【作者】 陈果；

【导师】 王国英； 郑军；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 本研究前期从玉米Mu突变体库中筛选干旱胁迫差异表达基因的插入突变体,获得其中19个基因的候选突变体。在此基础上,在大田种植这些候选突变体的后代,并继续进行分子鉴定,保留阳性植株,对其自交或与B73回交,以期获得背景较为一致的纯合突变体材料进行表型鉴定。通过PCR鉴定,保留阳性植株,在大田对其自交,与B73回交,到目前为止,获得了BC1F3、BC2F2或BC3F1代,共17个目的基因,43个插入株系的可遗传Mu转座子插入突变体；通过对突变体材料的基因型检测,获得了6个基因的Mu插入纯合突变体,其中5个为BC1F3代,1个为BC1F4代；对19个目的基因,共63个插入位点的候选突变体材料的PCR产物测序,确定了Mu插入的具体位置：28个Mu插入位点定位在目的基因的5’非翻译区,17个在内含子区,6个在外显子区,12个插入在非目的基因序列当中。在获得Mu插入纯合突变体的基础上,对M9-2G4和M25-37811进行了干旱胁迫处理,并检测了基因M9,M25在B73以及突变体中的表达情况。在正常浇水条件下,突变体M9同B73相比,表型没有明显差别,而在干旱胁迫条件下,突变体M9的存活率以及叶片保水能力低于B73,表现出对水分更敏感；M9基因的表达检测表明,该基因在突变体和B73中都有表达,不过在突变体中表达相对较弱。同样的,突变体M25在水分胁迫条件下,同B73相比,叶片枯黄程度较重,表现出对水分更敏感,不耐旱；M25基因的表达检测表明,该基因在突变体中没有表达。通过RT-PCR方法,克隆了4个玉米蛋白磷酸酶基因。将这4个基因构建了用35S启动子驱动的植物超表达载体,并转化了野生型的拟南芥。通过PPT筛选,获得了M25转基因纯合株系19个。M25转基因纯合株系的ABA, NaCl,甘露醇胁迫处理表明,转基因株系与WT的表型没有明显差异。更多还原

【Abstract】 The genes with significant differential expression were selected from the gene chip of maize seeding under drought stress, then19genes containing the Mu transposonal insertion were identified from the Mu mutant library. On the basis, the mutants are planted in the field. Each generation of maize is identified by PCR in the lab, and these positive plants are self-pollinated and back cross with B73in the field to obtain the mutants of the same background and homozygous for identification of phenotype.The positive plants identificated by nest PCR are self-pollinated and back cross with B73in the field. So far, the BC1F3,BC2F2,BC3F1generation of mutants containing17genes,43insertional sites are obtained. The experiments confirmed that the muants are heritable.The genotype of the mutants are identificated.9ears are the homozygotes from the5genes,5lines in the BC1F3generation and3ears from the1gene,1lines in the BC1F4generation. The63insertional sites in19interesting genes containing Mu insertion are sequenced. The results indicated that28sites are located in the5’-UTR,17sites located in the introns,6sites located in the exon and12sites located not in the sequence of interesting genes.The Mu insertional homozygous mutants of M9-2G4and M25-37B11are treated by the drought stress, and detected the expression of the gene M9,M25in them. The results indicated that the phenotype of the mutant M9and B73are not different under the normal conditions, however the leaves are extended, lower level of wilt in mutant M9than B73, so the mutant M9shows more sensitive to water than B73under the drought stress, then the expression of M9is detected. The result is that the gene M9expressed in all the mutant and B73, but more weaker in mutant than in B73. the leaf scorch is more severe in mutant M25than in B73under the drought stress.So the mutant M25shows more sensitive to water than B73. the gene M25expressed in mutant M25, not in B73.The4protein phosphatase genes are cloned by the RT-PCR in maize. The plant over-expression vectors used35S promoter are constructed for the4genes, and transformated into the wild type Arabidopsis. The19transgenic homozygous lines of M25are obtained by PPT.Under ABA, NaCl,Mannitol treatments,the phenotypes of homozygous of M25transgenic Arabidopsis are not significant difference,compared with the WT Arabidopsis.更多还原