Molecular Mechanism Of MADS-box Transcription Factor SOC-like4 (SlSL4) Regulating Tomato Fruit Ripening

Fruit ripening represents a summation of physiological and biochemical processes of fleshy fruits including degreening and accumulation of colored pigments for attraction,textural changes associated with cell wall metabolism and cell turgor variation leading to softening,and metabolic changes related to flavor and nutrient composition,generally associated with accumulation of sugars,acids and volatiles culminating in a diverse array of tastes and smells varying among species.These changes not only make fruit assisting in seed dispersal,but also provide essential nutrition for human and animal diets.In recent years,research on fruit maturation,especially the regulation of the maturity of fleshy fruit,has become a hot topic for scientists.In our previous study,the RNA of AC⁺⁺,Cnr,rin and nor at different developmental stages were used in gene chip analysis,and established signal regulation network model for tomato fruit ripening by artificial neural network?ANN?.Through this neural network model,we found a lot of specific genes closely related to the ripening regulation of tomato fruit,especially the low abundance transcription factors expressed during fruit ripening.A new SOC-Lile 4?SlSL4?gene,which is closely related to tomato fruit ripening and plays important role in immature tomato mutants,has been discovered.In order to further analyze the possible function of SlSL4 in tomato fruit,the following main results have been obtained in this study.1.The SlSL4 gene was isolated and cloned?GenBank accession number:XM₀04234224?.The full length of CDS region of SlSL4 gene is 498bp,encoding 165amino acids.SlSL4 localizes to the nucleus and has multiple nucleic acid and protein binding sites.SlSL4 belongs to the MADS-box gene family and has a typical MADS domain.2.Analysis of the expression pattern of SlSL4 gene by Quantitative real-time PCR technology?QT-PCR?,we found that SlSL4 is expressed in the flowers,fruits,sepals,and roots of tomato,and is expressed in high level in flowers and fruits,but is less expressed in leaves.Further analysis showed that the expression level of SlSL4 in AC⁺⁺,rin,nor and Nr were significantly different.The expression pattern analysis of SlSL4 in the leaves of tomato after treatment with various hormones showed that the gene might be involved in the regulation of ethylene.3.To further analyze the function of SlSL4 in the regulation of tomato ripening,we constructed the overexpression and silencing vector of this gene,and transformed the wild tomato AC⁺⁺by Agrobacterium-mediated method,and obtained transgenic tomato mutant lines with over expression and silence of SlSL4 gene.Through the study of SlSL4 transgenic tomato fruit,we found that the maturity of transgenic tomato fruit is delayed compared with wild tomato fruit under the same environment,and the silenced transgenic tomato plants are susceptible to shedding and are not easily fruited.4.The function of the SlSL4 gene was analyzed at the mRNA level and the results showed:The expression of ethylene synthesis related genes?ACO1,ACO3,ACS2?in transgenic tomato decreased significantly,and the ethylene responsive genes?E4,E8,ERF1?were inhibited in transgenic tomato.And the carotenoid synthesis gene PSY1 in the fruits of transgenic tomato is significantly different from that of the wild type fruit.These results suggest that SlSL4 gene may delay fruit ripening by participating in the regulation of the expression of ethylene related genes.5.Since MADS-box proteins are usually regulated by interaction,therefore,this study explored the interaction between SlSL4 protein and other MADS-box proteins involved in tomato fruit regulation.Yeast two hybrid results show that,SlSL4 protein can interact with SlMADS-RIN,SlMADS1 and SlTAGL1 protein respectively,indicating SlSL4 possibly interacts with SlMADS-RIN,SlMADS1 and SlTAGL1 to influence ethylene thereby influence tomato fruit ripening.However,there is no interaction between SlSL4 protein and tomato absicission zone development related protein?SlMADS-MC and JOINTLESS?,indicating that SlSL4 protein may not control the loss and results of flowers by interacting with SlMADS-MC and JOINTLESS proteins to form complexes.