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Background: Identification of the motifs bound by a transcription factor (TF) is important to reveal the function of TF. Previously, we built a transcription factor centered yeast one hybrid (TF-Centered Y1H) that could identify the motifs bound by a target TF. However, that method was difficult to comprehensively identify all the motifs bound by a TF., Results: Here, we build an improved TF-Centered Y1H to comprehensively determine the motifs bound by a target TF. Recombination-mediated cloning in yeast was performed to construct a saturated prey library that contains 7 random base insertions. After TF-Centered Y1H screening, all the positive clones were pooled together to isolate pHIS2 vector. The insertion regions of pHIS2 were PCR amplified and the PCR product was subjected to high-throughput sequencing. The insertion sequences were then retrieved and analyzed using MEME program to identify the potential motifs bound by the TF. Using this technology, we studied the motifs bound by an ethylene-responsive factor (BpERF2) from birch. In total, 22 conserved motifs were identified, and most of them are novel cis-acting elements. Both the yeast one hybrid and electrophoretic mobility shift assay verified that the obtained motifs could be bound by BpERF2. In addition, chromatin immunoprecipitation (ChIP) study further suggested that the identified motifs can be bound by BpERF2 in cells of birch. These results together suggested that this technology is reliable and has biological significance., Conclusion: This method will have wide application in DNA-protein interaction studies., (© 2023. The Author(s).)

Background: The paper mulberry (Broussonetia papyifera) is one of the multifunctional tree species in agroforestry system and is also commonly utilized in traditional medicine in China and other Asian countries. To identify the transcription factors (TFs) and comprehensively understand their regulatory roles in the growth of the paper mulberry, a global transcriptomics TF prediction and the differential expression analysis among root, shoot and leaf were performed by using RNA-seq., Results: Results indicate that there is 1, 337 TFs encoded by the paper mulberry and they belong to the 55 well-characterized TF families. Based on the phylogenetic analysis, the TFs exist extensively in all organisms are more conservative than those exclusively exist in plant and the paper mulberry has the closest relationship with the mulberry. According to the results of differential expression analysis, there are 627 TFs which exhibit the differential expression profiles in root, shoot and leaf. ARR-Bs, ARFs, NACs and bHLHs together with other root-specific and highly expressed TFs might account for the developed lateral root and unconspicuous taproot in the paper mulberry. Meanwhile, five TCPs highly expressed in shoot of the paper mulberry might negatively regulate the expression of 12 LBDs in shoot. Besides, LBDs, which could directly or indirectly cooperate with ARFs, bHLHs and NACs, seem to be the center knot involving in the regulation of the shoot development in the paper mulberry., Conclusions: Our study provides the comprehensive transcriptomics identification of TFs in the paper mulberry without genome reference. A large number of lateral organ growth regulation related TFs exhibiting the tissue differential expression may entitle the paper mulberry the developed lateral roots, more branches and rapid growth. It will increase our knowledge of the structure and composition of TFs in tree plant and it will substantially contribute to the improving of this tree.

The paper mulberry is one of the multifunctional tree species in agroforestry systems and is also commonly utilized in traditional medicine in China and other Asian countries. However, little is known about its molecular genetics, which hinders research on and exploitation of this valuable resource. To discern the correlation between gene expression and the essential properties of the paper mulberry, we performed a transcriptomics analysis, assembling a total of 37,725 unigenes from 54,638,676 reads generated by RNA-seq. Among these, 22,692 unigenes showed greater than 60% similarity with genes from other species. The lengths of 13,566 annotated unigenes were longer than 1,000 bp. Functional clustering analysis with COG (Cluster of Orthologous Groups) revealed that 17,184 unigenes are primarily involved in transcription, translation, signal transduction, carbohydrate metabolism, secondary metabolism, and energy metabolism. GO (Gene Ontology) annotation suggests enrichment of genes encoding antioxidant activity, transporter activity, biosynthesis, metabolism and stress response, with a total of 30,659 unigenes falling in these categories. KEGG (Kyoto Encyclopedia of Genes and Genomes) metabolic pathway analysis showed that 7,199 unigenes are associated with 119 metabolic pathways. In addition to the basic metabolism, these genes are enriched for plant pathogen interaction, flavonoid metabolism and other secondary metabolic processes. Furthermore, differences in the transcriptomes of leaf, stem and root tissues were analyzed and 7,233 specifically expressed unigenes were identified. This global expression analysis provided novel insights about the molecular mechanisms of the biosynthesis of flavonoid, lignin and cellulose, as well as on the response to biotic and abiotic stresses including the remediation of contaminated soil by the paper mulberry.