BpERF1.1基因在低温下的表达特性及过表达载体构建

Expression Characteristics of BpERF1.1 Gene under Cold Stress and Construction of Overexpression Vector

分析白桦BpERF1.1基因不同组织部位以及低温胁迫后的表达模式,定位基因表达部位,并为获得转基因白桦做初步准备。通过实时荧光定量PRC技术测定BpERF1.1基因的相对表达量;利用基因枪法在洋葱表皮细胞瞬时表达35S::BpERF1.1-GFP,激光共聚焦显微镜观察GFP发光情况,获得BpERF1.1基因表达产物的亚细胞定位信息;此外,利用双酶切法将BpERF1.1基因连接到pRokⅡ过表达载体,以及热激法转化到大肠杆菌感受态细胞中。BpERF1.1基因在叶片中相对表达量最高;低温胁迫后的24 h内,白桦BpERF1.1基因持续上调表达;BpERF1.1基因的表达产物定位在细胞核;pRokⅡ-BpERF1.1植物过表达载体菌液PCR的目的条带位置正确,测序结果BLAST比对后与序列吻合。白桦BpERF1.1基因主要表达部位在叶片,其可以响应低温逆境,且过表达载体pRokⅡ-BpERF1.1构建成功。

Analyze the expression pattern of birch BpERF1.1 gene between different tissues and during low-temperature stress,determine the gene location,and then make initial preparations for transgenic birch.Quantitative real-time PCR(PT-qPCR)was used to determine the relative expression of the BpERF1.1 gene.35 S::BpERF1.1-GFP was transiently expressed in onion epidermal cells by particle bombardment,then we observed the luminescence of GFP by using laser scanning confocal microscope and obtained the subcellular localization information of BpERF1.1 gene expression product.We used the double digestion technique to link the BpERF1.1 gene to the pRokⅡoverexpression vector,and transformed it into E.coli.The BpERF1.1 gene has the highest relative expression in leaves and the gene continued to be up-regulated within 24 h after cold stress.The expression product of BpERF1.1 gene is located in the nucleus.The electrophoretic bands position of pRokⅡ-BpERF1.1 overexpression vector is accurate,and the sequencing results match with the sequence after alignment on the BLAST website.Leaves is the main expression site of BpERF1.1 gene,and the gene authentically responds to low temperature stress.Finally,the overexpression vector pRokⅡ-BpERF1.1 was successfully constructed.