miR156靶向SPL26负调控苹果矮化砧木不定根发生的机制解析

Leafy cutting is one of the important methods using in the propagation of apple dwarf self-rooted rootstocks. But the apple dwarf rootstocks gradually decrease the ability of adventitious root formation during the development and transition from juvenile to adult phase. Our previous studies showed that low expression of miR156 in adult cuttings could induce the high expression of SPL26 and furthermore inhibit adventitious roots’ formation. However, the direct downstream targets of SPL26 and the molecular mechanism of hard rooting are still unclear. Thus, here we would take M26 in which rMsSPL26 is overexpressed as material to identify the potential direct target genes which are regulated by MsSPL26 through ChIP-seq method and compare the data libraries with our previous RNA-seq data. In the meanwhile, ChIP-PCR and EMSA will be conducted to confirm the coupling effects among MsSPL26 and its targeted genes and then disclose the biological function of the targeted genes of MsSPL26 during adventitious roots’ formation by utilizating the virus silencing ACLSV vector. After all we try to illustrate the regulatory network of how MsSPL26 can supress the formation of adventitious roots and provide the theoretical basis for the establishment of fast vegetative propagation of apple dwarf rootstocks.

绿枝扦插是重要的自根砧苗木繁育方式，随着童期到成龄期的阶段转变，苹果矮化砧木丧失生根能力。项目组前期研究表明，miR156的低表达及其导致的SPL26高水平表达，是造成成龄期矮化砧木绿枝插穗难生根的重要原因，但SPL26的下游靶基因及其抑制不定根发生的调控机理仍不清楚。本项目首先通过酵母双杂交系统，明确MsSPL26的转录活性；然后，以过表达rMsSPL26-GFP的转基因植株M26为试材，进行ChIP-seq测序分析，进一步与项目组已有的童期和成龄期矮化砧木生根过程转录组数据库进行关联分析，筛选MsSPL26候选靶基因；再利用ChIP-PCR和EMSA技术进行验证，确定MsSPL26调控不定根发生的直接下游靶基因；最后，借助ACLSV病毒沉默载体，研究MsSPL26靶基因功能，旨在初步阐明MsSPL26抑制不定根发生的分子机制，为易生根矮化砧木资源创制及良种繁育提供理论依据。

绿枝扦插是无性系苹果砧木重要的繁殖方式，但砧木童性丧失导致扦插生根障碍限制了其在生产中的应用。项目组前期研究表明，miR156的低表达及其导致的SPL26高水平表达，是造成成龄期矮化砧木绿枝插穗难生根的重要原因，但SPL26的下游靶基因及其抑制不定根发生的调控机理仍不清楚。本项目首先通过酵母双杂交系统，明确SPL26的转录活性；然后，以过表达rSPL26的转基因苹果植株为试材，通过系列分子生物学手段，筛选并验证SPL26下游靶基因；然后通过转基因技术，研究SPL26下游靶基因功能，旨在初步阐明SPL26抑制不定根发生的分子机制，为易生根矮化砧木资源创制及良种繁育提供理论依据。取得如下结果：.（1）SPL26显著抑制苹果不定根发生。35S:3xFlag-rSPL26转基因‘GL-3’试管苗生根率17.6%，每茎段生根0.4条；未转基因‘GL-3’植株生根率75.0%，每茎段生根3.8条。.（2）酵母单杂交、电泳迁移率试验和染色质免疫共沉淀结果证实SPL26蛋白直接与HB13启动子结合，SPL26和外源IBA共同负调控HB13基因转录。.（3）35S:HB13转基因烟草生根率、生根数量和根长度均显著高于野生型（WT）。外源20 μM 的1-N-萘基邻氨甲酰苯甲酸完全抑制WT烟草生根，但35S:HB13转基因烟草仍然能够生根，说明HB13可能参与调控极性生长素运输而影响不定根发生。.综上所述，Mx-A插穗中miR156表达水平低，致使SPL26转录丰度高，SPL26蛋白直接结合于HB13启动子，使HB13表达下调，影响PAT抑制不定根发生。