【作者】 陈书霞；

【导师】 程智慧；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2003， 博士

【摘要】 ETEC（Enterotoxigenic Escherichia Coli，肠毒性大肠杆菌）是引起腹泻的一种主要病原菌，其主要致病因子是定居因子抗原（Colonization Factor Antigens，CFAs）和肠毒素（Enterotoxin，LT）。它在致病时是通过定居因子蛋白表面结构粘附到肠上皮细胞表面，然后分泌肠毒素致病。一个有效的ETEC口服疫苗必须能够激起抗定居以及抗毒素两方面的免疫反应。如果一个疫苗包含有定居因子抗原和LT肠毒素，则理论上可使服食者免受80％的ETEC菌株的侵害。CS3是ETEC定居因子之一，临床研究结果表明口服CS3可引起免疫应答。本研究期望通过农杆菌转化系统将CTB（霍乱毒素B亚基，可作为佐剂和载体）基因和CTB-CS3融合基因分别转入胡萝卜植株，使CTB和CTB—CS3融合基因稳定整合到胡萝卜基因组内，希望以此获得以胡萝卜为受体的ETEC口服疫苗，使服食者在进食的同时就可获得腹泻免疫。通过研究ETEC定居因子基因对胡萝卜的稳定转化探索了转基因口服疫苗生产的潜在途径，这对国内研究廉价实用腹泻口服疫苗具有重大的理论和实践意义。同时本研究还通过构建LTB—ST融合基因PVX病毒表达载体，并用农感染法转化植物，探索了暂态表达系统生产疫苗的可能性。 在胡萝卜的再生体系建立阶段，从4个品种中筛选出了适合于本实验的2个胡萝卜品种“新透心红”和“AC93”，并确定了它们通过胚状体发生途径的激素条件及培养基类型。“新透心红”品种是通过MS₀+0.1mg／L 2,4-D诱导愈伤组织，然后转到MS₀培养基上诱导胚状体的发生；而“AC93”是通过B₅+0.2mg/L 2，4-D诱导胚性愈伤组织，然后转至B₅₀培养基上诱导胚状体的形成。两品种愈伤组织上发生的胚状体接种到不含激素的基本培养基上后都能长成健康小植株。同时还研究了本实验激素组合条件下胡萝卜胚的发生情况。2，4—D浓度相同KT浓度不同的激素组合随着KT浓度的升高，诱导的胚性愈伤转至不含激素的培养基上后胚状体发生量及发生胚状体的块数都随KT浓度增加而减少，但愈伤组织块的生长量却随KT的浓度升高而增加。 为了对转化苗加以较为严格的筛选，界定了“新透心红”组培过程中各个阶段的Kan筛选压。把愈伤组织诱导阶段的Kan浓度定为somg/L;愈伤组织继代的Kan浓度定为100m叭;小植株长根的培养基的Kan浓度定为SOm叭。在所进行的转化实验中，分别对幼苗进行4oC不同天数的处理，观察抗性愈伤组织的形成情况。其中幼苗经4aC处理4天，从其下胚轴所获得的抗性愈伤最多，其抗性愈伤组织的诱导频率为80%，高于其它各处理的抗性愈伤组织的诱导频率。说明外植体的不同生理状态对农杆菌转化有一定的影响。 以“新透心红，，品种无菌苗的下胚轴切段和农杆菌PBinCTBS为试材进行了共培养天数的确定。将“新透心红”品种无菌苗的下胚轴切段和农杆菌PBinCTBS分别共培养ld、3d、sd、7d，以确定本研究中共培养的天数。发现外植体在共培养2一3d四周出现较明显的菌液时在含有抗生素的培养基上生长良好，所以确定了共培养2一3d为本实验较适宜的共培养时间。分别研究了胡萝卜不同苗龄（7d、lld、30d）的抗性愈伤形成率。在相同条件下的转化实验中，发现不同苗龄的外植体的抗性愈伤的诱导频率基本相同:还进一步研究了胡萝卜同一苗龄不同部位的抗性愈伤形成率，同时用3Od苗龄（照光3周）的叶柄和下胚轴做转化实验，发现叶柄的抗性愈伤形成率高于下胚轴的抗性愈伤形成率，进一步的实验发现，由叶柄诱导的苗子的阳性率高于由下胚轴诱导的苗子的阳性率，此结果和Pawlicki的结论是一致的。 经过PCR一Southem检测以及southem杂交等实验手段初步证明CTB一CS3和CTB基因己分别整合到部分胡萝卜基因组中去，RT一PCR可分别证明外源基因在RNA的水平上有初步转录。 另外，构建了LTB一ST的PVX病毒表达载体的重组克隆，并将重组质粒转化入农杆菌MOG101和LBA4404中，利用农感染法侵染烟草，得到发病的植株，通过RT一PCR检测了目的基因表达，对利用病毒表达系统表达外源基因作了初步的探索和研究。更多还原

【Abstract】 ETEC (Enterotoxigenic Escherichia coli) is the most common cause of diarrhea in human being and Enterotoxin and Colonization Factor Antigens (CFAs) play the main factor to cause diarrhea. ETEC can adhere to the epithelial by means of proteinaceous surface structures called colonization factors (CFs) to cause diarrhea. An effective ETEC oral vaccine can evoke responses of anti-colonization and anti-toxin. If ETEC vaccine contains Colonization Factor Antigen such as CFA/I and CS1-CS6, associating with Enterotoxin (LT), it can, theoretically, protect the user from diarrhea by percent 80 ETEC attacking. CSS is the one of the colonization factor antigens which is a protective antigen can cause immune reaction by the means taking orally. In this study, carrot was separately transformed with Agrobacterium tumefacience strain LBA4404 that contains CTB or CTB-CS3 fused gene in order to get oral diarrhea vaccine. This is a potent strategy to produce ETEC oral vaccine. In addition, we also focused on constructing a plant virus-based gene vector PVX to express LTB-ST fused gene by means of agroinfection. The possibility of producing vaccine was probed through plant virus-based gene vector expression system.The hormone condition and medium type of two cultivars "New Touxinhong" and "AC93" were established via somatic embryogenesis. The embryogenic callus of "New Touxinhong" was induced on the medium MSo supplemented with 0.1mg/L 2, 4-D, and embryo could produce only when the calli were transferred to MSo medium. The embryogenic calli of "AC93" were induced on the medium 850 supplemented with 0.2mg/L 2,4-D, and embryo could produce only when the calli were transferred to B50 medium. The embryo of these two varieties can grow into healthy plantlet after being transferred to medium with no hormone. The condition of somatic embryogenesis under different hormone conditions was discussed. The amount of embryo decreased but the amount of calli increased as the increase of KT concentration under the same 2,4-D level after embryogenesis calli were transferred to medium with no hormone. The suitable concentration of kanamycin for selecting transgenic explants in the course of carrot tissueculture was screened in order to strictly select transformant. The optimum concentration of kanamycin was different at different stage of culture, which was 50mg/L at calli induction stage,100mg/L at embryogenic calli selecting stage, and 50mg/L at plantlet root elongating stage, respectively. In all treatments of aseptic seedlings under 4℃ for different days, the treatment under 4℃ for 4 days produced the most amount (80%) of resistant embryogenic calli induced from hypocotyls. It has been obvious that the physiological state of explants has an effect on transformation.The optimum duration of co-cultivation of "New Touxinhong" hypocotyls together with Agrobacterium tumefacience PBinCTBS was determined among 1,2, 3, 5,7days, and which was 2-3 days when the hypocotyls was obvious surrounded by the Agrobacterium. The age of the explants was also studied to analyze the effect of the explants age on the genetic transformation. Hypocotyls were excised from 7, 11 and 30 days old aseptic seedling to transform under the same condition. It showed that the induction efficiency of the resistant calli were the same among all explants of different age, but the embryos on the calli from explants of 7 days appeared earlier than those from explants 11 and 30 days. In order to compare the resistant calli induction efficiency, petioles and hypocotyls were excised from 30days aseptic seedlings to transform with PBinCTBS under the same condition. The resistant calli induction efficiency of petioles was higher than those of hypocotyls. A more detailed analysis of the different types of explants showed that the number of PCR positive plants induced from petioles was higher than that from hypocotyls. This conclusion is corresponding with that of Pawlicki’s.The result of PCR amplification, PCR-Southern, Southern blotting showed that CTB or CTB-CS3 was stabl更多还原