园艺植物生物技术1植物遗传转化现状及应用课件

1、园艺植物生物技术1植物遗传转化现状及应用园艺植物生物技术1植物遗传转化现状及应用22013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 42013年事件美国孟山都公司首席技术官福瑞里、比利时人Ma第七章Genetic transformation in plant第七章Genetic transformation in4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6目录转基因技术现状及其应用5第一节植物遗传转化现状

2、及其应用7第一节植物遗传转化现状及其应用6Nature has a rich source of variation8Nature has a rich source of v7What controls this natural variation?Allelic differences at genes control a specific traitGene - a piece of DNA that controls the expression of a traitAllele - the alternate forms of a gene9What controls this na

3、tural va8But nature does not contain all thegenetic variation man desiresFruits with vaccinesGrains with improved nutrition10But nature does not contain 91110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。19

4、94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。12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11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ha2012年全球转基因作物种植面积170.3 million ha13转基因植物重要里程碑1983年首例转基因植物培育成功122012年全球转基因作物种植142012年全球转基因作物种植13按作物分-201215按作物分-201214按性状分16按性状

5、分15几种作物种植GMO比例17几种作物种植GMO比例16全球种GMO国家分布18全球种GMO国家分布17我国批准6个转基因作物商品化抗病毒甜椒（北京大学）耐储存番茄（华中农大）抗病毒番茄（北京大学）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）19我国批准6个转基因作物商品化抗病毒甜椒（北京大学）耐储存18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20转基因研究涉及的部分园艺作物番木瓜番茄19Production of transgenic plants resistant to disease

6、sApplication 121Production of transgenic pla20Diseases/viral diseasesLimit productionAffect qualityInfluence growth22Diseases/viral diseasesLimit21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研究成果23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22Fresh papaya production in state of Hawaii and in Puna distr

7、ict from 1992 to 2001YearTotalPuna (x 1,000 lb)(PRSV enters Puna) 199255,80053,010199358,20055,290199456,20055,525199541,90039,215199637,80034,195199735,70027,810(transgenic seeds released) 199835,60026,750199939,40025,610200050,25033,950200152,00040,000The yield and quality of Rainbow was exception

8、al, amounting annually to 125,000 L/acre vs. 5,000 L/ acre of non transgenic ones 24Fresh papaya production in s23Transgenic papaya resistant to PRSV25Transgenic papaya resistant 24Fresh papaya productiona in state of Hawaii and in Puna district from 1992 to 2001YearTotalPuna (x 1,000 lb)(PRSV enter

9、s Puna) 199255,80053,010199358,20055,290199456,20055,525199541,90039,215199637,80034,195199735,70027,810(transgenic seeds released) 199835,60026,750199939,40025,610200050,25033,950200152,00040,00026Fresh papaya productiona in 25过量表达CTV-CP蛋白的来檬抗CTVCTV symptoms of different intensity in young leaves a

10、fter 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).27过量表达CTV-CP蛋白的来檬抗CTVCTV s

11、ympt26Sweet orange overexpressing PR protein from tomato: resistant to phytophthora28Sweet orange overexpressing 27Apple fire blightfire blight:1000ha apple were killed by FB in Michigan in 2000.Lytic protein (LP)：Royal Gala, Galaxy 和M26转avian LPs SB-37,T-4 lysozyme：Gala转harpin（来于Fire blight细菌）NPR1蛋

12、白：过量表达Silencing the DIPM kinase which is related to the occurrence of the disease.29Apple fire blightfire blight28Transgenic Royal Gala with attacin gene: resistant to fire blight30Transgenic Royal Gala with a29Production of transgenic plants resistant to pests and insectsApplication 231Production o

13、f transgenic pla30Pests and insectsNegative impact on yield, growth and qualityGlobal loss: up to 15% of potential production, amounting for 10 bRMBNormal control：farm chemical (resistance and pollution)32Pests and insectsNegative im31Aftermath 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

14、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。 33Aftermath of farm chemicals我32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%研究成果34利用苏云金芽孢杆菌（Bt）杀虫蛋白基因CryI和CryI33Bt-cottonChina：1991年单价抗虫棉，1993年，开展双价抗虫棉的

15、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 35Bt-cottonChina：1991年单价抗虫棉，1934Non-Bt cottonBt cottonSource: CAAS36Non-Bt cottonBt cottonSource35抗虫转基因棉花获大面积推广37抗虫转基因棉花获大面积推广36Numbers of pesticide applications in Bt and

16、non-Bt cotton in Hebei and Shandong in 1999- reduced by 13 applications In 2000: by 12 applications In 2001: by 14 applications 38Numbers of pesticide applica37Percentage (%) of poisonings reported as numbers of farmers interviewed in Henan in 200039Percentage (%) of poisonings38Cabbage looper粉蚊夜蛾 t

17、est 40Cabbage looper粉蚊夜蛾 test 39Diamondback moth小菜蛾 test41Diamondback moth小菜蛾 test40Production of transgenic plants resistant to herbicideApplication 342Production of transgenic pla41GMO by traits-201243GMO by traits-201242Transgenic soybean1994年5月美国Monsanto公司耐除草剂“镇草宁”大豆Round up Ready（农达安）。44Transge

18、nic soybean1994年5月美国M43 Move to greener herbicide Benefits of Glyphosate 草甘磷Tolerance in Crops Can use at any time - can wait until there is a problem Reduced herbicide use Very effective - Weeds very sensitive - GM crop very resistantGM canola surrounded by weeds- glyphosate+ glyphosate45 Move to g

19、reener herbicide B44Production of transgenic plants with altered development progressApplication 446Production of transgenic pla45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

20、个商品化生产的转基因产品47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464847对照转基因苹果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CKSSC：高于CK比CK耐贮藏3个月后49对照转基因苹果乙烯生成减少的苹果转基因苹果：3个月后48不变褐的苹果50不变褐的苹果49Production of transgenic plants tolerant to abiotic stressesApplication 551Production of transgenic pla50Approximately 70% of the genetic potent

21、ial yield of major crops is lost by environmental factors. (Boyer, Science, 1982, 218: 443-448)Abiotic stress52Approximately 70% of the gen51Enhanced growth in transgenic aspen53Enhanced growth in transgeni52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

22、；耐贮藏、利于通风透光，适于保护地栽培，产量可达50008000kg。 54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53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。CONTROL对照组WITH GENE 含基因CONTROL对照组WITH GENE 含基因孟山都与巴斯夫合作53抗旱玉米2008年农业进步展览会：含抗旱基因 对照组 转基因55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54抗溃疡病的柑橘56抗溃疡病的柑橘55抗脱水的枳57抗脱水的枳56Production of transgenic plants w

23、ith special value or traitsApplication 658Production of transgenic pla57Transgenic 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，59Transgenic flowers with diff58改良作物营养品质植物类胡萝卜素代谢工程Shewmaker et al., Plant J, 1999; Lindgren et al., P

24、lant Physiol, 2003; Paine et al., Nat Biotechnol, 2005; Rosati et al., Plant J, 2000; DAmbrosio 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, 200960改良作物营养品质植物类胡萝卜素代谢工程Shewmaker59生产特殊类胡萝卜素植物类胡萝卜素代谢工程Mann et al., Nat

25、 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, 200961生产特殊类胡萝卜素植物类胡萝卜素代谢工程Mann et 60Golden rice62Golden rice61The Golden Rice Story Vitamin A deficiency is a major heal

26、th problem Causes blindness Influences severity of diarrhea, measles 100 million children suffer from the problem Improved vitamin A content in widely consumed crops isan attractive alternativeOur bodies cannot make vitamin A, we get it in our diet in 2 ways:- 50% from pro-Vitamin A carotenoids from

27、 plants are converted into vitamin A in the intestine- rest from ingesting vitamin A itself in animal products or supplementsAll carotenoids that contain a -ring can be converted into retinol, and one of the most important carotenoid pro-vitamins is -carotene63The Golden Rice Story Vitami62-Carotene

28、 Pathway in PlantsIPP异戊二烯焦磷酸GGPPPhytoeneLycopene -carotene(vitamin A precursor)Phytoene synthasePhytoene desaturaseLycopene-beta-cyclase-carotene desaturase Problem:Rice lacksthese enzymesNormalVitamin A“Deficient”Rice64-Carotene Pathway in Plants63Two T-DNAs encoding 3 genes for pro-vitamin A synth

29、esis (plus selectable marker gene) introduced together via Agrobacterium by co-transformationproterDaffodil 1proterHyg resisproterDaffodil 2protpter ErwiniaLBLBRBRBDaffodil 1 = phytoene synthaseDaffodil 2 = lycopene -cyclaseErwinia = Erwinia double desaturase - with added transit peptide, expressed

30、from 35SCaMV promoterWith own native transit peptides and endosperm-specific promoter from rice glutelin (GT1 promoter) Grain of resulting transgenic rice has light golden-yellow colourEngineering provitamin A ( -carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Ye et al., Scienc

31、e 287,303-305 (2000).- best line had 85% of its carotenoids as -carotene水仙花欧文氏菌65Two T-DNAs encoding 3 genes 64GA-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 GA3.66GA-20氧化酶部分正义抑制的苹果Reduced con65LFY encodes a plant specific tra

32、nscription 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.Genes involved in floral development67LFY encodes a plant specific66Overexpression o