(块根/块茎）储藏器官特异启动子 β-胡萝卜素高含量马铃薯 Golden Potato.

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1 (块根/块茎）储藏器官特异启动子 β-胡萝卜素高含量马铃薯 Golden Potato

2 功能基因（目的基因）：原始基因；改进版的基因，如单子叶植物密码子偏好基因；双子叶植物密码子偏好基因；含信号肽基因（定位蛋白在细胞中表达的位置）等
终止子：基因转录终止信号 基因表达盒可以自己构建，也可以公司合成

3 第二步：转基因及转基因植株的培育 农杆菌法 基因枪法

4 第三步：转基因植株的鉴定、评价与筛选 ——转基因植株鉴定：报告基因、PCR，Southern Blot（DNA杂交） ——转基因植株评价（栽培与分析）：不同转基因植株的表现不一样 —— 将符合要求的转基因植株培育成新的品种

5 植物转基因技术在农作物大豆、油菜、玉米、棉花、番茄等植物上得到成功应用
苏云金芽胞杆菌毒素蛋白基因（BT） 对照烟草 对照番茄 转Bt 番茄 Bt 烟草

6 抗虫棉花 将苏云金杆菌杀虫蛋白（Bt）基因转入植物，培育出抗虫新品种 转基因 对照 转基因 对照 抗虫烟草

7 抗虫转基因棉花获大面积推广

8 转基因水稻抗性栽培试验

9 Ingo Potrykus: Golden Rice 金色稻米
β-胡萝卜素=维生素A Science （2000）

10 Paine et al. Nat. Biotechnol.  23, 482 - 487 (2005) ：优化了基因，β-胡萝卜素含量提高23倍

11 转基因植物生产药物蛋白 植物基因工程技术的发展为利用植物作为生物反应器生产有用蛋白成为可能，诞生了分子农业（分子农场）=Molecular Farming 利用基因工程技术将特定的基因转入植物中，通过栽培转基因植物生产具有治疗、诊断、或者工业用途的蛋白或多肽。

12 分子农业示意图

13 药物蛋白（人、家禽家兽用） ：疫苗、抗体、生长因子、生长激素等。
——微生物体系：产量高、成本低，但有时无活性 ——动物细胞体系：有活性；细胞培养环境和产品分离纯化苛刻，成本高，纯化难（病原、过敏原）

14 ——植物体系： 可行：可以进行蛋白翻译后的加工、形成有活性的物质）； 安全性好：人与植物没有共患疾病，人对常用作物不过敏；
成本低：农作物种植投入少，可机械化大规模种植； 可储藏：1-3年以上 使用方便：疫苗可以直接使用（香蕉、番茄等）

15 20多年来转基因植物生产药物蛋白的论文数量（近1000篇，100多项专利）
学术结构和商业公司对分子农业兴趣浓厚，近20年发展迅速 20多年来转基因植物生产药物蛋白的论文数量（近1000篇，100多项专利）

16 药物蛋白的种类：疫苗、抗体、生长激素、细胞因子、膜蛋白、酶等

17 美国2002-2005年批准进行试验的植物来源的重组蛋白

18 浏览以下网站可了解更多转基因植物生产的药物蛋白和工业用蛋白的研发情况：http://www. molecularfarming
浏览以下网站可了解更多转基因植物生产的药物蛋白和工业用蛋白的研发情况：

19 Transgenic Research 13: 245–259, 2004
Recombinant protein expression plasmids optimized for industrial E. coli fermentation and plant systems produce biologically active human insulin-like growth factor-1 in transgenic rice and tobacco plants Mitra Panahi1, Zaman Alli1, Xiongying Cheng1, Loubaba Belbaraka2, Jaafar Belgoudi2, Ravinder Sardana1, Jenny Phipps2 & Illimar Altosaar1,* 1Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Canada 2Steacie Institute for Molecular Sciences, National Research Council of Canada, Ottawa, Ontario, K1A OR6,Canada

20 human insulin-like growth factor-1=hIGF-1，人胰岛素样生长因子-1
a single-chain peptide of 70 amino acid residues sharing 50% homology with human insulin. Synthesis of hIGF-1: Mainly in the liver and regulated by several factors such as human growth hormones and insulin. Main functions of hIGF-1: 1 Being essential for normal fetal growth and development; 2 Stimulating proliferation and survival of many cell types; 3 Promoting differentiation of a limited cell types.

21 hIGF1 plays an important role in childhood growth
hIGF1 plays an important role in childhood growth. Inability to make or respond to hIGF1 produces a distinctive type of growth failure termed Laron dwarfism. hIGF1 continues to have anabolic effects in adults.   Laron dwarfism

22 The clinical importance of hIGF-1
hIGF-1 can increase insulin sensitivity and possesses hypoglycemic effects similar to insulin, so hIGF-1 may become a substitute therapeutic agent for the people whose insulin receptors have functional defects, especially for those who have one or more of the following problems: 1. Growth hormone-sensitive (Laron-type) syndrome; 2. Type A insulin resistance syndrome ; 3. Diabetes; 4. Osteoporosis; 5. Acquired immunodeficiency syndrome (AIDS). The demand for hIGF-1 is likely to increase in the future.

23 Production of hIGF-1 Recombinant hIGF-1(rthIGF-1) has been expressed in several different host–vector systems: In yeast: 8 to 55 mg/L culture (Vai et al, 2000); In E. coli: 8.5 g/L culture, but almost all the IGF-1 were not soluble (in aggregates) (Joly et al., 1998) ; In transgenic rabbits: a homozygous offspring produced 678 mg /L milk (Zinovieva et al., 1998).

24 Limitations in the present systems expressing hIGF-1:
1. Not soluble in bacteria; 2. Formation of non-native proteins having different biological activities in yeast; 3. Transgene induced instability of certain cell lines in mammalian cell cultures; 4. Contamination of animal-based products with human pathogens in mammalian cell cultures; 5. High costs of mammalian cell cultures.

25 Potential advantages of expressing recombinant proteins in plants:
1. Minimal risk of contamination by pathogenic microorganisms; 2. The ability to effect post-translational modifications resulting in authentic products; 3. High yields of stable products by adding plant signal sequences and increasing G+C content of the coding sequences; 4. Low scale-up costs; 5. Edible tissues can be targeted for precise tissue-specific accumulation of recombinant proteins.

26 表达载体 适合大肠杆菌的基因 普通基因 适合植物的基因
Figure 1-1. The rthIGF-1 expression constructs used for transformation of tobacco and rice plants. The gene constructs containing the hIGF-1 sequences (210 bp) were placed under the control of the maize ubiquitin 1 promoter (Panels A–C).

27 Production of tobacco and rice via Agrobacterium-mediation
The transgenic plants and hIGF-1 gene expression in the plants were confirmed by Southern blots, RT-PCR and Western Blots.

28 Did the transgenic plants produce hIGF-1?
Yes. hIGF-1detection by ELISA showed that all the transgenic tobacco or rice plants produced hIGF-1 in their leaves, but the yields varied dramatically among them.

29 Production of hIGF-1 in transgenic plants
Rice Tobacco Figure 1-2. Amounts of rthIGF-1 (ng of rthIGF-1/mg of total protein) in each plant extract.

30 Plants Groups Promoter Coding sequence pUIGF-1 ubiquitin 1 No SS
Signal sequences Coding sequence Range of hIGF1 levels Means of hIGF1 levels Tobacco pUIGF-1 ubiquitin 1 No SS E.coli-codon optimized 4–129 ng/mg 26±7 ng pULam B-IGF-1 Bacterial Lam B SS 5–73 ng/mg 22±4 ng pUSYN-IGF-1 Rice prolamin SS plant-codon optimized 8–241 ng/mg 70±13 ng Rice 15–371 ng/mg 113±24 ng