【作者】 陈焕丽；

【导师】 尹钧； 李永春；

【作者基本信息】 河南农业大学 ， 作物栽培学与耕作学， 2007， 硕士

【摘要】 干旱是影响小麦高产稳产的重要逆境因子,抗旱相关基因的克隆及转基因研究是作物抗旱分子育种的重要研究内容。本研究在克隆构建了海藻糖-6-磷酸合成酶和海藻糖-6-磷酸磷酸化酶的融合基因TPSP植物表达载体的基础上,进行了小麦转基因研究,并获得了经分子鉴定的转基因小麦材料;同时,在分析了水分胁迫过程中小麦根系基因表达谱特性的基础上,利用RT-PCR技术克隆了与小麦水分胁迫反应密切相关的转录因子基因TaSNAC1,并初步分析了该基因在水分胁迫过程中的表达特性。主要内容如下:1.以豫麦18,豫麦34,豫麦49,豫麦70和郑麦9023的幼胚为受体材料,通过组织培养技术筛选适合基因枪法转化的优良受体基因型。结果表明,郑麦9023和豫麦18的小麦幼胚的再生能力较强,是较好的受体材料;继代次数不同对转化后愈伤组织分化率有一定影响,以诱导培养继代两次时愈伤组织的分化率较高;分化培养基中加入0.5mg/L的IAA和1.5mg/L的多效唑后可以有效促进根系的分化,提高再生植株的移栽成活率。2.小麦品种豫麦34和豫麦18经浓度为0～120 mg/L PPT的溶液叶片涂布,确定100 mg/l的PPT作为转bar基因小麦植株对除草剂抗性的一个选择标准;转基因小麦幼苗叶片用100mg/l的PPT涂抹后表现出一定的抗性,而非转基因对照则在涂抹5天后部位开始萎蔫和死亡,转化植株的叶片对PPT的抗性明显高于对照植株。3.在基因转化与植株再生培养条件优化的基础上,以大面积推广的小麦品种“豫麦18”幼胚来源愈伤为受体,利用基因枪法进行了TPSP基因的小麦遗传转化。1286块愈伤组织经基因枪轰击后,通过抗性愈伤的筛选及分化,获得54株抗性再生植株。经PPT抗性筛选和PCR分子鉴定,最终获得转基因小麦TO代植株5株。4.以豫麦34、豫麦49、豫麦70、豫麦18和郑麦9023作为受体材料,用花粉管通道法进行了TPSP基因转化。共处理5个小麦品种的5848朵小花,收获种子3726粒,平均结实率为63.7%;对转化所得TO代种子单粒点播成株行,T1代共出苗3526株,平均出苗率为94.63%;对豫麦34的T1代幼苗,进行PPT抗性检测及叶片基因组总DNA的PCR检测,421株植株中有4株呈阳性反应。5.利用RT-PCR方法,克隆了受水分胁迫诱导的转录因子基因TaSNAC1,该基因序列长度1169 bp,其中编码区1029 bp,推测翻译出的蛋白质有342个氨基酸组成,其等电点为6.26,分子量为38.14 KD。基因推测的氨基酸序列包含有NAC类蛋白的保守功能域,与小麦NAC类基因(AY625682、DQ022842和DQ022843)的同源性达到96%左右,其定名缩写为TaSNAC1。半定量RT-PCR分析表明,TaSNAC1在水分胁迫不同时期的小麦根系中存在差异表达,而在叶片中未检测到其表达。更多还原

【Abstract】 Drought is a major factor which limited the high and stable yield of wheat,the cloning of drought-resistance related genes and the transgentic study is an important branch of crop molecular breeding for drought tolerance.In this study,based on the constructed plant expression vector,which harboring a fusion gene TPSP for trehalose synthesis,the genetic transformation of several wheat varieties was performed and some transgenic wheat materials were obtained.In addition,based on the gene expression profile in root tissues of wheat under the water stress,a candidate gene TaSNACl,which closely related to the water-stress response and coding a putative transcription factor,was cloned via RT-PCR method and its expression patterns were characterized during the wheat dehydration.The main content of the study was summarized as follows:1.Immature embryos of several wheat varieties Yumai 18,Yumai 34,Yumai 49, Yumai 70 and Zhengmai 9023 were used to study on the genotype screening for genetic transformation via microprojectile bombardment.The results showed that the properties of proliferation,differentiation and regeneration of Zhengmai 9023 and Yumai 18 were better than other varieties;Subcultivation times affected the differentiation of the calli to some extent and subcultivate 2 times of induced calli could significantly enhance the differentiation rate;a proper plant hormone concentration,IAA（0.5mg/L）and CCC （1.5mg/L）,in the differential medium can effectively promote the root differentiation and is very benefit to transplanting of regenerated plantlets.2.The test of different PPT concentrations（0-120 mg/L）showed that 100mg/L was the proper concentration for transgenic assay on wheat leaves of Yumai 18,Yumai 34. Under this concentration,transgenic leaves can survive and grow normally,while the no-transgenic leaves become wither and finally died after 5 days.3.Based on the optimized methods of genetic transformation,the wheat variety Yumai 18 was transformed with the fusion gene TPSP for for trehalose synthesis via microprojectile bombardment.1286 embryo generated calli were used as the target tissues,and 54 putative transgenic plantlets were obtained after the ppt resistant screening,differentiation and plant regeneration.After the PCR identification and the ppt resistant assay,5 transgenic plantlets（T₀）were obtained.4.Wheat cultivars,Yumai 18,Yumai 34,Yumai 49,Yumai 70 and Zhengmai 9023, were transformed with the fusion gene TPSP by the method of pollen-tube-pathway.In total,5848 flowers from the 5 cultivars were treated and 3726 seeds were obtained with a seed-set percentage of 63.7%.T₀ seeds were sowed in the field individually and 3526 T₁ plantlets were got with a seedling percentage of 94.63%.Tests of T₁ Yumai 34 offspring were carried out,including PPT resistence and PCR identification,and finally 4 plantlets were selected as candidate transgenic plantlets from 421 tested plantlets.5.A transcriptional factor gene TaSNACl,which strongly induced by the the water stress,was cloned by Reverse Transcription-Polymerase Chain Reaction（RT-PCR） from wheat root.The cloned gene including a sequence of 1169bp in length and a coding region 1029bp,the deduced amino acids sequence（342 aa）of this gene is about 38.14KD and the pI is 6.26.Anylysis showed that the conserved function domains from amino acid sequence belongs to NAC genoid and the homology of it to NAC genes from wheat（AY625682、DQ022842 and DQ022843）is up to 96 or so.The expression pattern of the cloned gene was characterized via RT-PCR and the results indicated that the TaSNACl gene was induced in roots during the water stress accumulation,while the expression of the gene was not detected in leaves.更多还原