Fine Mapping And Gene Cloning Of The Small Grain And Narrow Leaf Gene GSNL4 In Rice

ARGONAUTE1(AGO1)proteins mediate plant development and growth by acting as recruiters binding mature small interfering RNAs(si RNAs)or micro RNAs(miRNAs).It has been previously demonstrated that rice OsAGO1 a,b,c,and d predominately bind to known miRNAs,and among the four OsAGO1 s,OsAGO1 b may be the leading player regulating rice growth and development.However,the study of OsAGO1 b has not been reported by forward genetics.ARGONAUTEs(AGOs)In this study,a small grain size and narrow leaf mutant was obtained by EMS mutagenesis of the japonica rice Wuyunjing21;the mutant was named gsnl4(grain size and narrow leaf 4).The main results were as follows:1.The gsnl4 plants showed a pleiotropic mutation characterized by small grain size,narrow leaf,low pollen fertility,low seed setting and fewer roots.2.Histological analysis showed that the gsnl4 mutants had a significantly reduced number of vascular bundles in leaves and culms,size and number of culm and spike cells.3.Genetic analysis indicated that the gsnl4 phenotype of small grain size and narrow leaf was controlled by a single recessive nuclear gene.4.Using map-based cloning,we mapped the GSNL4 to a 42 kb interval on chromosome 4.5.Sequence analysis of the wild-type and gsnl4 showed that a single-base mutation from G to A at position 13746 was found in LOC＿Os04g47870(OsAGO1b)gene of the gsnl4 plants,which resulted in a Ser to Asn the substitution in the Piwi domain of the OsAGO1 b protein.Intriguingly,we found the mutational site of OsAGO1 b in the gsnl4 plants was the same as the mutational site of At AGO7 in Arabidopsis mutant ago7-15.6.Knockout of LOC＿Os04g47870 gene in wild-type japonica rice Wuyunjing21 generated obvious small grain,narrow leaf and low seed setting rate indicating that it was a candidate gene for GSNL4.7.Subcellular localization indicated that GSNL4 was expressed in cytoplasm and nucleus.8.Through GUS staining and real-time quantitative PCR demonstrated that GSNL4 was ubiquitously expressed in all the tissues but predominantly in young panicles.9.The miRNA-seq analysis showed that about 29 significantly upregulated miRNAs and 23 significantly downregulated miRNAs in the gsnl4 plants,and most of the target genes of miRNAs expressed differently between the WT and gsnl4 plants were related to plant hormone signal transduction pathway,sugar metabolism pathway,anther and pollen development.10.Through stem-loop PCR demonstrated that the several miRNAs were down-regulated,and their target genes were upregulated in gsnl4 mutants.11.The content of endogenous IAA in the gsnl4 plants was significantly lower than that in wild-type.Through q RT-PCR analysis,the expression of most auxin transporting and synthesizing genes and leaf development related genes were disturbed in the gsnl4 plants.In this study,OsAGO1 b mutant is first obtained by forward genetics method,which not only regulates organ development by controlling cell division and expansion,but also plays an important role in regulating IAA synthesis and transport in rice.S810 N residues in Piwi was conserved in rice and Arabidopsis,which provides genetic evidence for further analysis of the chemical function of AGO1/7,and provided good genetic materials for further research on the function of OsAGO1 b in the future.