Identification And Characterization Of R2R3Myb Gene Responding To Abiotic Stress In Citrullus Lanatus

MYB transcription factor gene is one of the largest family genes in plants, which involved in regulation of secondary metabolism responding to hormones, environmental factors, also regulation of cell differentiation, cell cycles and the formation of leaves and other organs. In this study, watermelon varieties97103(C. lanatus (Thunb.) Matsum.&Nakai var. lanatus) was used as tested material. The watermelon R2R3MYB transcription factor gene family were identified and classified in the genome-wide level by bioinformatics, and analyzed its gene structure, protein domain characteristics, phylogenetic relationships. Then gene expression was researched under abiotic stresses, including cold, drought, salt and ABA using RT-PCR. Transcription factor ClMYB55was selected to construct over-expression vector. While the wild watermelon W001which preserved by laboratory was used as experimental material to optimized watermelon regeneration and genetic transformation system. The main results were obtained as follows.1. Watermelon MYB transcription factor family was identified at the genome-wide level using Hidden Markov Model, watermelon genome database. It contained234genes, and79of them were R2R3MYB transcription factor. Although each of the11watermelon chromosomes contained some watermelon R2R3MYB genes, the distribution seemed to be uneven. The large number of R2R3MYB genes were found on chromosomes2,3,6,10. Most of genes located in top and bottom of chromosome; small part of genes located in the middle of chromosome. The intron-exon structure of79R2R3MYB genes is similar. The number of exon ranged from one to four and76%of genes contained three exons. Two kinds of exons were highly conserved, the fragment sizes were130bp,133bp, respectively, they distributed at both3’ends of the gene. Within the79watermelon R2R3MYB proteins, all the R2repeat sequences contained three tryptophan residues. However, in the R3repeats, the first tryptophan residue was replaced by phenylalanine. The second and third tryptophan residues were conserved in all the members.2. When the watermelon seedlings were at the two-true-leaf stage, four treatments were conducted respectively:100mM NaCl,100μM ABA, low temperature (10℃), drought (15%PEG). The leaves and roots were collected separately used for gene expression. One pair of specific primers was designed for each gene, to amplify the fragments of79R2R3MYB genes by RT-PCR. The results showed that only44genes involved in stress response, and5genes are constitutively expressed.37genes responded to a variety of abiotic stresses.2genes were able to respond to all four treatments in leaf and21genes to two or three treatments.14genes could respond to only one abiotic stress.16genes responded to low temperature;18genes responded to drought;20genes responded to ABA;15genes responded to salt.3. Cotyledon at5days seedlings induced by seeds of wild watermelon was used as explants to establish an efficient plant regeneration system. Four benzyladenine (BA) concentrations were tested using cotyledon explants. Cotyledon explants were cultured on Murashige and Skoog medium (MS) containing test concentration of benzyladenine(1.0,2.0,3.0and4.0mg/L). Adventitious shoot organogenesis was initiated in all induction media and the differences among BA concentration were significant. The data were analyzed by ANONA. The results showed that explants cultured in MS+BA (1.0mg/L) achieved the highest regenerated rate and6shoots regenerated from per explant were observed. Adventitious shoots were able to elongate both on MS medium with0.1mg/L KT,0.2mg/L KT and0.3mg/L KT.62%adventitious shoots were successfully rooted on a MS medium with0.05mg/L NAA. Rooted plants were successfully acclimatized and gradually hardened-off to green-house conditions and subsequently established in soil with a survival rate of80%. A system of genetic transformation for cotyledon of watermelon was established preliminarily.4. Specific primers of transcription factor genes ClMYB55were designed to clone full-length cDNA sequence and coding sequence. The fragment of ClMYB55coding sequence was ligated into expression vector PBI121and constructed over-expression vector, named PBI55.5. The agrobacterium-mediated genetic transformation system of watermelon was optimized through orthogonal design using the watermelon cotyledon as materials. The genetic transformation percentage was determined by GUS staining. The results showed that the optimized system included:2d pre-culturing, bacteria cell density (OD600)1.0,15min infection incubation,4d co-culture time.