The Transcriptome Analysis Of Hard Wood Cutting In Mulberry And Comprehensive Analysis Of R2R3-MYB Family Genes

In this research subject, a genome-wide gene expression profiling was conducted by Solexa next generation sequencing to investigate the transcriptome dynamics and molecular biology analysis during hardwood cutting rhizogenesis in mulberry combined with related physiological experiments. Meanwhile, we also lucubrate the comprehensive functions of MYB gene families(Myeloblastosis) in mulberry. The main results and conclusions were as follows:1. According to the developmental condition and physiological character of cuttings, 3 transcriptome database were established by using the cortex of hardwood cutting base in late autumn. They were respectively named as S0( negative control),S1( root primordium formation), S2( adventitious root elongation). We have obtained total 46,970,598 to 47,055,500 high-quality contigs after preprocess. 18,72919,044 and 19,987 genes were annotated by Uniprot with an annotation rate of58.68% to 60.53%. After combination of contigs, 327 different expressed genes were obtained in S0 vs. S1 and 6,380 in S1 vs. S2(FDR ? 0.001, |log2Ratio| ? 1). 10 significantly differentially expressed genes were randomly selected to verify the transcriptome data, and the results are in line with our expectations. By systematically processing those DEGs by GO and KEGG analysis. The major pathways involved with functional groups of genes were identified, including the energy metabolism,carbonhydrate metabolism, hormone signal transduction pathway, cell division and differentiate. Summarization above results, we have preliminary discussed the mechanism of adventitious root formation in cuttings of mulberry.2. In this study, a preliminary comprehensive analysis of MYB family genes during hardwood cutting rhizogenesis in mulberry was obtained. As a typical transcription factor, the N-terminal contains conservative structures with incomplete duplications of 50 ~ 53 amino acids, named as R1, R2 and R3 in sequence, which can form Helix-turn-helix( HTH) to combine with DNA spiral structure to play the corresponding role. The candidate genes were obtained by comparison of MYB model( DNA-binding domains DBDs, PF00249) in Pfam and Mulberry Genome Database,manual reviews were applied to make sure the existence of tryptophan( W). The MYB genes with repetitive structures were screened and identified by MEME(version 4.8.1), and by which the Hidden Markov Model( HMM) was built. After further comparing, the MYB family genes of mulberry were obtained. Classificationwere done by the number of repetitive structures, including 117 MYB-related genes,123 R2R3-MYB genes, 4 3R-MYB genes, 3 atypical MYB genes.3. Based on the detailed research of R2R3-MYB genes in mulberry, we have cloned three typical ones relating to adventitious root formation from stem cortex,and respectively named MmMYB1, MmMYB2, MmMYB3( GenBank accession number: KT323919, KT323920, KT323921). They all play important roles during hardwood cutting rhizogenesis in various directions. MmMYB1 has the same function of AtMYB44( Arabidopsis thaliana), through the regulation of AHLs( N-acyl-homoserinelactones) pathway to stimulate the formation of auxin and cytokinin in gram-negative bacteria, which promote the adventitious root growth.MmMYB2 is almost like GmMYB39( Glycine max), it can cause the inhibition of flavonoids synthesis, which can improve the root formation. MmMYB3 is a gibberellin regulation gene, with the regulation of bud, it also can influence the formation of adventitious roots. The mulberry cuttings were then treated with Auxin( IAA) and gibberellin( GA), the real-time fluorescent quantitative PCR( Actin genes: MaACT1) results showed that most of the randomly selected R2R3-MYB genes in base of the cortex of cuttings correspondingly fast up-regulated or down-regulated. According to the different stages in hardwood cutting rhizogenesis,the subclass analysis of C-terminal structures and clustering analysis based on differentially expressed RPKM were accomplished, showing that majorities of those genes significantly up-regulated, but smoothly regulated during adventitious root elongation, speculating that the R2R3-MYB genes play important active roles in differentiation of parenchymatous tissue and formation of adventitious roots in cuttings of mulberry.