2013年事件 美国孟山都公司首席技术官福瑞里、比利时人Marc Van Montagu 和美国人Mary-Dell Chilton获得2013年“世界粮食奖”，这是27年来该奖项首次授予基因改良作物研究人员 世界粮食奖于 1986 年由诺贝尔和平奖得主诺曼•E•博洛格 (Norman E. Borlaug) 博士创立。诺曼的杰出工作引发了绿色革命，并为他赢得了“有史以来拯救生命最多的人士”的美誉。1990年，艾奥瓦商人兼慈善家约翰•鲁安 (John Ruan) 成立了世界粮食奖基金会。

第七章 Genetic transformation in plant

目录 转基因技术现状及其应用 植物遗传转化技术 载体构建 遗传转化操作 转基因鉴定

第一节 植物遗传转化现状及其应用

Nature has a rich source of variation As we are all aware, most species have an abundance of variation. The photograph of bean seeds is a great illustration of the variation in nature. You can notice not only many different colors, but also many different patterns. A large array of interacting genes are responsible for this variation. But man can always dream of a new use for an organism. These dreams often involve asking the species to do something it does not now normally do. Biotechnology involves added new traits to a species.

What controls this natural variation? Allelic differences at genes control a specific trait Gene - a piece of DNA that controls the expression of a trait Allele - the alternate forms of a gene Before we can understand who man goes about using biotechnology approaches to modify a species, we must understand basic genetic principles and terminology. All traits are controlled by genes. A gene can have different forms. These forms are called alleles. It is important that you become fluent with these terms and the differences they imply. It is simple as remember that genes have alleles. Or, alleles are alternate forms of a gene.

But nature does not contain all the genetic variation man desires Fruits with vaccines Grains with improved nutrition Two of the most dramatic examples of man’s dreams of improving the utility of plants is shown here. At the top is the picture of bananas. The goal is to express vaccines in these fruits so that individuals who eat them will receive the vaccine and become immune to the disease. The rice photograph illustrates a recent invention called “Golden Rice.” This strain of rice has been engineered to express enzymes required for the vitamin A pathway that don’t normal exist in this species. By the goal is to provide this as crop as a dietary product that will improve the nutrition and health of those who eat it.

转基因技术发展 1983年华盛顿大学成功将卡那霉素抗性基因导入烟草，同年4月美国威斯康星大学成功将大豆基因转入向日葵，标志着植物转基因技术的诞生。 1985年，第一批抗病毒、抗虫害和抗细菌病的转基因植物进入田间试验。 1986年，美国环保署允许世界第一例转基因作物——抗除草剂烟草进行种植。 1994年，转基因延熟保鲜番茄——“FlavrSavr”获得美国食品药品管理局的批准进入市场销售，成为世界上第一个获许进行销售的转基因食品。

转基因植物重要里程碑 1983年首例转基因植物培育成功 1986年转基因植物获准进入田间试验 1994年转基因番茄在美国批准上市 1996年GMO面积170万ha 2012年全球转基因作物种植面积170.3 million ha

2012年全球转基因作物种植

按作物分-2012

按性状分

几种作物种植GMO比例

全球种GMO国家分布

我国批准6个转基因作物商品化 转CHS基因矮牵牛（北京大学） 抗虫棉（中国农科院研制） 抗虫棉（孟山都公司研制） 抗病毒番茄（北京大学） 耐储存番茄（华中农大） 抗病毒甜椒（北京大学）

转基因研究涉及的部分园艺作物 番木瓜 苹果 柿 荔枝 龙眼 红薯 草莓 梨 柑橘 桃 番茄 西瓜 葡萄 Pepper 花菜 大白菜 李 马铃薯

Production of transgenic plants resistant to diseases Application 1 Production of transgenic plants resistant to diseases

Diseases/viral diseases Limit production Affect quality Influence growth

研究成果 利用植物病毒外壳蛋白基因、病毒复制酶的部分基因或反义核糖核酸抑制病毒编码的加工酶，已获得多种抗植物病毒的转基因植物

Fresh papaya production in state of Hawaii and in Puna district from 1992 to 2001 Year   Total Puna (x 1,000 lb) (PRSV enters Puna) 1992   55,800 53,010 1993   58,200 55,290 1994   56,200 55,525 1995   41,900 39,215 1996   37,800 34,195 1997   35,700 27,810  (transgenic seeds released) 1998   35,600 26,750 1999   39,400 25,610 2000   50,250 33,950 2001   52,000 40,000 The yield and quality of Rainbow was exceptional, amounting annually to 125,000 L/acre vs. 5,000 L/ acre of non transgenic ones

Transgenic papaya resistant to PRSV

Fresh papaya productiona in state of Hawaii and in Puna district from 1992 to 2001 Year   Total Puna (x 1,000 lb) (PRSV enters Puna) 1992   55,800 53,010 1993   58,200 55,290 1994   56,200 55,525 1995   41,900 39,215 1996   37,800 34,195 1997   35,700 27,810  (transgenic seeds released) 1998   35,600 26,750 1999   39,400 25,610 2000   50,250 33,950 2001   52,000 40,000

过量表达CTV-CP蛋白的来檬抗CTV CTV symptoms of different intensity in young leaves after graft-inoculation with CTV T-305. Severe leaf distortion and stunting symptoms in a non-transgenic control plant (left), delay in virus infection and symptom attenuation in a transgenic plant (centre), and resistance against CTV T-305 in other transgenic plant (right).

Sweet orange overexpressing PR protein from tomato: resistant to phytophthora

Apple fire blight fire blight:1000ha apple were killed by FB in Michigan in 2000. Lytic protein (LP)：Royal Gala, Galaxy 和M26 转avian LP’s SB-37,T-4 lysozyme：Gala 转harpin（来于Fire blight细菌） NPR1蛋白：过量表达 Silencing the DIPM kinase which is related to the occurrence of the disease.

Transgenic Royal Gala with attacin gene: resistant to fire blight

Production of transgenic plants resistant to pests and insects Application 2 Production of transgenic plants resistant to pests and insects

Pests and insects Negative impact on yield, growth and quality Global loss: up to 15% of potential production, amounting for 10 bRMB Normal control：farm chemical (resistance and pollution)

Aftermath of farm chemicals 我国化学杀虫剂约占农药总量的70%以上，年产量22万吨左右，30%以上用于防治棉铃虫。 1992年至1996年， 24万余人农药中毒。1992年中毒人数就高达7万多人，直接经济损失达100多亿元。

研究成果 利用苏云金芽孢杆菌（Bt）杀虫蛋白基因CryI和CryIII，分别获得抗鳞翅目和鞘翅目害虫的转基因植物（番茄、马铃薯、烟草、杨树、水稻和棉花等） 在美国，转Bt基因作物的推广减少了26.9亿美元的杀虫剂的使用 1986-1998年转基因作物田间试验中，抗虫作物占29%

Bt-cotton China：1991年单价抗虫棉，1993年，开展双价抗虫棉的创新研究 2005年，国产抗虫棉已累计推广9000多万亩，创造经济效益150多亿元,2006年种植2000万亩 India: Bt cotton covers 8.1 m acres in 2006 每亩可减少化学农药0.5公斤，每亩增加的收益约为140元

Non-Bt cotton Bt cotton Source: CAAS

抗虫转基因棉花获大面积推广

Numbers of pesticide applications in Bt and non-Bt cotton in Hebei and Shandong in 1999 -- reduced by 13 applications In 2000: by 12 applications In 2001: by 14 applications

Percentage (%) of poisonings reported as numbers of farmers interviewed in Henan in 2000

Cabbage looper粉蚊夜蛾 test

Diamondback moth小菜蛾 test

Production of transgenic plants resistant to herbicide Application 3 Production of transgenic plants resistant to herbicide

GMO by traits-2012

Transgenic soybean 1994年5月美国Monsanto公司耐除草剂“镇草宁”大豆‘Round up Ready（农达安）’。

Benefits of Glyphosate 草甘磷Tolerance in Crops Can use at any time - can wait until there is a problem Move to greener herbicide Very effective - Weeds very sensitive - GM crop very resistant Reduced herbicide use GM canola surrounded by weeds - glyphosate + glyphosate

Production of transgenic plants with altered development progress Application 4 Production of transgenic plants with altered development progress

—延迟果实成熟 利用反义RNA技术抑制果实软化过程中的关键酶（聚半乳糖醛酸酶或乙烯前体合成酶），可使番茄果在储存期的软化延迟。 1993年美国食品和药品管理局（FDA）批准上市的第一个转基因食品——Calgene公司的转基因延熟番茄Flavr Savr（保味） 华中农业大学园艺系叶志彪教授获得转基因番茄——百日鲜（Bioscience)华番一号是我国第一个商品化生产的转基因产品

乙烯生成减少的苹果 转基因苹果： 乙烯生成减少70% 需更长时间软化 硬度：高于CK SSC：高于CK 比CK耐贮藏 对照 转基因苹果 3个月后 对照 转基因苹果

不变褐的苹果

Production of transgenic plants tolerant to abiotic stresses Application 5 Production of transgenic plants tolerant to abiotic stresses

Abiotic stress Approximately 70% of the genetic potential yield of major crops is lost by environmental factors. (Boyer, Science, 1982, 218: 443-448)

Enhanced growth in transgenic aspen

耐盐番茄 适宜保护地栽培的或露地早春栽培；抗寒、抗盐、无限生长型单株，果高桩型；每果序4-8果、坐果率高、单果重70-100g，糖度5.0-6.0%，Vc含量为41.4mg/100g鲜重；耐贮藏、利于通风透光，适于保护地栽培，产量可达5000～8000kg。

抗旱玉米 孟山都与巴斯夫合作 将先进的抗旱产品送入温室进行筛选；田间试验中领先的产品表现优异；继续评估以评价抗旱性方面的表现。 2008年农业进步展览会： 含抗旱基因 对照组 转基因 WITH GENE 含基因 CONTROL对照组 WITH GENE 含基因 CONTROL对照组 将先进的抗旱产品送入温室进行筛选；田间试验中领先的产品表现优异；继续评估以评价抗旱性方面的表现。 53 53 53 53

抗溃疡病的柑橘

抗脱水的枳

Production of transgenic plants with special value or traits Application 6 Production of transgenic plants with special value or traits

Transgenic flowers with different flower color 第一个转基因园艺植物是淡紫色的康乃馨Moondust：把编码类黄酮羟化酶和二氢类黄酮还原酶的基因转到白色康乃馨中，在转基因植物中积累了翠雀素，使康乃馨呈淡紫色，

植物类胡萝卜素代谢工程 改良作物营养品质 Shewmaker et al., Plant J, 1999; Lindgren et al., Plant Physiol, 2003; Paine et al., Nat Biotechnol, 2005; Rosati et al., Plant J, 2000; D‘Ambrosio et al., Plant Sci, 2004; Fujisawa et al., 2008; Zhu et al., PNAS, 2008; Apel and Bock, Plant Physiol, 2009; Maass et al., Plos One, 2009

植物类胡萝卜素代谢工程 生产特殊类胡萝卜素 Mann et al., Nat Biothechnol. 2000; Morris et al., Metab Eng, 2006; Jayaraj, et al., Transgenic Res, 2007; Suzuki et al., Plant Cell Rep, 2007; Zhu et al., PNAS, 2008; Fujisawa et al., J Exp Bot, 2009

Golden rice

The Golden Rice Story Vitamin A deficiency is a major health problem Causes blindness Influences severity of diarrhea, measles >100 million children suffer from the problem Improved vitamin A content in widely consumed crops is an attractive alternative Our bodies cannot make vitamin A, we get it in our diet in 2 ways: - 50% from pro-Vitamin A carotenoids from plants are converted into vitamin A in the intestine - rest from ingesting vitamin A itself in animal products or supplements The second major plant biotechnology product is more recent and was developed to address the vitamin A deficiency problems prevalent throughout the world. This vitamin deficiency is very critical because it can cause blindness and affects the severity of many diseases including diarrhea and measles. This is a severe problem that affects more than 100 million children worldwide. A simple solution would be to distribute vitamins to the affected children. Unfortunately, many countries where the deficiency is chronic do not have the necessary infrastructure to deliver the vitamin tablets to the most needed. The solution that is currently being promoted is to improve the vitamin content in widely-consumed, and readily available to the consumer. Transgenic rice plants were developed that contain elevated levels of the precursor to vitamin A. This GMO is called “Golden Rice” because of its color: it is yellow rather than white. It is yellow because β-carotene, a yellow precursor to vitamin A is abundant in the seed. All carotenoids that contain a -ring can be converted into retinol, and one of the most important carotenoid pro-vitamins is -carotene

Lycopene-beta-cyclase -Carotene Pathway in Plants IPP异戊二烯焦磷酸 GGPP Phytoene synthase Problem: Rice lacks these enzymes Phytoene Phytoene desaturase Unlike the single-step RoundUp Ready pathway, the β–carotene synthesis pathway involves multiple enzymes. This important vitamin A precursor cannot be synthsized in rice because it lacks four of the key enzymes. Therefore, the precursor is not made, and the plant contains white kernels. ξ-carotene desaturase Lycopene Lycopene-beta-cyclase Normal Vitamin A “Deficient” Rice  -carotene (vitamin A precursor)

‘Engineering provitamin A (ß -carotene) biosynthetic pathway into (carotenoid-free) rice endosperm’. Ye et al., Science 287,303-305 (2000). Two T-DNAs encoding 3 genes for pro-vitamin A synthesis (plus selectable marker gene) introduced together via Agrobacterium by co-transformation 水仙花 欧文氏菌 LB RB pro Daffodil 1 ter pro tp Erwinia ter LB RB pro Daffodil 2 ter pro Hyg resis ter Daffodil 1 = phytoene synthase Daffodil 2 = lycopene ß-cyclase Erwinia = Erwinia double desaturase - with added transit peptide, expressed from 35SCaMV promoter With own native transit peptides and endosperm-specific promoter from rice glutelin (GT1 promoter) Grain of resulting transgenic rice has light golden-yellow colour - best line had 85% of its carotenoids as ß-carotene

GA-20氧化酶部分正义抑制的苹果 Reduced concentration of GA1 and GA20 in shoot tips and young leaves. The dwarf type can be reversed by GA3.

Genes involved in floral development LFY encodes a plant specific transcription factor and is considered a master regulator of floral meristem development. AP1 is a member of the MADS-box gene family of transcription factors ,which play critical roles in development processes across the plant, animal and fungal kingdoms. FLOWERING LOCUS T (FT) is one of the flowering-time genes in Arabidopsis genes and is characterized as a floral pathway integrator.

Overexpression of LEAFY in citrus transgenic plants. (A) Transgenic shoot grafted in vitro on a nontransgenic rootstock showing a precocious terminal flower five weeks after regeneration. (B) Transgenic plant showing a weeping growth habit. (C) Leaves from transgenic plants showing various degrees of curling (top) compared to leaves from nontransformed control plants (bottom). (D) Vegetative shoot from a transgenic plant showing the reduction of thorns and small curled leaves (right) compared to a vegetative shoot from a nontransformed control plant of the same age (left). (E) Transgenic plant flowering 16 months after its transfer to the greenhouse. (F) Ripened fruit from a transgenic plant grown in the greenhouse.

不引起过敏的苹果

Transgenic tomato producing insulin 按照植物偏爱的密码子设计并合成了人胰岛素的A链和B链的编码序列，并通过以某种方式连接成为一个ORF的人胰岛素基因（180bp） ，依据软件分析使其产物模拟天然的胰岛素结构。该基因受控于番茄果实专一性启动子，在番茄果实中高效表达。通过生吃番茄的口服途径可改善胰岛素依赖型糖尿病人自体免疫状况，达到治疗效果。亦可从番茄果实中纯化出人胰岛素，做成注射剂型。

转基因作物三处性状 Input traits Output traits Value-added traits