Cloning And Functional Analysis Of SlMYB1R-1 Gene In Tomato

The large MYB gene family has diverse functions and plays crucial roles in plant development and stresses response.The role of tomato MYB-related TFs in drought and salt stresses tolerance are largely unknown.Tomato(Solanum lycopersicum)is relatively vulnerable to abiotic stress conditions such as drought and salt,but the tolerance mechanisms for such stresses are still unclear in tomato.In this research,we identified the role of the SlMYB1R-1 gene in regulating salt and drought tolerance in tomato.SlMYB1R-1 was cloned from cDNA,and the gene structure and protein conserved domain were analyzed by bioinformatics methods.The temporal and spatial expression patterns of the SlMYB1R-1were studied in tomato.Phylogenetic analysis of MYB-related proteins was performed.We constructed the overexpression vectors and RNAi suppression expression vectors for tomato transformation.Moreover,we analyzed the promoter information of the SlMYB1R-1 and investigated the gene expression patterns under hormones and abiotic stress treatments.Since stress conditions generate reactive oxygen species(ROS),its production affects the antioxidant system of plants,several genes involved in ROS scavenging and plant defense were monitored in WT and transgenic plants under both normal and stress conditions by q RT-PCR.Moreover,the SlMYB1R-1 gene fuction was assessed by physiological parameters including MDA and soluble protein.We preliminaryly explored the biological function of SlMYB1R-1 gene in tomato growth and development.The main results are as follow:(1)To identify stress-related factors in tomato,we previously carried out bioinformatics methods to analyze the gene structure of SlMYB1R-1 was 1523 bp,contained 3 extrons and 2 introns and encoded a polypeptide of 302 amino acids.Amino acid sequence alignment results showed that SlMYB1R-1 encoded a putative R1-type MYB-like transcription factor.Evolutionary relationship analysis showed that tomato SlMYB1R-1 had high homology with StMYB1R-1 in potato(Solanum tuberosum)plants.(2)Promoter components analysis showed that there were abscisic acid and ethylene response components as well as some adversity related response components.WT seedlings were treated with different hormones and stress,and then qRT-PCR analyses were performed.SlMYB1R-1 transcripts were significantly up-regulated by drought and salt stress in leaf and root and the fold change was higher in root than leaf.Meanwhile,SlMYB1R-1 transcripts were up-regulated by all five hormones,including ABA,Eth,KT,IAA,and SA.(3)To further investigate the potential role of SlMYB1R-1 in tomato development,samples from roots,stems,leaves,flowers,and fruits at different stages were collected to perform qRT-PCR.The results showed SlMYB1R-1 gene expressed in variety of tissues.These data suggested that SlMYB1R-1 might have important roles in vegetative growth and reproductive development.(4)Based on GUS staining and qRT-PCR results,three down-regulated transgenic tomato lines were obtained.When wild type and RNAi transgenic tomato were treated with salt and drought,we found that down-regulated transgenic tomato grew better growth status.(5)The expression levels of several genes involved in ROS generation and scavenging,including CAT,SOD,GST,POD,LOX,and ascorbate peroxidase(APX),were monitored in Wild type and transgenic plants under both normal and stress conditions by q RT-PCR.Expression levels of three genes,CAT,LOX and PR1,were activated in both WT and transgenic tomato plants.(6)Several physiological parameters were measured in leaves from treated plants.After salt and drought treatments,soluble protein content was obviously increased in down-regulated transgenic tomato.Meanwhile,transgenic plants had less MDA contents.Taken together,our results suggest that SlMYB1R-1 gene plays a negative role under salt and drought stresses.Hence,it may serve as a useful biotechnological tool for the genetic improvement of stress tolerance in crops.