【作者】 张媛媛；

【导师】 江昌俊；

【作者基本信息】 安徽农业大学 ， 作物遗传育种， 2004， 硕士

【摘要】 小麦是重要的粮食作物之一，利用基因工程进行遗传改良的研究一直倍受人们关注。本研究以安徽本地小麦栽培品种为材料，通过建立起良好的再生休系，利用基因枪，将含有抗除草剂Bar基因的植物表达载体，导入安徽优良品种小麦幼胚愈伤组织，并通过GUS瞬时表达情况对基因枪轰击参数进行了优化。用除草剂对轰击后的材料进行分化生根筛选，共获得33株抗性再生苗。通过PCR检测和涂叶检测，初步证明其中有4株已将Bar基因整合到小麦基因组中并且得到表达，平均转化率为0.33%。此外通过回交将抗Basta（主要成分为PPT）除草剂的Bar基因转育到这些小麦品种，通过对转Bar基因小麦人工杂交与回交后代除草剂抗性的连续跟踪鉴定，研究了Bar基因在小麦杂种后代的遗传表现。结果表明：1 小麦再生体系的建立小麦幼胚离体培养中，基因型和培养基是建立良好再生体系的主要因素。从愈伤组织的诱导率、生长质量、分化率和再生率来看，均有扬麦158>安农98005>皖麦33>安农92484。小麦幼胚在MS+2.0 mg/L 2,4-D+0.5 mg/L ABA培养基诱导愈伤组织，经过4℃低温处理，转入分化培养基MS+1.0 mg/L KT+0.2 mg/L NAA中， 在1/2 MS+0.4 mg/L NAA+15 mg/L PPT+0.6 mg/L PP333培养基中壮苗生根。在以上再生条件下，幼胚愈伤再生频率显著提高。2 小麦基因枪转化体系的建立并通过GUS基因的瞬时表达优化了转化条件。结果表明：在沉淀剂为50 μl 2.5 mol/L CaCl2+20 μl 0.1 mol/L亚精胺，金粉/DNA用量为250 mg/0.5 μl，轰击距离在9 cm时，GUS基因具有较好的表达效果，受体材料分别在轰击前4 h和轰击后16 h作渗透培养(0.4 mol/L渗透剂)。轰击压力为1100Psi，真空度为91.4×103-94.9×103 Pa，每皿轰击一次。 3 转化体的筛选和抗性植株的获得确定了不同筛选阶段的选择压浓度：愈伤组织继代、分化、生根时PPT分别为3 mg/L、5 mg/L和15 mg/L。抗性植株经PCR检测后最终得到了扬麦158和安农98005两个品种的4株转基因植株，最高转化频率为0.66%。对其叶片作除草剂PPT的表型性分析，结果有2株表现完全抗性，2株表现部分抗性，表明Bar基因已在小麦植株中得到表达，转化率在0.33%~0.66之间。转化周期为3~4个月左右。4 杂交育种 <WP=4>以含Bar基因的抗除草剂小麦基因遗传工程体为基因供体，与安徽品种进杂交，获得的F1后代均为抗性植株。进行自交获得的F2代中抗除草剂植株与感除草剂植株的分离比基本符合3:1的分离比，与安徽品种进行回交得到的B1F1代中，分离比为1:1。结果表明，抗除草剂转基因小麦杂交与回交后代Bar基因的遗传，符合1对显性基因的分离比例和孟得尔遗传规律。更多还原

【Abstract】 Wheat ,a major world crops, is primary target for thre application of genetic engineering to improving agronomic characteristic. In this paper, the regeneration system of four genetypes wheat was established. the immature embroys calli used as receptor to be bombardment with gold partical coated with herbicide resistance of Bar gene and the parameters of bombardment were optimized by GUS transient expression. After callus induced regeneration and shooted elongation on selection with PPT, total of 33 resistant plants were achieved. By PCR analysis and spreads the leaf examination with PPT in T0 regeneration, 4 transgenic plants were identified with a transferent frequency of 0.33%. In addition, we transfer the wheat Bar gene into wheat cultivated varieties with backcross way. Then by the tracking investigation of the herbicide resistance of generations crossed or backcrossed with Bar gene transgenic wheat and the wheat hybrid descent’s heredity performance were studied. The result indicated that: 1 Development of regeneration systemsWheat genotypes and medium are the key factores influencing the development of regeneration systems for immature embryo cultivation. As far as the frequency of callus induction、quality of callus induction、frequency of compact differentiation and regeneration were concerned, there was a trend of Yangmai 158>Annong 98005>Wanmai 33>Annong 92484. A effection regenaration system had been established : callus induction medium was MS+2.0 mg/L 2,4-D+0.5 mg/L ABA. After beeing treated in 4℃ low temperature, the callus were regenerated on MS+1.0 mg/L KT+0.2 mg/L NAA medium. Then shoot were induced on 1/2 MS medium supplemented with 0.4 mg/L NAA、and 0.6 mg/L PP333. In such system, the callus regeneration frequency had the remarkable elevation.2 Development of bombardment transformation systems Under the vacum pressure of 91.4×103~94.9×103 Pa and bombarded once per dish with the Helium pressure of 1100Psi, conditions of bombardment were optimized by GUS transient expression. The results indicated that when the precipitants for coating particles containing 50 μl 2.5mol/L CaCl2 and 20 μl 0.1mol/L spermidine, the suitable dosage of DNA and gold powder per bombardment was 250 mg/0.5 μl and the bombardment distance was 9 cm, the GUS expression get the higest expression. The acceptor materials were cultured on osmotic medium (0.4 mol/L osmotic agents) 4 hours before bombardment and 16 hours after bombardment. <WP=6>3 Transformation and selection Selection system of high efficiency was determined by different PPT concentration. Callus growth inhibition 、regeneration and rooting were 3 mg/L、5 mg/L and 15 mg/L. 4 plants were proved transgenic including Yangmai 158 and Annong 98005 by PCR anlysis. The herbicide-resistance test on the plants leaves indicated that two plants were more resistant than the other two. It suggested that Bar gene were expressed on different level. The wheat transformation frequency was between 0.33~0.66%, and cultivated time was only about 3~4 months.4 Through the hybridization of Anhui local wheat cultivated variety and the herbicide resistance variety which is including Bar gene by genetic engineering, the herbicide resistance F1 generation plant were obtained which was self-crossed or backcrossed with Anhui variety. The separation in F2 between the resistance and sensistive basically according 3:1, while in B1F1 generation is 1:1. The result showed that the inheritance of Bar gene was a single dominant gene and accorded with the law of Mendelian segregationbasically.更多还原