Function Alanalysis Of OsFBX13 Involved In Grain Size Regulation In Rice （Oryza Sativa L.）

Rice（Oryza sativa L.）is the largest food crop in China,so it is very important to stabilize and improve rice yield for food security.Grain size is one of the important factors for rice yield.In recent years,many progresses have been made in the identification,cloning and functional verification analysis of rice grain size regulation related genes.However,because grain size is a very complex quantitative trait,its genetic and regulatory molecular mechanisms need further in-depth analysis.According to the information of the common transcriptome database in rice grain development,we screened the genes that may be related to the regulation of grain size,and obtained gene knockout mutants to explore the effects on rice grain size and possible regulatory mechanisms.The main results are as follows:1.Based on the information from the public transcriptome database of rice grain development,897 genes possibly related to grain size were screened.The grain size of the corresponding mutant in CRISPR/cas9 gene knock-out mutant library of "Baige Gene Technology Co.,Ltd." was investigated.It was found that the grain width and thickness of the mutants for OsFBX13（F-box gene with unknown domain 13）were significantly increased compared with wild type "Zhonghua 11",indicating that the gene may be a negative regulation role in grain size.By separating and identifying the mutant offspring,two independent homozygous mutant lines were obtained.When grown in Lingshui,Hainan Province,osfbx13 mutants significantly increased in grain width,grain thickness and grain weight,but no significant difference in grain length compared with wild type;the mutants showed relatively shorter panicle length and plant height,but no significant difference in the number of effective tillers compared with wild type.The osfbx13 mutants grown in Nanjing,Jiangsu Province,displayed similar phenotypes to those in Lingshui,except a significantly lower seed setting rate.Further observation found that the decrease in seed setting rate of the mutants might be caused by anther indehiscence,which indicates that OsFBX13 was also involved in the regulation of environment-mediated seed setting rate.2.Bioinformatics analysis showed that OsFBX13 encodes a member of the F-box protein family,which participates in SCF（Skp1-Cullin-F-box）E3 ligase complex and plays a role in substrate recognition.OsFBX13 has an F-box domain and an NTF2（Nuclear transport factor 2）-like domain,the latter is the first found in F-box proteins.The yeast twohybrid experiment confirmed the interaction of OsFBX13 with OSK1,the main SKP1（Sphase Kinase-associated Protein 1）protein in rice,through the F-box domain,indicating that OsFBX13 participates in the formation of SCF E3 ligase complex.By screening yeast twohybrid library,we identified 14 proteins that possibly interact with the NTF2-like domain.Subsequenly,five of the alternative interacting proteins were confirmed by one-to-one of yeast two-hybrid tests.Through bioinformatics analysis,two of the interacting proteins were suggested as the substrates of OsFBX13,one is the nucleoporin OsALADID,which might be related to the regulatory function of OsFBX13 in grain size,the other is the transcription factor OsARF8/12,which might be involved in the regulation of OsFBX13 in seed setting rate.In this study,OsFBX13,a new grain size regulatory gene in rice,was discovered and identified,and two proteins that interact with OsFBX13 were obtained,which is helpful to reveal the new node of grain size regulatory network and provides theoretical basis and gene resources for grain size genetic improvement in rice.