Cloning And Expression Analysis Of A Rice Spikelet Development Gene OsSP1

Rice ear is the harvesting site of rice,and its morphological development is closely related to yield.In this study,that Kitaake derived from Japanese methane-type conventional rice（Oryza sativa L.）,was induced by Ethyl methane sulphonate（EMS）,and we selected a spikelet abnormal mutant in M₂ generation.The mutant trait presented stable by several generations of selfing,and was temporarily named ossp1.According to agronomic traits survey,growth and development observation,exogenous hormone treatment,endogenous hormone determination,cytological analysis,gene mapping,map cloning,four-primer hindered molecular markers,bioinformatics analysis and qRT-PCR quantitative analysis,the results showed that the gene controlling the mutant trait was cloned and its expression pattern was analyzed.The specific research results are as follows:1.By phenotypic identification of mutant,it was found that compared with wild-type KTK,mutant traits for plant height,tiller number,seed setting rate,1000-grain weight and effective kernel number presented in significantly decreasing except for increasing in grain length.The contents of endogenous hormones GA,IAA and CTK in the stem and ear showed a downward trend.The spikelets of the mutant ossp1 were not significantly different from the wild-type at the ear development stage.However,in the maturity stage of the grain wax,the inner and outer guards of the mutant showed a significant increase and broadening,and the shape of the caryopsis became slender,and the 1000-grain weight decreased by 33.1%.Cytological observation showed that the mutant ossp1 sterile lemma cells and glume cells were significantly larger than in the wild-type,and the cell structure showed obvious silicification,and there were more hairy,similar to the structure of the outer cell.2.The contents of endogenous hormones GA,IAA and CTK in the jointing stage,early filling stage and maturity stage were determined and found to decrease in the mutant ossp1.At the same time,exogenous IAA treatment experiments were carried out to confirm that ossp1 was sensitive to IAA sprayed from external sources.Thus,we speculated that OsSP1may be involved in the extensive regulation of plant growth and development activities by IAA.3.The F₂ population obtained by crossing the mutant ossp1 with YXB was used for gene localization.Genetic analysis indicated that this trait was controlled by a nuclear recessive single gene（χ²=3:1）.Using the F₂ targeting population constructed by ossp1/YXB,the target gene was located between the SSR markers RM22422 and RM22475 of the short arm of chromosome 8,and the physical distance was 212 kb.Analysis and sequencing of the localization interval revealed that at the 10th exon of the gene LOC_Os08g06480,a single base substitution occurred at the 1562th base,and G（guanine）was replaced by A（adenine）,resulting in its coding.The amino acid at position 521 is replaced by G（glycine）to E（glutamate）,and the genetic traits of this site are co-segregated.This gene has been reported to regulate spikelet development（the mutation site of this gene differs from the reported mutation site in this study）,it is confirmed that LOC_Os08g06480 is the target gene of OsSP1.4.A homologous alignment of the gene and construction of a phylogenetic tree revealed that the mutation occurred in a highly conserved domain and correlated with the transcriptional repressor TOPLESS in gramineous plants such as short anther wild rice,maize and sorghum.There is a high degree of similarity in the protein TPR2.5.To analyze the expression of candidate genes for tissue specificity,four organs including roots,stems,leaves and ears were selected for quantitative analysis by qRT-PCR.The results showed that the gene was expressed in roots,stems,leaves and ears,with the highest expression in the panicle and the stem.Quantitative expression of four genes MFS1,OsMADS3,OsMADS4 and OsMADS16 in the regulation of florescence development was detected in the mutant ossp1.It was found that they expressed different levels in different developmental stages of young ears,and they were suggested to participate in the early flower development of rice.This study found that OsSP1 is the allele of the reported gene ASP1,both encode a TPR/TPL transcriptional co-repressor,but there are still similarities and differences:ASP1and OsSP1 are located at the same gene locus,but the conserved structure of the mutation site.The source and mutation patterns of the mutant materials are different,and OsSP1 has other incidental phenotypes.Therefore,the study of OsSP1 not only can analyze the biological function of the gene in more detail,but also provide a new germplasm resource for the mutant library.The results of this study have deepened our understanding of the function of ASP1 gene and the regulation mechanism of rice spikelet development.