Cloning and expression characteristics of a Calcineurin B-like protein gene(PbCBL10) from Pyrus betulaefolia

【Objective】 The objective of this study was to illuminate sequence features and expression characteristics of CBL gene family in birch-leaf pear(Pyrus betulaefolia Bunge).【Method】 The full-length cDNA and DNA sequences of a CBL gene were cloned from birch-leaf pear seedlings by reverse transcription polymerase chain reaction(RT-PCR),rapid amplification of cDNA ends(RACE) and PCR methods.Its expression patterns under different stresses were analyzed by semi-quantitative RT-PCR using cross-introns primers.【Result】 The results showed that PbCBL10 cDNA sequence contained an 801 bp length coding region which encoded 266 amino acid residues.PbCBL10 predicted isoelectric point and relative molecular mass were 4.56 and 30.45 ku,respectively.PbCBL10 genomic DNA sequence length was 1 983 bp which consisted of 9 exons(1-72,189-363,504-587,731-791,1156-1262,1363-1420,1551-1624,1709-1824,1930-1983 bp) and 8 introns(73-188,364-503,588-730,792-1155,1263-1362,1421-1550,1625-1708,1825-1929 bp).Subcellular localization result indicated that PbCBL10 protein was most probability localized in plasma membrane through the PSORT software analysis.PbCBL10 deduced polypeptide had four EF-hand structure domain(101-112,135-146,173-184 and 217-228 amino acids) which was absolutely necessary for calcium-binding and one calcineurin A subunit binding sites(196-214 amino acids).Homology analysis exhibited that it had the highest similarity with tomato SlCBL10(NP_001239045) and Arabidopsis AtCBL10(NP_195026) which were 82% and 77%,respectively.Furthermore,PbCBL10 and AtCBL10 belonged to the same branch in the CBL phylogenetic tree.PbCBL10 gene expression was inducible and its transcription abundances climbed up quickly after 50 to 200 mmol·L-1 CaCl2,20 μmol·L-1 ABA,100 mmol·L-1 NaCl,10%(w/v) PEG6000 or 180 mmol·L-1 mannitol treatments.【Conclusion】 The studies suggested that PbCBL10 gene has the inherent characteristics of the CBL gene family in plants,which can respond to changes of intracellular calcium concentration.Furthermore,PbCBL10 transcription was response to ABA,salt,drought and osmotic stresses.