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林业科学 ›› 2018, Vol. 54 ›› Issue (2): 52-59.doi: 10.11707/j.1001-7488.20180206

• 论文与研究报告 • 上一篇    下一篇

毛白杨PtoWOX4a基因过表达对次生生长的影响

李建波1,2, 贾会霞1, 张进1, 刘伯斌1, 胡建军1, 王丽娟1, 卢孟柱1   

  1. 1. 林木遗传育种国家重点实验室 中国林业科学研究院林业研究所 北京 100091;
    2. 中国林业科学研究院华北林业实验中心 北京 102300
  • 收稿日期:2017-05-03 修回日期:2017-08-08 出版日期:2018-02-25 发布日期:2018-03-30
  • 基金资助:
    中央级公益性科研院所基本科研业务费专项资金"WOX4WUSa基因在杨树形成层分化调控中的互作机制"(CAFYBB2014ZX001-1)。

Effect of Overexpression of Populus tomentosa WUSCHEL-related homeobox 4 (PtoWOX4a) on the Secondary Growth of Poplar

Li Jianbo1,2, Jia Huixia1, Zhang Jin1, Liu Bobin1, Hu Jianjun1, Wang Lijuan1, Lu Mengzhu1   

  1. 1. State Key Laboratory of Tree Genetics and Breeding Research Institute of Forestry, Chinese Academy of Forestry Beijing 100091;
    2. Experimental Center of Forestry in North China, Chinese Academy of Forestry Beijing 102300
  • Received:2017-05-03 Revised:2017-08-08 Online:2018-02-25 Published:2018-03-30

摘要: [目的]WUSCHEL-related homeobox(WOX)转录因子是植物特有的一类转录因子,参与干细胞维持、侧生器官发育和不定根再生等过程,对植物生长发育有至关重要的作用。本文通过对毛白杨PtoWOX4a基因表达模式的分析和过表达植株表型的观察,研究PtoWOX4a在杨树生长发育中的作用,为全面了解不同物种中WOX基因的作用机制奠定基础。[方法]利用qRT-PCR技术分析PtoWOX4a基因在茎顶端分生组织、幼叶、成熟叶、嫩茎、老茎、木质部、韧皮部和根中的相对表达量;利用GUS染色技术对pPtoWOX4aGUS转基因植株进行染色,通过GUS信号观察,进一步分析PtoWOX4a在不同组织中的表达模式。对过表达PtoWOX4a植株和对照植株的不定根、茎和叶片进行形态观察和生长指标测定,并通过组织切片对其解剖结构进行观察比较,分析过表达PtoWOX4a对杨树不定根、茎和叶片维管发育的影响。[结果]qRT-PCR结果显示,PtoWOX4a在茎中表达量最高,且主要集中在木质部和韧皮部,在根中表达水平次之,而在叶片等其他组织中的表达量较低;pPtoWOX4aGUS转基因植株的GUS染色结果与qRT-PCR结果一致,并发现PtoWOX4a主要在根、叶及茎的维管组织中表达。对过表达PtoWOX4a转基因植株表型观察发现,过表达PtoWOX4a抑制了不定根的伸长,3周苗龄的转基因植株不定根长度约为3~4 cm,仅为对照植株的1/3~1/2,不定根直径变粗,根部木质部区域增加。3月苗龄的过表达PtoWOX4a植株变矮,与对照植株相比,株高减少了14%~20%,节间数减少了16%~22%;对第10节间茎段的解剖结构分析发现,过表达PtoWOX4a植株茎部髓心部分变窄,比对照植株减少了10%~20%,木质部区域变宽,比对照植株增加了24%~35%,而形成层区域宽度无明显变化。此外,过表达PtoWOX4a导致植株叶片生长异常,叶片两侧边缘向上卷曲。[结论]毛白杨PtoWOX4a基因主要在杨树的维管组织中表达,过表达PtoWOX4a可影响杨树不定根、茎和叶片维管的发育。

关键词: 毛白杨, PtoWOX4a, 基因表达, 次生生长, 解剖结构

Abstract: [Objective] WUSCHEL-related homeobox (WOX) is a class of plant specific transcription factors. They play important roles in plant growth and development including stem cells maintaining, lateral organs development, de novo root regeneration of the explant, and other development processes. In this study, the expression patterns and phenotypic traits were analyzed to explore the gene function of PtoWOX4a and also to provide a basis for comprehensive research on the mechanism of WOX genes in different species.[Method] The relative expression of PtoWOX4a in shoot apical meristem, young leaf, mature leaf, tender stem, old stem, phloem, xylem and root in Populus tomentosa were analyzed by qRT-PCR. GUS staining was further analyzed the expression patterns of PtoWOX4a in different tissues. The genetic effects of PtoWOX4a on plant development were explored by studying adventitious root (AR), stem and leaf morphology and histological anatomy structure in the PtoWOX4a overexpression poplar.[Result] qRT-PCR result showed that the highest accumulation of PtoWOX4a transcripts was observed in stem, followed by root and leaf. The result of GUS staining was consistent with the qRT-PCR result, revealing that PtoWOX4a was mainly expressed in the vascular tissue of AR, leaf and stem. In 3-week-old seedlings, overexpression of PtoWOX4a shortened the AR length to 3-4 cm, which was only 1/3-1/2 compared with the control; overexpression of PtoWOX4a increased AR diameter and promoted xylem cell differentiation in AR. In 3-month-old plants, the overexpression of PtoWOX4a transgenic plants showed dwarf phenotype. Compared with the control, they exhibited 14%-20% decrease in stem height, 16%-22% decrease in internode number. In the tenth internode, the transgenic plants showed 10%-20% decrease in the width of pith parenchyma cells, 24%-35% increase in the width of xylem, but the width of cambium did not change significantly. Furthermore, overexpression of PtoWOX4a caused the upward leaf curling.[Conclusion] PtoWOX4a mainly expresses in the vascular tissue of poplar. Overexpression of PtoWOX4a affects the development of vascular tissues of poplar root, leaf and stem.

Key words: Populus tomentosa, PtoWOX4a, gene expression, secondary growth, anatomical structure

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