Response Of Phosphorus Transporter McPht To Phosphorus In Transgenic Rice

Phosphorus is an essential element required for plant growth and development,have a positive effect on the physiological and biochemical processes of plant.Phosphorus transport proteins play an important role in phosphorus uptake and transport in plants.In order to better understand the function of phosphorus transporters in plants,a Mc Pht gene was isolated from the Mesembryanthemum crystallinum L and transferred into rice kitaake.The bioinformatics analysis showed that Mc Pht gene was 1071 bp and encodes 357 amino acids with the molecular weight of 37.77 k Da,and there are six transmembrane domains.In order to further determine the classification of McPht protein in phosphorus transporters,subcellular localization experiments were carried out,the result showed that McPht is localized in mitochondria,indicating that Mc Pht gene is a typical Pht3 subfamily member.The expression the McPht gene in transgenic rice and Mesembryanthemum crystallinum L.under low-phosphorus stress was studied by q PCR,the results showed that under low-phosphorus stress the expression of Mc Pht gene in the roots and shoots of transgenic rice plants was higher than the normal treatment.Under low-phosphorus stress,the expression of Mc Pht gene in shoots of Mesembryanthemum crystallinum.L was higher than the normal treatment.In order to further explore the physiological function of phosphorus transporter gene McPht under low-phosphorus stress,we carried out hydroponics and tissue culture experiments on transgenic and wild type rice.The results showed that biomass,root activity,chlorophyll content,relative water content,total nitrogen content and total phosphorus content of transgenic rice was increased under low-phosphorus stress compared with wild type.Root scanning was performed on roots of hydroponics and tissue culture rice,the results showed that root length,root area,projection area and lateral root number of transgenic rice were higher than wild type under low-phosphorus stress.In order to investigate the gene expression characteristics of phosphorus transporter gene McPht under low-phosphorus stress,sequencing of the transcriptome was used to analyze the transgenic and wild-type rice shoots genes change under low-phosphorus stress,a total of 198 differentially expressed genes were screened.The wild type was used as control,154 genes were up-regulated and 44 genes were down-regulated in transgenic rice.GO function enrichment analysis showed that the highest number of up-regulated genes was found in the cytoplasmic membrane-bounded vesicle,the highest number of down-regulated genes was found in the mitochondrion and cytoplasmic membrane-bounded vesicle.Pathway analysis showed that differential genes were mainly enriched in plant secondary metabolisms and plant-pathogen interaction,such as phenylpropanoid synthesis,phenylalanine metabolism,and secondary metabolites synthesis and so on.These results provide a basis for further study on the molecular mechanism of transgenic rice in response to low-phosphorus stress.In order to further verify the function of the phosphate transporter protein gene McPht,in this study,a CRISPR/Cas9 gene editing technology was applied to knock out the phosphate transporter protein Os Pht gene in rice,which shares the highest homology with the phosphate transporter protein Mc Pht gene in Mesembryanthemum crystallinum,to provide a reliable material for scientifically elucidating the function of McPht transporter protein in rice mutant with Os Pht deletion.The results showed that: 1)The mutants from gene deletions were divided into two categories,which one is that 6 bases were deleted in the target editing fragment with 20 bases,and the other one is that 8 bases were deleted or mutated in target editing fragment with 20 bases.2)The mutants missing 6 bases accounted for 28.6% of the total mutant lines,and the muatnts missing 8 bases accounted for 42.9% of the total mutant lines,and the mutants with mutation of GT bases accounted for 28.6% of the total mutant lines.3)The plant height,fresh weight and root activity of mutant rice were less than wild type.The root length,shoot area,surface area and lateral root number were higher than those of wild type.The content of chlorophyll and soluble sugar of Cas-2 were higher than that in wild type,the difference was significant,the chlorophyll content and soluble sugar content of Cas-7 were less than that of wild type,but the difference was not significant.