| MYB proteins are widely distributed in higher plants and comprise a large family of plant transcription factors, members of which perform a variety of functions in plant biological processes, such as secondary metabolism, hormone signal transduction, disease resistance, organ development, etc. Flavonoids are a diverse group of secondary metabolites derived from the phenylpropanoid pathway. These compounds are distributed predominantly in plants and play important roles in plant-environment interactions and human health. Consequently, the protective effects of the flavonoids against hormone dependent cancers, cardiovascular disease, osteoporosis, and menopausal symptoms have been generated considerable interest within the genetic and metabolic engineering fields to enhance the dietary intake of these compounds for disease prevention.Soybean is an important oil crops, and also a major source of protein and flavonoids. Thus, it will has an important academic significance and wide application prospects for cloning and indentification of transcription factor genes that are involved in soybean flavonoids metabolic pathway. Moreover, the recent advent of large-scale sequencing projects provides a valuable resource for genomic analyses of this gene family and also has facilitated an understanding of the evolution of the MYB gene family in land plants.Given its well known role in the flavonoid pathway, in the present study, we performed a comprehensive computational analysis of the MYB gene family and also the flavonoid pathway in land plants, including the phylogeny, the evolution relationships, the expression patterns, the synteny analysis, and also the structural and functional characteristics. The aim of this study is to answer the following questions:1). the molecular evolution mechanisms of the MYB gene family and flavonoid pathway in land plants; 2). identification the MYB genes involved in the flavonoid pathway; 3). subsequently, we identified the roles of candidate genes in flavonoid pathway by different experiments. The main results of this study are as follows:1. We performed a comprehensive phylogenetic analysis of the 2R-MYB proteins in the major lineages of eukaryotes, with particular emphasis on the plants. The results revealed that:(1) the MYB proteins are not plant-specific, but found throughout eukaryotic organisms; (2) the MYB genes expanded together with the differentiation of plants into organisms of increasing complexity after the divergence of angiosperm from the vascular plants, where the genome-wide segmental and tandem duplication contribute to the expansion of MYB genes. (3) fifty-six subfamilies of eukaryote 2R-MYB proteins were defined, with 6 subfamilies existed in the common ancestors of moss and/or vascular with highly conserved structure and number; and there are also species-specific subfamilies as well; it was suggested that eukaryote 2R-MYB proteins are monophyletic, whereas, the angiosperm 2R-MYB genes are like to be a common origin. (4) a molecular evolutionary investigation of the mode and tempo of selection acting on the angiosperm 2R-MYB subfamilies revealed site-specific patterns of shifting evolutionary constraint in the first helix 1 region of each MYB repeat. (5) The gene structure, such as intron/exon structures and motifs outside the MYB domain were highly conserved in each subfamily, indicating functional conservation.2. We present a genome-wide analysis of MYB-related proteins from 16 species of flowering plants, moss, Selaginella, and algae. (1) We identified many MYB-related proteins in angiosperms, but few in algae. (2) Phylogenetic analysis classified MYB-related proteins into 5 distinct subgroups, including CCA1-like/R-R, TBP-like, CPC-like, I-box-like and TRF-like. (3) The intron patterns, consensus motifs, and protein domain architecture are highly conserved in each subgroup. Despite the divergence of individual MYB-like domains, the 2 consensus sequences, SHAQK(Y/F)F and LKDKW(R/K)(N/T) are highly conserved in the MYB-like domains of CCA1-like/R-R and TBP-like subgroups, respectively. (4) Phylogenetic and functional analyses revealed that the CCA1-like/R-R and TBP-like subgroups are more than 1 billion years old, whereas the I-box-like and CPC-like subgroups appear to be newly derived in angiosperms. (5) Similar to the 2R-MYB gene family, we further demonstrated that the MYB-like domain has evolved under strong purifying selection, indicating the conservation of MYB-related proteins. Expression analysis revealed that the MYB-related gene family has a wide expression profile in maize and soybean development and plays important roles in development and stress responses. (6) We hypothesize that MYB-related proteins initially diversified through three major expansions and domain shuffling, but remained relatively conserved throughout subsequent plant evolution.3. We performed a detailed genome-wide analysis of the 2R-MYB gene family in soybean and maize, respectively, including the gene structures, phylogeny, chromosome locations, conserved motifs, and expression patterns, as well as a comparative genomic analysis with Arabidopsis. Genome chromosomal location analyses revealed that MYB genes were distributed on almost all chromosomes. The gene density per chromosomewas uneven, with the highest density observed at the top and bottom of chromosomes. The phylogenetic relationships of each subgroup of MYB genes were well supported by the highly conserved intron/exon structures and motifs outside the MYB domain. Expression profile study indicated that 2R-MYB genes exhibit a variety of expression patterns in both of maize and soybean, imlying that members of MYB proteins may involved in the whole process of plant growth and development.4. We explored the molecular evolution of the flavonoids pathway in land plants, as well. We found that the CHS, CHI genes are only distributed in angiosperms which may imply that the flavonoid and anthocyanin biosynthesis pathways are originated in angiosperms. Whereas, the isoflavone synthase genes (CYP93C) are likely to be lineages-specific, as it was just found in leguminous plants, that is why the isoflavonlids are synthesized predominantly in leguminous plants.5. Based on the phylogenetic analysis, co-expression analysis and synteny analysis, we focused on the roles of GmMYB042/Z2 gene in the flavonoid biosynthesis pathway. The experiments include (1) the expression analysis of the GmMYB042 gene in root, stems, leaves, flowers and immature seeds of soybean; (2) the transcriptional activation of GmMYB042 protein was confirmed by yeast one-hybrid assay; (3) in order to investigate the mechanism of GmMYB042 gene in signal pathway, constructs including detection of the conserved motif (PDLNLELTIS) and the domain of the zinc finger were made. The effect of the altered proteins were monitored using yeast one-hybridize. The yeast one-hybrid assay showed that GmMYB042 had transcriptional activation function. But the transcriptional activity was lower when the intron existed. The result showed that the PDLNLELTIS motif and zinc finger may repress the transcriptional activity of GmMYB042. (4) GmMYB042 and its mutants were transformed into tobacco NC89 with Agrobacterium LBA4404, respectively. Semi-quantitative RT-PCR analysis revealed that GmMYB042 could improve the expressions of some enzyme genes in the flavonoid biosynthesis pathway, such as PAL, CHS, CHI, and FLS, resulting in the increase of the total flavonoid levels. These results indicated that it may involved in the regulation of tobacco flavonoid biosynthesis pathway. (5) We cloned the promoter sequence of GmMYB042 gene, and analyzed the transcription start site of it, as well. |
PDF Full Text Request