【作者】 梁欣欣；

【导师】 刘录祥；

【作者基本信息】 中国农业科学院 ， 作物遗传育种， 2004， 硕士

【摘要】 盐害是小麦生产的重要胁迫因素，我国一些地区土壤盐碱化严重，所以通过基因工程方法提高小麦的耐盐性具有重要的理论意义和应用价值。 本研究利用分子生物学的实验手段，构建了包括来源于植物拟南芥的DREB1A（Dehydration Responsive Element Binding protein，一种逆境诱导转录因子，诱导相关抗逆基因的表达）基因和用于筛选转化植株的Bar基因（编码除草剂抗性）的表达载体。 以综合农艺性状较好的小麦品种H6756的932块幼穗愈伤组织为受体，用高压氦气基因枪（PDS1000／He）将DREB1A基因导入小麦，经愈伤及分化筛选成功获得了205株再生植株。经除草剂Basta抗性筛选，其中有89株植株表现除草剂抗性。89株抗性植株再经PCR扩增和Southern杂交鉴定，其中50株植株的基因组中整合有外源基因DREB1A，转化频率达到5.36％。取31株转基因小麦的后代种子继续种植，获得的T₁代植株经分子鉴定，结果显示20个后代株系的基因组中整合有DREB1A基因，证明DREB1A基因可以在转基因后代植株中相对稳定遗传。T₁代转基因植株后代种子继续种植，获得的T₂代植株经PCR扩增和Southern杂交鉴定，4个株系中没有检测到DREB1A基因，而4个株系中DREB1A基因没有发生遗传分离，其它各株系表现不同的分离比例，进一步证明了DREB1A基因在后代中可以相对稳定遗传，并且以显性形式遗传给后代。转DREB1A基因的小麦植株经初步耐盐试验表明，其耐盐性明显高于对照植株。在盐胁迫条件下，转DREB1A基因小麦植株叶片的细胞表面糖蛋白层较为致密，而对照植株叶片的细胞表面糖蛋白层出现细胞壁外缘脱落现象。 将DREB1A基因利用农杆菌介导法导入小麦H6756中。以小麦幼苗的茎尖分生组织为转化受体，与农杆菌共培养后共获得247棵转化幼苗，经除草剂初步筛选，有66棵转化幼苗存活。经PCR扩增鉴定，其中30棵幼苗证明整合有DREB1A基因，转化率达到12.1％。 本研究利用优化的基因枪和农杆菌介导两种基因转化体系，较好地克服了小麦转化受体的基因型障碍，提高了小麦的基因转化效率，获得了耐盐性增强的转DREB1A基因小麦。更多还原

【Abstract】 The salt stress is an important factor influencing production of wheat. The soil of some areas of our country salinizes seriously, so it is very significance to improve the wheat’s salt tolerance by genetic engineering.The plasmid expression vector including DREB1A gene and bar gene was constructed. DREB1A (dehydration responsive element binding) is a transcription factor and introduced the expression of a series of stress-related genes. The bar gene encodes the resistance to herbicide and is used to select the transgenic plants.The DREB1A gene in the plasmid pAHC25-DREB1A was transferred into 932 calli induced by young spikes of wheat cultivar H6756 by microprojectile bombardment, and 205 plantiets were regenerated through screening on the medium containing Basta. According to the resistance test by daubing with 100mg/l Basta solution on the seedling leaves 89 transformed plants survived. By PCR amplification and Southern Blotting, 50 plants were identified to be the transgenic ones containing the target gene of transcription factor DREB1A gene, and the frequency of transformation reached 6.72%. Seeds from 31 transgenic plants were planted and selected for molecular characterization (PCR and Southern Blotting), it was found that progeny of 20 plants integrated DREB1A gene. The results showed that the DREB1A gene can be transmitted to the next generation relatively steadly. Further molecular characterization in Ta generation showed that DREB1A gene wasn’t monitored in progeny of 4 TI plants, while it existed in all progeny of 4 T1 plants and segregated in different rates in progny of other T1 plants, which comfirmed that DREB1A gene can be dominantly inherited. Germination tests in 2.0% NaCl concentration showed that the salt tolerance of transgenic wheat was higher than the controls. In addition, the glycoproteins layer at the cell surface of transgenic wheat became more compact in the condition of 2.0% NaCI salt stress, while those of control plants were destroyed or dropped.The DREB1A gene in the plasmid pC3300IS-DREB1A was also transferred into the wheat cultivar H6756 by Agrobacterium-mediated transformation. The stem apical meristem of wheat seedling was cultured together with Agrobacterium, and 247 seedlings were obtained. While 66 plants survived by selection with 100mg/l Basta. Monitoring the target gene amplified by PCR, 30 plants integrated the DREB1A gene, the frequency of transformation reached 12.1%The transformation systems of microprojectile bombardment and Agrobacterium-mediated were perfected and used to produce wheat lines with salt tolerance in this study. Transgenic wheat lines with DREB1A gene were obtained succesfully in an improved transformation efficiency.更多还原