Phenotype Analysis,Gene Identification And Function Study Of Small Grain Mutant Sg3 In Rice(Oryza Sativa L.)

As China’s urbanization rate continues to increase,the area of arable land is decreasing year by year,and the situation of maintaining national food security is severe.Although China strictly adheres to the bottom line of 1.8 billion mu of cultivated land and fully implements the permanent basic farmland protection policy,how to use limited land resources to increase food production has been the research direction of agricultural workers.Rice,as an important monocotyledon model plant,is one of the most important food crops in China.Its yield consists of ears per unit area,total grains per ear,seed setting rate and thousand-grain weight,in which thousand-grain weight is affected by grain type factors（grain length,Grain width,grain thickness）regulation.Therefore,analyzing the genetic regulation mechanism of rice grain shape is helpful to understand the molecular mechanism of yield and provide a strong theoretical basis for improving rice yield.In this study,a small grain mutant sg3（small grain 3）was obtained by screening the EMS mutant library of the heavy ear hybrid rice backbone parent Shu Hui 498（R498）,and the phenotype analysis,genetic analysis and gene identification were performed.The main results obtained are as follows:1.Phenotypic analysis showed that,compared with R498,the sg3 mutant showed a reduction in plant height of 8.70%,a decrease in grain length of 12.59%,no significant difference in grain width,a decrease in ear length of 6.64%,and a decrease in thousand-grain weight of 17.50%.The number of primary branches was not significantly different,but the number of secondary branches increased by 14.8%,resulting 15.16%in an increase in the number of grains per ear.There was no significant difference in seed setting rate and effective panicle number per plant,and there was no significant difference in final yield per plant.2.Scanning electron microscope analysis of cytology revealed that the cell length of the sg3 mutant glume outer epidermal cells was significantly shorter than that of R498.In addition,quantitative analysis found that the expression of multiple cell expansion-related genes was significantly down-regulated in the sg3 mutant,suggesting that SG3 may affect cell expansion-related gene expression,regulate cell elongation,and ultimately affect particle length.3.F₂ isolated population was constructed by crossing sg3 with R498.Genetic analysis showed that the phenotype of the sg3 mutant was controlled by a pair of semidominant nuclear genes.In the F₂ segregated population,30 extremely small single plants were selected for sequencing by pooling,and gene mapping was performed using the Mut Map method.An obvious linkage segment was identified on chromosome 10.A total of three SNPs with a SNP index of 1 were identified in this segment,of which only SNP3 was located in the coding region of the gene LOC＿Os10g42110 resulting in an amino acid substitution（Ala302Thr）,which encodes a BSK family kinase Os BSK2.Analysis of expression patterns found that the gene was highly expressed in developing young ears and glumes,consistent with its biological function of regulating grain development.The knockout mutant of Os BSK2 in the Nipponbare background showed a consistent change trend with sg3,indicating that this gene is a candidate gene that leads to the sg3 phenotype.4.The BR sensitivity test experiment confirmed that Os BSK2 knockout mutants are consistent with BR in susceptibility to WT.It is speculated that the Os BSKs family has functional redundancy in the BR signal transduction pathway,and Os BSK2 is not the main gene of the family in the BR transduction pathway.5.Subcellular localization analysis found that Os BSK2 is localized to the cell membrane,and the N-terminal myristoylation site is essential for its membrane localization.BIFC experiments confirmed that Os BSK2 can form homodimers by itself and interact with Os BSK3 and Os BSK4 to form heterodimers.The grain size of the interfering plants of Os BSK3 and Os BSK4 also became significantly smaller.It is speculated that the Os BSK gene in rice may regulate the grain size by forming homo or heterodimers.6.Under the same background of R498,we hybridized and polymerized the sg3mutant with 08sg2/Osbak1 and NIL-GS3^R3551,respectively,to obtain the bidendrial lines sg3/08sg2 and sg3/NIL-GS3^R3551.Phenotypic analysis revealed that the grain shape of the double-stranded lines was a simple accumulation of the single-stranded lines,and there was no genetic interaction effect,indicating that SG3 and Os BAK1 and GS3 regulate the grain type through independent pathways.