关键词: 光皮桦, BlMYB基因, 应拉木, 表达模式, 共表达

Abstract: [Objective] MYB protein is one of the important transcription factors, which play critical roles in regulating the syntheses of cellulose and lignin. In order to identify those MYB genes involved in wood formation of Betula luminifera, four BlMYB genes were cloned and their sequence characteristics, expression patterns and downstream regulatory genes were also analyzed. These results would provide a theoretical basis and genetic resources for further gene function analysis and molecular assisted breeding.[Method] cDNA fragments of four BlMYB genes of B. luminifera were isolated by using RACE technology. Their sequence features were analyzed by bioinformatic tools, and then, the relative expression levels of these BlMYB genes and seven downstream regulatory genes in different organs/tissues (female inflorescence, male inflorescence, bud, young leaf, mature leaf, stem, phloem, cambium, inner and outer xylem) and early stage of tension wood(TW) were analyzed by real-time quantitative PCR. Furthermore, two xylem specific expressed BlMYB were used as the guide genes to construct a co-expression network by mutual ranking method and Cytoscape software. Cis-elements in the promoter regions of the putative downstream genes were analyzed by using the Plant CARE website.[Result] The full-length cDNA of four BlMYB genes were isolated and were named BlMYB1, BlMYB2, BlMYB3 and BlMYB4, respectively. These four BlMYB genes encode 395, 252, 258, 320 amino acid residues, respectively, and all these proteins contain R2R3 domains near the N-terminus. The four BlMYB protein sequences show 27%-37% identity with each other, but exhibit higher identity (39%-55%) with their homologs in Arabidopsis thaliana. There are one or two introns in these BlMYB genes. The phylogenetic analysis revealed that the four BlMYB belonged to different branches, and BlMYB2 and BlMYB4 belonged to two branches which contained MYB genes involved in the formation of secondary cell wall. BlMYB1 and BlMYB3 dominantly expressed in mature leaf, and their expression levels increased with the maturation of leaf, which suggested that they may be associated with development of leaf. BlMYB2 strongly expressed in the xylem of lignified stem, while weakly expressed in leaves, phloem and other tissues. During the early stages of TW formation, the expression of BlMYB2 in TW was down-regulated, especially after 48 hours and 7 days bending treatment. BlMYB4 predominantly expressed in the xylem and male inflorescence, and its expression levels were relatively low in root and leaves. And, its expression was up-regulated in TW and down-regulated in OW (Opposite Wood). In addition, according to co-expression analysis and specific AC element screening, seven Unigenes annotated as cellulose and lignin biosynthesis enzyme genes (FRK, COMT, HCT, CesA3, CesA4, 4CL and CCoAOT) might be regulated by BlMYB2 and BlMYB4.[Conclusion] Four BlMYB genes belonging to the R2R3-MYB family were cloned from B.luminifera and showed different gene structures. The expression patterns of BlMYB indicated that they might participate in different metabolic pathways. In consideration of tension wood feature and the result of co-expression analysis, it could be speculated that the BlMYB2 and BlMYB4 might regulate the wood formation of B. luminifera.

Key words: Betula luminifera, BlMYB genes, tension wood, expression pattern, co-expression