Cloning and characterization of PdDHN2b gene from Populus deltoides×Populus nigra.
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摘要: 以欧美杨为材料,通过RT-PCR方法从欧美杨叶片中克隆到了抗旱脱水素DHN2b基因(命名为PdDHN2b),该基因开放阅读框为1 440 bp,编码379个氨基酸,绝大多数氨基酸组成属于亲水性氨基酸,无明显跨膜结构域。亚细胞定位表明PdDHN2b基因定位在细胞质或细胞膜上。荧光定量PCR分析表明:该基因在顶端中表达量最高,功能叶、根和茎中则无表达。同时该基因受NaCl、ABA和干旱等诱导表达。另外,构建了原核表达载体pET-PdDHN2b,并在大肠杆菌BL21中表达融合蛋白,诱导纯化后免疫小白鼠,制备抗体。SDS-PAGE电泳结果表明,融合蛋白分子量为58 ku。最后利用Ni-NTA纯化的PdDHN2b-his重组蛋白用于Western blot分析。Abstract: With a RT-PCR method we cloned a drought tolerant DHN2b gene (designated PdDHN2b) from Populus deltoides×Populus nigra. The open reading frame of PdDHN2b is 1 440 bp and encodes 379 amino acids of which most are hydrophilic, without an obvious transmembrane domain. A subcellular localization assay showed that PdDHN2b is localized in the membrane orcytoplasm. Real time fluorescence quota PCR expression analysis suggests that PdDHN2b is strongly expressed in top leaves, but not in functional leaves, roots or stems. PdDHN2b is induced by NaCl, ABA and drought. A recombinant pET-PdDHN2b vector was constructed for prokaryotic expression and expressed in E. coli BL21. Fusion proteins were purified and used to immunize white mice to obtain antiserum. SDS-PAGE electrophoresis showed that the molecular weight of the fusion protein was 58 ku. In the end, we used the Ni-NTA purified recombinant PdDHN2b-his protein for Western blot analysis.
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Key words:
- Populus deltoides×Populus nigra /
- PdDHN2b /
- clone /
- express /
- stress
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