欧美杨脱水素PdDHN2b的克隆与表达分析.pdfVIP

第37卷摇 第1期 北 京 林 业 大 学 学 报 Vol.37,No.1 2015年1月 JOURNAL OF BEIJING FORESTRY UNIVERSITY Jan. ,2015 DOI:10.13332/ ki.jbfu.2015.01.017 欧美杨脱水素PdDHN2b 的克隆与表达分析 1 2 1 1 郭摇 鹏 摇 邢摇 鑫摇 张万筠摇 姜摇 健 1大连民族学院环境与资源学院摇 2 山东省威海市农业局 摘要:以欧美杨为材料,通过RT鄄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1440bp,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鄄 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鄄PAGE 电泳结果表明,融 合蛋白分子量为58ku。 最后利用Ni鄄NTA纯化的PdDHN2b鄄his重组蛋白用于Western blot分析。 关键词:欧美杨;PdDHN2b基因;克隆;表达;逆境 鄄鄄 鄄鄄 鄄鄄 中图分类号:S792郾11;S718郾43;Q943郾2摇 摇 文献标志码:A摇 摇 文章编号:1000 1522 2015 01 0022 07 1 2 2 1 GUO Peng ; XING Xin ; ZHANG Wan鄄jun ; JIANG Jian . Cloning and characterization of PdDHN2b genefromPopulusdeltoides 伊Populus nigra. Journalof Beijing Forestry University 2015 鄄鄄 37 1 22 28 [Ch,18 ref.] 1College of Environment and Resources,Dalian Nationalities University,Dalian 116600,Liaoning; 2 Weihai Agricultural Bureau,Weihai,264200,Shandong. With a RT鄄PCR method we cloned a drought tolerant DHN2b gene designated PdDHN2b from Populus deltoides伊Populus nigra. The open reading frame of PdDHN2b is 1440 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鄄PdDHN2b vector was constructed for prokaryotic expression and expressed in E. coli BL21. Fusion proteinswere purified and used to immunize white mice to obtain antiserum. SDS鄄PAGE electrophoresis showed thatthemolecularweightofthefusionproteinwas58ku. Intheend,weusedthe Ni鄄NTA purified recombinant PdDHN2b鄄his protein for Western blot analysis. Key words摇 Populus deltoides伊Populus nigra;PdD