Research Article

Isolation and characterization of Calcineurin B-like gene (PbCBL1) and its promoter in birch-leaf pear (Pyrus betulifolia Bunge)

Published: December 14, 2015
Genet. Mol. Res. 14 (4) : 16756-16770 DOI: https://doi.org/10.4238/2015.December.14.3
Cite this Article:
(2015). Isolation and characterization of Calcineurin B-like gene (PbCBL1) and its promoter in birch-leaf pear (Pyrus betulifolia Bunge). Genet. Mol. Res. 14(4): gmr5296. https://doi.org/10.4238/2015.December.14.3
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Abstract

Calcium plays a critical role in regulating abiotic stress responses in plants. Calcineurin B-like (CBL) proteins are calcium sensors in calcium signaling pathways. However, the molecular mechanisms underlying calcium signaling remain to be elucidated. In this study, the CBL1 gene, which codes for the CBL protein, was isolated from the birch-leaf pear. One 2,969-bp sequence was cloned using PCR, and using the cloned 2,027-bp sequence was isolated from pear genomic DNA via genome walking. Sequencing analysis revealed that the 4,996-bp sequence was a PbCBL1 gene consisting of eight exons and seven introns, and the 2,027-bp sequence was identified as the promoter of the PbCBL1 gene, which contains the basic promoter elements TATA and CAAT boxes. In addition, some other cis-acting elements including heat, cold, drought, and hormone responsive elements were also present. To further investigate the activity of this promoter, the sequence was used to drive a GUS fusion gene into leaf discs of tobacco (Nicotiana benthamiana) with Agrobacterium-mediated transformation method. GUS gene expression could be regulated by the PbCBL1 promoter following induction by GA, ABA, SA, and MeJA. Furthermore, the results of real-time RT-qPCR indicate that the PbCBL1 gene can respond to changes in the intracellular calcium concentration, and that it can be induced by cold, heat, drought, and stress by several hormones including GA, ABA, SA, and MeJA. PbCBL1 gene may be involved in several signal transduction pathways, and play an important role in the condition of adversity stress in pear.

Calcium plays a critical role in regulating abiotic stress responses in plants. Calcineurin B-like (CBL) proteins are calcium sensors in calcium signaling pathways. However, the molecular mechanisms underlying calcium signaling remain to be elucidated. In this study, the CBL1 gene, which codes for the CBL protein, was isolated from the birch-leaf pear. One 2,969-bp sequence was cloned using PCR, and using the cloned 2,027-bp sequence was isolated from pear genomic DNA via genome walking. Sequencing analysis revealed that the 4,996-bp sequence was a PbCBL1 gene consisting of eight exons and seven introns, and the 2,027-bp sequence was identified as the promoter of the PbCBL1 gene, which contains the basic promoter elements TATA and CAAT boxes. In addition, some other cis-acting elements including heat, cold, drought, and hormone responsive elements were also present. To further investigate the activity of this promoter, the sequence was used to drive a GUS fusion gene into leaf discs of tobacco (Nicotiana benthamiana) with Agrobacterium-mediated transformation method. GUS gene expression could be regulated by the PbCBL1 promoter following induction by GA, ABA, SA, and MeJA. Furthermore, the results of real-time RT-qPCR indicate that the PbCBL1 gene can respond to changes in the intracellular calcium concentration, and that it can be induced by cold, heat, drought, and stress by several hormones including GA, ABA, SA, and MeJA. PbCBL1 gene may be involved in several signal transduction pathways, and play an important role in the condition of adversity stress in pear.

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