Functional Studies Of SlRCHY1 And SlPL In The Regulation Of Tomato Fruit Development And Ripening

Tomato,native to South America,is an important vegetable crop.The Incas and Aztecs began cultivating tomato plants as early as 700 A.D.Tomato fruit is rich in protein,vitamins,antioxidants and trace elements,and it is also an excellent model biomaterial for the study of fleshy fruit development,ripening and post-harvest softening.In this study,the effects of zinc finger protein transcription factor(SlRCHY1)and pectin lyase(SlPL)gene on fruit ripening and softening in Chinese tomato(Solanum lycopersicum Mill.var.Ailsa Craig,AC++)tomato were studied.The results showed that the two genes play an important role in the process of tomato fruit development and maturation.Excavating the key genes that control the development and ripening of tomato fruit and studying its biochemical metabolism and molecular regulation mechanism are of great significance for the breeding of high-quality and storage-tolerant tomato fruits in the future.Zinc finger protein transcription factor SlRCHY1(RCHY1,Solyc08g067960.2)belongs to C3H2C3 Ring-H2 transcription factor family.It exists mainly in eukaryotes and achieves its goal by regulating the transcription and translation of target genes.In higher plants,C3H2C3 transcription factors play an important role in cell differentiation,apoptosis and stress.Gene expression pattern analysis showed that SlRCHY1 gene was mainly expressed in mature and senescent plant tissues,indicating that the gene might play a role in the mature stage.In order to understand the role of the gene in the maturation of the fruit and to explore the molecular mechanism of the RING-H2 type zinc finger protein,we use the tissue-specific promoter AcMADS1 to drive the target gene in tomato fruit development at a high level.Overexpression of the SlRCHY1 gene resulted in a significant shortening of the tomato fruit from flowering to break.Compared with wild-type tomatoes,the chlorophyll content of the pericarp of SlRCHY1 overexpression(SlRCHY1-OE)was significantly lower than that of the wild type,and the carotenoid content was significantly higher than that of the wild type.The results of Realtime Fluorescence Quantitative PCR showed that over-expression of SlRCHY1 could induce the regulation of ABA synthesis and signal transduction pathway gene expression of fruit senescence hormone abscisic acid and increase the content of abscisic acid in fruit compared with wild type.At the same time,transgenic plants also showed a series of physiological and biochemical changes,such as tomato fruit decreased hardness,increased soluble solids content and high level expression of carotenoid synthesis gene PSY1,PDS,ZDS.The subcellular localization of SlRCHY1 gene in the nucleus indicates that it may play a functional role in the growth and development of tomato fruit.In conclusion,SlRCHY1 gene plays an important role in tomato fruit development and ripening.Hardness is an important index to affect the storage tolerance of tomato fruit.Pectin lyase(PL)is one of the members of pectin degrading enzyme family.It mainly cleaves ?-1,4-galactoic acid group in pectin by ?-elimination,and achieves the purpose of cell wall disintegration,cell separation and fruit softening.In order to clarify the specific mechanism of pectin lyase(PL)in the ripening and softening of tomato fruit,we carried out the function study of the corresponding gene of the pectin lyase(SlPL)that specifically expressed the development of tomato fruit.The expression pattern of SlPL(soly064g083580.2)gene was analyzed,and it was found that the slpl gene was expressed in the young stems,flowers and young fruits of the tomato,indicating that the gene might be closely related to the growth and development of the stem,the flower and the fruit.In order to further explore and analyze the function and regulation mechanism of SlPL gene,we construct the SlPL silencing vector based on the rnai technology and obtain the transgenic line with better silencing effect.After silencing SlPL gene,tomato fruit firmness increased,pericarp thin,mesocarp cells arranged dense and cell volume increased,from exocarp to endocarp cells closely arranged.The transgenic tomato fruits silenced by SlPL showed more tolerance to storage.Further studies on cell level,molecular level,physiological level,and post harvest storage showed that silencing SlPL resulted in the decrease of Pectinase activity in fruits.The genes of pectin synthesis,extensin and glucosyltransferase were significantly lower.But there was no difference between transgenic tomato and wild-type tomato fruit in apparent index,In addition,the results of Realtime Fluorescence Quantitative PCR also confirmed the related phenotypic changes of fruits.These results suggest that RNAimediated specific silencing of SlPL gene can effectively enhance fruit firmness,decrease softening rate and prolong shelf life of tomato fruit,which provides important theoretical reference for artificial directional regulation of tomato and fruit of other species.