草莓PHO2基因功能解析及其基因编辑研究

Sugar content is the important character of fruit quality. In our previous study, we found that sugar content is positively correlated with phosphorus content in strawberry fruits. And the overexpression of miR399 can significantly improve the phosphorus and sugar contents in fruits of woodland strawberry. PHO2 is the target of miR399 and a key negative regulator in phosphorus uptake and transport. So, PHO2 is an ideal candidate gene for breeding of P-efficiency and high-quality cultivars base on the method of gene editing. But the function of PHO2 has not yet completely elucidated. To investigate the function of PHO2 in strawberry, we try to obtain the PHO2-overexpression and silencing plants by using diploid woodland strawberry (Fragaria vesca) as materials in this project. In addition, PHO2 promoter-GUS will be performed to analyze PHO2 gene expression pattern. And the mechanism of effect of PHO2 gene in strawberry will by analyzed by transcriptomics and proteomics technologies. The effect of PHO2 gene knockout on the growth of diploid and octoploid strawberry will be analyzed. At the same time, we will explore the efficiency of CRISPR technique on octoploid strawberry. The purpose of this project is to reveal the function of PHO2 gene in strawberry and provide insights for breeding strawberry cultivars with P-efficiency and high-sugar strawberry varieties by gene editing technology.

糖含量是果品重要品质性状。前期研究中我们发现，草莓果实中磷含量与糖含量呈显著正相关，在二倍体森林草莓中过量表达miR399能够显著提高果实中磷含量和糖含量。miR399的靶基因是PHO2，对磷吸收和转运具有负调控作用，因此PHO2是基于基因编辑来培育磷高效利用进而实现品质提高的理想候选基因，但是PHO2的功能尚未完全解析。基于此，本研究首先以二倍体森林草莓为试材，获得PHO2基因过量表达植株和沉默植株并在此基础上全面揭示PHO2的功能，通过启动子驱动GUS来阐明PHO2基因表达特性，利用转录组学和蛋白质组学技术解析PHO2基因的作用机制。然后以二倍体和八倍体栽培草莓为试材，分析PHO2基因敲除对草莓植株生长发育的影响，同时探讨CRISPR技术对八倍体植物的基因敲除效率。本研究旨在全面揭示草莓PHO2基因的功能，为通过基因编辑技术培育磷高效利用的高糖草莓品种奠定基础。

草莓果实中磷含量与糖含量呈显著正相关。PHO2基因对植物磷吸收和转运具有负调控作用，是基于基因编辑来培育磷高效利用进而实现品质提高的理想候选基因。本项目组成功组装出二倍体野生森林草莓资源‘Ruegen’的高质量基因组和八倍体栽培凤梨草莓品种‘艳丽’高质量单倍型基因组，在此基础上，全面解析了二倍体森林草莓和八倍体凤梨草莓中的PHO2基因结构的序列和结构，揭示了草莓PHO2基因的转录特性，证实了在草莓中PHR1通过miR399来负调控PHO2基因表达的机制，获得了草莓PHO2基因被敲除的植株、RNAi植株、过表达植株，全面揭示了草莓PHO2基因的功能，筛选到与草莓PHO2互作的蛋白。本项目研究结果为通过基因编辑技术培育磷高效利用的高糖草莓品种奠定基础。项目组完成了研究目标，培养5名博士研究生和5名硕士研究生，已发表SCI收录论文2篇，另有1篇论文已被接受。