【作者】 杨成丽；

【导师】 刘德立； 周吉源；

【作者基本信息】 华中师范大学 ， 生物化学与分子生物学， 2002， 硕士

【摘要】 甜蛋白Thaumatin是一种高甜度、低热量、安全无毒、甜味纯正的非糖类新型甜味剂，具有很高的经济价值。然而该蛋白的来源植物只产于西非Thaumatococcus danielli的果实中，该植物对生活环境要求苛刻，在世界各地的引种尚不成功，不能满足人们对甜蛋白Thaumatin的大量需求。研究人员试图用微生物表达甜蛋白，结果不如人意。因此，本试验构建了植物表达载体pBI₁₂₁-th，并将其转入根癌农杆菌LBA₄₄₀₄中，然后通过农杆菌的介导将thaumatin基因导入烟草中，并对转基因植株进行了检测，借以初步探讨甜蛋白在植物中的转化和表达情况，为其今后在更多的植物上应用提供依据。实验结果如下： 1．甜蛋白thaumatin基因植物表达质粒pBI₁₂₁-th的构建与鉴定 利用DNA重组技术，将植物甜蛋白thaumatin基因克隆至植物表达载体pBI₁₂₁中，通过酶切、电泳，鉴定thaumatin基因已成功构建到植物表达质粒pBI₁₂₁中。 2．pBI₁₂₁-th质粒转化根癌农杆菌 重组质粒pBI₁₂₁-th通过直接转化法导入农杆菌LBA₄₄₀₄中，卡那霉素（Km）平板筛选阳性克隆，有单菌落生长，证明重组质粒已转进农杆菌LBA₄₄₀₄中。 3．植株再生体系的建立 采用无菌苗烟草叶片为外植体，以MS为基本培养基，筛选出MS+6-BA2.0mg／L+NAA0.3mg／L以诱导不定芽的分化，并于MS+NAA0.3mg／L的 PA 硕士学位论文 V沉了砂 “工队nk” 主根培养基上生根，获得完整再生植株。其叶片分化率达65石％，生根率达 95％。 4．转化体筛选及植株再生 将预培养2天的外植体与农杆菌菌液浸染5～6分钟后，共培养2～3天， 然后转化到含Km75m旮 的选择培养基上进行分化筛选，再转入K。为 75mg几、100mg／L的培养基上进行生根筛选，生根率为82．5％。转入外源基 因的转化体将会在这一系列筛选过程中发芽、生根，获得转基因植株。 5．转基因PCR检测 PCR扩增 nPt 11基因，证明 thaumatin基因己导入烟草中，转化率为 引．3％c更多还原

【Abstract】 Sweet protein Thaumatin has great economic value because thaumatin is a no caloric sweetener that has many advantages such as high sweet intensity, low calories, no toxicity and so on. However, this protein can’t meet people’s dem--ands because of lacking of resources. Therefore, in this paper, the recombinant plant expression vector pBIi2i-th was constructed successfully and introduced into Agrobaterium tumefaciens LBA4404. Subsequently thaumatin gene was transformed to tobacco through Agrobaterium-mediated system. And transgenic plants were confirmed by a series of examinations. The results were as following:1. Construction and identifcation of recombinant plant expression vector pBI!2i-thBy DNA recombination technology, sweet protein thaumatin gene was cloned into plant expression vector pBIi2i. Recombinant plasmid pBIi2i-th was constructed successfully by enzyme cutting and electrophoresis.2. Introduction of pBI121-th plasmid into Agrobacterium tumefaciens . The pBI121-th was introduced into Agrobacterium LBAncn by direct transformation .By screening in the medium containing kanamycin , it was confirmed that the result was successful .3. Regeneration system of plants.The regeneration system of tobacco was established. The adventitious shoots were induced from leaf explants of tobacco based on MS basal medium supple--mented with 2.0mg/L 6-BA and 0.3mg/L NAA . Then the regenerated plants were rooted on MS medium containing 0.3mg/L NAA. And the frequency was about 65.6%, 95% respectively.4. Transformation and selection of plants.Leaf explants had been pre-cultured for 2 days, then immersed in Agroba--cterium suspension for 5~6minutes. The co-cultivation had been carried out for 2~3 days. After that, the transformants were obtained by transferring explants to selection medium containing 75mg/L kanamycin and rooting medium contain--ing 75mg/L, 100mg/L kanamycin .And its rooted frequency was 82.5%.5. Detection of transgenic plants.The PCR assay of kan-resistant plants showed that the target gene had been integrated into tobacco accompanying with npt II gene. The frequency of transf--ormation was 31.3%.更多还原