Function Analysis Of OsSCL30 In Regulating Rice Phosphorus Homeostasis

Phosphorus is one of the indispensable elements in the growth and development of plants.Phosphate is widely involved in photosynthesis,respiration,energy metabolism and biosynthesis-Since the available phosphorus content in the soil is very low,it is particularly important to increase the absorption and utilization of phosphorus in plants.At present,great progress has been made in the study of the regulation mechanism of phosphorus uptake and transport in plants.Alternative splicing(AS)is a ubiquitous mechanism in higher eukaryotes for enhancing the complexity and variability of the transcriptome and consequently the proteome.SR protein is an important splicing factor that regulates the process of biological alternative splicing.It is widely involved in various abiotic stresses,but there has been no report on the stress of nutrients.The mechanism of action of the SR gene in response to abiotic stress has not been studied in detail.This paper mainly studies the function of OsSCL30 in regulating rice phosphorus homeostasis.OsSCL30 belongs to the SCL subfamily.The protein structure of this subfamily is highly conserved.It contains a RRM domain at the N-terminus and an RS construct at the C-terminus.The special structure of the SR protein determines its function of regulating the splicing of the precursor mRNA.In general,SR gene can be spliced variably by itself.We found a new transcript by rt-pcr and TA cloning and sequenced,and intron retention events occurred.And through subcellular localization experiments in tobacco and rice protoplasts,it was confirmed that OsSCL30 is localized in the cell nucleus.In order to study the biological function of OsSCL30,we found that the phosphorous content of many mutants of the SR family was significantly different in the pre-laboratory screening.we performed time gradients(10 d,17 d,24 d,31 d)and different phosphorus concentrations(10μM,91μM,450μM)on knockout mutant E4,overexpressing OE5,OE6 of wild type rice Nipponbare and OsSCL30.We found that OsSCL30 mutant E4 showed high phosphorus accumulation in both normal and phosphorus treatment environment.Under normal culture and high-phosphorus treatment,the content of inorganic phosphorus in the over-expressed OE5 and OE6 was significantly reduced.However,there was no significant difference in phosphorus content in the roots.The old leaves of E4 under high phosphorus treatment will have some brown spots,but the leaves of overexpressing OE5 and OE6 have fewer or no leaf spots.The above results indicate that OsSCL30 can affect the translocation of available phosphorus from rice to the shoots and respond to high phosphorus stress,which can inhibit the phosphorus toxicity of high phosphorus to rice.In addition,we found that the leaf angle of mutant E4 of OsSCL30 was significantly larger than that of wild type,and the plant morphology was loose;while the leaf angle of OsSCL30 overexpressing OE5 and OE6 was significantly smaller than that of wild type,and the plant morphology was compact.It shows that OsSCL30 also affects the growth and development of rice.In the wild-type rice Nipponbare and OsSCL30 mutant E4,the root transcriptome sequencing analysis of overexpressing OE6,we found that SPX2 was significantly inhibited in the mutant and the results were verified by quantitative PCR.Because SPX2 is an important negative regulator of phosphorus transport,we predicted that OsSCL30 mutants inhibited the expression of SPX2 and caused the accumulation of phosphorus in the shoots,which affected the phenotypes such as plant height,leaf spots and leaf inclination.In the OsSCL30 overexpression material,the expression of SPX2 was not different from that of the wild type.High-throughput sequencing results also include PT4,PT6,PT13,PT19,SPX-MFS2,SPX1 and other phosphorus transport regulatory factor genes,and their expression levels were also regulated.Therefore,we speculate that OsSCL30 regulate the regulation of phosphorus homeostasis in rice by regulating the phosphorus transporter.Because OsSCL30 is an RNA-binding protein,in order to screen its interaction RNA,we used the principle of TRIBE(Targets of RNA-binding proteins identified by editing)to fuse the OsSCL30 CDS and the catalytic domain coding region of Drosophila ADAR editing enzyme.Construction of OsSCL30-ADAR transgenic rice.By RNA-seq analysis of transgenic plants,we found that OsPGK2,OsRSZ23,and OsSR45a are potential target genes for OsSCL30,suggesting that OsSCL30 may cause alternative splicing of other SR genes.In conclusion,OsSCL30 plays an important role in regulating phosphorus homeostasis in rice.