插穗倒插对滇杨扦插苗生长影响的机制研究

Organ polarity plays an irreplaceable role in plant growth and development. In our previous research, it was found that the inverted cuttings of Populus yunnanensis could root at the original morphological top position and could sprout at the original morphological bottom position, and then developed into complete plant. However, the cuttings were collected from rootsuckers of same tree, which had same genetic background, and eliminated the effects of age and position, but both the height and diameter growth of shootings and roots showed significant differences between inverted cutting seedlings and upright cutting seedlings. So, when the cuttings were inverted cut, what changes have taken place in growth rhythm, physiological and biochemical characteristics, transcriptional regulatory network and DNA methylation modification of cutting seedlings, which lead to the growth differences between upright and inverted cutting seedlings? In this connection, one-year-old shooting stems of rootsuckers from plus tree of P. yunnanensis will be used as cuttings to propagate inverted and upright seedlings, and the growth traits, photosynthetic and fluorescence properties will be observed. Endogenous hormone content and oxidase activity in stem tips, stem basic bark, root tips and basic bark of grade 1 root system will be measured, as well as transcriptome of the four organs and whole genome DNA methylation will be sequenced and analyzed. Through the comprehensive analysis of the above research results, the influence mechanism of inverted cuttings on the growth of inverted cuttings seedlings can be clarified systematically and deeply. Our research results could enrich the theoretical understanding about the polarity of plant organs, and will provide a scientific basis for further elucidation of the regulation mechanism of organ polarity on plant growth and development.

器官极性对植物生长发育具有不可替代的作用。前期研究发现，滇杨枝条插穗倒插后能够在原形态学上端生根，原形态学下端萌芽抽枝，进而发育为完整植株。但来自于同一棵母树的根萌苗插穗，遗传基础完全相同，且消除了年龄效应和位置效应的影响，而正、倒扦插苗的生长存在明显差异。那么，倒插插穗发育而成的苗木在生长节律、生理性状、转录调控网络及DNA甲基化修饰等水平发生了什么样的变化，从而导致正、倒插苗之间生长的差异？为此，本项目以滇杨优树根萌苗1年生主干为插穗繁育正、倒扦插苗，通过对扦插苗的生长规律观测、光合荧光特性分析，以及速生期主枝茎尖、基部皮层和根尖、根基部皮层的内源激素含量和氧化酶活性测定、RNA-seq分析及全基因组DNA甲基化BSP-seq分析，深入地揭示滇杨插穗倒插对扦插苗生长影响的机制，从而丰富对植物器官极性的理论认识，进而为深入阐明器官极性对植物生长发育的调控机理提供科学理论依据。

以滇杨当年生和1年生正、倒扦插苗为对象，对苗木生长量、4种酶活性、内源激素含量及光合和荧光特性观测分析，以及当年生正、倒扦插苗生长初期和快速生长期茎尖组织的RNA-seq和甲基化差异进行了比较分析，研究结果如下：.1. 当年生和1年生滇杨正插苗主枝长度、主枝粗度、叶生长量及其干/鲜重、根系生长指标均表现为正插苗高于倒插苗，正插苗表现出了较突出的生长优势；.2. 在7月-10月共计4个月的生长期内，当年生扦插苗的嫩叶和成熟叶，正插苗叶的POD、XET及PME活性均高于倒插苗，而IAAO活性表现为倒插苗高于正插苗，1年生滇杨倒插苗的嫩叶IAAO活性在7-8月高于正插苗，成熟叶IAAO酶活性在8-9月高于正插苗，POD和PME活性均表现为正插苗高于倒插苗，XET活性表现为倒插苗嫩叶高于正插苗嫩叶，而正插苗成熟叶中的活性高于倒插苗成熟叶；.3. 在7-9月生长期内，正插苗的IAA、ABA及JA-IlE含量高于倒插苗，而倒插苗7-9月嫩叶的SA含量及9月嫩叶的JA含量均高于正插苗；.4. 当年生滇杨正插苗净光合速率、气孔导度及胞间二氧化碳浓度日变化均值高于倒插苗，但7月和8月倒插苗蒸腾速率日变化均值较高，而到了9月和10月正插苗显著高于倒插苗。1年生扦插苗的光合特性表现为正插苗净光合速率、胞间二氧化碳浓度、蒸腾速率及气孔导度日变化均值均高于倒插苗。.5. 叶绿素荧光参数中，于O相，当年生和1年生滇杨倒插苗荧光产值均高于正插苗，而J、I及P相，正插苗荧光产值高于倒插苗，7月和8月，正插苗φPo、φEo、Ψo高于倒插苗，而倒插苗Mo高于正插苗。.6. 以滇杨正、倒插苗生长初期与快速生长期茎尖组织的RNA-seq分析表明，经KEGG通路富集数据库对比分析显示，在JZT_vs_JDT比较组中DEGs主要富集于植物激素信号转导通路，DEGs在该通路主要集中于IAA、CTK、GA、ABA、BR和JA等合成通路中，且通路中多数基因均表现为上调表达。.7. 将ONT甲基化数据与转录组数据进行联合分析，筛选到PyMORG1、PySOD1、PyUNC、PyDET2、PyMYBP和PyfrmA共8个候选目标基因。.8. 以滇杨愈伤组织为受体材料，利用农杆菌介导法建立了滇杨遗传转化体系，将PyDET2基因在滇杨中过表达，由GUS染色分析表明，PyDET2基因在滇杨转基因植株的所有组织均有表达。