Optimizing the binding activity of the AP2/ERF transcription factor with the GCC box element from Brassica napus by directed evolution

Jin, Xin; Zhu, Bo; Peng, Ri‐He; Jiang, Haihua; Chen, Jianmin; Zhuang, Jing; Zhang, Jian; Yao, Qizheng; Xiong, Ai‐Sheng

doi:10.5483/bmbrep.2010.43.8.567

Cited by 15 publications

(11 citation statements)

References 22 publications

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“…The four proteins (BnaERF-B3, Mu1, Mu2, and Mu3) were investigated using yeast one-hybrid assays, and the binding activity to the GCC box relatively varied (Jin et al, 2010a). …”

Section: Resultsmentioning

confidence: 99%

“…The yeast cells were examined with X-gal after growing on SD/-Ura /-Trp- medium for 2 d at 30°C. GCC: yeast reporter cells carry GCC box (as a control); Mu1–Mu3: the mutants by Jin et al (2010a). The information of the substitution site and amino acid of all Mu4 to Mu16 were as follows: Mu4: Met19Leu; Mu5: Gln51Leu; Mu6: Ala57Val; Mu7: Phe62Ser; Mu8: Met70Thr; Mu9: Thr78–, Thr79–; Mu10: Gly156Arg; Mu11: Tyr171Cys; Mu12: Asn184Cys; Mu13: Thr192Val, Thr193Ala; Mu14: Thr192Ile, Thr193Ala; Mu15: Gly156Arg, Thr192Val; Mu16: Gly156Arg, Thr192Ile.…”

Section: Resultsmentioning

confidence: 99%

“…The BnaERF-B3-hy15 gene encoding the AP2/ERF protein was cloned from B . napus L. Huyou15 as previously described, and three mutants were obtained by Jin et al (2010a). Two-month-old plants of A. thaliana (L.) Heynh.…”

Section: Methodsmentioning

confidence: 99%

“…AF394909) was inserted into the vector G222, which could synthesize Ura; the vector also contained the reporter gene LacZ under the control of the iso -1-cytochrome C (CYC1) minimal promoter. This recombinant plasmid was used as a bait plasmid in the yeast one-hybrid system (Zhang Y. et al, 2009; Jin et al, 2010a,b). …”

Section: Methodsmentioning

confidence: 99%

“…Huyou15 and obtained three BnaERF-B3 mutants by DNA shuffling (Jin et al, 2010a). The yeast one-hybrid system showed that the three modified proteins exhibit strong binding activity to the GCC box, but the original protein showed almost no binding activity.…”

Section: Introductionmentioning

confidence: 99%

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Arg156 in the AP2-Domain Exhibits the Highest Binding Activity among the 20 Individuals to the GCC Box in BnaERF-B3-hy15, a Mutant ERF Transcription Factor from Brassica napus

Zhuang¹,

Li²,

Wu³

et al. 2016

Front. Plant Sci.

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To develop mutants of the ERF factor with more binding activities to the GCC box, we performed in vitro directed evolution by using DNA shuffling and screened mutants through yeast one-hybrid assay. Here, a series of mutants were obtained and used to reveal key amino acids that induce changes in the DNA binding activity of the BnaERF-B3 protein. With the BnaERF-B3-hy15 as the template, we produced 12 mutants which host individual mutation of potential key residues. We found that amino acid 156 is the key site, and the other 18 mutants host the 18 corresponding individual amino acid residues at site 156. Among the 20 individuals comprising WT (Gly156), Mu3 (Arg156), and 18 mutants with other 18 amino acid residues, Arg156 in the AP2-domain is the amino acid residue with the highest binding activity to the GCC box. The structure of the α-helix in the AP2-domain affects the binding activity. Other residues within AP2-domain modulated binding activity of ERF protein, suggesting that these positions are important for binding activity. Comparison of the mutant and wild-type transcription factors revealed the relationship of protein function and sequence modification. Our result provides a potential useful resource for understanding the trans-activation of ERF proteins.

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“…The four proteins (BnaERF-B3, Mu1, Mu2, and Mu3) were investigated using yeast one-hybrid assays, and the binding activity to the GCC box relatively varied (Jin et al, 2010a). …”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Arg156 in the AP2-Domain Exhibits the Highest Binding Activity among the 20 Individuals to the GCC Box in BnaERF-B3-hy15, a Mutant ERF Transcription Factor from Brassica napus

Zhuang¹,

Li²,

Wu³

et al. 2016

Front. Plant Sci.

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Genome-wide analysis of AP2/ERF transcription factors in carrot (Daucus carota L.) reveals evolution and expression profiles under abiotic stress

Huang

et al. 2015

Mol Genet Genomics

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AP2/ERF is a large transcription factor family that regulates plant physiological processes, such as plant development and stress response. Carrot (Daucus carota L.) is an important economical crop with a genome size of 480 Mb; the draft genome sequencing of this crop has been completed by our group. However, little is known about the AP2/ERF factors in carrot. In this study, a total of 267 putative AP2/ERF factors were identified from the whole-genome sequence of carrot. These AP2/ERF proteins were phylogenetically clustered into five subfamilies based on their similarity to the amino acid sequences from Arabidopsis. The distribution and comparative genome analysis of the AP2/ERF factors among plants showed the AP2/ERF factors had expansion during the evolutionary process, and the AP2 domain was highly conserved during evolution. The number of AP2/ERF factors in land plants expanded during their evolution. A total of 60 orthologous and 145 coorthologous AP2/ERF gene pairs between carrot and Arabidopsis were identified, and the interaction network of orthologous genes was constructed. The expression patterns of eight AP2/ERF family genes from each subfamily (DREB, ERF, AP2, and RAV) were related to abiotic stresses. Yeast one-hybrid and β-galactosidase activity assays confirmed the DRE and GCC box-binding activities of DREB subfamily genes. This study is the first to identify and characterize the AP2/ERF transcription factors in carrot using whole-genome analysis, and the findings may serve as references for future functional research on the transcription factors in carrot.

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Analysis of Brassica napus ESTs: gene discovery and expression patterns of AP2/ERF-family transcription factors

Zhuang

Zhu

2013

Mol Biol Rep

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Starting from expressed sequence tag sequences and using the conserved amino acid sequence of the Arabidopsis thaliana AP2/ERF domain as a probe, we used in silico cloning to identify 87 genes that encode putative AP2/ERF transcription factors (TFs) from the Brassica napus. Almost all of the putative AP2/ERF factors from B. napus were similar to genes previously defined as AP2/ERF genes from A. thaliana. Based on the number of AP2-domains and the function of the genes, the AP2/ERF TFs from B. napus were classified into four subfamilies, named the AP2, DREB, ERF, and RAV subfamilies. We then predicted and analyzed cDNA sequences and amino acid sequences, amino acid compositions, physical and chemical characteristics, phylogenetic trees, conserved domain sequences, functional domains, molecular models, and folding states of the proteins they are predicted to encode. Expression analysis showed that four factors, which belonged to the ERF and DREB subfamilies, were induced by abiotic stresses, as well as by hormone treatment. This suggests that those AP2/ERF factors may be involved in signaling pathways responsive to abiotic and biotic stresses. The results from this study, reported herein, form a basis for future functional analyses of B. napus TFs that belong to the AP2/ERF family.

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Optimizing the binding activity of the AP2/ERF transcription factor with the GCC box element from Brassica napus by directed evolution

Cited by 15 publications

References 22 publications

Arg156 in the AP2-Domain Exhibits the Highest Binding Activity among the 20 Individuals to the GCC Box in BnaERF-B3-hy15, a Mutant ERF Transcription Factor from Brassica napus

Arg156 in the AP2-Domain Exhibits the Highest Binding Activity among the 20 Individuals to the GCC Box in BnaERF-B3-hy15, a Mutant ERF Transcription Factor from Brassica napus

Genome-wide analysis of AP2/ERF transcription factors in carrot (Daucus carota L.) reveals evolution and expression profiles under abiotic stress

Analysis of Brassica napus ESTs: gene discovery and expression patterns of AP2/ERF-family transcription factors

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