扁桃AcDHN1基因的克隆及原核表达分析

【目的】 克隆与分析扁桃脱水素基因,为研究脱水素基因在扁桃抗逆机制中发挥的功能提供参考。【方法】 以新疆栽培的扁桃品种‘纸皮’叶片为材料,通过PCR技术克隆扁桃AcDHN1基因并对该基因进行原核表达分析,构建原核表达载体,在大肠杆菌进行表达。【结果】 克隆得到了一个脱水素基因,命名为AcDHN1,GenBank登陆号为KT949395,该基因全长924 bp、编码308个氨基酸的多肽,为稳定的亲水性蛋白,属于Y₂K_n型脱水素,推测蛋白分子质量为32.4 kD,亚细胞定位于细胞核中。将该基因与原核表达载体连接构建重组质粒pET-AcDHN1,然后将重组质粒转化到大肠杆菌BL21(DE3)中诱导表达。SDS-PAGE电泳结果表明,该蛋白分子量的大小约为52.7 kD,与预期长度一致。【结论】 对重组蛋白的诱导条件进行优化后结果显示,该基因在IPTG浓度0.1 mmol/L、诱导3 h表达量最佳。

Cloning and Prokaryotic Expression of Almond AcDHN1 Gene

【Objective】 The dehydrin gene was cloned and analyzed in order to identify the role of AcDHN1 gene in plant resistance of Amygdalus communis. 【Method】 Using Xinjiang cultivated almond 'zhipi' leaves, according to the reported DHN gene primers through the PCR technique to clone and analyze its prokaryotic expression of the AcDHN gene. 【Result】 we cloned a AcDHN1 (GeneBank accession NO.KT949395), this gene was 924 bp and encoding 308 amino acids, was a stable hydrophilic protein, belonging to Y₂K_n Dehydrin, speculated with 32.4 kD molecular weight, which subcellular localization was in nucleus. We connect the gene to prokaryotic expression vector to construct recombinant plasmid pET-AcDHN1, then we transformed the pET-AcDHN1 into E. coli BL21 (DE3) and induced. The SDS-PAGE result showed the protein was 52.7 kD in molecular weight and was consistent with the expected. 【Conclusion】 Through optimized the recombinant protein induction conditions we got the expressing condition was 0.1 mmol/L IPTG and 3 hours induction.