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The motifs putatively involved in plant growth and development, phytohormone responses, and stress responses are summarized.
Nested gene matchmakiny primers were designed according to the predicted Wx sites and are listed in Supplementary Table S4. Mafchmaking least eight clones were taken and confirmed by Sanger sequencing. KM was used as a reference gene as described previously Liu et al. Matchmkaing curve was used to evaluate amplification specificity. Normalization of matchmaikng expression data from three biological replicates was performed as described Willems et al. Expression Analysis of BlmiR Expression of BlmiR was analyzed using specific reverse transcription primers as the method described in the previous studies Varkonyi-Gasic et al.
The 5. The BD fused constructs and AD fused constructs were transformed into Y2HGold cells and Y cells by the lithium acetate-mediated method, respectively. The yeast two-hybrid assay was performed by yeast mating according to the user manual. The BD fused constructs and AD fused constructs were tested for their autoactivation and toxicity, and those constructs without autoactivation and toxicity in yeast cells were used for yeast two-hybrid assay Supplementary Figure S1. These predicted gene models were further verified and corrected through molecular cloning and sequencing. These results revealed the diversity of SPL genes features in B. The overall height of each stack represents the degree of conservation at this position, while the height of the letters within each stack indicates the relative frequency of the corresponding amino acids.
These 81 SPLs were selected from four plant species, including 18 from B.
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Only the highly conserved SBP domains 76 aa sequences were used for phylogenetic analysis Riese et al. As reported in previous studies, this phylogenetic tree indicated the evolutionary diversification and differentiation of BlSPL genes as well as other plant SPL genes. Phylogenetic analysis of B. Phylogenetic tree was constructed with SBP domain protein sequences from B. SPL genes from B. SBP domain sequences of all genes used for phylogenetic tree construction, as well as the accession numbers or locus IDs and data sources of SPL genes from different species are listed in Supplementary Tables S7S8.
A A phylogenetic tree was constructed with B. B Exons and introns are indicated by green boxes and black horizontal lines, respectively. In addition, motif 8 also widely exists in several SPL groups. Interestingly, several motifs are found to be group-unique, such as motif 12 specifically existing in group VII, and motifs 3 ANK domain and 7 are found exclusively in group II Figure 4. Functions of these group-unique motifs are currently unclear, but should be tightly related to specific roles of SPLs in Wz 131 matchmaking institute group. Distribution of conserved motifs in BlSPLs. Motifs represented with boxes are identified using MEME online tool.
The number in boxes 1—12 represents motif 1-motif 12, respectively. The position and length of each colored box represents the actual motif size. Consensus sequences of 12 motifs identified in BlSPLs. Sequence alignment indicated that the miR complementary sites were quite conserved across these BlSPL genes Figure 5Aand the sequence divergence was mainly restricted to the first, second and last nucleotide of complementary sequences Figure 5B. It means that the complementary sites are actually under strong selection pressure during the evolution, even for those locating in the 3'-UTR.
These results are consistent with the previous studies from other plants, suggesting the importance of miRmediated posttranscriptional regulation for functions of SPL genes in plant. The 50 nucleotides upstream and downstream of the miR complementary sequences were included in the alignment. Vertical arrows indicate the cleaved nucleotide position, with the frequency of clones shown. Important sites include start codon, SBP domain, and binding sites, cleavage sites of the miR complementary sequences with their nucleotide positions. Heavy gray lines represent open reading frames ORFs. SBP domains are indicated in blue. Distances between these sites are measured and indicated by base pair bp.
Interestingly, both of these two cleavage sites were detected in BlSPL1. Moreover, by comparing the distances between important sequence sites of these BlSPL genes, three types of distance between SBP domain and complementary site were revealed. It could be roughly classified into short-distance less than bpmedium-distance approximately bp and long-distance more than bp Figure 5D. Similar distance type was observed for the BlSPL genes within the same phylogenetic groups. This implies that a common ancestral gene may have experienced different evolutionary events resulting in the differentiation of these miRregulated BlSPL genes. According to their putative functions, these elements were categorized into eight classes, which are shown in the Figure 6.
As the results, light-responsive elements had the largest number and were present in all BlSPL gene promoter regions Supplementary Table S9Figure 6. And, the hormone responsive elements, plant tissue-specific elements, and stress responsive elements are also found to be present in promoter regions of all BlSPL genes Supplementary Table S9Figure 6. In addition, other rarely distributed cis-elements in BlSPLs were found to be functionally involved in transcription regulation, circadian control, protein binding, elicitor responsiveness Supplementary Table S9Figure 6.
Thus, the transcription of BlSPL genes could be regulated by various environmental and developmental changes, which implied that BlSPL genes were involved in important physiological processes and developmental events. These observations suggest that the differentiations in the promoter regions may promote the neofunctionalization of BlSPL genes during their divergence and evolvement. Cis-elements in promoter regions of 18 BlSPLs. Pittsburgh pa dating sites. Top dating apps on android. Types of radioactive dating. Crazy dating advice. Radiocarbon dating creationism. Inventor of online dating. Best dating sites for uk. Grande prairie alberta dating site. Matchmaking institute southeast asia.
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