【作者】 房玮；

【导师】 卢孟柱；

【作者基本信息】 中国林业科学研究院 ， 林木遗传育种， 2010， 博士

【摘要】 木材形成是一个受到严格调控的复杂的生物学过程。程序性细胞死亡(Programmed cell death, PCD)是细胞在一定的生理或病理条件下,为了维持内环境稳定,更好地适应生存环境而采取的一种由基因控制的细胞主动的、有序的死亡。大量研究表明,PCD对维持生物体正常生长、发育等具有非常重要的作用。PtCDD是一种Ca2+依赖型DNase,在杨树维管形成层的细胞程序化死亡中发挥关键作用,为找到与PtCDD互作的因子,我们构建了杨树形成层区域的cDNA文库,以PtCDD为诱饵蛋白,通过酵母双杂交在已构建的文库中筛选可能的互作因子。最终得到两个DNA片段,分别为AD1-10和AD12-2,在GenBank中对其进行生物信息学分析,前者是银桦线粒体的核糖体RNA小亚基基因(mtSSU),后者是杨树叶片在干旱胁迫条件下得到的一种EST,功能尚不明确,结构与杨树26S核糖体RNA基因相似。由于AD1-10为核糖体RNA小亚基基因,本研究利用凝胶阻滞实验在体外验证了PtCDD蛋白与AD1-10的作用。初步说明了PtCDD参与杨树细胞PCD可能由线粒体所介导。锌指蛋白PtSZF基因是次生维管系统发育中发生特异性表达的基因,其功能有待进行深入研究。根据杨树数据库PopulusDB和JGI中基因全长的信息,克隆了其cDNA全长。通过将PtSZF基因蛋白序列输入Swiss-Model网站和EMBL-EBI网站对其进行分析比较,发现PtSZF蛋白与植物光信号传导途径调控开花的关键因子CO(constans)蛋白相类似。本研究构建了PtSZF基因的超表达载体,并转入烟草中。发现PtSZF基因超表达的烟草同野生型相比较,超表达烟草的表型发生了非常明显的改变,主要有以下几个方面:植株明显变粗,开花时间延后;节间明显变短;髓部细胞大量减少;PtSZF基因大量表达(为野生型植株的40倍)的转基因植株,无法进入生殖生长,植株矮小,不断分生出芽。这些表型显示PtSZF蛋白基因不只是调控了植株的某个单一的发育过程,而是参与了对生长发育各个阶段的调控。其作用同过去CO-like基因在拟南芥、水稻草本植物中所获得研究结果有很大不同,这说明CO家族基因很可能在木本植物发育中参与了更广泛、更系统的调控过程。更多还原

【Abstract】 Wood formation is a complex process composing many biological events. Programmed cell death (PCD) is the active process of cell death which occurs during secondary vascular system (SVS) development in response to environmental cues. Ca2+ dependent DNase from Populus tomentosa (PtCDD) play a critical role in PCD during the development of secondary vascular system in woody plants. To find out possible factors which interacts with PtCDD, we prepared cDNA from the cambial zone and subsequently amplified the full length of PtCDD and construct a cDNA yeast library to perform yeast two-hybridization analysis. We have screened the potential interacting factors of PtCDD in poplar cDNA library from cambium zone and finally got two potential targets named AD1-10, AD12-2. After sequencing and BLAST in GenBank, AD1-10 has been identified as a homolog of small subunit ribosomal RNA (mtSSU) gene, while AD12-2 is a function-unknown EST, induced by drought stress. The interaction between PtCDD and AD1-10 is verified in vitro by Pierce Lightshift EMSA, suggesting PtCDD likely participate in regulation of mitochondrion-mediated PCD of secondary vascular regeneration in woody plants.The function of zinc-finger protein PtSZF involved in SVS development needs further study. We have cloned the full-length cDNA of PtSZF gene from poplar through blasting protein sequence of PtSZF in Swiss-Model and EMBL-EBI online, and shown that PtSZF is a homology of CO(constans), a critical switch in light signaling-mediated flowering process. We made PtSZF overexpression construct, and transformed it into tobacco. The transgenic tobacco displayed several significant phenotypic changes when compared with wild type: thicker stem and delayed-flowering; Dramatic decrease of pith cells in number; Shorter Internode; extremely overexpressed (40-fold) line gave short and continual tilleringand failed to reproductive transition, suggesting that PtSZF might participate in many stages of tobacco development and regulate its overall growth cycle. It is significant to reveal the distinct function between PtSZF and its homolog in Arabidopsis and Oryza, which suggests that CO family genes likely play more ubiquitous and versatile roles in developmental process of woody plants except flowering control.更多还原