Genome-wide Identification Of R2R3MYB Gene Family And Functional Analysis Of SlMYB41 And SlMYB64 In Tomato

Tomato(Solanum lycopersicum L.)is not only one of the most important vegetable all over the world,but is also a model system for study.In the process of tomato development,biotic and abiotic stress had serious impact on tomato yield and quality.The R2R3 MYB subfamily is the most abundant type of the MYB superfamily and plays regulatory roles in developmental processes,secondary metabolism and stress responses in plants.To date,in tomato,only few R2R3 MYB genes were cloned and identified.The completion of tomato genome sequence provides an opportunity for using reverse genetics to study genes function.In this study,the comprehensive genomic and functional analyses of 121 R2R3 MYB genes were systematically carry out in tomato.The expression profiles during plant development and in response to abiotic conditions were compared among the 51 selected R2R3 MYB genes which may involve in stress responses by quantitative real-time PCR.Finally two genes were chosen to further studied.The main results are as follows:1.One hundred and twenty-six R2R3 MYB protein sequences in Arabidopsis were used to identify tomato R2R3 MYB proteins and finally 121 typical R2R3 MYB genes were confirmed by Pfam and SMART program.The detailed information on the domain sequence features,genomic structures,chromosomal locations and phylogenetic analysis among R2R3 MYB genes in tomato,Arabidopsis,rice,soybean,poplar,grape,cucumber and apple were presented.2.According to the Arabidopsis functional clades where SlR2R3 MYB genes were in the phylogenetic tree,we selected 51 putative tomato R2R3 MYB genes responding to abiotic stress to gain insights into their role in abiotic stress tolerance and in tomato growth and development.Among these genes,10 genes respond to NaCl treatment,11 genes to ABA and 14 genes to low temperature.Tissue specificity or different expression levels of SlR2R3 MYBs in different tissues suggested differential regulation of tissue development as well as metabolic regulation.3.The coding sequence of SlMYB41 gene was cloned into the pBI121 vector driven by CaMV35 s promoter and the resulting construct was transform into tomato.The transgenic lines were chosen for gene expression and morphology analysis.The results revealed that overexpression of SlMYB41 gene could influence plant development,including plant height,leaf size and seed size.4.To further investigate the effect of SlMYB41 on tomato development,transcriptomic analysis was performed using RNA from the seedlings of the wild type and SlMYB41-OE line.The differential expression genes associated with stress response were significantly enriched,suggesting that SlMYB41 may involve in stress resistence.5.The promoter of SlMYB41 was used to screen its interaction proteins with yeast one-hybrid approach.As a result,an ERF protein similar to Arabidopsis ERF105,was obtained.In addition,full-length CDS sequences of SlMYB41 were subcloned into pGBKT7 vector and transformed into yeast strain AH109.Result showed that SlMYB41 has obvious transcriptional activation activity.Further study revealed that the domain of transcriptional activation activity may between 269 aa and 334 aa.6.To investigate the effect of SlMYB64 in tomato,phylogenetic analysis was performed using SlMYB64 and its homologs from the published annotated genomes and papers.In this clade,the function of SlMYB64 involved in pollen development is new.7.Compared with the non-transgenic plant,SlMYB64-OE plants exhibited decreased growth vigor at both seedlings and anthesis stage,including plant height,root length,leaf size indicating that SlMYB64 is negatively correlated with the plant growth.8.To examine in detail the defects of anther development in SlMYB64-OE line,both wild-type and SlMYB64-OE line anthers were examined by microscopy of anther semi-thin sections to further clarify its role in pollen developmental process.These results showed that the anther development was abnormal in the SlMYB64-OE line,particular tapetal development,PCD and pollen development.The qRT-PCR analysis showed that the expression of SlMYB64 was gradually decreased as the anther development.Whereas,in SlMYB64-OE line,SlMYB64 expression was 6-fold at all stages.In vivo pollen germination assay revealed that SlMYB64 also reduce the male fertility.9.To further investigate the effect of SlMYB64 on tomato pollen development,transcriptomic analysis was performed using RNA from the anthers of the wild type and SlMYB64-OE line at stage 13.The GO term ?pollen wall assembly? and ?pollen exine formation? were significantly enriched,implying that SlMYB64 could play a direct or indirect regulation role in regulating pollen wall development.The DEGs in SlMYB64-OE line and in sldyt1 were compared,a total of 26 genes were commonly variation between them,implying that SlMYB64 and SlDYT1 may have common downstream regulatory pathway for anther development.Yeast two-hybrid assays revealed that SlMYB64 was unable to directly interact with SlDYT1.These results indicated that SlMYB64 and SlDYT1 act distinct pathways to regulated anther development in tomato.