PowerPoint 演示文稿

Size: px

Start display at page:

Download "PowerPoint 演示文稿"

需勇时
7 years ago
Views:

1 第十章植物基因工程 Plant Genetic Engineering Transgenic Plants Genetically Modified Organism (GMO) Genetically Engineered Organism (GEO) 1

2 第一节植物遗传转化的基础一植物组织培养技术 1. 愈伤组织培养从植物体上切取的小片组织 ( 外植体, explant) 放在含有低浓度植物激素的培养基上培养, 会在切口的表面长出未分化的细胞团愈伤组织在含有不同激素的培养基中, 愈伤组织可分化成根芽花或整个植株 2

3 3

4 2. 细胞悬浮培养当愈伤组织被转入液体培养基振荡培养时, 细胞团会分散成为单细胞小细胞团大细胞团的悬浮培养物悬浮培养物可无限培养, 但会发生聚集遗传不稳定性导致突变的积累, 细胞异源化 3. 原生质体培养植株的再生 4

5 二植物基因转移方法 Agrobacterium tumefaciens Gene Gun Protoplast transformation Electroporation Microinjection 5

6 Agrobacterium tumefaciens Ti plasmid with the new gene + cell s DNA Transformation Agrobacterium Plant cell The new gene Transgenic plant Cell division 6

7 Gene Gun Technique DNA coated golden particles Cell s DNA Gene gun Plant cell A plant cell with the new gene Transgenic plant Cell division 7

8 Electroporation Technique Power supply Plant cell Duracell Protoplast DNA containing the gene of interest DNA inside the plant cell The plant cell with the new gene 8

9 Plant Transformation Systems 9

10 三植物转化的标记基因系统 Selectable Markers Neomycin phosphotransferase (NPT) gene Hygromycin phosphotransferase (HPT) gene Bar 基因 : 链霉菌的 bar 基因编码的膦丝菌素乙酰转移酶可使膦丝菌素 ( 除草剂 ) 失去毒性 Reporter Genes Beta-Glucuronidase (GUS) GFP 10

11 Plant Promoter Systems The Cauliflower mosaic virus 35S (CaMV)promoter is commonly used as a strong constitutive promoter in plants Promoter strength is effected by enhancers, introns, plant species, and the site within the plant genome where the T-DNA is inserted 11

12 Organ/ tissue specific promoters Vicilin( 豌豆球蛋白 ) and phytohemaglutinin ( 植物凝集素 ), glutenin( 麦谷蛋白 )promoters for seed specific expression α-amylase promoter for expression in the aleurone( 糊粉 ) of cereal grains Patatin promoter for tuber stem specific expression in potatoes and the RuBisco promoter for green tissue specificity 12

13 第二节根癌农杆菌介导的遗传转化一根癌农杆菌与 Ti 质粒 1. 根癌农杆菌 (Agrobacterium tumefaciens) 13

14 Agrobacterium tumefaciens A.tumefaciens is a gram negative soil bacterium A.tumefaciens infects dicotyledonous plants causing the formation of crown gall tumors ( 冠瘿瘤 ) 14

15 15

16 冠瘿瘤细胞的特征不需要补充植物激素便可进行离体培养合成肿瘤特有的化合物冠瘿碱 (opine) Octopine( 章鱼碱 ), nopaline( 胭脂碱 ), which are unusual amino acid derivatives not found in normal plant tissues) 根癌农杆菌能利用冠瘿碱作为唯一的碳源和氮源 16

17 Opines 17

18 Crown gall tumor formation is the consequence of the transfer, integration, and expression of genes from the Ti plasmid (Tumor inducing) of A. tumefaciens 18

19 二 Ti 质粒 (Tumor induced plasmid) DNA 复制区 Ti 质粒结合转移区参与不同农杆菌间 Ti 质粒的接合转移冠瘿碱分解区毒性区 (vir) T-DNA 区边界区致瘤区冠瘿碱合成区 19

20 Virulence (vir) Genes Small molecules secreted by wounded plants (i.e., acetosyringone 乙酰丁香酮 ) induce the activity of the vir genes encoded on the Ti plasmid The vir genes are essential for the transfer and integration of the T-DNA region 20

21 T-DNA (Transferred DNA) Ti 质粒中仅有一部分进入植物细胞,T-DNA T-DNA 包括植物激素及冠瘿碱合成区基因及两侧的正向重复序列, 长约 23kb T-DNA 可整合到植物染色体上, 整合位点不具特异性整合了 T-DNA 的植物细胞合成冠瘿碱 (octopine and nopaline), 根癌农杆菌利用冠瘿碱作为碳氮源生长 21

22 Left and Right Borders The left and right borders of the T-DNA are the sites where a single strand of the T-DNA is cut from the Ti plasmid The borders contain a repeating unit of 25 base pairs Only the right border is needed for transfer, however, both borders are usually used in cloning vectors 22

23 Auxin and Cytokinin The T-DNA region encodes a number of genes that synthesize the plant hormones auxin and cytokinin Auxins and cytokinins regulate plant cell growth and cause the plant to develop tumorous growths such as crown galls 23

24 Ti 质粒作为载体的优点宿主范围广, 能转化所有的双子叶植物整合到染色体上后成为染色体的正常组分永远保留冠瘿碱合成酶基因启动子是强启动子 24

25 天然 Ti 质粒作为载体的问题 Auxin and cytokinin prevents regeneration of plants grown in culture, therefore, these genes must be removed from the Ti plasmid Opine synthesis diverts plant resources from the final plant yield, therefore, the opine synthesis gene should be removed 25

26 Ti plasmids are large (200 kb) so nonessential DNA must be removed to allow recombinant DNA to be inserted Ti plasmids do not replicate in E. coli, so an E. coli origin of replication must be added Plant and bacteria selectable marker genes must be added 26

27 Ti Plasmid Cloning Vector 27

28 二 Ti 质粒载体 Disarmed Ti vectors( 非致瘤载体 ) Cointegrate vectors( 共整合载体 ) Binary vectors( 双元载体 ) 28

29 1. Disarmed Ti vectors 去除 T-DNA 中的肿瘤基因在 T-DNA 中插入用于转化植株筛选的遗传标记基因 29

30 2. Intermediate vectors A small portion of T-DNA was subcloned in a conventional E.coli plasmid vector (i.e. pbr322) for easy manipulation, producing intermediate vectors Intermediate vectors are incapable of replication in A.tumefaciens and also lack conjugation functions. Homologous recombination between the T- DNA of the disarmed Ti plasmid and the intermediate vector results the formation of a large cointegrate plasmid. 30

31 3. Cointegrate vectors 31

32 4. Binary vector T-DNA does not need to be physically attached to the rest of the Ti plasmid Use separate plasmids to supply the disarmed T-DNA and the virulence functions 32

33 P nos Kan T nos P camv GUS T nos pbi121 ( 13kb) Kan r Vir pal kb The small plasmid can be used to inserted foreign gene When the two plasmids present together in the same A.tumefaciens cell, the T-DNA carried by pbi121 is transferred to the plant chromosomal DNA by proteins coded by genes carried by pal

34 三根癌农杆菌转化的一般步骤 Leaf-disc transformation( 叶盘法 ) 34

35 35

36 General protocol Small discs were punched from leaves, surface-sterilized Inoculated in a medium containing A.tumefaciens transformed with the recombinant disarmed T-DNA (cointegrate or binary vector) 36

37 The discs were cultured for 2 days and transferred to medium containing antibiotic (i.e. carbenicilin to kill the Agrobacterium and kanamycin to select for transformants) After 2~4 weeks, developing shoots were excised from callus and transplanted to root-induced medium. After 4~7 weeks, rooted plantlets were transplanted to soil. 37

38 四转基因植物的鉴定外源基因是否进入受体细胞? 报告基因的检测 (GUS, GFP), PCR 检测外源基因是否整合到染色体上? Southern blot 整合的方式如何? 整合到染色体上的外源基因是否表达? RT-PCR, Northern blot, Western blot, 表型分析 38

39 39

40 五根癌农杆菌与单子叶植物 Agrobacterium infection of most monocots is inefficient Wounded monocot tissues do not produce phenolics, such as acetosyringone( 乙酰丁香酮 ), at sufficient levels to introduce vir gene express 40

41 Pretreating Agrobacterium and discs with wound exudate from dicotyledonous or acetosyringone helps transformation of monocots. Many monocots, including rice, maize, wheat, barley and sugar cane have been succesfully transformed by Agrobacterium 41

42 第三节其它的植物转化方法一原生质体转化 Polyethylene glycol (PEG) 聚乙二醇 Electroporation The major limitation of protoplast transformation is the ability of the protoplast regeneration 42

43 二基因枪 (particle bombardment) Gold or tungsten particles (0.4 to 1.2 um in diameter) are coated with precipitated DNA The coated particles are accelerated to meters/second with a particle or gene gun The DNA can be transiently expressed or integrated into the plant genome 43

44 Maize streak virus 三植物病毒 Cauliflower mosaic virus Tobacco mosaic virus 44

45 Advantage: Viruses are able to absorb to and introduce their nucleic acid into intact plant cell Infected cells yield large amounts of virus, so recombinant viral vectors have the potential for highlevel transgenic expression The viral spreads throughout the plant, allowing transgenic expression in all cells. Viral infections are rapid, so large amounts of recombinant protein can be produced in a few weeks. All known plant viruses replicate episomally; therefore the transgene are not subject to position effects. Disadvantage Since plant viruses neither integrate into nor pass through the germ line, plants cannot be stably transformed by virial infection and transgenic lines cannot be generated 45

46 四叶绿体转化 Chloroplast Transformation Thousands of chloroplasts present in photosythetic cell,and this can result 50 times higher expression level of transgene than in nuclear genome. Transgenes integrated into chloroplast DNA do not appear to undergo silencing of suffer from position effect. The transgene cannot be transmitted through pollen 46

47 Agrobacterium are not suitable for chloroplast transformation because the T-DNA is targeted to the nucleus. Particle bombardment and PEG-mediated transformation has been achieved. 47

48 Vectors Foreign genes can be targeted to the chloroplast or mitochondria genomes by homologous recombination 48

49 Chloroplast transformation 49

50 Selection of homogenous transgenic chloroplasts. Plant cells contain chloroplasts, each chloroplast contains nucleoids, each nucleoid contains 5-10 chloroplast genomes. Therefore, each plant cell may contain >10000 chloroplast genomes. Transformation may result in the alteration of just one of these genomes. A non-homogenous genome content in a chloroplast appears to be unstable, so multiple rounds of regeneration and selection are required to produce a homogenous population of transgenic chloroplasts (homoplasmic) within the plant cell. 50

51 第四节转基因技术在植物中的应用 51

52 Glyphosate, 草甘磷 EPSP 合成酶基因 Roundup Ready by Monsanto Co. GAT (Glyphosate N-acetytransferase) GAT from B. licheniformis PAT (Phosphinothricin acetytransferase) Streptomyces spp. Herbicidal Resistance 52

53 Insect Resistance Intracellular crystalline bodies of Bacillus thuringiensis contain insecticidal protein called the δ-endotoxins Highly poisonous: 80,000 times Relatively selective δ-endotoxin Type Effective Against CryI CryII CryIII CryIV CryV CryVI Lepidoptera (moth and butterfly) larvae Lepidoptera and Diptera (two-winged fly) larvae Lepidoptera larvae Diptera larvae Nematode worms Nematode worms 53

54 Other Insecticidal Proteins 植物蛋白酶抑制剂基因植物 α- 淀粉酶抑制剂基因植物凝集素基因一种结合碳水化合物的蛋白质, 雪莲凝集素 ( GNA) 信息素蛋白 Alarm pheromone: some species of wild plant have evolved to mimic the chemical warning signals put out by aphids a major crop pest in the temperate zones when they are under attack. 54

55 Viral Resistance 将编码病毒外壳蛋白的基因转入植物其他病毒的复制酶基因 (posttranscriptional gene silencing) 抗病毒蛋白等病毒抗体等 55

56 Fungal Resistance 几丁质酶与葡聚糖酶 56

57 Bacterial pathogens resistance Blight of rice: a resistance gene Xa21 57

58 Abiotic stresses Resistance 抗旱 : BADH (betaine aldehyde dehydrogenase) 抗冻耐盐重金属抗性 58

59 Golden Rice increased Vitamin A content 改良作物品质 Stoneless Plum By starting with genes from a mostly stoneless, conventionally bred plum, Scientists are engineering a fruit with no stone at all Delayed Ripening tomatoreduce the expression of polygalacturonase gene or ethylene synthase gene Arctic Apple - produce lower polyphenol oxidase, a key enzyme in the chain of biochemical events that cause browning 59

60 Golden Rice Carotenoids are a group of plant pigments important in the human diet as the only precursors of vitamin A Certain carotenoids, most importantly β-carotene, are cleaved to vitamin A within the body and are referred to as pro-vitamin A Vitamin A deficiency, a major problem in parts of the developing world, can result in permanent blindness and increase the incidence and severity of infectious diseases 60

61 Carotenoid Biosynthesis in plants 八氢番茄红素脱氢酶环化酶番茄红素 lcy β-carotene 叶黄素玉米黄素 Rice (Oryza aativa L.), the major food staple for more than two billion people, contains neither β-carotene (provitamin A) nor C 40 carotenoid precursors in its endosperm. Investigation of the biochemical properties of immature rice endosperm using 14 C- labelled substrates revealed the presence of geranyl geranyl diphosphate (GGDP), the C 20 general isoprenoid precursor necessary for C 40 carotenoid biosynthesis.

62 Engineering the Provitamin A (β-carotene) Biosynthetic Pathway into (Carotenoid-Free) Rice Endosperm X Ye et al. Science 2000;287: Precultured immature rice embryos (n=800) were inoculated with Agrobacterium LBA4404/pB19hpc. Hygromycin-resistant plants (n=50) were analyzed for the presence of psy and crti genes Immature rice embryos (n=500) were inoculated with a mixture of Agrobacterium LBA4404/pZPsC and LBA4404/pZLcyH. 12/60 lines were cotransformed plants identified by Southern hybridization. Figure 1. Structures of the T-DNA region of pb19hpc used in single transformations, and of pzpsc and pzlcyh used in co-transformations.

63 I-SceI 由酿酒酵母线粒体 rrna 基因中的内含子编码的限制酶 5 -TAGGGATAA CAGGGTAAT-3 TATT 63

64 Figure 2 Phenotypes of transgenic rice seeds. Mature seeds from T0 transgenic lines and from control plants were air dried, dehusked, and, in order to isolate the endosperm, polished with emery paper. The pb19hpc single transformants, engineered to synthesize only lycopene (red), were similar in color to the pzpsc/pzlcyh co-transformants engineered for b-carotene (yellow) synthesis. control A: pb19hpc single transformants lines B: pzpsc/pzlcyh co-transformants lines

65 Figure 3 The carotenoid extracts from seeds (1 g for each line) were subjected quantitatively to HPLC analysis. (A) Control seeds (B) Line h2b (single transformant) (C) Line z11b (co-transformant) (D) Line z4b (co-transformant).

66 Expression of a psy transgene increases the carotenoid content of maize callus Nature Biotechnology 23, (2005)

67 Carotenoid enhancement of the rice endosperm by transformation with psy orthologues and crti

68 Immunoblots of grain protein extracts from wild-type (WT) and transgenic plants. Anti-CRTI Anti-maize PSY

71 Humanitarian Project for Golden Rice. Syngenta has no commercial interest in Golden Rice. The reported transgenic rice events are experimental. Consistent with Syngenta s support of the Humanitarian Project for Golden Rice, Golden Rice 2 transgenic events will be donated for further research and development through license under certain conditions. Such conditions include being governed by the strategic direction of the Golden Rice Humanitarian Board and full regulatory compliance. Please direct requests to Adrian Dubock in the first instance (adrian.dubock@syngenta.com). 71

72 转基因作物的专利保护 Terminator technology, Genetic use restriction technology (GURT) Terminator gene technology creates plants that make suicide seeds. 72

73 The history of the terminator gene Developed and patented in 1998 by Delta & Pine Land Company with the help of the United Stated Department of Agriculture Monsanto purchased D & PL 73

74 How it works? TetR: the gene encoding the tetracycline repressor under the control of a constitutive promoter (35S), produces a repressor protein Cre: Cre recombinase gene. Between the promoter (35S) and the recombinase gene, scientists place a DNA fragment which is a binding site for tetracycline repressor 74

75 RIP: the gene for a ribosome inactivating protein, a toxin lethal to embryos. It is controlled by a promoter that is only active during late embryogenesis (P lea ; the promoter from the late embryogenesis abundant gene). Between P lea and RIP gene, scientists place a piece of DNA called a Blocker, which interferes with the ability of the promoter to turn on the gene 75

76 76

77 全球转基因作物概况 Nature, Vol 497,

78 78

79 79

80 80

81 根据农业部农业转基因生物安全管理办公室发布的信息,, 我国先后共为 7 种转基因植物发放了农业转基因生物安全证书 : 耐贮存番茄抗虫棉花改变花色矮牵牛抗病辣椒抗病番木瓜抗虫水稻和转植酸酶玉米目前商业化种植的转基因农作物主要是转基因棉花和转基因番木瓜, 其余已发放安全证书的转基因植物并未大规模应用批准了 4 种可进口的转基因作物 : 棉花大豆玉米和油菜除进口的转基因棉花可种植外, 进口的转基因大豆玉米油菜均不能种植, 仅限于加工原料消费者真正能接触到的转基因食品, 主要就是番木瓜大豆油玉米油菜籽油及其调和油, 其他诸如土豆大米玉米西红柿都不是转基因, 消费者在市场上通常是买不到的 81

82 第五节转基因植物的问题一转基因沉默 (transgene silencing) 导入并整合到植物细胞和基因组上的外源基因, 在当代或后代转基因植株中表达活性受到抑制的现象 82

83 染色体水平的转基因沉默 : 转基因插入染色体的重复序列或异染色质区引起 ---- 位置效应 (position effect) 转录水平的基因沉默 : 外源基因的多拷贝整合, 导致重复序列之间发生自发配对, 从而发生甲基化或异染色质化, 转录受到抑制转录后水平的转基因沉默 : 外源基因能够转录成 mrna, 但 mrna 不能积累转录后共抑制 : 在外源基因沉默的同时, 内源基因表达也受到抑制 83

84 二昆虫对 BT 转基因作物的抗性 Production of two toxins Temporal control over toxin synthesis Mixed planting 84

85 三选择基因的安全性 Kan R (nptii) gene express in all cells of an engineered plant. Is the product of Kan R gene toxic to human? Could the Kan R gene contained in a GM food be passed to bacteria in the human gut? Could the Kan R gene be passed to other organisms in the environment and would this result in damage to the ecosystem? 85

86 四对生态环境的影响 Unknown ecological impacts, super virus Gene flow to related species Outcrossing to neighboring crop fields 86

87 五转基因食品安全性 New food safety hazards, such as allergens Evaluation of the safety of GM foods 87

88 六转基因作物的专利保护特殊性状的遗传利用限制技术 V (variety)-gurt T (trait)-gurt T-Gurt seeds are fertile. However, in order to benefit from the genetically modified traits inserted into the genome, the farmer must purchase a proprietary chemical each year to spray on the seeds or young plants to activate the trait if he saves seeds for replanting. 88

89 第六节植物安全转基因技术安全标记基因法标记基因剔除与基因叠加防止基因漂移的叶绿体转化法和基因拆分法基因定点修饰技术 89

90 1. 安全标记基因糖代谢相关基因 : 磷酸甘露糖异构酶基因 (phosphomannose isomerase,pmi) 木糖异构酶基因 (xylose isomerase,xyla) 氨基酸代谢相关基因 : 激素相关基因抗逆相关基因 90

91 2 标记基因删除和外源基因累加技术共转化法剔除标记基因位点特异性重组法介导的标记基因删除和外源基因累加 Cre/loxP 重组系统 91

92 3. 叶绿体转化技术 92

93 4. 基因拆分技术 93

94 5. 植物基因定点修饰技术 ( 打靶技术 ) 锌指核酸酶 (ZFNs) 介导的定点修饰技术 TALEN 介导的基因组定点修饰技术 CRISPR/Cas9 系统介导的基因组定点编辑技术 94

95 Molecular Plant 8, , August

Microsoft PowerPoint - 基因改造作物 [唯讀]

Microsoft PowerPoint - 基因改造作物 [唯讀] 基改作因造物 1 基因重組作物 (transgenic crops) 又稱基因改造作物 (genetically modified crops) 係屬基因改造生物 ( g enetically modified organisms; GMO) 的一種 2 所謂的 GMO 係指利用操作基因之方式, 計劃性的選擇所