Identification Of Mutant Response To Low Phosphorus Stress And Cloning Of Related Genes In Arabidopsis Thaliana

Phosphorus is one of the necessary nutrient elements for plants and plays an important role in the growth and development of plants.There is much total phosphorus in the soil,but most of the phosphorus is insoluble phosphorus,leading to the majority of phosphorus can not be directly used by plants,so plants often face low phosphorus stress.Therefore,improving the use of insoluble phosphorus is greatly significance to agricultural production.Plant rhizosphere acidification can activate insoluble phosphorus and provide more effective phosphorus for their own,to a certain extent,which can alleviate the low phosphorus stress.Experiments have shown that plants are able to undergo rhizosphere acidification faster under low phosphorus induction,which can be shown by the acid-base indicator bromocresol violet.In order to study the association between low-phosphorus stress and rhizosphere acidification,we screened the EMS library and wanted to screen the relevant mutants to establish the relation.Based on the low phosphorus induced rhizosphere acidification,we established two screening methods.Method 1:EMS mutants were cultured on MS medium for 9 days,the seedlings were placed on low-phosphorus medium containing bromocresol purple,by contrasting with the WT rhizosphere acidification phenotype,we screened two phenotypically stable mutant plants,respectively,named p328,p250.Method 2:plant rhizosphere acidification is mainly root secretion of protons,organic acids and so on,these substances can reduce the pH of the surrounding environment,to a certain degree,this can activate the insoluble phosphate around the roots,which is absorbed by plants.By simulating the soil environment,the KH2PO4 in the MS medium was replaced with FePO4,we prepared into a poorly soluble phosphate medium,The EMS mutants were grown on MS medium for 6 days,the seedlings were placed on a poorly soluble phosphate medium for 12 days,we screened the phenotypically stable mutant plants,named p1-31.p328 is a mutant that reduce rhizosphere acidification under low phosphorus.The results showed that the rhizosphere acidification of p328 was weaker than that of WT by bromine cresol violet medium pH in situ display experiments and acidification quantitative experiments.In addition,anthocyanin accumulation,lateral root and root hair increased are important features of low phosphorus stress.The results showed that the content of anthocyanin in WT and p328 was not different under normal conditions.The content of anthocyanin in p328 was 1.2 times higher than that of WT under low phosphorus treatment,indicating that the content of anthocyanin in p328 is higher than that of WT.Under normal condition and low phosphorus treatment,the lateral root density of p328 was 0.96 times and 0.85 lower than that of WT,respectively,which indicated that the lateral root density of p328 was lower than that of WT.Under normal conditions,the root density of p328 was 0.67 lower than that of WT and the length of root hair was 0.63 lower than that of WT;Under the condition of phosphorus deficiency,the root hair density of p328 was 0.77 lower than that of WT and and the length of root hair 0.56 lower than that of WT,it shows that the root density and length of p328 are lower than that of WT.In order to study the effect of P328 mutations on the phenotype,mutation of the site was located.Genetic analysis showed that p328 was a single gene recessive mutant.Through the method of map cloning,the preliminary localization indicated that the mutation of site was at the midpoint of chromosome 4,and the fine locus indicated that the gene was between 4-9.85M and 4-10.08M.Sequencing results and tair site comparison search,it was initially determined that the mutant p328 was changed from A to T at base 10044454 on chromosome 4,resulting in the amino acid encoded by the gene from phenylalanine to isoleucine.p250 is a mutant that enhances rhizosphere acidification under low phosphorus.The results showed that p250 was a low-phosphorus-induced rhizosphere acidification-enhancing mutant by bromine cresol violet medium pH in situ display experiments and acidification quantitative experiments.The main difference between p1-31 and WT was the change of leaf color under low phosphorus stress,which indicated that the accumulation of anthocyanin in mutant p1-31 and WT under low phosphorus stress was different.The results showed that there was no difference in anthocyanins under normal conditions.The anthocyanin accumulation of p1-31 under low phosphorus and FePO4 medium were 0.83 and 0.77 times higher than that of WT,respectively,the results showed that the content of p1-31 anthocyanin was lower than that of WT under lowphosphorus stress.