Cloning And Functional Analysis Of Tomato GRAS Family Gene SlFSR

GRAS family proteins are plant-specific transcription factors, which are typicallycomposed of400-770amino acid residues and exhibit conserved motifs in theircarboxy-terminal, including LHR I, VHIID, LHR II, PFYRE and SAW. Theamino-terminal of GRAS proteins vary highly in sequence and length.There are a largenumber of GRAS proteins in higher plants. GRAS proteins have been found to play aregulatory role in plant development, meristem maintenance, signal transduction anddisease resistance.However, most of GRAS proteins will still need to be clarified.Fruit constitute important components of human and animal diets and nutrients.Tomato has long served as the model of fruit ripening research, primarily because it hasa small genome, advanced genetic transformation technology and rich genomicresources. In recent years, the characterization of tomato mutants at the molecular levelhelps us to understand the physiological and biochemical processes of fruit ripening.In our study, we have searched PlantTFDB and NCBI to get51tomato GRASgenes. These genes were divided into nine subfamilies of SHR, SCR, SCL9, PAT1,SCL3, DELLA, SCL4/7, Ls, and HAM according to the results of phylogenetic analysis,which was performed using the Neighbor-Joining method. Tomato GRAS genes weremapped in the terminals of12chromosomes by BLAST tomato genome database. Andeach GRAS gene had only one copy in the chromosome. Then six cDNA libraries ofTIGR database were used to search for ESTs of predicted GRAS genes. We conducted adigital expression profile analysis to excavate a GRAS gene, SlFSR, which wasspecifically expressed in fruit ripening.SlFSR was a member of SHR subfamily and located on chromosome7.SlFSRencoded a protein composed of429amino acid residues. There were severalpotential Ser/Thr phosphorylation sites and Tyrphosphorylation sites in SlFSR protein.It indicated that post-translational modification might affect the biological activity ofSlFSR in vivo. We also predictedseveral plant hormone response elements and threepotential RIN binding sites within the promoter of SlFSRgene.We found that SlFSR gene was specifically expressed in the tissues of ripeningfruit by Real-time PCR analysis. The expression levels of SlFSR were down-regulatedin Nr fruit and werealmostabolished in rin fruit. The expression ofSlFSR could beinduced by exogenous ethylene.These results suggest thatSlFSRmight function at the downstream of RIN and ethylene signal.RNAi silence vector targeting SlFSR was conducted and transferred toAgrobacteriumtumefaciens strain LBA4404. Then the construct was used to transformtomato cotyledon explants. We obtained five transgenic tomato lines. The expressionlevels of SlFSR in B+4fruits of1-4transgenic lines were down to97.1%,96.6%,94.4%and96.9%respectively. We also found that cell wall metabolism related genes of fruit,such as PG, CEL2, and TBG4were inhibited in B+4fruits of transgenic lines,accompanied with increased tomato fruit storability. However, the expression level ofPE1was up-regulated in B+4fruits of transgenic lines. These results suggest that SlFSRmight be involved in the regulation of fruit softening process during fruit ripening.