杜梨CBL1和CBL7基因对非生物逆境的响应

Comparison of two CBL genes on stress tolerance functions from Pyrus betulaefolia

【目的】克隆2个杜梨类钙调磷酸酶B亚基蛋白(Calcineurin B-like protein,CBL)基因,并对其序列特征、表达特点及抗逆功能进行比较研究。【方法】采用电子克隆、RT-PCR和长片段PCR克隆了PbCBL1和PbCBL7的cDNA和DNA序列,利用生物信息学方法进行序列分析,半定量RT-PCR检测它们的表达特点,原核表达初步比较研究上述2个基因的抗逆功能。【结果】杜梨PbCBL1 cDNA编码区为642 bp,编码213个氨基酸,对应基因组DNA序列长2 969 bp;PbCBL7 cDNA编码区为639 bp,编码212个氨基酸,对应基因组DNA序列长1 788 bp;PbCBL1和PbCBL7均由8个外显子和7个内含子组成。PbCBL1和PbCBL7编码的多肽均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PbCBL1和PbCBL7的表达非常微弱,NaCl、PEG6000、甘露醇和ABA处理后,它们在根和叶中的表达均上调,且PbCBL1在叶片中的表达量显著增加。2个CBL基因分别转入大肠杆菌BL21(DE3)后,均能够明显减轻NaCl、甘露醇和PEG6000对菌株的生长抑制,而转PbCBL1菌株的抗逆效果较为明显。【结论】PbCBL1和PbCBL7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,上述基因的转入能够提高大肠杆菌BL21(DE3)对盐胁迫和渗透胁迫的耐受能力,转PbCBL1菌株的抗逆功能强于转PbCBL7菌株。

【Objective】 To investigated the molecular mechanism of pear calcineurin B-like protein(CBL)genes participating in the stress resistance progress preliminarily, two cDNAs designed as PbCBL1 and PbCBL7 were isolated from Pyrus betulaefolia Bunge and the sequence characteristics were analyzed. Furthermore, expression patterns and tolerance functions of the Escherichia coli cells conferred above genes were studied respectively. 【Method】 The open-reading frame(ORF) of cDNA and DNA sequences were isolated by EST sequence splice, RT-PCR and PCR techniques step by step. Bioinformatics methods were employed in the analysis of nucleotide sequences and their deduced protein structure. Semi-quantitative RT-PCR was used to reveal the expression modes of above genes via cross-introns primers and prokaryotic expression was applied in the research of stress tolerance of E. coli cells mediated by above genes apart.【Result】 The cDNAs of PbCBL1 and PbCBL7 were 642 bp and 639 bp in length, respectively, and encoded 213 and 212 amino acids. The DNAs of PbCBL1 and PbCBL7 were 2 969 and 1 788 bp in length,separately, and both consisted of 8 exons and 7 introns. The deduced protein of either PbCBL1 or PbCBL7contained four EF-hand structure domains and one calcineurin A subunit binding site, which was the hallmark of the CBLs of plants. The mRNA abundance of either of these genes was induced in leaves and roots by the stress of salt, drought, osmotic and ABA dramatically. Especially what deserves to be mentioned is the transcription level of PbCBL1 increased most visible in leaves in comparison with normal condition. The tolerance of E. coli BL21(DE3)cells conferred PbCBL1 or PbCBL7 was enhanced under either salt stress or osmotic stress compared to the control cells. It is worth nothing that the stress tolerance ability of E. coli mediated by PbCBL1 is stronger than that of PbCBL7. 【Conclusion】 The results of our experiments show that both these two genes belong to the CBL gene family of P. betulaefolia and they may play important roles in the regulatory mechanism of abiotic stress.