Cloning And Functional Analysis Of SlGRAS1 In Tomato

TFs(Transcription factor)are also known as transacting factor,which could specifically bind to cis acting elements in promoter region of eukaryotic and activate or repress gene transcription process.They take interactions to regulate the expression of target genes.GRAS protein family is one of the specific transcription factors in higher plants.According to researches,this family plays verious important roles in gibberellin(GA)signal pathway,maintain of meristems,phytochrome signal pathway and plant resistance process.Typical GRAS family member is usually composed of 400 to 770 amino acids,whose C-terminal are highly conserved,N-terminal are complex and varied which determine the function of protein.So far,the functions of a majority of GRAS proteins remain undiscovered.This study chose SlGRAS1 protein from GRAS family as the research object.SlGRAS1 gene was first isolated from tomato genome for the first time,and the gene structure,promoter region,physical and structural features of the protein were identified,as well as phylogenetic tree analysis;The expression pattern of SlGRAS1 gene in different tissues and stages and response to stress and exterior hormones treatments were analyzed by Real-time PCR method;Overexpression vectors and antisense vectors were constructed and transformed into tomato mediated by Agrobacterium tumefaciens,meanwhile,phenotypic observation and the expression of mature related genes were obtained.We preliminary explored the biological functions of SlGRAS1 gene in fruits ripening.The main results of this study are as followings:1.With the help of bioinformatics methods,we found the gene sequence length of SlGRAS1 was 1629 bp,which coding 542 amino acids as a complete exon,containing a typical feature of GRAS family.And its promoter region had varieties of function elements,such as light response elements,hormone response elements and stress response elements.2.According to the predicted analysis of SlGRAS protein,the molecular weight and the isoelectric point of SlGRAS1 protein were 60.5 KDa and 5.52,respectively.SlGRAS1 protein was a kind of unstable fat soluble hydrophilic protein,without signal peptide and transmembrane domain,which meant that it could not be secretory protein or transmembrane protein.And SlGRAS1 belonged to PAT1 subfamily,had 83% similarity with At SCL13 in amino acid sequence.3.On the basis of gene expression pattern,SlGRAS1 gene had high expression level in flower and stem,and expressed significantly high in fruits set stage and breaker stage compared with other stages.Under stress treatments,low temperature and high temperature treatments had more obvious influence in gene expression rather than oxygen stress,osmotic stress and salt stress.Exogenous treatments showed that the expression level of SlGRAS1 gene was regulated by hormones in the transcriptional level,and that IAA and ethylene could significantly increase the expression level of SlGRAS1 gene.4.Over-expression vector K303-35S-SlGRAS1-GFP and antisense vector pCambia1301-AsSl GRAS1 were constructed and transformed into tomato mediated by Agrobacterium tumefaciens to acquire the transgenic lines,the overexpression transgenic plants present higher height,longer internode length and longer fruit ripening period.5.Considering the phenomenon of delayed fruits ripening,we acquired cDNA through RT-PCR by using extracted RNA in overexpression plant fruits from four periods: MG,Br,Br+4 and Br+7.We also detected the relative expressions of genes related to fruit ripening in tomato fruits via qRT-PCR,and found that the overexpression of SlGRAS1 gene affects the expression level of tomato genes relative to ripening,such as RIN,NOR,ACO1 and PSY1.They were all in downward trend,thus affect the ethylene synthesis and regulation in fruit development and led to the delayed fruit ripening.