Screening And Cloning Of Key MYB Transcription Factor Of Athocyanin Biosynthesis In Eggplant (Solanum Melongena L.)

The R2R3 MYB proteins comprise one of the largest family of transcription factors and play regulatory roles in developmental processes and defense responses in plants. A genome-wide analysis of this family has been carried out with the draft of complete genome sequence of several plant species. Now members of R2R3 MYB transcription factors related to anthocyanins synthesis have been identified in some model species, such as Arabidopsis, rice, and soybean. However, limited comprehensive genomic and functional analyses of these genes in eggplant(Solanum melongena L.) have been performed. The eggplant peel has certainly color agronomic trait such as purple, white, red and orange, which provides the foundation for the study of anthocyanin biosynthesis. The completion of the eggplant genome sequencing provided a powerful advantage to identify of the R2R3 MYB family and screen the R2R3 MYB gene related to anthocyanin biosynthesis in eggplant. In this study, we identified and cloned SmMYB6 related to anthocyanin synthesis by genome-wide analysis of the eggplant MYB transcription factor and transcriptome analysis, which provided a theoretical foundation for eggplant genetic improvement and created new germplasm with high anthocyanin content. The main results were as follows:1. 126 Arabidopsis R2R3 MYB gene sequences and the HMM(HiddenMarkov Model) for the MYB binding domain(PF00249) used as queries in multiple database were searched against genome files that were downloaded from eggplant genome batabase(http: //eggplant. kazusa. or. jp/), and 73 eggplant R2R3 MYB genes were screened.2. To get insight into the eggplant R2R3 MYB genes, phylogenetic comparisons of this superfamily's members among eggplant, tomato, Arabidopsis, grape, rice, poplar, soybean, cucumber and apple were conducted. Intron-exon structure and the sequence features of the MYB DNA-binding domains were also analyzed.3. In order to study the regulation function of eggplant R2R3 MYB genes in the anthocyanin biosynthesis, 9 eggplant R2R3 MYB genes associated with anthocyanin biosynthesis were identified from 73 R2R3 MYB genes and their expression patterns, which conducted under different LED light treatment and in specific eggplant tissue, were analyzed based on the qRT-PCR. The transcript abundances of SmMYB4, SmMYB6, SmMYB13, SmMYB21 and SmMYB22 under R/B=1:1 treatment were higher than that under single LED blue or red light. The expression level of SmMYB2, SmMYB6, SmMYB13, SmMYB22 and SmMYB6 under the red light blue 1:1 combination treatment was significantly higher than that under LED white light. The transcript abundances of SmMYB13 and SmMYB22 had significant difference under LED white light. By analyzing the expression patterns of these genes in different color tissue, it was found that the transcript abundance of SmMYB6 was high in purple tissues, such as stems, leaves, calyxes, petals and peels, while it was very low in white tissues, such as roots and pulps. The transcript abundance of SmMYB4 in calyxes and that of SmMYB1 and SmMYB13 in peels and pulps were also very high. The results indicated that transcript abundance of SmMYB6 was higher in purple tissues.4. Illumina sequencing technology was used to characterize the transcriptome of eggplant peels. Approximately 89823 transcripts were identified including 86095 known transcripts and 3728 novel transcripts.5. The total differential expression genes were 1121 including 390 up-regulated genes and 731 down-regulated gens. GO, KEGG, InterProScan analysis of the differential expression genes were also conducted. 475 genes belonged to biological process, 496 genes belonged to molecular function, 246 genes belonged to cellular component. 6 pathways and total 45 genes related to anthocyanin synthesis were found.6. Through the analysis of the transcriptome data, three R2R3 MYB transcription factors and eight structural genes associated with eggplant anthocyanin biosynthesis were screened. To further analyze the result of qRT-PCR, SmMYB6 expression was significantly higher than SmMYB10 and SmMYB18 in the purple peel. The transcript abundances of four structural genes, SmF3'5'H, SmDFR, SmF3 GT and Sm5 GT, were higher in the purple peel. Comprehensive bioinformatics and transcriptome data showed that SmMYB6 was a key transcription factor involved in eggplant anthocyanin biosynthesis.7. The full-length CDS 771 bp of SmMYB6 was cloned; then the SmMYB6 was constructed into the plant expression vector pRII and transient expression vector IL-60 successfully based on one step cloning method.