Functional Analysis Of Antisence Transcript Of OsPHO1in Rice(Oryza Sativa L.)

Phosphorus is one of the macro-essential mineral elements for plant growth. It is important component of many biochemical compounds, such as nucleic acids, phospholipids, ATP, ADP etc. It is also involved in fundamental physiological and biochemical processes, such as photosynthesis, cell signal transduction, energy metabolism etc. Phosphorus uptake and transport is a complex process. Previous researches have elucidated a regulatory pathway of phosphate (Pi) signaling under control of PHR2, miR399and PHO2. However, as an ubiquitin coupling enzyme, how PHO2regulates its downstream target to increase plant’s absorption of phosphate and transport Pi to the above-ground part is still not clear. In this study, by screening an EMS mutant library under background of mutation of OsPHO2(pho2), which showed leaf necrosis phenotype, a phenotype recovery mutant was identified. According to the phenotype, the mutant was named pho2-supl (pho2-suppressorl). The leaf necrosis phenotype of pho2mutant was rescued in pho2-supl mutant under solution culture with50μM Pi, and partial rescued with200μM Pi. The cellular Pi concentration in leaves was much lower in pho2-supl than that in pho2mutant.By using map-based cloning, we cloned the mutant gene. It is located on the second chromosome which was annotated as OsPHO1(LOC_Os02g56510). It is one of the three homologous genes of Arabidopsis PHO1in rice. Sequencing analysis revealed a mutation in the fifteenth exon in OsPHO1gene, resulting in an amino acid change from histidine to proline in EXS domain. The complementation experiment proved that the single mutation in OsPHO1causes the mutant phenotype of pho2-supl.The analysis of genomic data indicated that there exist cis-natural antisense transcripts (NATs) in the5’end of the three PHO1homologous genes. In addition, OsPHO1(LOC_Os02g56510) contains a cis-natural antisense transcript with no intron. Transgenic plants overexpressing the PHO1NATs showed lower Pi concentration in leaves compared with the wild type plants. Realtime quantitative PCR (qRT-PCR) analysis indicated that the transcript level of OsPHO1is lower in the transgenic plants than that of the wild type plants. This study provides novel knowledge of the function of PHO1natural antisense transcripts which would down regulate the transcript level of endogenous PHO1and further regulate phosphate homeostasis in rice.