【作者】 刘玲；

【导师】 刘桂丰；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2013， 硕士

【摘要】 植物突变体是开展遗传研究,确定基因功能的良好材料。因此,人们采用物理的化学的方法诱导植物突变,获得突变体。但这些突变体的突变基因位点随机性强,很难确定具体位置并克隆得到基因。随着新技术的发展,对于全基因组已知的植物采用插入诱变而导致性状变异,再进行基因克隆,使过去很难完成的事情变得非常容易了。人们可通过TAIL-PCR技术扩增其插入片段的侧翼序列,找到相近的基因,并对基因的功能进行分析,从而确定突变基因。本研究就是针对这样一个矮化突变体进行研究的。在前期的小黑杨转TaLEA基因研究中,获得了11个转基因株系,其中1个株系表型为矮化突变,命名为XL11。以11个转基因株系（包括矮化突变体）及野生型对照小黑杨为试材,开展生长性状、叶片解剖结构比较研究。发现在叶面积、气孔密度、叶片厚度等方面矮化突变株系XL11株系明显低于其他参试株系；角质层厚度、栅海比,气孔大小等方面XL11株系显著高于其他参试株系。进而以XL11株系为材料,采用TAIL-PCR方法克隆插入位点的旁侧序列得到AP2/PthRAV （XM₀02311402.1）。荧光定量分析发现AP2/PthRAV的相对表达量在XL11株系中显著高于其它转基因株系及野生型平均值的7.78倍,初步认为该株系在生长及叶片解剖结构等性状的改变,可能与AP2/PthRAV上调表达有关。进而对矮化株与与野生型的AP2/EREBP转录因子超家族进行表达谱分析,深入分析该家族基因的结构与功能,发现其与植株的生长发育紧密相关,影响根部的发育。随后构建了植物超达载体35S::AP2和RNA干扰植物表达载体35S::amiR-AP2,开展小黑杨的遗传转化,并获得抑制表达的抗性植株。综合分析得出,该基因的转化株成活率低,株系缺绿,根系生长缓慢,有侧根较多,短小粗壮,生根困等特点。更多还原

【Abstract】 Plant genetic mutants is the good material to carry out studies and to determine gene function. Therefore, it is the use of physical and chemical methods of plant mutations induced obtained mutants. However, these mutants mutant loci strong randomness, it is difficult to determine the specific location of the cloning of genes. With the development of new technologies for genome-wide insertional mutagenesis using the known plant traits caused by mutation, then clone, so in the past difficult to accomplish things very easy. It can be amplified by TAIL-PCR sequences flanking the inserted fragment, found similar genes and gene function analysis to determine the mutant gene.Eleven TaLEA overexpressed Poplus simonii×P. nigra transgenic lines and wild-type control were studied for growth traits and leaf anatomy. Among the transgenic lines, one line performed dwarf trait, which was given a name of XL11.The results showed that XL11lines were significantly lower than the other tested lines on leaf area, stomatal density and leaf thickness. On the contrary, XL11lines were significantly higher than in the other lines on epidermis, palisade and spongy tissue ratios and pore size. Focusing on XL11lines, TAIL-PCR method was used for cloning flanking sequences of insertion position, and found AP1/PthRAV （XM₀02311402.1）. Fluorescent quantitative analysis of the relative expression of the AP2/PthRAV lines in dwfl was7.78times significantly higher than other transgenic lines. A preliminary view of dwfl lines in the growth and anatomical structure of leaf traits change may be related to the upregulation of AP2/PthRAV genes. Furthermore, expression profiling of the AP2transcription factor superfamily to the dwarf strain with the wild type. With structure and function analysis of the family genes, the results were closely related to plant growth and development. Then we build a overexpressed vector35S::AP2and RNA interference plant expression vector35S: Amir-AP2. We carried out Poplus simonii×P. nigra genetic transformation, and obtained resistant plants with inhibiting the expression.According to above results, we found that transgenic plants performed low survival rate, lack of green, slow root growth, more lateral root, short and stout, difficulty on rooting.更多还原