miR1507调节大豆耐缺磷胁迫机理的研究

Phosphorus (P) is an essential macronutrient for plant growth and development. Despite the abundance of P in the soil, P available for plants is usually present at a low level due to its precipitation with cations and conversion to organic matter. Soybean is one of the most important economic crops. However, P deficiency is becoming one of the major limiting factor for soybean production. Although the roles of miRNAs in P deficiency signaling and regulation of phosphate homeostasis have been revealed, miRNAs of which the related functions have been characterized are also limited, especially in legume crops. miR1507 is one of the legume-specific miRNA and its expression is induced by P deficiency. In this study, we will study the roles of miR1507 and its target genes in the regulatory mechanism of tolerance response to P deficiency in soybean. Promoters of miR1507a/b/c are isolated and fused to GUS, to generate miR1507a/b/c promoter-GUS transgenic plants for the analysis of expression pattern; and the P responsive elements in the promoters are deleted for the analysis of the regulatory roles of P responsive elements in the promoters of miR1507. The target genes of miR1507 are predicted by computational analysis and validated by 5′-RACE analysis. To elucidate the potential function of miR1507 in the tolerance response to P deficiency, constructs of miR1507 overexpression, target-gene overexpression and miR1507-resistant target-gene overexpression are used to transform soybean plants. Transgenic lines with different constructs are then used to evaluate the physiological and biochemistry responses to P deficiency. The possible genes downstream miR1507 which are responsible for P deficiency response are to be identified further. This study may provide significant evidence for the elucidation of regulatory mechanism of P deficiency tolerance of legume plants.

磷是植物生长必需的营养元素，但土壤中磷的有效性差是当今农业生产中的一个主要问题。大豆是我国主要经济作物，缺磷是限制大豆产量提高和品质改良的一个主要因素。miRNA在植物磷素代谢与平衡的调节上具有重要作用，但目前清楚的具有相关功能的miRNA还不多，尤其在豆科作物中的研究更是非常有限。本项目拟研究一个受缺磷诱导的豆科特异的miRNA，miR1507，在调节大豆耐缺磷胁迫上的作用机理。通过启动子融合GUS、启动子磷响应元件缺失等方法分析miR1507对缺磷响应的调控机制。为探明miR1507及其靶基因在调控缺磷胁迫耐性反应中的具体功能，通过RACE方法鉴定miR1507所调控的靶基因，再创建大豆miR1507及其靶基因过量表达株系，然后分析不同株系对缺磷胁迫的生理生化反应，并鉴定下游可能受调控的基因。本项目将对阐明豆科特异miRNA调节豆科植物缺磷胁迫耐性反应的机制具有重要的理论意义。

大豆是一种重要的经济作物也是一种潜在的能源植物，缺磷是影响大豆产量和品质的一个主要因素。miRNA在植物磷素代谢与平衡的调节上具有重要作用，但目前大豆中调节磷素代谢与平衡的miRNA的功能的研究还很少。在本项目中，我们通过数字表达谱测序在全基因组范围内挖掘了大豆根系中对缺磷响应的基因，包括了某些缺磷响应miRNA的靶基因，然后对缺磷响应基因的可能的生物学功能进行分类，并通过qRT-PCR验证了它们对缺磷的响应。我们然后在拟南芥中挑选了两个缺磷响应的miRNA，miR408和miR447，进行功能研究。研究结果表明大豆根系中总共有1612个基因对缺磷响应，其中727个基因上调，885个基因下调。这些缺磷响应基因包含了一些可能参与磷酸盐平衡、养分吸收和运输、活性氧平衡、钙信号、激素信号和基因转录的基因，其中6个基因为miRNA的靶基因，比如miR399、miR1510和miR3508。基因本体富集分析发现，总共有17个生物学过程的条例显著富集，包含光合作用、铁离子运输、dUPT代谢、细胞壁组成、脂肪酸代谢和胁迫反应。我们从这些缺磷响应基因中随机挑选了30个进行qRT-PCR验证，发现绝大部分基因（29个）的表达模式与数字表达谱的结果类似。因为考虑到大豆遗传转化体系还不够成熟，而拟研究的miR1507又为豆科植物特有的miRNA，在拟南芥等植物中还没有。因而miR1507与缺磷胁迫反应的功能不便于在大豆中研究，也不能转移到拟南芥中进行研究。为此，我们在拟南芥中选择了两个缺磷响应，但具体功能仍不明确的miRNA，miR408和miR447，进行功能上的研究。我们通过构建miR447和miR408过表达转基因植物并分析它们对缺磷胁迫的反应，研究结果表明所构建的过表达植株中miRNA的表达量升高，而其靶基因的表达量下降，并且在缺磷条件下miR447和miR408过表达植株的根长和生物量相较于野生型明显增加，说明miR477和miR408通过调控靶基因参与了植物对缺磷胁迫的抗性。以上实验结果可为大豆缺磷反应调控网络的鉴定提供信息，也为大豆缺磷胁迫的分子适应机理提供依据。