维管发育相关基因TFOS的克隆和功能鉴定

Woods are the secondary vascular tissues of vascular cambium activity. Many genes are involved in the highly complex and orderly process of wood formation, in which regulation of vascular tissue polarization is the key step and to identify key genes in the process by taking a rapid and effective method is the molecular basis of improving timber properties. To better understand the molecular mechanism of wood formation, it is necessary to develop a quick and efficient method to identify the function genes. Herbaceous plants Arabidopsis undergo a certain degree of secondary growth and form certain amount of “wood tissues” under short day conditions, thus it can be used to study wood formation. Using the secondary growth induction method, we screened out a vascular development mutant named tfos (tail-fins-on-stem) in our previous work. The most remarkable characteristic of tfos is that there are three to four ridges emerging from the base and extend till to the middle of stem. Microscopic observation shows that there are tubular molecular structures inside the ridged bumps and genetic analysis indicates that mutant traits are controlled by a single recessive gene and the mutant is mapped within approximate 190kb of chromosome Ⅰby map-based cloning. However, the specific mutated gene’s locus remains to be verified by further narrowing the candidate region..Based on the coarse positional cloning, fine mapping, DNA sequencing, gene compensation, expression and analysis are used to clone and identify the gens’ function and polar homologous genes are further cloned so as to identify its function in vascular development regulation network. This study would provide a novel gene for wood formation so as to explore a feasible, effective and rapid new method to dissect gene functions.

树木维管形成层的活动产生大量的次生维管组织，即木材。木材形成过程高度复杂而有序，期间有许多基因参与，维管组织两极分化的调控是关键步骤。采取快速、有效的方法鉴定该过程中的关键基因是进行木材材性改良的分子基础。拟南芥在短光照条件下，也能形成类似“木材”的维管组织，可用来研究木材形成。前期实验利用该方法筛选到一个尾翼茎突变体，其显著特点是茎中下部有脊状突起附着。显微观察显示脊状突起内部有管状分子结构；遗传分析表明突变性状受隐性单基因控制；利用图位克隆将突变位点定位在Ⅰ号染色体约190kb区域内。但具体突变基因有待进一步缩小候选区域并验证。. 本研究以此为出发点，利用精细定位、DNA测序、基因补偿和表达分析等实验，克隆并鉴定该基因功能，并进一步克隆杨树同源基因并鉴定其在维管发育调控网络中的作用。本项目的完成将为木材发育的研究提供新基因，探索一条可行、有效且快速的解析基因功能的新途径。

维管组织发育和分化是高等植物形态建成的基础，研究其调控机制具有重要理论价值。维管发育高度复杂而有序，木质部和韧皮部分化的调控是关键步骤，鉴定该过程中的关键基因是进行林木改良的分子基础。模式植物拟南芥是研究维管发育过程中一些基本问题如维管组织的发生和轴向分化等的一个较好的模型和辅助手段。.本项目利用图位克隆、分子克隆和转基因等现代遗传和分子生物学技术从拟南芥维管组织发育突变体tfos中分离和克隆到AGO1等位基因TFOS，发现是单碱基替换（CCA→TCA）进而引起编码氨基酸的改变（Pro→Ser）。AGO1蛋白Pro479位点在不同物种间高度保守，表明它在进化过程中起到非常重要的作用。鉴于AGO1是植株发育进程sRNA调控通路的关键基因，利用转录组方法检测了tfos中基因表达总体状况，获得298个差异表达基因。为进一步研究杨树AGO基因在维管发育过程中的作用，利用生物信息学初步分析了杨树AGO家族，并克隆了美洲黑杨和毛白杨AGO1，分别构建了过量表达载体并转化‘84K’杨树，获得了转基因植株。本项目的完成，为维管组织发育分子机制提供了新基因，并为进一步研究该基因在sRNA调控作用过程中的机理提供了基础。