杜梨类钙调磷酸酶B亚基蛋白基因PbCBL2的克隆和功能初探

Isolation of a Calcineurin B-like Protein Gene PbCBL2 from Pyrus betulaefolia and Preliminary Study of Gene Function

类钙调磷酸酶B亚基蛋白(Calcineurin B-like protein,CBLs)是植物中一类重要的钙离子传感器,参与调控植物生长发育及逆境胁迫响应过程。为了探明杜梨CBLs家族成员PbCBL2的序列特征和表达模式,以杜梨(Pyrus betulaefolia Bunge)幼苗为试材,运用EST搜索结合RACE技术、染色体步移法对PbCBL2的cDNA、DNA和启动子进行克隆,采用半定量RT-PCR和原核表达研究该基因在非生物胁迫下的表达模式。结果表明,PbCBL2基因cDNA序列长681bp,编码一个含有226个氨基酸残基的蛋白。基因组DNA序列长1927bp,包括8个外显子和7个内含子,启动子序列包含光反应元件、厌氧诱导必需顺式作用元件、赤霉素反应元件和水杨酸响应顺式作用元件。PbCBL2编码的多肽具有植物类钙调磷酸酶B亚基蛋白结合Ca2+所必需的4个EF手型结构和1个典型的植物钙调磷酸酶A亚基结合位点。未经处理的杜梨幼苗(对照)根和叶中未检测到PbCBL2的表达,PbCBL2的表达受NaCl、PEG6000、甘露醇和ABA诱导上调。PbCBL2转入大肠杆菌BL21(DE3)后,能够明显减轻NaCl、甘露醇和PEG6000对该菌株的生长抑制。PbCBL2基因具备植物CBLs基因家族的固有特征,对盐碱、干旱、渗透胁迫和ABA处理均存在转录响应,大肠杆菌转入该基因后能够提高对盐胁迫和渗透胁迫的耐受能力。

Calcineurin B-like protein（CBLs）,as a plant calcium sensor,plays critical role in the regulation of plant growth and stress response process. However,CBL2 gene sequence feature,expressioncharacteristic and physiological function in birch-leaf pea（rPyrus betulaefolia Bunge）are largely unknown. In this study,we isolated the cDNA,genomic DNA and its responding promoter sequences of PbCBL2 gene from birch-leaf pear seedlings by EST database mining,rapid amplification of cDNA ends（RACE） and genome walking approaches. The results have showed that PbCBL2 cDNA sequence contained a 681 bp open reading frame which encoded 226 amino acid residues. The length of genomic DNA sequence was 1 927 bp which consists of 8 exons and 7 introns. The promoter region of PbCBL2 harbored some specific regulatory elements or motifs,such as light responsive element,cis-acting regulatory element essential for the anaerobic induction,gibberellin-responsive element and cis-acting element involved in salicylic acid responsiveness. The deduced PbCBL2 polypeptide had four EF-hand structure domain（s58–71,95–106, 132–143 and 176–187 amino acids）which was necessary for calcium-binding and one calcineurin A subunit binding sites（156–171 amino acids）. Semi-quantitative RT-PCR and prokaryotic expression analyses validated that the mRNA abundance of PbCBL2 is responsive to different abiotic stresses. However,PbCBL2 expression was barely detected in roots and leafs of birch-leaf pear seedling without abiotic stresses treatment. The inhibition effects on BL21（DE3）growth causing by NaCl,mannitol or PEG6000 were significantly alleviated after PbCBL2 gene transformation. Our studies have suggested that PbCBL2 gene has the inherent characteristics of the CBLs gene family in plants,which transcription level is respond to salt,drought,osmotic stresses and ABA treatment. E. coli BL21（DE3）tolerance to salt stress and osmotic stress was enhanced by transferred PbCBL2.