| Rice as a main grain crop,the research about how to improve the quality and yield of rice has became the focus of more and more studies.Seed size directly affect rice yield.In recent years,there have been a lot of reports about mi RNA regulating plant height and grain weight,but it is not very clear which AGO protein participateing the small RNA pathways to regulate these important agronomic traits.In this study,we created genetic material of these AGO genes,and did a deep research of AGO17.The main results are as follows:Using RNAi and CRISPR techniques,we created RNAi mutants of 10 AGO genes and 4 CRISPR mutants of them,and created 3 overexpression mutants using 35 S promoter.Preliminary phenotype observations showed that down-regulated of these AGO genes resulted in defects in plant height,floral organ morphology and seed development in rice,suggesting that they may be involved in regulating rice growth and development.According to phylogenetic analysis,AGO17 was belongs to AGO1 clade.It is a peculiar AGO protein in rice,and was missing in other plants.Protein sequence analysis revealed that AGO17 deleted N terminal Gly-rich which AGO1 clade generally possessed,and catalytic triad DDH in PIWI domain was mutated into HDR.The expression analysis showed that AGO17 was expressed in the vegetative organs and reproductive organs of rice,and the expression level was higher in the internode and node at booting stage,young panicle,glume and early developing seeds.Subcellular localization revealed that AGO17 was localized in the nucleus.CRISPR mutant ago17 were dwarf compared with wild type ZH11,and grain length,grain width and grain weight significantly reduced;phenotype of RNAi transgenic plants was similar with ago17,and the severity of phenotype was consistent with the reduced expression level of AGO17;the grain length and grain width of AGO17 overexpression material OE increased,and as well as grain weight.The results showed that AGO17 can positively regulate the seed size.Cytological observation showed that cell size of sheath,stem and glume of ago17 and RNAi-lines were obviously smaller than that of WT,but the cell number showed no significant change;instead,the cell size of OE increased significantly,and the cell number didn’t change.The results showed that AGO17 mainly regulated cell size to affect stem development and seed size of rice.From transcriptome sequencing of ago17 and OE,we found some cell wall development related genes showed different expression,these results confirmed that AGO17 mainly through regulating cell expansion and to affect organ size in rice.In addition,transcription analysis found that there are some hormone-related genes showed different expression,and some GA biosynthesis and metabolism genes showed opposite expression in ago17 and OE by q RT-PCR,concentration of endogenous activity GA3 decreased in ago17 and increased in OE,exogenous GA3 can partially recovered dwarf phenotype of loss-of-function mutants.Therefore,AGO17 may regulate rice plant height by regulating the content of active GA.Small RNA sequencing found that 5’-U 22 nt s RNA decreased in ago17 and increased in OE,and 5’-U 22 nt s RNA mi R397 b decreased in ago17 and increased in OE by stem-loop RT PCR and northern blot;additionally,phenotype of AGO17 overexpression plants were similar to mi R397 b overexpression plants,and phenotype of RNAi-lines and ago17 were similar to mi R397 b down expression mutant;so we speculated AGO17 may through accumulating mi R397 b to regulated grain size of rice.Based on the genome-wide association studies(GWAS)on whole panel(533accessions),we found four SNP loci associated with grain width and grain weight at AGO17 promoter region.Promoter element analysis found two core promoter elements TATA box at these SNP loci,and AGO17 expression was varied in different haplotypes and positively related with grain width and grain weight.This future indicated that AGO17 was a key factor in controlling grain size and weight in rice.Our previous studies found that Os EMF2 b,Os FIE2 and Os CLF down regulated transgenic mutants showed a dwarfing phenotype,we carefully observed these RNAi transgenic lines,and studied how they regulated plant height development in rice.Histological observation showed that cell expansion and cell division were suppressed when Pc G genes expression reduced.In emf2 b transcriptome analysis,expression of 484 genes were up-regulated and 399 genes were down-regulated,GO analysis showed that the cell wall structure and biosynthesis,cell biosynthesis(membrane components)and cell division related elements(intermediate filaments spindle body)were enrichment.And the expression of some cell cycle proteins and cell expansion proteins were changed,which confirmed the cell expansion and cell division changed.Based on report of fie2 transcriptome analysis,we found the expression of IAA transporters,IAA induced protein and GA receptor genes were down-regulated,GA2-oxidase gene and CK-oxygen glycosyltransferase gene were up-regulated in fie2 and emf2b.q RT-PCR results showed in Os EMF2 b,Os FIE2 and Os CLF RNAi-lines GA oxidase genes and GA synthase genes were up-regulated,GA receptor genes were down-regulated.Endogenous GA3 concentration were aslo reduced in RNAi-lines,and exogenous GA3 can partially recovered the dwarf phenotype,so Pc G gene regulated GA pathway to affect rice plant height.We made the co-localization analysis of differently expressed genes between emf2 b and fie2,and found 109 genes both up-regulated in emf2 b and fie2,95 of them have H3K27me3 modification sites.The expression of 6 genes was detected and up-regulated in Os EMF2 b,Os FIE2 and Os CLF RNAi-lines,and their H3K27me3 was decreased in emf2 b according to Ch IP analysis,these results suggested the regulation of these genes may involved in PRC2 function.Os EMF2 b physically interacts with Os CLF in yeast,so we hypothesized that the Pc G gene might through the PRC2 complex to regulate rice plant height. |
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