快速筛选拟南芥受精和早期胚胎发生相关基因的方法

阐明拟南芥受精和早期胚胎发生过程对理解被子植物生殖发育有着重要的指导意义,而利用正向遗传学方法研究拟南芥突变体的表型及其分子机理是探究植物基因功能最常用的一种方法。基于常规的插入突变(包括T-DNA和转座子)、化学诱变(如ethylmethane sulfonate,EMS)和高能射线方法构建的突变体库中假阳性突变体多,难以高效筛选到受精和早期胚胎发生相关基因的突变体。为解决这一难题,本研究建立了一种构建T-DNA插入突变体文库的新方法。即在载体p CAMBIA1302的T-DNA元件上增加花粉特异荧光标记基因(p LAT52∷EGFP),并遗传转化具有四分体花粉的Columbia野生型拟南芥突变体qrt1-2;对获得的突变体库可利用花粉荧光快速排除假阳性突变体,并采用反向PCR(inverse-PCR)扩增技术确定突变位点。此方法在筛选拟南芥受精和早期胚胎发生相关基因突变体上的成功应用表明,其是一种效率高、针对性强、操作相对快捷方便的拟南芥突变体筛选方法。

A Convenient Method for Screening Genes Related to Fertilization and Embryogenesis in Arabidopsis

Research on early embryogenesis and fertilization is a central issue for understanding sexual reproduction in higher plants. The forward genetic method e.g. mutation, is commonly used to explore the physiological functions of genes in plants. However, the high false positive rate in mutant libraries based on regular mutagenesis methods, for example T-DNA insertion, transposon, ethylmethane sulfonate and high-energy rays, retard the screening of mutants related to fertilization and early embryogenesis. To solve this problem, we developed a new method to construct mutant stock. We introduced a pollen-specific fluorescence marker (pLAT52::EGFP) into the T-DNA element of the plasmid pCAMBIA1302, then transferred it into the qrt1-2 mutant, in which the four products of microsporogenesis remained fused and pollen grains were released as tetrads, to construct a T-DNA insertion mutant library. This allowed us to discard the false positive mutants rapidly by simply visualizing the fluorescence of pollen. In addition, we could determine the mutated gene locus of candidate mutants through inverse-PCR. Results showed that this method could help screen mutants related to fertilization and early embryo development rapidly and identify the mutated gene locus efficiently.