毛白杨PtoWOX11/12a对杨树扦插苗生长发育的影响

Effects of PtoWOX11/12a Gene from Populus tomentosa on the Growth and Development of Cutting Seedlings in Poplar

【目的】WOX转录因子家族是一类植物特有的转录因子家族,在植物胚胎建成、干细胞分裂和分化的维持以及器官的形成过程中发挥重要调控作用。本研究分析毛白杨Pto WOX11/12a基因对转基因84K杨叶形、茎等生长发育的影响,分析毛白杨不定根诱导过程中及过表达和抑制表达Pto WOX11/12a转基因84K杨中Pto WOX11/12a基因、生长素合成基因YUCCA1和YUCCA8的表达模式,为深入研究WOX基因对植物生长发育的调控机制提供参考。【方法】对在温室生长1~3个月的过表达和抑制表达Pto WOX11/12a转基因以及非转基因84K杨(对照)的叶形、株高和地径进行比较;通过组织切片对转基因和非转基因84K杨的第5、9和13节间的解剖学特征进行分析,并对各节间的形成层和木质部宽度进行比较;利用实时荧光定量PCR(q PCR)技术,分析Pto WOX11/12a、生长素合成基因YUCCA1和YUCCA8在毛白杨不定根产生过程中及在过表达和抑制表达Pto WOX11/12a转基因84K杨中的表达模式。【结果】在叶形上,过表达Pto WOX11/12a转基因84K杨叶片长度与非转基因84K杨(对照)没有显著差异,但是宽度显著大于对照;抑制表达Pto WOX11/12a转基因84K杨的叶长和叶宽都明显小于对照,且叶边缘呈现锯齿状缺刻。在植株的株高和地径方面,过表达和抑制表达Pto WOX11/12a转基因84K杨的株高和地径都明显低于对照,且存在显著差异;茎解剖分析发现,过表达Pto WOX11/12a转基因84K杨木质部宽度小于对照,形成层细胞层数多,而抑制表达Pto WOX11/12a转基因84K杨木质部宽度同样小于对照,但与过表达植株相比,木质部宽度相对变大,形成层细胞层数变少。q PCR结果显示Pto WOX11/12a基因在毛白杨不定根产生过程中被诱导并持续表达,而生长素合成基因YUCCA1和YUCCA8在诱导培养3天后表达量才迅速增加,但在过表达Pto WOX11/12a转基因植株中表达量升高,在抑制表达植株中表达量降低。【结论】Pto WOX11/12a基因的过表达或者抑制表达都影响了转基因84K杨叶的发育、茎的高生长和径向生长(次生木质部发育);Pto WOX11/12a基因在杨树不定根形成和生长过程中发挥作用涉及到生长素合成基因YUCCA1和YUCCA8的参与。在生根过程中,Pto WOX11/12a基因和生长素合成基因YUCCA1及YUCCA8在表达时间上存在时间间隔。高水平的Pto WOX11/12a抑制了生长素合成相关基因的表达,导致过表达Pto WOX11/12a转基因84K杨由于更容易形成分生组织,促进了根原基的形成,产生比较多的不定根。在不定根生长过程中,YUCCA1及YUCCA8基因上调表达,可促进生长素合成和根部分生组织细胞的分裂;在茎中,二者上调表达促使过表达Pto WOX11/12a转基因84K杨形成层活动的增强,继而影响木质部的分化,木质部变窄。

【Objective】 WOX transcription factor family is one of the plant-specific transcription factor families,which have been demonstrated playing an important role in the embryonic patterning,stem cell maintenance and proliferation and lateral organ development. This study analyzed the roles of PtoWOX11/12 a gene from Populus tomentosa in the development of leaves and stems of transgenic 84 K poplar（ Populus alba × P. glandulosa cl. 84 K）. The gene expression level during the process of adventitious roots formation of Populus tomentosa,and in overexpression and suppressed expression of PtoWOX11/12 a transgenic 84 K poplars were analyzed. Our research provides a foundation for further studies of the roles of WOXs gene in plant development.【Method】 The leaf shape,height and basal diameter of one-month-old tothree-month-old transgenic lines and non-transgenic 84 K poplar（ control） in the greenhouse were measured and compared. The anatomy of stem sections of the 5 th,9 thand 13 thinternodes of transgenic lines and control plants was analyzed and the width of xylem and cambium were compared. The expression of PtoWOX11/12 a,YUCCA1 and YUCCA8 during the process of adventitious roots development in P. tomentosa and in overexpression and suppressed expression of PtoWOX11/12 a transgenic 84 K poplars was examined using quantitative real-time PCR（ q PCR）.【Result】 There were no significant differences between overexpression of PtoWOX11/12 a and control plants in leaf length,but the leaf width of overexpression of Pto WOX11/12 a was sharply larger than the control. The leaf length and width of suppression expression PtoWOX11/12 a transgenic poplars were less than control,and the leaf margin had serrate incision. The height and diameter of overexpression and suppression expression PtoWOX11/12 a transgenic poplars were significantly less than the control poplars. The anatomy analysis indicated that the xylem, the number of cambium layers of overexpression PtoWOX11/12 a transgenic poplars was thinner than the control and the xylem of the suppression expression of PtoWOX11/12 a was also thinner than the control,but it is wider than overexpression PtoWOX11/12 a transgenic poplars. q PCR analysis showed that Pto WOX11/12 a was strongly induced and expressed in the stem during the process of adventitious roots development. The expression levels of auxin synthesis genes,YUCCA1 and YUCCA8,were increased at the third day. Furthermore,their expression levels were increased in overexpressed while decreased in suppressed PtoWOX11/12 a transgenic poplars.【Conclusion】 Overexpression or suppression expression of PtoWOX11/12 a gene has effects on plant leaf shape, height and stem radial growth（ secondary xylem development）. Pto WOX11/12 a gene participates in adventitious roots formation and elongation involved in the expression of auxin synthesis genes YUCCA1 and YUCCA8. In the process of root formation,expression time interval was existed among PtoWOX11/12 a,YUCCA1 and YUCCA8 genes.Higher expression of PtoWOX11/12 a may inhibits the expression of auxin synthesis genes, thus overexpression PtoWOX11/12 a transgenic poplars are easier to form meristem,promote root primordium formation and produce large number of adventitious roots. During adventitious root growth,the increased expression level of YUCCA1 and YUCCA8 promote auxin synthesis and root meristem cell division. In stem the increased activities of cambium affected the xylem differentiation in overexpression PtoWOX11/12 a transgenic poplars,thus the xylem of transgenic poplars is thinner in size than the control.