Functional Analysis Of Fruit-Weight2.2-Like Gene Family In Rice

Tomato fruit-weight 2.2 (FW2.2) was reported to control up to 30% fruit weight. It was demonstrated that the homologs of FW2.2, named FW2.2-like (FWL), also play important roles in plant growth and development. For instance, a maize homolog of FW2.2, named cell number regulator 1 (CNR1), negatively regulates plant and organ size. Rice is an important human food crops. However, FWL genes in rice have not been characterized yet. In this study, we focused on molecular characteristics and functional analysis of OsFWL gene family. Using bioinformatics, genetic transformation, and T-DNA insertion mutant phenotype identification we have studied gene structure, expression and function of OsFWL. Main results are shown as following:Based on sequence homology and conserved domain analysis, we found that there are eight OsFWL genes in rice genome. We conducted a series of bioinformatics analysis of OsFWL gene family, including chromosomal localization, gene structure, protein conserved domains, evolutionary relationships. We found that 8 OsFWLs are distributed on 3 rice chromosomes. Among them OsFWL4,6,7, and 8 form a gene cluster at the end of the long arm of chromosome 3. The aa sequences of OsFWL proteins range from 136 to 186 aa. OsFWL proteins contain a conserved CCXXXXCPC motif except for OsFWL 1 and 3.Cooperation lab found a rice grain weight QTL located at the end of the long arm of chromosome 3. It was indicated that OsFWL gene cluster may be the effective genes of this QTL. By screening a Zhenshan 97 BAC library, we have obtained a Zhenshan 97 BAC clone containing the OsFWL gene cluster. Using shotgun sequencing, we found three polymorphic markers. The BAC clone was digested with Not I and the fragment containing the OsFWL gene cluster was ligated to a BIBAC vector. The BIBAC was used to transform the rice variety Zhonghua 11.The RT-PCR result and microarray data revealed that OsFWL genes exhibited diverse expression patterns. OsFWL3 is expressed only in the panicle in heading stage and the OsFWL4 expression is very low throughout the growth period. The detailed expression patterns of OsFWL5,6, and 7 negatively correlated with leaf growth activity. Co-expression analysis showed that OsFWL 1,2,5 co-expressed with many zinc finger proteins and ubiquitin-related proteins. Rice protoplast transient transformation experiment showed that most OsFWL proteins locate at cell membrane but OsFWL8 is present in the nucleus. Prokaryotic expression and purification of OsFWL 1,2,4 proteins for in vitro enzyme activity assay are performed.Over-expression and RNAi vectors of some OsFWL genes were constructed and transformed into rice and the independent transgenic plants were obtained. In TO generation the expression of the target gene was checked. The results showed that in overexpression and RNAi plants, expression of the target gene has been increased and decreased, respectively. Overexpressing CNR1 or OsFWL1 resulted in phenotypes of small plant, no tiller and no seed. Insertion mutant analysis showed that compared to wild-type the grain length of osfwl3 mutant was increased by 5.8% and grain weight was increased by 5.3%. We also found that the increase in grain length of osfwl3 mutant was due chiefly to incremental cell number, not cell size and the expression of OsFWL3 negatively correlated with glume growth activity. We found that the plant height of osfwl5 mutant was increased by 12.5%. Further analysis revealed that the increase was mainly occurred in the first internode.In rice, there are two casein kinase II (CKII) regulatory subunit homologous proteins. The bimolecular fluorescence complementation assay proved that OsFWL1 interacted with the encoded protein of OsO7g31280, but not with that of Osl0g41520.These results provide a comprehensive foundation of further study OsFWL...