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Functional Analysis of the Maize C-Repeat/DRE Motif-Binding Transcription Factor CBF3 Promoter in Response to Abiotic Stress.

Liu J, Wang F, Yu G, Zhang X, Jia C, Qin J, Pan H - Int J Mol Sci (2015)

Bottom Line: PZmCBF3 was activated by cold stress.The MYCCONSENSUSAT elements (CANNTG) were responsible for the ability of PZmCBF3 to respond to cold stress.The results of the present study suggest that PZmCBF3 might play a role in cold tolerance in maize.

View Article: PubMed Central - PubMed

Affiliation: College of Plant Sciences, Jilin University, Changchun 130062, China. jlliu@jlu.edu.cn.

ABSTRACT
The ZmCBF3 gene is a member of AP2/ERF transcription factor family, which is a large family of plant-specific transcription factors that share a well-conserved DNA-binding domain. To understand the regulatory mechanism of ZmCBF3 gene expression, we isolated and characterized the ZmCBF3 promoter (PZmCBF3). Three deletion fragments of PZmCBF3 were generated, C1-C3, from the translation start codon at position -1079, -638, and -234, and fused to the GUS reporter gene. Each deletion construct was analyzed by Agrobacterium-mediated stable transformation and expression in Arabidopsis thaliana. GUS expression assays indicated that the PZmCBF3 exhibited root-specific expression activity. A 234-bp fragment upstream of the ZmCBF3 gene conferred a high level of GUS activity in Arabidopsis. Some cis-acting elements involved in the down-regulation of gene expression were detected in the promoter, encompassing positions -1079 to -234. PZmCBF3 was activated by cold stress. The MYCCONSENSUSAT elements (CANNTG) were responsible for the ability of PZmCBF3 to respond to cold stress. The results of the present study suggest that PZmCBF3 might play a role in cold tolerance in maize.

No MeSH data available.


Related in: MedlinePlus

Transformation of Arabidopsis plants using PZmCBF3:GUS constructs. (A) Schematic representation of the PZmCBF3 promoter constructs used to analyze GUS expression in Arabidopsis leaves. The serial 5′-deletion constructs of the PZmCBF3 promoter were fused to the GUS reporter gene in the vector, pCAMBIA1301; (B) Schematic representation of the PZmCBF3:GUS construct. The insertion position of the ZmCBF3 promoter in the vector is indicated with restriction enzyme sites (XbaI or EcoRI and NcoI). LB, left border; RB, right border; 35S-ter, CAMV 35S terminator; 35S pro, CAMV 35S promoter; GUS, β-glucuronidase gene; HPTII, hygromycinphosphotransferase (II) coding region; NOST, nopaline synthase terminator; PZmCBF3, ZmCBF3 promoter.
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ijms-16-12131-f002: Transformation of Arabidopsis plants using PZmCBF3:GUS constructs. (A) Schematic representation of the PZmCBF3 promoter constructs used to analyze GUS expression in Arabidopsis leaves. The serial 5′-deletion constructs of the PZmCBF3 promoter were fused to the GUS reporter gene in the vector, pCAMBIA1301; (B) Schematic representation of the PZmCBF3:GUS construct. The insertion position of the ZmCBF3 promoter in the vector is indicated with restriction enzyme sites (XbaI or EcoRI and NcoI). LB, left border; RB, right border; 35S-ter, CAMV 35S terminator; 35S pro, CAMV 35S promoter; GUS, β-glucuronidase gene; HPTII, hygromycinphosphotransferase (II) coding region; NOST, nopaline synthase terminator; PZmCBF3, ZmCBF3 promoter.

Mentions: To determine the minimal necessary region of PZmCBF3, three promoter deletion fragments were fused with the GUS reporter gene in pCAMBIA1301 for agro-infiltration into Arabidopsis (Figure 2). The expression of each PZmCBF3:GUS fusion gene was examined in the leaves of 15 independent T3 transgenic Arabidopsis plants. As shown in Figure 3, deletion constructs with C3 showed much higher GUS activity than that of the C1 and C2 promoters in the leaves of transgenic Arabidopsis plants (Figure 3 and Figure 4). C1 and C2 promoter-mediated GUS activity showed no significant difference. Compared to GUS activity driven by cauliflower mosaic virus (CaMV) 35S promoter (as positive control), PZmCBF3 showed lower activity in leaves. In addition, C3-mediated GUS activity was accounting for only 13% of the CaMV 35S promoter-mediated GUS activity. These results indicated that the leaves of the T3 transgenic Arabidopsis plants had very low PZmCBF3 activity.


Functional Analysis of the Maize C-Repeat/DRE Motif-Binding Transcription Factor CBF3 Promoter in Response to Abiotic Stress.

Liu J, Wang F, Yu G, Zhang X, Jia C, Qin J, Pan H - Int J Mol Sci (2015)

Transformation of Arabidopsis plants using PZmCBF3:GUS constructs. (A) Schematic representation of the PZmCBF3 promoter constructs used to analyze GUS expression in Arabidopsis leaves. The serial 5′-deletion constructs of the PZmCBF3 promoter were fused to the GUS reporter gene in the vector, pCAMBIA1301; (B) Schematic representation of the PZmCBF3:GUS construct. The insertion position of the ZmCBF3 promoter in the vector is indicated with restriction enzyme sites (XbaI or EcoRI and NcoI). LB, left border; RB, right border; 35S-ter, CAMV 35S terminator; 35S pro, CAMV 35S promoter; GUS, β-glucuronidase gene; HPTII, hygromycinphosphotransferase (II) coding region; NOST, nopaline synthase terminator; PZmCBF3, ZmCBF3 promoter.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490434&req=5

ijms-16-12131-f002: Transformation of Arabidopsis plants using PZmCBF3:GUS constructs. (A) Schematic representation of the PZmCBF3 promoter constructs used to analyze GUS expression in Arabidopsis leaves. The serial 5′-deletion constructs of the PZmCBF3 promoter were fused to the GUS reporter gene in the vector, pCAMBIA1301; (B) Schematic representation of the PZmCBF3:GUS construct. The insertion position of the ZmCBF3 promoter in the vector is indicated with restriction enzyme sites (XbaI or EcoRI and NcoI). LB, left border; RB, right border; 35S-ter, CAMV 35S terminator; 35S pro, CAMV 35S promoter; GUS, β-glucuronidase gene; HPTII, hygromycinphosphotransferase (II) coding region; NOST, nopaline synthase terminator; PZmCBF3, ZmCBF3 promoter.
Mentions: To determine the minimal necessary region of PZmCBF3, three promoter deletion fragments were fused with the GUS reporter gene in pCAMBIA1301 for agro-infiltration into Arabidopsis (Figure 2). The expression of each PZmCBF3:GUS fusion gene was examined in the leaves of 15 independent T3 transgenic Arabidopsis plants. As shown in Figure 3, deletion constructs with C3 showed much higher GUS activity than that of the C1 and C2 promoters in the leaves of transgenic Arabidopsis plants (Figure 3 and Figure 4). C1 and C2 promoter-mediated GUS activity showed no significant difference. Compared to GUS activity driven by cauliflower mosaic virus (CaMV) 35S promoter (as positive control), PZmCBF3 showed lower activity in leaves. In addition, C3-mediated GUS activity was accounting for only 13% of the CaMV 35S promoter-mediated GUS activity. These results indicated that the leaves of the T3 transgenic Arabidopsis plants had very low PZmCBF3 activity.

Bottom Line: PZmCBF3 was activated by cold stress.The MYCCONSENSUSAT elements (CANNTG) were responsible for the ability of PZmCBF3 to respond to cold stress.The results of the present study suggest that PZmCBF3 might play a role in cold tolerance in maize.

View Article: PubMed Central - PubMed

Affiliation: College of Plant Sciences, Jilin University, Changchun 130062, China. jlliu@jlu.edu.cn.

ABSTRACT
The ZmCBF3 gene is a member of AP2/ERF transcription factor family, which is a large family of plant-specific transcription factors that share a well-conserved DNA-binding domain. To understand the regulatory mechanism of ZmCBF3 gene expression, we isolated and characterized the ZmCBF3 promoter (PZmCBF3). Three deletion fragments of PZmCBF3 were generated, C1-C3, from the translation start codon at position -1079, -638, and -234, and fused to the GUS reporter gene. Each deletion construct was analyzed by Agrobacterium-mediated stable transformation and expression in Arabidopsis thaliana. GUS expression assays indicated that the PZmCBF3 exhibited root-specific expression activity. A 234-bp fragment upstream of the ZmCBF3 gene conferred a high level of GUS activity in Arabidopsis. Some cis-acting elements involved in the down-regulation of gene expression were detected in the promoter, encompassing positions -1079 to -234. PZmCBF3 was activated by cold stress. The MYCCONSENSUSAT elements (CANNTG) were responsible for the ability of PZmCBF3 to respond to cold stress. The results of the present study suggest that PZmCBF3 might play a role in cold tolerance in maize.

No MeSH data available.


Related in: MedlinePlus