PHO1基因在草莓果实磷输出中的作用及其表达特性分析

We found that both the contents of soluble sugar and phosphate in fruits of a white flesh mutant of strawberry cultivar 'Sachinoka' were significantly higher than those in wild type. And then we analyzed the contents of soluble sugar and phosphate in many strawberry cultivars and found a significant correlation between the content of soluble sugar and the content of phosphate. Data of transcriptome sequencing showed that the expression of PHO1 gene, which mediates phosphate efflux, was significantly down-regulated in white flesh mutant. So we speculate that the PHO1 gene has a significant effect on the content of phosphate in strawberry fruits. To verify this hypothesis, the dynamic change of contents of phosphate and sugar at the different ripen stages of strawberry fruits will be investigated, and the evidence of phosphate efflux during strawberry fruit ripening will be searched. The relationship between the expression level of PHO1 gene and the content of phosphate will be analyzed, and the expression model of PHO1 gene will be elucidated. The role of PHO1 gene on phosphate efflux from fruits will be illustrated by over-expression and silencing the PHO1 gene in strawberry fruits. The effects of ABA, low phosphate and high phosphate on the expression of PHO1 will be analyzed, and the regulation role of miR399 on the expression of PHO1 will be explored. The aim of this project is to elucidate the effect of PHO1 gene on the phosphate efflux from strawberry fruits, and to lay a foundation for regulating the contents of phosphate and sugar in strawberries.

我们发现'幸香'草莓白肉突变体果实中可溶性总糖和全磷含量均显著高于野生型，进一步对多个草莓品种进行分析，发现果实中可溶性总糖含量与全磷含量呈极显著正相关。转录组高通量测序结果显示，在草莓粉熟期白肉突变体果实中介导磷输出的PHO1基因表达水平比野生型显著下调。因此我们推测PHO1基因对草莓果实中磷含量具有重要调控作用。为了验证这一假设，本项目拟全面调查草莓果实发育过程中磷含量与糖含量的动态变化，寻找草莓果实发生磷输出的证据，分析PHO1基因表达水平与果实磷含量变化的关系，通过基因过量表达和沉默技术来验证PHO1基因在果实磷输出中的作用，揭示PHO1基因的时空表达特性，分析ABA、低磷、富磷等对PHO1基因表达的影响，探讨miR399对PHO1基因表达的间接调控作用。本项目旨在揭示PHO1基因在草莓果实磷输出中的作用，为调控草莓果实磷含量，进而调控糖含量奠定重要基础。

尽管长期以来植物的磷素营养一直是植物营养学研究的热点之一，但是磷含量与果实品质关系的研究却非常少。在发现‘幸香’草莓白肉突变体果实中可溶性总糖和全磷含量均显著高于野生型的基础上，我们推测磷含量对草莓果实品质具有重要影响。为了验证这一假设，本项目调查了草莓果实发育过程中磷含量的动态变化，分析了果实中磷含量与糖含量的相关性，克隆了PHO1基因家族的多个基因并通过稳定遗传转化揭示了它们的功能，分析了PHO1基因的转录特性，揭示了miR399对磷吸收和转运的影响。项目研究结果表明：（1）随着草莓果实发育，果实中磷含量不断下降；（2）以24个草莓品种为试材证明成熟草莓果实中可溶性固形物含量与磷含量存在显著正相关性（r=0.95）；（3）施用6mM磷酸可以显著提高草莓果实中磷含量和可溶性固形物含量；（4）克隆了草莓PHO1、PHO1;H1、PHO1;H9基因的完整编码区，在拟南芥中过量表达草莓PHO1基因可以显著提高磷含量，而沉默草莓的PHO1;H9基因则显著降低植株中磷含量；（5）在草莓中过量表达miR399能够显著提高叶片和果实中磷含量，转基因果实中糖和Vc含量显著高于非转基因对照；（6）揭示了PHR1-miR399-PHO2模块调控草莓磷饥饿反应的作用机理。项目研究结果为草莓磷高效利用分子育种及通过施用磷肥提高草莓品质提供了理论指导。项目组完成了研究目标，培养博士研究生4名、硕士研究生6名，发表SCI收录论文6篇。