection against European corn borer (Ostrinia nubilalis) under insect pressure several hundred-fold higher than natural infestations, and transformed potatoes are resistant to Colo-rado potato beetle larvae (36). Although Bt maize has been a major biotechno-logical success, the insects that attack this crop differ depending on the geographical region, and field trials are necessary to evaluate their effective-ness for insect control in different environments. Corn plants resistant to beetle rootworms were also generated by the expression of two novel proteins from Bacillus thuringiensis(47). Elite hybrid Bt rice lines have been produced that show resistance to two of the most important lepidopteran rice pests (leaffolderand yellow stem borer) without reduced yield (48). Transgenic lines of insect-resistant sugarcane, an important crop in developing countries, have also been developed(49). Several insect-resistant transgenic crop plants including maize, potato, and cotton are currently produced commercially.The production of Bt endotoxins is the most widely used strategy to produce insect-resistant transgenic plants; however, these proteins are not effec-tive against all pests and alternative insecticidal proteins are required to avoid the development of resistance in the target pest. Therefore, it has been necessary to find alternative insecticidal and nematicidal proteins, such as proteinase inhibitors, which are part of the defense system of many plants. Proteinase inhib-itors of plant origin effective against certain target insects have been engineered into different plant species, such as canola, potato, alfalfa, lettuce, petunia, and tomato; however, they have not been commercialized (38). Transgenic Ara-bidopsis plants expressing a gene encoding a proteinase inhibitor have been shown to suppress the growth and egg production of two root nematodes (50).Avidin, a glycoprotein that sequesters the vitamin biotin, was expressed in maize at levels that prevent the development of insects that damage grain dur-ing storage. This toxicity is caused by biotin deficiency and suggests that avi-din could be used as a biopesticide in stored grains (51). Fertile transgenic tobacco plants with leaves expressing avidin in the vacuole have been pro-duced and shown to halt growth and cause mortality in larvae of two nocturnal lepidopterans,Helicoverpa armigera and Spodoptera litura(52).Many virus-resistant transgenic plants have exploited genes derived from viruses themselves, in a concept referred to as pathogen-derived resistance (PDR). The first example of this strategy was the expression of the Tobacco mosaic virus(TMV) coat protein gene in tobacco plants. These transgenic plants were found to have effective resistance against TMV (53). Using a simi-lar strategy, transgenic yellow squash resistant to Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus II (WMVII) have been produced (54).Papaya ringspot virus(PRSV) transmitted by aphids causes one of the most important diseases in papaya. Several attempts using conventional breeding failed to produce PRSV-resistant papaya varieties. However, the use of plant biotechnology succeeded in producing PRSV-resistant, transgenic plants by expressing the coat protein gene from this virus (55). The future of the papaya crop in Hawaii and other regions of the world effectively rests on the develop-ment of virus-resistant plants, an aim already achieved by transgenic technol-ogy(56) (see Chapter 27). Transgenic tomatoes resistant to Tomato mosaic virus (ToMV), transgenic potatoes resistant to Potato virus X(PVX) and Potato virus Y(PVY), and transgenic cucumbers resistant to Cucumber mosaic virus (CMV) have all been produced (57). In China, watermelon has been transformed with the WMV-II coat protein gene showing high resistance to the infection bythis virus (58). To date, transgenic, virus-resistant papaya and yellow squash have been commercialized. The protective mechanism conveyed by the coat protein or other viral genes is not yet completely understood, and an RNA-mediated cosuppression model has been proposed (59).Because the potyvirus genome is initially translated into a polyprotein, the completion of the life cycle of this virus depends on the site-specific process-ing of this precursor by the action of self-processing viral cysteine protein-ases. It has been shown that the expression of a rice cysteine proteinase inhibitor in tobacco induces resistance against two important potyviruses, Tobacco etch virus (TEV) and Potato virus Y viruses (PVY). This represents an alternative method to control this agriculturally important group of aphid-transmissible plant viruses (60).4.3. Resistance to Phytopatogenic Fungi and BacteriaFungal pathogens cause some of the most devastating diseases of crop plants; therefore much effort has been spent on producing resistant plants. Suc-cess in the production of transgenic resistant plants has been limited. Expres-sion in transgenic plants of genes encoding enzymes capable of degrading the major constituents of fungal cell walls (chitin and G-1,3-glucan) have been used as a strategy to control these organisms. Expressing the genes of two of these enzymes in tomato showed a useful level of resistance to a Fusarium wilt disease (54).Production of toxins by phytophatogenic bacteria is an important virulence factor. For instance, Xanthomonas albilineans produces a family of toxins that lead to characteristic chlorotic symptoms by blocking chloroplast development. The introduction and expression of the albicidin detoxifying gene (albD) from the bacterium Protoea dispersa resulted in sugarcane in a significant reduction of disease symptoms and decreased multiplication of the pathogen (61). The bac-teria Pseudomonas syringae pv phaseolicola produces phaseolotoxin, which inhibits ornithine carbamoyltransferase (OCTase), an enzyme involved in the biosynthesis of citrulline. As P. syringae also produces citrulline, this bacterium harbors a gene encoding a phaseolotoxin-resistant OCTase, which when intro-duced into the tobacco genome provides resistance against this pathogen (62).The identification, characterization, and understanding of the mode of action of plant disease resistance genes will provide new avenues to generate disease resistance plants. Several genes encoding key components of the machinery that recognizes avirulance factors produced by viral, bacterial, and fungal patho-gens have been cloned and characterized in the past 10 yr. Although the mecha-nisms by which these disease resistance genes work is still not completely understood, some of them have been successfully used to engineer disease resistance. For instance, the Xa21gene from the wild rice Oryza longistaminata,。
转基因植物中的标记基因及其消除
转基因植物中的标记基因及其消除
陆晓春;薛庆中
【期刊名称】《浙江农业学报》
【年(卷),期】2001(013)001
【摘要】随着转基因植物的商业化，植物遗传转化技术将为农业生产带来一场新的革命。
新的转化程序要求把基因导入农艺性状优良的品种中，呈单拷贝、不带有辅助序列，并在不同的转化体中表达一致，稳定遗传。
本文主要讨论标记基因的安全性及标记基因消除的方法等问题。
【总页数】6页(P49-54)
【作者】陆晓春;薛庆中
【作者单位】浙江大学农学系,;浙江大学农学系,
【正文语种】中文
【中图分类】S330
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植物基因转化及转基因植物的分析与鉴定
植物基因转化及转基因植物的分析与鉴定1. 引言植物基因转化是一种重要的生物工程技术，利用这种技术可以引入外源基因或修改内源基因，从而改变植物的性状和功能。
转基因植物是通过植物基因转化技术获得的具有外源基因的植物，具有重要的应用价值。
本文将介绍植物基因转化的基本原理和方法，并探讨转基因植物的分析与鉴定方法。
2. 植物基因转化的基本原理和方法2.1 基本原理植物基因转化利用穿透细胞壁的技术，将外源DNA导入植物细胞，通过细胞的内源机制使其稳定地表达。
常用的植物基因转化方法包括农杆菌介导的转化、生物弹射法和基因枪法等。
2.2 基本方法2.2.1 农杆菌介导的转化农杆菌介导的转化是最常用的植物基因转化方法之一。
基本步骤包括构建表达载体、感受剂的处理和遗传转化的选择和鉴定。
构建表达载体时，将目标基因插入适当的载体上，并添加转录和翻译的调控序列，如启动子和终止子，以确保目标基因的表达。
感受剂的处理是将表达载体导入农杆菌中，并通过培养条件的优化，使农杆菌中的表达载体得到高效表达。
遗传转化的选择和鉴定是将感受剂经过适当的处理后，转化到植物细胞中，并通过筛选和鉴定来确定转化成功的细胞株。
2.2.2 生物弹射法生物弹射法是将DNA以高速撞击植物细胞，使其穿透细胞的质壁和细胞膜，进而将外源基因导入细胞内。
生物弹射法通常使用微粒子加速器或毛发管射击法进行。
微粒子加速器是一种将金属微粒或微球与外源DNA一起加速，并将其发射到目标细胞上的设备。
通过微粒的高速撞击，外源基因能够穿透细胞的质壁和细胞膜。
毛发管射击法是将DNA包裹在微小的金属颗粒上，然后使用高压气体将金属颗粒射击到目标细胞上。
这种方法也能够使外源基因穿透细胞膜进入细胞。
2.2.3 基因枪法基因枪法是将外源DNA包裹在金粒或微米级金属颗粒上，并使用高压气体或炮发射器将其穿过细胞质，进入植物细胞。
基因枪法不需要依赖转化菌或细胞融合等辅助手段，直接将外源DNA送入目标细胞，因此具有较高的成功率。
转基因植物的筛选与鉴定
转基因植物的筛选与鉴定随着植物转基因技术的不断进步，它对于分子遗传学研究和植物改良都具有特别重要的意义。
转基因植株的筛选在转基因技术中起着关键性的作用。
将含有35S启动子的基因载体PMDC150经农杆菌介导，利用农杆菌侵染拟南芥花序的方法转入拟南芥后得到T1代种子。
文章通过组织培养来筛选含有Kan抗性的转基因植株。
标签：拟南芥；转基因植物；筛选1 文献综述1.1 转基因植物的研究进展植物的遗传转化是指利用重组DNA，细胞组织培养等方法，将外源基因导入到植物的组织或细胞中，获得转基因植物的技术[1]。
从转基因植株成功之后，转基因技术飞速发展，如今，植物在抗病、抗虫和抗药等方面都有了转基因植株。
这种技术对改进农作物品质、提高产量等方面都有非常大的帮助。
1.2 基因转化技术随着人们对基因转化技术不断地深入研究和探索，现已发现多种基因转化的方法，例如农杆菌介导的基因转化、花粉管通道法等。
相比较其他植物基因的转化方法，农杆菌介导法有着减少成本、容易操作等优点，但对宿主细胞有着严格的要求。
每种方法都有其优缺点，所以根据植物的不同种类，我们要选择合理的转化方法，达到理想的转化效果。
1.3 本论文的目的和意义植物的转基因技术日新月异迅速发展，已经成为许多国家重点发展的新目标新领域，它所产生的巨大的社会和经济效益促使各个国家对其进行更加深入的研究，由其产生的巨大的生产潜能一定会推动社会的进步，改善人类现有的生产、生活质量以及健康水平。
本文以拟南芥为主要研究对象，通过转化后的农杆菌侵染拟南芥花序的方法及组织培养的方法进行了轉基因植物的筛选。
借由本次实验研究，可深入了解基因工程等相关领域的理论，可熟练掌握相关技术例如无菌操作技术、植物转基因技术、植物组织培养技术等等。
2 转基因植物的筛选与鉴定2.1 实验材料2.1.1 植物材料拟南芥2.1.2 载体与菌株重组表达载体PMDC150-35S（由实验室提供）、农杆菌菌株GV31012.1.3 主要试剂75%酒精、84消毒液、侵染缓冲液、限制性内切酶、卡那霉素、壮观霉素（重组表达载体含有的抗性）、利福平2.1.4 培养基LB培养基：5g/L酵母提取物，10g/L蛋白胨，10g/L NaCl，15g/L琼脂（只固体培养基添加）1/2MS培养基2.2 实验方法2.2.1 拟南芥种植：将种子放在浸过水的滤纸上，放在4℃的冰箱中，不见光培养24小时，然后取出种子种在营养土里，在光照强度8000Lux，温度25℃的培养箱中经过光照16小时/黑暗8小时培养[2]。
tk基因作为筛选标记基因的原理
tk基因作为筛选标记基因的原理引言随着科学技术的不断发展，基因工程和生物技术在生物学领域的应用日益广泛。
其中，基因筛选在疾病治疗、农业生产和环境保护等方面发挥着重要作用。
t k基因作为一种常用的筛选标记基因，在基因工程研究中具有重要的意义。
本文将介绍tk基因作为筛选标记基因的原理，以及其在实际应用中的优点和局限性。
1. t k基因的背景t k基因全称为胸腺激酶（t hy mi di ne ki n as e）基因，是一种编码酶的基因。
该酶参与细胞内嘌呤和嘧啶代谢途径，具有催化转化胸腺嘧啶核苷酸为胸腺嘧啶核酸的能力。
由于tk基因在多种细胞类型中无法表达，它成为筛选标记基因的理想选择。
2. t k基因作为筛选标记基因的原理2.1原理概述t k基因作为筛选标记基因的原理是利用其特殊的生理功能，通过特定的筛选方法，识别和选择带有tk基因的转基因细胞或生物体。
2.2t k基因与筛选方法的结合基于tk基因的筛选标记系统主要包括增强型tk（H SV-t k）系统和细菌质粒t k（pS V2tk）系统。
这些系统使用特定的筛选方法来实现对t k 基因的识别和选择，如酶标记法、细胞培养法和P CR扩增法等。
2.3筛选标记基因的工作流程1.将期望转导的基因与t k基因连接构建成转基因表达载体。
2.将转基因表达载体导入宿主细胞或生物体中。
3.利用相应的筛选方法选择tk基因的表达细胞或生物体。
4.分离和培养经过筛选的细胞或生物体，以获得目标基因表达的细胞群体。
3. t k基因作为筛选标记基因的优点可靶向筛选-：利用t k基因的特异性表达，可以实现对特定基因的选择。
筛选效率高-：基于t k基因的筛选方法较为简便且有效，能够高效地筛选出带有t k基因的细胞或生物体。
广泛应用-：t k基因作为筛选标记基因已广泛应用于基因工程研究和生物技术领域。
4. t k基因作为筛选标记基因的局限性产生毒性物质-：一些筛选方法需要使用抗代谢药物，这些药物可能会产生毒性物质，对宿主细胞或生物体造成损害。
消灭转基因的方法
消灭转基因的方法
消灭转基因的方法包括以下几种：
1. 避免种植转基因作物：政府或组织可以采取立法或政策措施，禁止或限制种植转基因作物。
2. 检测和追踪：建立有效的检测方法，对农作物和食品进行转基因成分的检测，同时建立追踪系统，将转基因产品排除出市场。
3. 基因编辑技术：利用基因编辑技术，如CRISPR-Cas9，可以精准地修改植物基因组，使其恢复为非转基因状态。
4. 生物控制：利用天敌、寄生虫、病毒等生物来攻击转基因植物，从而降低它们在自然环境中的生存能力和种群数量。
5. 农业管理措施：采取一系列农艺措施，如旋转作物、间作、清除杂草等，来减少转基因植物的种植面积和数量。
6. 公众教育和意识提升：通过教育、宣传和科普活动，提高公众对转基因植物的认识和了解，激发公众对转基因食品的关注和拒绝。
需要注意的是，虽然有多种方法可以减少或消灭转基因，但各种方法的可行性和
效果存在差异，实施时应综合考虑各种因素。
转基因植物的筛选与检测
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• Western 印迹是将蛋白质从 SDS-PAGE胶 中电泳转移至固相支持体上 , 然后对固定化 蛋白质进行免疫学测定的方法。 • 特点：
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一、筛选的方法与原理
• 通过特定基因在转化体内的表达，利用特 定的试剂，选择出来转化细胞。
• 注： 1.特定基因又称“抗性基因” 2.表达方式：蛋白质的合成、酶的失活等
表“植物遗传转化中的选择试剂及其抗性基因”
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二、检测的方法与原理
• 利用特定基因在转化体内的表达，对其表 达产物进行检测。
扩增产物经琼脂糖凝胶电泳 ，染色后很容易观察， 不通过杂交分析就可以鉴定出基因组中的一些顺 序。材料用量少，检测方便，可以在试管苗阶段， 甚至对愈伤组织进行检测，了解转化早期的信息， 便于及时优化试验方案。DNA提取方便，适合于 大批量样品分析，又能检测目的基因的完整性， 是早期检测的一种较好方法。
• 注意： • 此过程最重要的是保持各DNA片段的转膜的固相支持物有多种，包括硝酸纤维素 膜（NC膜）、尼龙膜（Nylon） 、化学活化膜和 滤纸等。
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Western
• • • • • • 1.制胶及上样 2.电泳 3.转膜 4.封闭 5.抗体孵育 6.曝光显影
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Southern杂交
• • • • • • • 1.提取DNA 2.电泳 3.变性 转移 DNA 印迹 4.转膜 5.固定DNA 6.探针的标记 7.杂交与检测（NBT/BCIP显色）
膜
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转基因植物生物安全标记基因
转基因植物生物安全标记基因王兴春3 杨长登(中国水稻研究所农业部水稻生物学重点实验室 杭州 310006)摘要 在大量转基因植物被推向市场的同时,人们对转基因植物对环境及人类健康等许多方面可能存在的风险感到担扰。
标记基因的生物安全性成为人们普遍关注的问题之一。
新的标记基因不仅要求能够对转基因植物进行筛选和鉴定,而且必须对环境和生物都是安全的。
概述并评价了绿色荧光蛋白基因、核糖醇操纵子、62磷酸甘露糖异构酶基因、木糖异构酶基因以及谷氨酸212半醛转氨酶基因等生物安全标记基因及其最新研究进展。
关键词 基因工程 转基因植物 生物安全 标记基因收稿日期:2002209203,修回日期:20022112293电子信箱:wxingchun @ 自从1983年第一株转基因植物诞生以来,全球抗虫、抗病、抗除草剂和品质改良的棉花、水稻、大豆、玉米等转基因植物已达120多种,种植面积4700多万ha 。
为了加快作物遗传改良的进程,将外源目的基因导入植物体,并筛选出极少量的转化细胞,一套高效安全的选择方法极为重要。
到目前为止被广泛用于选择的标记基因主要有两大类:一类是抗生素类,包括潮霉素磷酸转移酶基因(hpt )、新霉素磷酸转移酶基因(npt )、卡那霉素抗性基因(npt Ⅱ)等;另一类是抗除草剂类,包括草丁膦(glu fosinate )抗性基因(bar )、草甘膦(glu fosinate )抗性基因(epsps )等。
这些存在于转基因植物中的具有抗生素或除草剂抗性的标记基因是否会对环境及人类健康有不良影响和损害引起了广泛关注。
这些问题主要集中在:(1)抗生素抗性基因会不会转移到微生物中,使病原菌获得抗性,从而导致目前临床使用的抗生素失效;(2)标记基因会不会传播到野生亲缘种中,使杂草获得这种抗性,变成现有除草剂无法杀灭的“超级杂草”;(3)具有抗生素或除草剂抗性的标记基因的应用,会不会破坏生态平衡。
然而,目前的试验水平还不能对这些问题进行准确的估计和评价。