生长素受体调控大豆根瘤发生机制研究

Root nodule is a specified lateral root organ of legume plants, which is formed through symbiosis between legume plant and rhizobia. The nodule could convert the atmospheric N2 into NH3 that can be utilized by legume plants under low nitrate conditions, which is benefit for the growth and production of plant. Auxin plays central roles in nodulation of soybean, and altered auxin accumulation and cellular response that are induced by nitrate status determine the nodule formation. TIR1/AFB2s are the main auxin receptors, which are critical for anxin signal response and transduction. However, it is still unknown about the role of TIR1/AFB2s in the auxin signaling pathway during soybean nodulation. In this project, six TIR1/AFB2 auxin receptors were cloned and named GmTIR1 and GmAFB2, respectively. To confirm the function of GmTIR1/AFB2 genes in soybean nodulation, the temporal and spatial expression patterns of those genes will be checked during nodule development. The nodule number phenotype will also be analyzed after over-expressing or knocking-down the GmTIR1/AFB2 genes in soybean. And the expression levels of auxin responsive genes downstream the auxin signaling pathway as well as several nodulation regulation marker genes will be checked in the transgenic soybean roots. This project will provide new evidence for the molecular mechanism in the auxin response regulating soybean nodulation.

根瘤是豆科植物与根瘤菌通过共生作用形成的特有组织，可通过共生固氮作用为植物提供氮源，有助于植物生长发育和产量提高。生长素在大豆根瘤发生发育过程中具有重要作用，TIR1/AFB2是主要的生长素受体，但它们如何参与生长素信号通路调控大豆根瘤的发生发育目前仍不清楚。本研究克隆了大豆TIR1/AFB2生长素受体家族基因，并分别命名为GmTIR1和GmAFB2。研究将采用基因转录水平检测及启动子-GUS组织化学染色的方法对这两个基因家族成员在大豆根瘤发生发育过程中的组织表达模式进行分析，利用基因过表达和基因沉默的转基因技术对它们在调控大豆根瘤数目方面的生物学功能进行验证。同时，在生长素受体基因异位表达的大豆根系中，检测生长素信号通路下游基因及调控结瘤过程关键基因的表达水平，探讨生长素信号通路调控大豆根瘤发生数目的内在分子机理，为大豆分子育种及提高大豆产量提供理论依据。

生长素在豆科植物根瘤形成过程中发挥重要功能，但其具体作用机制仍有待研究。生长素受体是生长素信号通路的重要组分之一，对生长素受体GmTIR1/AFB3家族在大豆结瘤过程中的功能进行研究具有重要意义。本项目克隆了6个GmTIR1/AFB3家族基因，荧光定量PCR结果显示这些基因受生长素诱导表达。启动子-GUS组织化学染色结果表明，这些GmTIR1/AFB3基因在大豆结瘤过程中呈现不同的组织表达模式。其中，GmTIR1A和GmAFB3A在根和根瘤中的表达水平较高，GmTIR1C在发育的根瘤中特异表达。此外，荧光定量PCR结果表明，GmTIR1C在根瘤菌侵染10天和28天后的大豆根中的表达水平远远高于侵染初期。利用RNAi基因沉默的方法下调表达全部GmTIR1/AFB3基因后，大豆根瘤数目显著降低；过表达GmTIR1A和GmTIR1C后结瘤数目显著增加，而过表达GmAFB3后根瘤数目则无明显变化。烟草共转化实验结果表明，miR393可负调控GmTIR1/AFB3基因的表达，在大豆中过表达不能被miR393沉默的突变型mGmTIR1C后，相比于过表达GmTIR1C时，大豆结瘤数目进一步增加。通过基因表达检测发现，抑制GmTIR1/AFB3基因表达后，GmIAA8、GmIAA9、GmIAA14显著下调表达，GmARF8A和GmARF8B显著上调表达。综上所述，GmTIR1A和GmTIR1C在生长素调控大豆结瘤过程中发挥重要功能，它们可能与GmIAA和GmARF8共同组成调控网络发挥功能。同时，GmTIR1C控制大豆结瘤的过程受到miR393的负调控作用。这些结果为深入解析生长素介导的大豆根瘤发生发育调控的分子机理奠定了基础。