Functional Analysis Of OsFWL2 Gene Involved In Grain Number Regulation In Rice

With the growth of world's population,food shortage has become a serious worldwide problem.Rice is one of the most important food crops,feeding more than half of the world's population.Hence,increasing the output of rice is of great significance to meet the increasing demand for food and ensure food security.fruit weight 2.2(FW2.2)is a major QTL controlling fruit size in tomato,accounting for 30%of tomato fruit size variation.Studies have shown that the FW2.2-like(FWL)genes are widespread in plant genomes and regulate the sizes of organs and/or fruits(seeds)by negatively regulating cell division.Some of them also participate in the response to metal ions such as cadmium.In this study,the novel CRISPR/Cas9 genome editing system was used to generate two gene knockout mutants of FWL2 in rice.Phenotypic analysis showed that traits such as plant height and flag leaf area of the mutants were not significantly different from that of the wildtype(WT);however,the grain numbers and yields were predominantly higher than that of the control.RNA-seq was further adapted to illustrate the molecular mechanism of the gene regulating grain number in rice.The main findings are as follows:(1)two CRISPR/Cas9 genome editting vectors Osfwl2a and Osfwl2b,targetting the coding sequence of OsFWL2,were constructed and transformed into japonica rice variety Zhonghua 11 using an Agrobacterium tumefaciens transformation method.Eight and 14 transgenic positive plants were obtained respectively.Mutant genotype analysis showed that the majority of mutation types were insertions or deletions of a few base pairs.Among the Osfwl2a transgenic plants,one was a homozygous mutant(accounting for 12.5%),six were biallelic mutants(accounting for 75%)and one was WT(accounting for 12.5%).Three of the Osfwl2b transgenic plants were homozygous mutants(accounting for 21.4%),nine were biallelic mutants(accounting for 64.3%)and three were WT(accounting for 21.4%).None of the vectors generated heterozygous mutants.(2)One homozygous mutant of Osfwl2a and Osfwl2b,respectively,was selected for further analysis.Among them,the mutant for Osfwl2a contains an 1-bp insertion and was named osfwl2a,while the mutant for Osfwl2b contains a 2-bp deletion and was named osfwl2b.Mutations carried by both mutants were transmitted stably in offsprings.Subsequently,a pair of HPT primers and a pair of sgRNA primers were used to identify the T-DNA construct in Ti generation mutants,and homozygous mutants without a T-DNA insertion were successfully isolated from both lines.(3)The plant height,flag leaf area,number of panicles per plant,panicle length and number of primary branches per panicle were not significant from that of WT.However,the grain number of the mutants increased by 14%to 18.6%and the yields per plant increased by 17.2%to 17.3%,comparing with WT.Further analysis showed that the numbers of secondary branches per panicle of the mutants increased by 10.9%,suggesting that the increase in grain numbers of the mutants may be caused by an increase in the numbers of secondary branches.(4)Analysis of gene expression using fluorescent quantitative RT-PCR showed that OsFWL2 was mainly expressed in developing leaves,but was also in developing roots,stems,panicles and seeds,and its expression in stems,panicles and seeds increased with the development of the tissues.In addition,the gene expression was not affected by cadmium treatment in both leaves and roots,indicating that it may not be involved in the response to cadmium stress.(5)RNA-seq analysis of WT and mutant young panicles at branch and spikelet differentiation stage identified a total of 3,406 differentially expressed genes,among which 1,383 were up-regulated genes and 2,023 were down-regulated genes.KEGG enrichment analysis showed that these genes might be involved in physiological processes such as plant hormone signal transduction and photosynthesis.In summary,this study successfully constructed genome editting mutants of the rice FWL2 gene using CRISPR/Cas9 technology and obtained transgene-free homozygous mutants at the T1 generation,suggesting that the CRISPR/Cas9 system is a powerful method for gene function studies in plants,especially in important crops.The secondary branch numbers,grain numbers and yields per plant of osfwl2 mutants increased significantly.Further RNA-seq analysis showed that the gene may negatively regulate the above yield traits by regulating plant hormone signalling.These results lay a foundation for the further functional study of the gene and its application in molecular breeding of rice.