bHLHx及其调控因子参与磷信号途径的分子机制研究

Cultivars with improved Pi uptake efficiency are required for future sustainability of agriculture. To develop such cultivars, we need to understand the mechanism of phosphate (Pi) signaling. PHR2-mediated transcriptional control plays a key role in Pi-starvation signaling in rice. Recently our lab reported that inhibition of PHR2 activity by SPX proteins is Pi dependent. The strength of interaction between SPX1 and PHR2 is directly influenced by Pi, providing a link between Pi perception and signaling. Whether SPX interacts with other factors involved in the Pi-signaling pathway is intriguing. By screening a yeast two-hybrid cDNA library with SPX1 bait, we identified a bHLH transcription factor (we named it as bHLHx here). We further confirmed the interaction between this protein and SPX1 by co-immunoprecipitation and BiFC assays. Transgenic rice overexpressing bHLHx displayed increased transcription of genes for certain phosphate transporters (PTs), and concomitant Pi accumulation. The bHLHx transcription factor has not previously been reported to be involved in the Pi-signaling pathway, and it likely represents a novel factor in Pi signaling. Moreover, we found the closest homologous of bHLHx in rice genome is a HLH protein lacking of basic region (we named it as HLHy here). Similar with bHLHx, HLHy also can interact with SPXs proteins but other tested bHLH protein can not. It has been reported that HLH proteins can function as negative regulators of bHLH proteins by forming non-DNA binding heterodimers with otherwise DNA binding bHLH proteins. We have already confirmed that bHLHx and HLHy interacted each other. In this project, we aim to reveal the mechanism of the SPX1/bHLHx and bHLHx/HLHy modules in Pi signaling.

对磷信号途径机制的了解是遗传改良作物磷吸收效率的基础。最近我们报道了SPX1/PHR2作用模块可将磷的感应与下游磷信号传导相联系。为探索SPXs是否还与其它因子发生互作并传递磷的感应信号，我们用酵母双杂筛选cDNA文库获得了一个与SPX1互作的bHLHx转录因子。bHLHx超表达转基因植株体内，一些磷运输体的基因表达升高了，同时体内磷含量超积累。由于尚未有bHLHx参与磷信号途径的报道，因此该基因很可能是磷信号途径的一个新成员。此外，我们发现水稻基因组中与bHLHx同源性最高的一个缺失basic区域的蛋白HLHy与 bHLHx之间可以形成二聚体。通常HLH蛋白与bHLH结合后形成的异源二聚体会使bHLH丧失与原靶基因结合，从而对bHLH转录因子起负调控作用。本课题拟揭示bHLHx在磷信号途径中的功能,并在此基础上进一步探索SPX1/bHLHx，bHLHx/HLHy这两个作用模块的分子机制。

本课题的初始目标为：对“bHLHx及其调控因子参与磷信号途径的分子机制研究”，这源于前期我们在对磷信号途径成员SPX基因的互作筛选中发现bHLH98与SPX存在互作,而水稻bHLH98的基因功能尚未有过报道。在本项目中我们构建了bHLH98超表达转基因水稻，还通过Crispr Cas9 构建了bhlh98突变体。利用一系列分子生物学、细胞学、遗传学等研究手段，我们对这个转录因子的功能进行了解析。由于bHLH98相关突变体与超表达材料并未表现出磷含量改变的表型，我们最终揭示的是bHLH98为一个调控水稻叶夹角的转录因子。水稻叶夹角是一个重要的农艺性状，水稻叶片的倾斜角度过大，会引起植株间相互遮蔽光线，影响单位面积的种植密度，从而导致单位面积光照养分利用效率变低。我们的结果表明bHLH98超表达转基因植株具有更直立的叶夹角表型，同时不影响分蘖数、籽粒大小、结实率等农艺性状。这表明，通过增强OsbHLH98的表达水平，可以调控叶夹角使得水稻适应高密度栽培，因此，OsbHLH98可以作为育种上的选择目标，助力水稻密植高产。该基因在分子育种中具有潜在的农业利用价值，本成果已经获得发明专利授权（专利号：ZL201911177057.8）。我们进一步揭示了bHLH98通过直接抑制下游靶基因BUL1的转录从而调控水稻叶夹角，相关文章即将发表于New Phytologist （Guo et al., 2021），本国家基金为第一标注，该文章已经于2021年2月22日被接收（DOI: 10.1111/nph.17303）。