玉米ZmLBD1响应缺磷胁迫和根系发育的机理研究

Maize is a multi-purpose crop which is utilized as food, feed and bio-energy, and contributes significantly to economies. Phosphorus deficiency is one of the major constraints limiting maize yield and quality. LBD transcription factors play important roles in regulating plant growth and development and stress response. However, distinct molecular mechanism of most of these LBD members remain unclear. Findings from our previous studies showed that the expression patterns of ZmLBD1 were significantly different between phosphorus deficiency tolerant and sensitive maize inbred lines. Expression of ZmLBD1 was significantly induced by phosphorus stress in roots of phosphorus tolerant inbred lines. Functional mutant complementarity experiments showed that ZmLBD1 could complement lateral root formation and growth in Arabidopsis. In this study, we intend to further validate the biological function of ZmLBD1 and its phenotypic differences between overexpression and CRISPR/Cas9 in response to phosphorus deficiency stress. In order to dissect the genetic basis of ZmLBD1 in response to phosphorus deficiency and the molecular mechanism of promoting root growth and development, the main approaches include gene clone, association and expression analysis, subcellular localization, Y1H and Y2H library screening, double luciferase detection and Co-IP. The results could enhance clarity on the functional roles of ZmLBD1. In conclusion, our research can enrich people's understanding of plant root morphogenesis, and offer theoretical scope to better understand the molecular mechanism of plant response to phosphorus stress, as well as, identifying and utilizing phosphorus-tolerance germplasm in maize.

玉米是集粮、经、饲及生物能源为一体的多元作物，磷的营养缺乏是限制其产量和品质的主要逆境之一。LBD转录因子对植物生长发育和逆境响应具有重要作用，但其调控机制尚未知晓。在前期研究中，我们发现ZmLBD1在玉米磷敏感自交系中的表达模式存在显著差异，且在耐低磷自交系根系中受缺磷胁迫显著诱导表达。在拟南芥中的功能互补实验发现，ZmLBD1能互补拟南芥突变体侧根的发生和生长。为了阐明ZmLBD1响应缺磷胁迫的遗传基础和促进根系生长发育的分子机制，本研究拟通过超表达和CRISPR/Cas9的方法进一步明确ZmLBD1的生物学功能。同时结合候选基因的关联分析、表达分析、亚细胞定位、酵母单/双杂交文库筛选、双荧光素酶检测和免疫共沉淀等技术方法解析其响应缺磷胁迫的遗传基础和促进根系生长发育的分子机制。这些研究能够丰富人们对植物根系形态建成的认识，也对玉米磷高效基因和种质的挖掘具有实践价值。

根系构型重建是植物适应逆境胁迫的重要调控机制，LBD转录因子是调控植物根系发育和逆境响应的重要成员，而ZmLBD1在玉米中对低磷胁迫的响应及其调控机制未见报道。本研究利用301份玉米自交系组成的自然群体，关联分析发现ZmLBD1与玉米苗期低磷胁迫的根系相关性状的显著关联，表明该基因可能具有响应低磷胁迫并调控根系发育的生物学功能。基于XP-CLR的选择扫描和Tajima’s D的中性检验分析，发现ZmLBD1受强烈的纯化选择，暗示该基因生物学功能高度保守，尚未受到人工驯化选择压力。表达分析发现ZmLBD1定位于细胞核和细胞质，且在耐低磷玉米自交系178根系中被低磷胁迫显著诱导表达，免疫组化（IHC）检测发现ZmLBD1在低磷胁迫后的侧根原基显著富集。过表达ZmLBD1能部分恢复拟南芥双突变体lbd16 lbd18的无侧根表型。正常和缺磷条件下，ZmLBD1过表达拟南芥均显著促进根系的生长发育，增强拟南芥对低磷胁迫的耐受性。利用CRISPR/Cas9技术获得多个玉米敲除突变体zmlbd1，与野生型相比，zmlbd1在根系形态上均表现出不同程度的生长发育缺陷，表明ZmLBD1在响应低磷胁迫的同时具有正向调节植物根系发育的功能。利用Y2H、Split-luc和Co-IP等技术筛选并验证互作蛋白ZmEXB7，过量表达发现能部分恢复三重突变体lbd16 lbd18 lbd29的侧根发育表型，表明ZmLBD1与ZmEXB7互作协同促进侧根发育。进一步利用Y1H技术并结合MESA和双荧光报告系统检测，筛选到ZmLBD1的上游调控因子ZmERF2直接结合GC box促进ZmLBD1的转录。本研究结果表明ZmLBD1是一个被低磷磷胁迫诱导表达，促进植物侧根发生的关键基因。

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