杜梨类钙调磷酸酶B亚基蛋白PbCBL10基因的克隆和表达特性研究

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

【目的】为了探明杜梨类钙调磷酸酶B亚基蛋白（CalcineurinB—likeprotein，CBL）基因的序列特征和表达特点，【方法】以杜梨幼苗为试材，运用同源克隆和半定量RT—PCR对PbCBLIO基因进行克隆和表达分析。【结果】结果表明．PbCBLIO基因编码区cDNA长度为801bp，编码的多肽由266个氨基酸组成。该多肽预测的等电点为4．56，估计的相对分子质量为30．45ku。其对应基因组DNA序列长1983bp，包括9个外显子和8个内含子。通过PSORT进行亚细胞定位分析发现PbCBLl0蛋白位于质膜上的几率最大。PbCBLIO基因编码的多肽具有4个钙离子结合域EF手形结构和1个钙调磷酸酶A亚基结合位点。PbCBLl0与番茄S1CBLIO（NP_001239045）和拟南芥AtCBLl0（NP_195026）蛋白间的同源性较高，分别为82％和77％，并与AtCBL10亲缘关系最近。PbCBL10基因在杜梨幼苗根、茎和叶片中均为诱导型表达，50～200mmol·L^（-1）CaCl2、20μmol·L^（-1）ABA、100mmol·L^（-1）NaCl、10％（w／v）PEG6000或180mmol·L^（-1）甘露醇处理后其表达量明显上调。【结论】PbCBLIO基因具备植物CBL基因家族的固有特征，能够响应胞内钙浓度变化，对ABA、盐碱、干旱和渗透胁迫均存在转录响应。

[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 fulllength eDNA 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 eDNA 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 PbCBLIO eDNA sequence contained an 801 bp length coding region which encoded 266 amino acid residues. PbCBLIO predicted isoeleetric point and relative molecular mass were 4.56 and 30.45 ku, respectively. PbCBLIO 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 ealeineurin A subunit binding sites （196-214 amino acids）. Homology analysis exhibited that it had the highest similarity with tomato S1CBL10 （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. PbCBLIO gene expression was inducible and its transcription abundances climbed up quickly after 50 to 200 mmol·L^（-1） CaC12, 20 μmol·L^（-1）ABA, 100 mmol. L^（-1） NaC1, 10% （w/v） PEG6000 or 180 mmol·L^（-1） mannitol treatments. [Conclusion] The studies suggested that Pb CBLIO 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.