烤烟植株磷对矿质养分转运基因表达的影响及调控途径

  【目的】  作物体内磷与其他矿质养分之间存在复杂的交互作用。分析不同供磷水平对作物体内养分转运相关基因(启动子中含P1BS顺式调控元件)的表达水平，从基因水平深入理解磷与其它养分间交互作用的机理。  【方法】  设置砂培试验，供试烟草品种为K326 (Nicotiana tabacum L. cv. K326)，营养液中设低磷(0.1 mmol/L)、正常(对照，1 mmol/L)和高磷(5 mmol/L) 3个磷供应水平，于烟草幼苗移栽4周后开始处理，5周后打顶，6周后取样，并分为根、茎、叶和腋芽，测定磷和其他元素浓度，鉴定启动子序列中含有磷调控顺式元件P1BS的基因，运用实时荧光定量PCR分析其中一些基因在缺磷和高磷条件下的相对表达量，采用生物学方法研究磷调控信号通路的中心转录因子NtPHR2调控钾、镁转运蛋白的途径。  【结果】  与对照相比，低磷和高磷处理均抑制烟株生长和地上部干物质量的积累；高磷处理植株各部位磷的积累均显著增加；地上部叶片中的钾、镁浓度随供磷水平的增加而提高，钙浓度在低磷和高磷处理下均不同程度地降低；根系中的铜、铁、锌浓度受供磷水平的影响不显著，而低磷显著降低了锰的吸收但促进锰向地上部转移。本研究在根系中共鉴定到86个含P1BS顺式调控元件的编码矿质营养转运蛋白的基因，选择其中磷、钾、钙、镁、铁、铜和锌转运蛋白基因12个进行RT-qPCR扩增分析，发现它们在低磷和高磷条件下的相对表达量不同。低磷和高磷处理均上调了磷、钾、钙、镁、铁转运蛋白基因表达量；低磷上调铜转运蛋白基因表达量而高磷下调其表达量；而两个锌转运蛋白基因一个在低磷和高磷时表达下调，另一个则表达上调。通过生物学方法证实了中心转录因子NtPHR2通过结合P1BS顺式元件直接调控钾和镁转运蛋白基因。  【结论】  缺磷和过量供磷均会抑制烟株生长，影响包括磷在内的养分的吸收和转运。增加磷供应水平可显著提高烟株各部位磷的浓度，提高钾和镁浓度，缺磷和过量供磷对地上部各组织其他养分浓度有负面影响。各营养元素转运蛋白基因的相对表达量受低磷和高磷的影响不同，可能与磷信号通路对其转运蛋白的调控途径有关。NtPHR2通过结合P1BS顺式元件直接调控钾和镁转运蛋白基因，因此，增加磷的供应提高了烟株磷的吸收，进而可能直接上调了钾和镁的吸收及转运。

Effects of phosphorus on the expression and regulatory path of nutrient transporter genes in tobacco

  【Objectives】  Phosphorus (P) has complex interactions with other mineral nutrients in plants. Here, we studied the effects of low and high P supply levels on the absorption and partition of mineral nutrients and nutrient transportation mechanism.   【Methods】  Three P concentrations were setup in the culture solution-low (LP, 0.1 mmol/L), normal control (CK, 1 mmol/L), and high (HP, 5 mmol/L) using four weeks old tobacco seedlings (Nicotiana tabacum L. cv. K326) as test materials. On the sixth week of treatment, the plant samples were collected and divided into root, leaves and bud, for the determination of mineral nutrient contents. The isolation and quantitative expression assay of transporter genes (containing the cis-regulatory motif P1BS) in the root were carried out using RT-qPCR technique. The binding of K+ and Mg2+ transporter genes by NtPHR2 through P1BS cis-element was evaluated using molecular methods.   【Results】  Both LP and HP inhibited tobacco growth and shoot biomass accumulation. Increasing P supply concentration enhanced the P, K and Mg concentrations in the root and shoot tissues of the crop. LP and HP treatment reduced the Ca concentration. The concentrations of Cu, Fe and Zn in the roots were not significantly affected by P supply, while LP significantly reduced the uptake of Mn and promoted the transfer to the shoot. We located 86 genes encoding transportation of mineral nutrients containing the cis-regulatory motif (P1BS). The relative expression of the selected 12 genes varied with HP and LP treatments. The LP and HP up-regulated the expressions of P, K, Ca, Mg, and Fe transporter genes, LP up-regulated and HP down-regulated the expressions of Cu transporter genes, and one Zn transporter gene down regulated and the other Zn transporter up regulated in LP and HP treatment. Meanwhile, we identified K+ and Mg2+ transporter genes directly regulated by NtPHR2 by binding to P1BS cis-element.   【Conclusions】  Both low and high P supply inhibit tobacco growth and absorption and partitioning of mineral nutrients to some extent. From the molecular perspective, P stimulates the expression and transportation of K and Mg genes directly by binding to P1BS cis-element of K+ and Mg2+ transporter genes. The strong interactions between P and other mineral elements suggest that nutrient transport and P-signaling appear to mediate tobacco growth.