Isolation And Functional Analysis Of Strawberry PHO1 Genes

There are many factors that directly impact the fruit quality of strawberry, including not only genetic factors but the environmental conditions and cultural practices such as planting at the right time and the management of water as well as fertilizers and so on. Among them, fertilizers are important factors in determining strawberry fruit quality and yield. However, there are few reports about the effect of phosphorus (P) on the fruit quality of strawberry and its mechanism at home and abroad. In this paper, the positive correlation between soluble solids content (SSC) and P content in strawberry fruits was revealed, using cultivated strawberries as the experimental materials. In order to elucidate the molecular mechanism of how P affects SSC of strawberry fruits, three PHO1 genes was isolated and cloned from strawberry (Fragaria×ananassa Duch.), the sequence characteristic as well as the expression profile of these genes was explored and their function was analyzed in Arabidopsis and strawberry through genetic transformation. The main results were as follows:1. The correlation between SSC and P content of 24 cultivars strawberry fruits and of ’Sachinoka’ fruits harvested at different dates was analyzed, respectively. The results showed SSC was both positively correlated with P content, and the correlation coefficient r equals 0.95 and 0.96, respectively. The positive correlation was verified in different parts (top, middle and bottom) of strawberry fruits through correlation analysis (r=0.87).2. Plants of ’Yanli’ strawberry were irrigated with different concentrations of P fertilizer (phosphoric acid) to provide further confirmation of the correlation and results showed that P content and SSC in fruits from plants treated with 6.0 mM phosphoric acid increased 45.0% and 16.7%, respectively.6.0 mM phosphoric acid treatment enhanced the photosynthetic efficiency of strawberry plants. Photosynthetic rate and water use efficiency of phosphoric acid treated plants increased 28.8% and 16.1% compared with the control plants.3. FaPHO1, FaPHO1;H1 and FaPHO1;H9 genes were all cloned from’Sachinoka’ strawberry through RT-PCR. Results showed that the full-length coding sequence (CDS) of these three PHO1 genes was 2340 bp,2481 bp and 2292 bp, respectively. The nucleic acid sequence identity of them is 63.44% and the amino acid sequence identity of them is 47.64%. Analysis showed that the encoded proteins of these three PHO1 genes both had the highest homology with the proteins of wild type strawberry, followed by apple and Chinese white pear, and other species had the relatively far relationship with strawberry.4. The expression of these three PHO1 genes in different organs and fruits at different developmetal stages was detected through real-time RT-PCR. Results showed that FaPHO1 and FaPHO1;H1 genes were expressed most in the root, while FaPHO1;H9 gene expressed in all organs and especially expressed most in the petiole. The expression of FaPHO1;H9 was decreased along with fruit development and ripening, besides the turning red stage.5. The distribution of P content in different organs of strawberry and P content in fruits at different developmental stages was studied. Results showed that the P content in the fruit was the lowest, while that in the leaf was the highest (4.3 times higher than P content in the fruit). Furthermore, the P content gradually decreased with the fruit development and ripening.6. Plants of’Yanli’strawberry were irrigated with different concentrations of P fertilizer (potassium dihydrogen phosphate) to reveal the relationship between the expreesion level of FaPHO1;H9 gene and P content. The gene expreesion level of FaPHO1;H9 in leaves and fruits from plants treated with 40 mM potassium dihydrogen phosphate increased significantly, 33.7 times and 5.4 times higher than expreesion level in control plants, respectively. At the same time, P content also increased. P content in leaves increased 64.8%, while P content in fruits increased 13.6% compared with the plants treated with water.7. Three over-expression vectors, namely pOE-FaPHO1, pOE-FaPHO1;H1 and pOE-FaPHO1;H9, were conducted. Transgenic lines of these three genes were both obtained through transforming Arabidopsis, which had significantly different phenotype in contrast with wild type, including lower plant height and less number of rosette leaves. A transgenic line of FaPHO1;H9 gene had the advanced flowering time about 3 days.8. Analysis of the expression specificity of three PHO1 genes and P content in these transgenic Arabidopsis plants showed the expression of three strawberry PHO1 genes was both boosted in the root and P content increased significantly at the same time. The P content increased most in the root of transgenic lines of FaPHO1 and FaPHO1;Hl genes, while P content of all organs was significantly higher than that in wild type plants. All these proved three PHO1 genes of strawberry founction in regulating P content in Arabidopsis.9. The RNAi vector of FaPHO1;H9, namely pRNAi-FaPHO1;H9, was conducted. Two transgenic lines were obtained through transforming ’Rugen’ strawberry, which had significantly different phenotype in contrast with wild type strawberry, including lower plant height, smaller leaf area, the less number of leaves, shorter petiole length. Further analysis of the gene expression of FaPHO1;H9 and P content in leaves of these transgenic lines showed the expression level of FaPHO1;H9 gene and the content of P decreased at the same time, proving FaPHO1;H9 founctions in regulating P content in leaves of strawberry.