植物基因转化的受体.ppt

1、第七章Genetic transformation in plant,2,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,3,第三节,载体构建,4,Transformation Cassettes,Contains,5,Lets Build A Complex Cassette,pB19hpc (Golden Rice Cassette),6,1. gene of interest,Based on what you are doing and what you want to get Quality Stress tolerance Secondary m

2、etabolism,7,A cloning site for the gene of interest,8,2. Selectable marker,Allows the transgenic plants to survive under stringent selection.,9,Effect of Selectable Marker,Transgenic = Has Kan or Bar Gene,Plant grows in presence of selective compound,Plant dies in presence of selective compound,Non-

3、transgenic = Lacks Kan or Bar Gene,X,10,A selectable marker gene,抗性素类 kanamycin resistance, nptII hygromycin resistance, hpt 除草剂类 bar gene (for resistance to herbicide phosphinothricin)草胺膦 DHFR gene (for resistance to methotrexate)甲氨蝶呤 ESPS gene (for resistance to glyfosate),11,常用抗生素抗性酶基因,新霉素磷酸转移酶基因

4、(NPTII)，抗卡那霉素 潮霉素磷酸转移酶基因（HPT），抗潮霉素(hygromycin),12,npt基因,在植物基因工程中被广泛使用，来源于细菌转座子Tn5上的ahpA2。该基因编码氨基糖苷-3-磷酸转移酶（aminnoglycoside3-phosphotransferase）。 Npt使ATP分子上的-磷酸基转移到抗生素分子上，影响抗生素与核糖体亚基的结合，从而使氨基糖苷类抗生素（新霉素、卡那霉素、庆大霉素、巴龙霉素和G418等）磷酸化失活。,13,HPT,潮霉素是一种很强的细胞生长抑制剂，对许多种植物都产生很高的毒性。 而潮霉素磷酸转移酶基因产物通过酶促磷酸化作用能使潮霉素失活，从

5、而产生抗性。,14,常用抗除草剂基因,bar基因，产生PPT乙酰转移酶，抗Bialaphos或glufosinate草铵膦 EPSP基因，产生5-烯醇式丙酮酸莽草酸-3-磷酸合酶，抗glyphosate草甘磷 GOX基因，产生草甘膦氧化酶,降解草甘膦,15,3. Reporter genes: scorable marker gene,具有两大特点 （1）便于检测； （2）其表达产物及产物的类似功能在未转化的植物细胞内并不存在。 optional, but very useful,16,Reporter genes,beta-glucuronidase activity (GUS) green

6、 fluorescent protein (GFP) chloramphenicolacetyl transferase activity luciferase activity,17,(1) GUS基因,Gus基因编码-葡萄糖苷酸酶（ -glucuronidase，GUS），能催化许多-葡萄糖苷脂类物质的水解。 能被许多葡萄糖苷诱导，即其表达受GUS底物的诱导。,18,GUS组织化学染色,以X-gluc（5-溴-4-氯-3-吲哚- -D-葡萄糖苷酸脂）为底物，在适宜条件下，底物进入检测组织。 经氧化二聚作用形成5，5-二溴-4，4-二氯靛蓝染料，使具有GUS活性的产位呈现蓝色。,19,(2)

7、 绿色荧光蛋白基因,GFP is a fluorescent protein isolated from Coelenterates, such as the Pacific jellyfish, Aequoria victoria, or from the sea pansy, Renilla reniformis.,Osamu Shimomura was the first person to isolate GFP and to find out which part of GFP was responsible for its fluorescence.,20,gfp,Its role

8、 is to transduce the blue chemiluminescence of the protein aequorin into green fluorescent light by energy transfer. The gene for GFP has been isolated and has become a useful tool for making chimeric proteins of GFP linked to other proteins where it functions as a fluorescent protein tag. 是一种活体标记。能够接受荧光辐射并发射荧光。GFP在原核或真核细胞中蓝光激发能发出绿光 即发射团的形成无物种特异性，也不需要特异的辅助因子。基于这一特点，gfp基因作为新型标记或报告基因可以广泛应用于各种生物。,21,22,gfp基因具有以下优点 适用于各种生物的基因转化 检测方法简便，只要有紫外光或蓝光照射，其表达产物即可发出绿色荧光 便于活体检测，有利于活体内基因表达调控研究 检测时可获得直观信息，有利于转基因植物安全性问题的研究及防范,23,荧光素酶基因 Luciferase,萤光素酶可催化生物体自身发光，荧火虫荧光素酶及细菌荧光素酶。 萤火虫萤光素酶催化6-羟基喹啉类，脱羧后