Construction Of Rice OsLPRs Mutant And Study Of Root Traits Under Low-phosphorus Stress

Rice(Oryza sativa L.)is one of the worldwide staple food crops.However,soil phosphorus deficiency has always been a key factor limiting rice yield.Although the application of phosphate fertilizer can increase rice yield,it will also cause a large amount of phosphorus resources consumption,environmental pollution and increase production costs.Therefore,it is of theoretical and practical significance to study the molecular and physiological mechanism of rice response to low phosphorus,and will be benefit to the breeding of low phosphorus resistant rice cultivars.In this study,the transcripome of low phosphorus sensitive rice TJ981,as the experimental material,was sequenced and analyaed and the results showed that the expressions of OsLPR1,OsLPR3,OsLPR4 and OsLPR5 genes significantluy up-regulated after 15d-low phosphorus treatment,suggesting that the OsLPRs gene family might regulate the steady equilibrium of phosphorus in rice.Then,using CRISPR/Cas9 gene editing technology,OsLPR1,OsLPR3,OsLPR4 and OsLPRS were mutated,and the difference of root morphology,physiology and iron plaque formation between in mutants and wild were determined.The expressions of OsLPRs gene and root growth regulation related genes were detected with qRT-PCR technique at transcription level.1.Transcriptome sequencing analysisThrough the transcription sequencing analysis of the test materials under low phosphorus stress after 15d treatment,it was found that phosphorus stress prompted the expression of OsLPR gene family in rice,and the results of qRT-PCR and proteomics verified the accuracy of transcriptome sequencing results.It is indicated that the OsLPR gene family might regulte the steady equilibrium of phosphorus in rice2.Construction and identification of mutantsUsing CRISPR/Cas 9 gene editing technique,4 genes of rice OsLPR1/3/4/5 were alltogether edited.From 102 tissue culture seedlings,39 positive seedlings were identified and after identification of the OsLPR3 genome target sites in the positive seedlings,16 positive seedlings were found to be edited at the OsLPR3 target site.Next,OsLPR1,OsLPR4 and OsLPR5 mutants were determined among the OsLPR3 mutant lines,and 12 strains of 4 OsLPR genes were finally identified,which indicated that it was practicable and effective to use the homologous sequence of CDS for multiple genes edit.In addition,1 plant with single OsLPR3 gene mutation was achieved.The detection of off-target sites showed that none of the 12 mutants occurred off-target.Mutation type analysis indicated that 2 lines of OsLPR1,OsLPR3,OsLPR4 and OsLPR5 were homologous.3.Changes of the root surface iron plaque of mutantsLow phosphorus(1/25 Pi)treatment of mutants and wild rice were carried out for 15d,and it was found that the mutant root surface red-brown iron plaque color is lighter than wild rice.DCB-Fe content detection of the iron plaque in rice root showed that the mutant root iron film accumulation is significantly weaker than the wild type.Meanwhile,the mutant root surface iron plaque under normal phosphorus culture was also significantly lower than wild type,which indicated that the OsLPR gene was related to the formation of iron plaque and the accumulation of trivalent iron.4.Differences in root morphology and physiological and biochemical characteristicsAlong with the prolongation of low phosphorus stress,the physiological response of mutants to phosphorus deficiency faded:the root/top ratio was significantly reduced,the root length was significantly shortened,the root dry-weight and plant height increased significantly,which indicated that the OsLPR family was an important component of rice root system in response to low phosphorus stress.After 15d of low-phosphorus stress,the total shoot phosphorus content of the mutant significantly increased compared with the wild type,and the activity of acidic phosphatase increased by 13.64%in the root secretion of mutant(no significant difference)which indicated that the OsLPR gene might not be involved in regulating the synthesis and secretion of acidic phosphatase.5.qRT-PCR analysis of OsLPRs gene and related genesResults of qRT-PCR detection showed that the transcription expression of OsLPRs(OsLPR1/3/4/5)still existed in mutant rice,suggesting that the edited OsLPRs did not bring earlier stop condon into the CDS.After low phosphorus treatment,at the transcription level,the expression of OsLPR1/3/4/5 in mutant rice was significantly decreased compared to wild type,and the transcription factors both SHR and SCR related to the differentiation activity and the?-1,3 glucanase gene(Os03g0221500)associated with the hydrolysis of corpus callosum also showed a significant decrease.These results indicated that the accumulation of corpus callosum might increase in the mutant root,and the activity of root apical meristem might be inhibited,which led to the production of short root traits in the mutant low phosphorus environment.In summary,based on the construction of OsLPRs mutant,root morphological difference,physiological analysis and qRT-PCR detection,the results above suggested that OsLPRs might participates in the formation of iron plaque under low phosphorus condition,which might indirectly regulate phosphorus absorption in rice and directly participate in the root morphogenesis.