新疆野生核桃JfDREB1A基因的克隆与原核表达分析  

Cloning and Prokaryotic Expression Analysis of JfDREB1A Gene in Xinjiang Wild Walnut

作　　者：韩立群 张捷[3] 赵钰 梅闯 马凯[2] HAN Liqun;ZHANG Jie;ZHAO Yu;MEI Chuang;MA Kai(College of Horticulture,Xinjiang Agricultural University,Urumqi 830052,China;Institute of Horticulture Crops,Xinjiang Academy of Agricultural Sciences,Key Laboratory of Genome Research and Genetic Improvement of Xinjiang Characteristic Fruits and Vegetables,Xinjiang Fruit Science Experiment Station,Ministry of Agriculture,Urumqi 830091,China;Xinjiang Forestry School,Urumqi 830026,China)

机构地区：[1]新疆农业大学园艺学院,乌鲁木齐830052 [2]新疆农业科学院园艺作物研究所,新疆特色果蔬基因组研究与遗传改良重点实验室,农业部新疆地区果树科学观测试验站,乌鲁木齐830091 [3]新疆林业学校,乌鲁木齐830026

出　　处：《西北农业学报》2023年第7期1050-1057,共8页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家自然科学基金(32060665);中央引导地方科技发展专项资金“特色果树种质创新与育种能力提升”。

摘　　要：为探究DREB1A基因在新疆野生核桃响应低温胁迫中的作用及表达特性,以新疆野生核桃群体中的‘小矩圆’类型为试验材料,根据同源克隆的方法得到JfDREB1A基因的cDNA全长序列,利用荧光定量PCR检测其在低温胁迫条件下的表达水平;将该基因片段重组到pET-28a(+)原核表达载体上,转化到大肠杆菌(DE3)感受态细胞中诱导表达。结果表明,获得了新疆野生核桃中的DREB1A,将其命名为JfDREB1A,该基因的开放读码框长度为645 bp,具有典型的DREB1/CBF转录因子结构特征,JfDREB1A蛋白与其他植物DREB1蛋白具有高度保守性,与核桃亲缘关系最近。RT-PCR分析显示在4℃低温胁迫下,JfDREB1A基因的表达量迅速上调,8 h时达到最大。成功构建了pET-28a-JfDREB1A重组表达质粒,得到大小约为29 ku的融合蛋白,与预测蛋白大小相符,该蛋白在大肠杆菌中最佳诱导条件是诱导剂IPTG浓度为0.5 mmol/L,诱导2 h。To investigate the role and expression characteristics of DREB1Agene in response to low temperature stress in Xinjiang wild walnut,the JfDREB1A gene from‘microoblonga’type of Xinjiang wild walnut was cloned by using homology cloning,and its expression levels were analyzed by quantitative real-time PCR under low temperature stress.The DNA fragment of JfDREB1A gene was inserted into the pET-28a(+)vector to construct the fusion vector,which was transformed into Escherichia coli Rosetta(DE3).The results showed that the DREB1Agene from Xinjiang wild walnut was cloned and was named as JfDREB1A.The open reading frame was 645 bp,with the structure features of the DREB1/CBFtranscriptional factors.Multiple sequences alignment indicated that the JfDREB1A shared a very high identity.Phylogenetic tree analysis revealed JfDREB1A was related to Juglans regia.Expression analysis displayed that the expression of JfDREB1A gene increased rapidly,and maximal expression was at 8 h under 4℃.The recombinant prokaryotic expressing plasmid of pET-28a-JfDREB1A was constructed.SDS-PAGE showed that the weight of expressed proteins was 29 ku,which was consistent with the predicted protein size.The optimum induction condition of the JfDREB1A in Escherichia coli was 0.5 mmol/L of IPTG for 2 hours.

关 键 词：新疆野生核桃 JfDREB1A基因 基因克隆 原核表达 

分 类 号：S664.1[农业科学—果树学]