【作者】 韩华；

【导师】 张守攻；

【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】 扦插生根率低是制约利用扦插进行无性系林业发展的瓶颈,因此,提高难生根植物插穗生根率和生根质量成为研究的热点。落叶松是我国重要速生用材和生态造林树种,属难生根植物。目前,对落叶松扦插生根的研究虽然已在生理生化水平上取得一定进展,但其生根机理仍不清楚。开展本项研究,为探索落叶松插穗不定根发育的分子机制奠定理论基础。本研究以杂种落叶松全同胞无性系为实验材料,从对插穗不定根发育的解剖学观察及内源激素分析入手,利用蛋白质组学技术研究不定根发育过程中蛋白差异表达及其功能,采用基因克隆技术获得部分蛋白全长基因并进行转录水平上的表达分析,探讨落叶松插穗不定根发育的分子机制。主要结果如下:1插穗不定根形成方式及发育关键时期通过对落叶松插穗不定根发育过程中外观形态和解剖观察发现,不定根原基发生于髓射线正对的形成层部分,属于诱导生根型。不定根发育的完整周期大约55-60天,其中扦插第14-18天、25-30天、35-40天是不定根发育的三个关键时期,分别是不定根诱导期、不定根原始细胞形成期、不定根原基形成伸长期。2内源激素与插穗生根性状的关系及复合激素的互作效应通过对不同无性系及IBA处理下同一无性系插穗不定根发育过程中内源激素含量的测定,认为采穗母株的激素水平,尤其是IAA和ABA,影响不同无性系插穗的生根性状,IAA越高、ABA越低,有利于生根;外源IBA对插穗不定根发育的促进作用主要是显著提高不定根诱导之前IAA含量、抑制ABA含量。不同无性系和IBA处理下同一无性系插穗不定根发育过程中IAA+GA+ZR /ABA的变化与最终生根性状一致,可以作为衡量插穗生根好坏的指标。通过测定复合激素处理下四类激素含量的变化,认为激素之间存在互作效应。与单独使用IBA相比,IBA+6BA和IBA+GA表现出增效作用,IBA+ABA表现出拮抗作用。因此,在生产中可以加入适当浓度的细胞分裂素或赤霉素配合生长素使用,可以更好地发挥促进生根的作用。3 LKCcO6b和LKMDH在插穗不定根发育过程中发挥调控作用通过对不定根发育过程中参与三羧酸循环和线粒体电子传递的两个关键蛋白LKMDH和LKCcO6b在蛋白及转录水平的分析,认为他们主要在不定根诱导期发挥调控作用,IBA诱导下蛋白和mRNA两个水平均上调表达,为此期髓射线和正对髓射线形成层的细胞分裂和新细胞的合成提供碳骨架和能量来源,促进不定根原始细胞的形成。通过对复合激素处理下LKCcO6b和LKMDH表达水平分析,认为适当浓度的细胞分裂素或赤霉素能增效生长素对LKCcO6b和LKMDH的调控作用,而脱落酸拮抗该作用。这与复合激素对内源激素水平的影响效果一致,表明LKCcO6b和LKMDH在不定根的形成发育过程中起到调控作用。4插穗不定根发育的分子机制通过对不同无性系(不同基因型)和IBA诱导下同一无性系(相同基因型)插穗不定根发育关键期差异表达蛋白功能分析,认为插穗不定根发育的分子机制表现在以下两个方面:1)碳和能量代谢调控不定根形成发育。无论是不同无性系还是IBA诱导下同一无性系插穗不定根发育过程中都能鉴定到大量碳和能量代谢相关蛋白,这些蛋白在生根较好的无性系或IBA处理下显著上调表达,尤其是在不定根诱导期,为形成层细胞分裂和新细胞合成提供稳定的碳水化合物和能量来源,促进不定根原始细胞的形成。2)信号转导调控不定根的形成发育。从不同无性系和IBA诱导下同一无性系插穗不定根发育过程中鉴定到生长素信号转导相关蛋白,这些蛋白在生根较好的无性系或IBA处理下显著上调表达。不定根诱导前期,内源IAA出现瞬时高峰,高于生长素信号转导中IAA的阈值;同时泛素降解相关蛋白上调表达,降解生长素信号转导途径中的抑制因子,共同促进IAA信号转导通路中下游基因的表达,调控不定根的形态建成。综上所述,落叶松插穗不定根形成和发育是一个非常复杂的多基因调控过程,上述研究结果为探索落叶松插穗不定根发育机制奠定理论基础,同时为其他针叶树不定根发育研究提供参考。更多还原

【Abstract】 Larix is an important fast-growing and ecological forestation species, but is difficult to root, which restricts the development of clonal forestry using cuttings, so how to improve the rooting rate and rooting quality become a research hotspot. Currently, the research on physiological and biochemical level of Larix rooting make some progress, but the molecular mechanism of cuttings rooting remains unclear.To make clear the molecular mechanism of cuttings rooting of hybrid Larix, anatomic development of adventitious roots was investigated, proteins expressed during adventitious roots development were extracted and identified using proteomics technique, and the expressions of some important proteins on transcription level were analyzed by gene cloning technology and qRT-PCR. The main results were as follows:1 The formation and the key development period of adventitious rootThe changes of morphology and anatomy were observed during adventitious root development, root primordia formed from cambium which opposite to pith rays, the formation of adventitious root was induce-to-root type. The period of adventitious roots development was about 55-60 days, the 14-18、25-30、35-40 days after cuttage were three key time-point for adventitious root induction, adventitious root primordia initiative cell formation and adventitious root primordia formation respectively.2 The relation between endogenous hormones and rooting traits, and the interaction between hormonesThe hormones content during adventitious roots development between different clones and treatments were determined. The hormones level of different donors, especially IAA and ABA, were important for cutting traits, the higher the IAA, the lower the ABA, the better the rooting traits. The effects of IBA on cuttings rooting were increasing IAA and decreasing ABA at the early stage of adventitious root development. The trend of (IAA+GA+ZR)/ABA between different clones or treatments were similar to rooting traits, so (IAA+GA+ZR)/ABA could be used as an index of rooting ability.The interaction between hormones was confirmed by measuring hormones content under different treatments. Compared with IBA using alone, IBA +6 BA and IBA + GA showed synergism, while the IBA + ABA showed antagonism. Therefore, the appropriate concentration of cytokinin or gibberellin could be used with auxin for promoting cuttings rooting.3 The regulation of LKCcO6b and LKMDH on adventitious root development The analysis on protein and transcription level of LKCcO6b and LKMDH which participated in the citric acid cycle and mitochondrial electron transport during adventitious root development showed these two proteins mainly played a regulation during adventitious roots induction, the protein and mRNA levels were up-regulated treated with IBA, which provided carbon skeletons and energy for cambium cells opposite to pith rays division and synthesis, promoted the formation of adventitious root initial cells.The expression analysis of LKCcO6b and LKMDH treated with combined hormones indicated that the appropriate concentration of cytokinin or gibberellic acid could increase the efficiency of auxin, while the abscisic acid could decrease the efficiency. These results were consistent with endogenous hormones changes treated with compound hormones. Therefore, LKCcO6b and LKMDH regulated adventitious roots formation and development.4 The molecular mechanism of adventitious root developmentThe function of differentially expressed proteins during key time-point of adventitious roots development between different clones (different genotype) or treatments (same genotype) were analyzed, the two-part research suggested the molecular mechanism of adventitious root development.1) The carbon and energy metabolism regulated adventitious root formation and development. Large amounts of carbon and energy metabolism related proteins were identified during adventitious root development of different clones or treatments, these proteins were significantly up-regulated in better-root clones or IBA treatments, especially at the adventitious root induction stage, which provided a stable source of carbohydrates and energy for cambium cells division and synthesis, promoted the formation of adventitious root initial cells.2) Signal transduction regulated adventitious root formation and development. Auxin signal transduction related proteins were identified during adventitious root development of different clones or treatments, these proteins were significantly up-regulated in better-root clones or IBA treatments. At the early stage of adventitious root induction, endogenous IAA appeared instantaneous peak higher than the threshold of IAA auxin signal transduction, while ubiquitin-related protein up-regulated for degradation the inhibitor of auxin signal transduction pathway, these changes jointly promoted the expression of genes down-stream of IAA signal transduction, regulated adventitious root morphogenesis.In summary, the formation and development of adventitious roots of Larix stem cuttings is a very complex and multi-gene regulation process, these results was a basis for study the mechanism of adventitious root development, while provided a reference for the other conifers.更多还原