Identification And Characterization Of Low Phytic Acid Mutant Genes In Rice (Oryza Sativa L.)

Phytic acid(PA,myo-inositol 1,2,3,4,5,6-hexakisphosphate)is the primary storage form of phosphorus(P)in cereal seeds,accounting for about 65-85%the total P.Phytates,the salt forms of PA chelating with cations such as K⁺,Ca²⁺,Mg²⁺and/or Zn²⁺,are mostly accumulated in the aleurone and embryo part of cereal seeds.Since PA and phytates are indigestible by human and non-ruminant animals;they not only affect the bio-availability of minerals and the absorption of protein,fat and starch,but also result in P pollution in water system due to the release of human and animal. Recently,breeding of low phytic acid(lpa)crops has opened a new approach to settle PA-related nutritional and environmental issues.In this study,we identified a lpa mutant through screening introduced T-DNA mutant lines;cloned and characterized two rice non-allelic lpa genes through fine mapping and candidate region analysis;we also investigated the yield and yield related traits of several lpa mutants and their wild types.The major results are as follows:1.From seven T-DNA mutant lines that were introduced from South Korea,a lpa mutant(originally T-DNA insertion line '1A-08603')was identified by testing seed inorganic P(Pi)content.Genetic analysis indicated that the ratio of normal and low phytic acids seed is 3:1,which illustrated the lpa trait was controlled by a recessive gene.Results of GUS histochemical detection and PCR testing suggested that the lpa trait in this mutant line did not result fromT-DNA insertion, implying that the mutation originated from somaclonal variation.The mutation could seriously affect seed vigour,resulting in homologous HIP seed not germinable in natural condition.2.The KBNT lpa1-1 mutation was previously fine mapped to a 47Kb region on chromosome 2L.However,we did not detect any DNA sequence differences between mutant KBNT lpa1-1 and wild type KBNT based on CEL 1 facilitated DNA mismatch cleavage and DNA sequencing.We then extended the 47 Kb region and searched for differences between KBNT lpa1-1 and KBNT using the same way as above.We found that there is a SNP(C/G→T/A)in one exon of the LOC_Os02g57400(TIGR V5)gene,which can create an Xspâ… restrictive enzyme site.Meanwhile,we detected a 1475-bp fragment deletion in this locus of another mutant Os-lpa-XQZ,which carried the lpa mutation allelic to KBNT lpa1-1.Two DNA markers-LPA1_CAPS and LPA1_InDel were developed based on sequence information.Analysis of two segregating populations,i.e.a recombinant inbred line populaton 'KBNT lpa1-1/Zhe733' and F₂ population of 'Os-lpa-XQZ/XQZ',showed that the two markers were completely co-segregating with the lpa phenotypes,respectively.There are 4 potential transcripts of LOC_Os02g57400 through alternative splicing.The two allelic mutations could affect the expression of LOC_Os02g57400 in the FGENESH model.BLAST of amino acid sequence showed that LOC_Os02g57400 was homolgous to the 2-phosphoglycerate kinase(2-PGK)in Arobidopsis thaliana, and there were two paralogs in the rice genome.3.Another lpa mutation 'Os-lpa-MH86-1' was first mapped using japanic/indica F₂ population,and first mapped to 350 kb region between RM5478 and RM17567 on chromosome 4 using BSA method.Os-lpa-MH86-1 mutation was finally delimited to a 112 kb region between markers CAPS3 and ID26 by applying available SSR markers and new CAPS and InDel markers,which developed based on polymorphisms between Nipponbare and 93-11.4.Eight genes were selected as potential site of the Os-lpa-MH86-1 mutation in the 112 Kb region after excluding other 6 genes that are less likely to be involved phytic acid metabolism according to their annotated function(TIGR V5).These 8 genes were directly sequenced for both Os-lpa-MH86-1 and MH86.A G deletion in the 12^thexon of LOC_Os04g55800 was found in Os-lpa-MH86, which results in non-sense mutation and a terminal protein.Sequencing of this gene of another mutant Os-lpa-Z9B,which carries an lpa mutation allelic to Os-lpa-MH86-1,revealed a 6-bp deletion in the first exon.This 6-bp deletion could lead to a deletion of two amino acids Lys and Ser.Analysis of the Os-lpa-Z9B/Nipponbare F₂ population showed that the 6-bp deletion co-segregated with the lpa mutation by new developed marker CAPS_Z.RT-PCR analysis at different development stages and tissures showed that the LOC_Os04g55800 was not expressed in roots,but in leaf blades and seeds at different development stages;The expression level in leaf blades and seeds was higher than that in leaf sheat;There were no significant differences of transcription determined between mutant and wild type;The expression level of this gene was lower.BLAST of amino acid sequence showed that LOC_Os04g55800 has homologs in rice genomes,and exists in a lot of plant species,which indicates that this gene has already existed before the divergence of monocotyledons and dicotyledons by constructing the phylogenetic tree.5.Different lpa mutants and their wild type parents were comparatively tested for yield and yield-related traits.The results showed that the yield of mutants was reduced by 12.5-25.6%compared with wild types,and grain weight was decreased by 5.0-10.7%,which might be the major reason of yield reduction. Germination experiments indicated that the vigor index of mutant lines were reduced by 7.8-26.3%and amylase enzyme activity decreased by 12.6-23.8% compared with respective parent lines.Artificial aging experiment showed the lpa mutant is more sensitive to storage than their corresponding WT parents.Results of field tests showed that the field emergence of lpa lines has a decreasing trend compared its parents.