Molecular Mechanism Of Mir399 Regulating Phosphorus Transportation In Rice

Phosphorus is one of the large amount of nutrients necessary for plants’ growth and development.The available phosphorous that can be absorbed by the plants seems to be extremely insufficient,which seriously affects the growth of plants,though there is plenty of total phosphorus in soil.microRNA,as a class of small RNA with regulatory roles,plays importantly regulatory functions in the nutrient metabolism of plants.It was firstly found to be induced by low phosphate in Arabidopsis thaliana to downregulate its target gene AtPH02,which is the mechanism of Arabidopsis thaliana response to low phosphate.Although the molecular mechanism of miR399 regulating phosphate transport in Arabidopsis thaliana has been extensively studied,relevant data and mechanism in rice are relatively vague.As staple food crop in China,one of the main reasons for the decrease of rice yield is insufficient available phosphate in the soil.It is of great significance to study the phosphate transport regulation mechanism mediated by rice miR399 practically and theoretically.Therefore,this study uses rice miR399 overexpression and STTM silent(Short Tandem Target Mimic,a technique used to silence microRNAs)lines as research materials.Under different phosphate levels of cultivation,the molecular mechanism of miR399 regulating phosphate transport in rice are studied by observing phenotype and analyzing molecular data,the main results obtained are as follows:1.Ti generation rice miR399 overexpression and STTM silent transgenic lines are propagated to T2 generation,the rice materials of T2 generation transgenic homozygotes are screened and identified.2.Under the condition of normal soil fertilizer,both transgenic lines could complete their life cycle,set seeds and get mature.Compared with wild type,The panicle length,spike length,grain length,grain width and thousand seed weight of miR399 overexpression lines and STTM silent lines decreased significantly,whereas the decrease in the latter is significantly smaller than that of the former.The plant height of miR399 overexpression lines are significantly reduced,while the STTM silent lines show no significant difference.3.Under low phosphate conditions,the accumulation of phosphate in the leaves and stems of miR399 overexpression lines was about 3.25 times of wild type,but phosphate in roots as no significant change,while the phosphate in the leaves of STTM silent lines was significantly lower than wild type,and the phosphate in the roots did not change significantly.The miR399 overexpression lines exhibited superior low phosphate tolerance,while the STTM silent lines is sensitized to low phosphate.4.The transcription level of the OsPHT1 gene responsible for phosphate uptake was basically the same as wild type in miR399 overexpression and STTM silent lines.The transcription levels of OsPHT1 in the three genotypes can be induced by low phosphate,suggesting that rice phosphate absorption gene OsPHT1 may not be controlled by the miR399-PHO2 module.However,phosphate content determination showed that the aboveground part with miR399 overexpression lines had the highest phosphate level.5.Another microRNA-miR827,which is associated with phosphate metabolism,was significantly downregulated in miR399 overexpression lines.The target gene of miR827,OsSPX-MFS1 is mainly responsible for the injection of phosphate into the vacuole,the downregulation of miR827 means the upregulated of OsSPX-MFS1 can transfer the aboveground phosphate accumulation in miR399 overexpression lines into vacuole,which is a stress response to phosphate accumulation in rice.6.Under low phosphate conditions,some physiological indexes were changed in miR399 overexpression and STTM silent lines.The chlorophyll,acid phosphatase activity and root activity of miR399 overexpression lines were significantly increased compared with wild type,while STTM silent lines was the opposite.The anthocyanin content of miR399 overexpression lines decreased significantly,and the anthocyanin content of STTM silent lines increased significantly.These changes of the physiological indexes may be affected indirectly by the difference phosphate content in the three genotypes.7.In this study,overexpressed rice miR399c is a family member induced by low phosphate,its target gene OsPHO2 is negatively regulated by miR399cIn summary,miR399 may be mainly mediating phosphate transport from the underground part to the aboveground part of rice without affecting the uptake of phosphate by the roots.Under normal soil fertilizer conditions,artificial intervention of miR399 expression didn’t conducive to the production of materials,but miR399 overexpression plants is obviously more suitable for soil fertilizer conditions with insufficient phosphate.These results not only contribute to a deeper understanding of the molecular mechanism of phosphate transport mediated by rice miR399,but the generation of transgenic materials adapted to specific soil fertilizer conditions.