【作者】 檀琮萍；

【导师】 江昌俊；

【作者基本信息】 安徽农业大学 ， 茶学， 2003， 硕士

【摘要】 杂草与农作物竞争水分、养料和光照，影响农作物的产量、品质，还是许多病害的中间寄主或越冬场所，通过除草剂来控制杂草已成为现代化农业不可缺少的一部分。但除草剂在消灭杂草的同时也对作物产生了伤害作用。目前，利用基因工程手段培育抗除草剂的作物品种解决了这一矛盾。 广谱、非选择性除草剂Basta的活性成分是草丁膦(Phosphinothricin)。草丁膦除草作用机理是抑制植物的氨基酸生物合成酶—谷氨酰胺合成酶(GS)，使杂草产生氨中毒。bar基因编码草丁膦乙酰CoA转移酶(PAT)，可催化草丁膦的自由氨基乙酰化，从而使除草剂草丁膦失活。而且bar基因也是迄今为止应用最为广泛的一个抗除草剂选择标记基因，克隆出bar基因对植物的遗传转化，基因表达和生理学研究都有重要的作用。 本实验从抗除草剂转基因Bobwhite小麦中，利用PCR克隆的方法扩增出bar基因全长，并在原核表达系统中表达，鉴定表达蛋白的活性，将能够正确编码PPT乙酰转移酶的bar基因片段，经过适当的修饰构建入真核表达载体。并将此构建的真核表达载体转化农杆菌获得工程菌，为基因转化及农杆菌转化做了前提工作。并对农杆菌转化小麦体系作了初步的研究，为选育出适宜在安徽麦区种植的高产、优质、抗除草剂小麦新品种做了基础工作。本实验研究结果表明： 1．利用PCR方法对已知序列基因的克隆是简单有效的方法，但很多因素影响PCR的特异性，其中退火温度起着关键性作用。本实验采用了高保真pfu DNA聚合酶，在退火温度61℃条件下从转基因Bobwhite品种基因组DNA中扩增出特异性片段，将此片段插入克隆载体pGEM-7fz(+)，经测序和序列分析表明，所扩增得到的片段含有bar基因完整的读码框，并且序列与GenBank中发表的序列完全一致。 2．选用pET32a表达载体和BL21trxB(DE3)宿主菌的原核表达系统，对扩增得到的片段进行表达鉴定。经IPTG诱导表达的融合蛋白的分子量39.6kDa，目的片段表达蛋白分子量20.1kDa，与预计的分子量20.0kDa相符。并且表达的蛋白酶(PAT)经DTNB比色法鉴定，具有酶活性。 3．为了目的基因在真核生物中有效的翻译和表达，通过PCR方法对bar基因起始密码子ATG旁侧序列进行改造。改造后的目的基因克隆于真核表达载体pBI121，构建表达载体pBI121-bar，通过三亲杂交法将真核表达载体pBI121-bar 转入农杆菌LBA4404，成功构建出工程菌。4．小麦离体培养中，基因型、培养基激素浓度与外植体类型是影响小麦胚性愈 伤组织诱导和再生频率的3个主要因素。基因型的影响主要影响到胚性愈伤组 织的发生频率，以幼胚为外植体杨麦87158可以达到sl．35％，而安农92484 只能达到 16．sl％。在所试的品种中对于成熟胚在 2，4D质量浓度为 4．omg／L 时出愈率和胚性愈伤组织诱导率达到最大值，而对于幼胚不同品种表现有所差 异，其中杨麦871 58和安农98005在2，4＊浓度为2．omg几时胚性愈伤组织诱 导率最高，而安农92484则是在4．omg几时效果较好。同时研究表明不同外植 体不仅影响到出愈率，更主要影响胚性愈伤组织的形成。5．在转化后抗性愈伤组织筛选中，适应的PPT筛选浓度为3刀mg几，Cef有效抑 菌浓度为 200pg／ml，乙酚丁香酮有效诱导浓度为 100pmol几，0．5％表面活性剂 Tween20，可以使GUS染色均匀，但并不是T－DNA转移所必需，盐的浓度对 T－DNA转移影响不大。更多还原

【Abstract】 Weeds compete with crops for moisture, nourishment and light, moreover, they are hosts of disease or the place where disease survives in the winter, which influences the output and quality of crops. It is indispensable for modernized agriculture to control weeds by using herbicide. But berbicide also injures crops while killing weeds. At present, cultivating berbicide resistant crops by genetic engineering has solved this contradiction.PPT(Phosphinothricin) is the active composition of broad-spectrum, non-selective hercicide Basta. It inhibits the activity of glutamine synthetase(GS) which is necessary for the production of glutamine and for ammonia detoxification. Inhibition of GS by PPT causes a rapid buildup of intracellular ammonia levels. Bar gene codes phosphinothricin acetyl- trasferase(PAT) which acetylizes the freedom amino of PPT and makes PPT lose activity. Bar gene also is one of widely used selective marker gene. The cloning of bar gene is important to plant transgenic engineering, gene expression and studying physiology.In the experiment, the full code sequence of bar gene was cloned by PCR from transgenic herbicide resistant Bobwhite wheat and checked. It was expressed in E.coli and its protein was determined. After having been properly modified, the bar gene which correctly codes PAT was cloned into binary vector pBI121 and then transferred into LBA4404 by triparantal crossing, which is the prerequisite work for genetic transformation. The system of wheat transformation mediated by Agrobacterium tumefaciens was preliminarily studied on, which make foundation for cultivating high yield, high-quality and herbicide resistant wheat in Anhui. The research suggested:1. PCR is a simple and effective method for cloning gene whose sequence is known. But a lot of factors influence PCR specificity, among which anneal temperature play a key role. In our experiment, the specific fragment was amplified from transgenic Bobwhite genome DNA at annealing temperature 61 by using high-fidelity pfu DNA polymerase and cloned into clone vector pGEM-7fz(+), then sequenced. The cloned sequence was completely identical to the sequencewhich was issued in GenBank.2. The sequence was cloned into express vector pET32a and expressed in BL21trxB( DE3). The molecular weight of fusion protein induced by IPTG was 39.6kDa, and the molecular weight of target protein was 20.1kDa, which accorded with the estimated molecular weight 20.0kDa. Then its enzyme activity was determined by DTNB spectra chromatography.3. In order that bar gene efficiently transcript and translate in eukaryote, the sequence next to ATG was modified by PCR. The modified sequence was cloned into binary vector pBI121. The constructed expression vector was named pBI121-bar, then transferred into Agrobacterium tumefaciens LBA4404 by triparental crossing. Thus the project bacterium has been successfully constructed.4.In the cultivation, wheat genotype, concentration of hormone in the medium and the kind of explant are the three key factors that influence frequency of induction embryogenic calli and plant regeneration. Genotype mainly influences the frequency of embryogenic calli formation, 81.35% immature embryo of Yangmai 87158 can form embryogenic calli, while only 16.81% immature embryo of Annong 92484 can form embryogenic calli. When mature embryo was used as explant, as for above three lines of wheat, the optimize concentration of 2,4-D for inducting calli and embryonic calli is 4.0 mg/L. However, when immature embryo was used as explant, the optimize concentration of 2,4-D is different, as for yangmai 87158 and Ann 98005,it is 2.0 mg/L,but as for Ann92484, it is 4.0mg/L. Research also indicate that explant influence the frequency of callus induction, especially influence embryogenic calli formation.5.The proper concentration of PPT is 3.0mg/L in screening resistant callus after transformation. Effective concentration of Cef restrain Agrobacterium tumefaciens is 200|xg/ml. The efficient density of AS is 100 umol/L in the co-cultivatio更多还原