miR159e-3p调节大豆应答低磷胁迫的分子和生理机制研究

Mi159s play important roles in flower development, and the responses to salt stress and ABA. But the roles of miR159s in response to phosphate (Pi) deficiency and underlying mechanisms remain elusive. We previously found that soybean miR159e-3p, one member of miR159 family, was repressed in roots under low Pi conditions, and target of which was transcription factor Myb-encoding gene, which was induced by Pi deficiency，indicating that miR159e-3p appear to regulates soybean’s responses to low Pi. . In this study, we employ soybean to carry out the following studies at physiology, molecular biology, and genetics levels: (1) to determine the expression patterns of miR159e-3p and its target gene through quantitative real-time PCR and GUS-fusion approaches, especially their responses to Pi starvation; (2) to explore the role of miR159e-3p and its target gene in P signaling via overexpression of them in soybean; (3) to decipher the functions of miR159e-3p and its target gene through overexpression of MIM159e-3p (miR159e-3p mimicry)and the mutated target gene (miR159e-3p resistant);(4) to determine the transcription factor activity of the target gene and the binding sites of target gene in genome-wide level through ChIP-seq techniques. We aim to reveal the molecular mechanisms of soybean's responses to low Pi regulated by miR159e-3p, and provide new clues for improving phosphorus nutrition efficiency in soybean in future.

miR159在花发育以及盐害和ABA响应方面起重要作用，但对miR159是否调节植物低磷胁迫响应及机制还不清楚。我们前期研究发现低磷抑制大豆miR159e-3p在根部表达，其靶基因是一个受低磷诱导的编码MYB类转录调控因子的基因, 表明miR159e-3p可能调节大豆低磷胁迫响应。. 我们以大豆为实验材料，通过分子生物学和遗传学等研究手段，开展如下研究：（1）定量PCR和融合报告基因方法确定miR159e-3p和靶基因的时空表达模式，特别是对低磷胁迫的响应模式；（2）过表达miR159e-3p和靶基因对大豆响应低磷胁迫的影响；（3）过表达大豆MIM159e-3p以及突变靶基因进一步研究其调节低磷胁迫响应的机理；（4）确定靶基因的转录因子活性和通过ChIP-seq技术在基因组水平上确定靶基因调控的下游基因。解析miR15e-3p调节大豆应答低磷胁迫响应的分子和生理机制。

本项目以粤春03-3（YC03-3）和过表达GmMIR159e的转基因大豆为实验材料，鉴定了大豆MIR159基因家族并预测其靶基因，分析了大豆MIR159基因家族对磷养分、ABA和NaCl的响应，以及对大豆GmMIR159e在调节磷养分平衡方面的功能进行了初步研究。主要结果如下：. （1）大豆基因组有6个MIR159家族成员，MIR159a和MIR159d位于大豆9号染色体，MIR159b和MIR159e位于大豆7号染色体，MIR159c和MIR159f位于大豆16号染色体；进化分析表明，大豆MIR159家族共分为2个亚组。. （2）确定miR159e靶基因是GmMYB33，其编码核蛋白，其N端和全长具有转录激活活性。. （3）长期低磷诱导MIR159a和MIR159e在叶表达，诱导MIR159b和MIR159c在根表达，诱导MIR159f在根、叶表达；短期低磷抑制MIR159c在叶和根表达，抑制MIR159e在根表达；长期低磷诱导MIR159a、MIR159b、MIR159c和MIR159f在花中表达，而抑制MIR159d和MIR159e表达；长期低磷抑制MIR159a、MIR159c和MIR159d在根瘤中表达，诱导MIR159b表达。. （4）ABA处理诱导叶中MIR159a表达；MIR159b在叶和根均被ABA诱导表达；MIR159c在叶诱导表达，在根被抑制；MIR159d在叶抑制表达，而在根被诱导；MIR159e在叶和根抑制表达，MIR159f在叶抑制表达，在根被诱导。. （5）NaCl处理诱导MIR159a、 MIR159b、MIR159c和MIR159e在叶和根中表达；MIR159d在根被诱导；MIR159f在叶抑制表达，在根被诱导。 . （6）高磷条件下，大豆MIR159e启动子在叶脉、花器官和种皮有活性；而低磷条件下在花药、胚根和种皮有较强活性，并且低磷条件下大豆MIR159e启动子主要在主根成熟区、主根根尖和侧根根尖分生区有活性。 . （7）在高磷高氮（HPHN）和低磷高氮（LPHN）条件下，过表达MIR159e对大豆生长发育和磷营养没有影响；但在HPLN条件下，过表达MIR159e导致根和叶中总磷含量下降，老叶和根瘤中可溶性磷浓度下降。

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