Isolation And Expression Analysis Of Transcription Factor Genes RsMYB28 And RsMYB29 In Radish (Raphanus Sativus L.)

Radish, belonging to the Brassicaceae family, is an important worldwide root vegetable crop and has widely grown in China and the world. Glucosinolates (GSL) are amino acid-derived natural plant products in the order Brassicaceae and well known for their degradation products role inducing resistance against generalist herbivores, microorganisms in plants and cancer-preventing properties in humans. In addition, GSL have gained increasing importance due to their function as flavour compounds and their potential use as biopesticides. MYB28、MYB29 and MYB76 are the main regulatory genes in the biosynthesis of glucosinolates. In radish, the content and distribution of GSL are directly controlled by the expression of these genes. Previous studies about regulatory genes were focused in A. thaliana, B. juncea and B. rapa. While, the molecular characterization and expression characteristics of GSL biosynthesis genes are remain largely unexplored in Brassicaceae species radish (Raphanus sativus L). In this study, we used ’NAU-ZQH’ as radish research material and obtained two GSL regulatory genes RsMYB28 and RsMYB29 through the Gene Blast. To study the characteristics, we researched their subcellular localization, transcriptional activation and expression characteristics. Laiding the foundation for producing new varieties of radish which hadhigher glucosinolate contents by means of genetically modified engineering. The main results were summarized as follow:1. Genomic DNA and cDNA of RsMYB28 IRsMYB29 genes were isolated from radish. The genomic DNA of RsMYB28 spanning 1836 bp have.The full-length cDNA of RsMYB28 was 1614 bp, and 1089 bp ORF encoding 363 amino acids. The genomic DNA of RsMYB29 spanning 1493 bp have 3 exons and 2 introns. The full-length cDNA of RsMYB29 was 1093 bp, and 1020 bp ORF encoded 340 amino acids. Amino acid sequence alignment and phylogenetic tree revealed that RsMYB28 and RsMYB29 proteins shared a close with MYB28/MYB29-\ike sequences from Brassicaceae species. The RsMYB28 proteins were grouped together with the BjuMYB28-1, Bo_MYB28-1, Bra_MYB28-3 protein, while RsMYB29 were grouped nicely with Bju_MYB29-2, Bo_MYB29. Promoter sequences analysis showed that RsMYB28 and RsMYB29 genes promoter sequence including a number of light-induced responsive elements. In addition RsMYB28 gene promoter also had a vinyl, GA, drought and others stress-related environmental stress response elements. The promoter of RsMYB29 gene also contained response elements drought, salicylic acid and circadian control elements. These results showed that these factors may be involved in these genes expression and regulation.2. Quantitative Real-Time PCR was carried out to analyze the dynamic expression pattern of RsMYB28 and RsMYB29.The results revealed that RsMYB28 and RsMYB29 were strongly expressed at the seedling and taproot thickening and the differential expression patterns of RsMYB28 induced by WO, ABA and glucose, while RsMYB29 induced by WO and MeJA. Meanwhile construct pCAMBIA1301-RsMYB28 and pCAMBIA1301-RsMYB29 overexpression vector were successfully transformed into Agrobacterium.3. Construct pGBKT7-RsMYB28 and pGBKT7-RsMYB29 vectors and transformed into Saccharomyces cereviseae strain Y2H Gold medium. Thereafter, all those transformed cell lines were plated on SD/-Trp and then streaked onto SD/-His-Ade and SD/X-a-gal. The results implied that both RsMYB28 and RsMYB29 function as transcription activators in yeast cell. The green fluorescent protein expression vectors were constructed and transformed into the epidermal cells of onion via a gene gun. The result showed that they were localized to the nucleus.