摘要
利用RT-PCR从野大麦cDNA扩增出两个基因HbCBL1和HbCBL2。蛋白结构分析表明,其具有典型的EF手臂,且HbCBL1的N-端含有豆蔻酰化结构域。为了明确野大麦HbCBL1和HbCBL2结构对其在植物体内亚细胞水平分布的作用,分别构建了以35S为启动子驱动的与绿色荧光蛋白基因GFP融合的植物表达载体,通过基因枪介导法将重组载体转入洋葱上表皮细胞,同时通过醮花法获得拟南芥稳定转基因株系,利用激光共聚焦扫描显微镜检测融合蛋白的表达部位。瞬时表达结果表明,融合蛋白HbCBL2::GFP在液泡膜上可见;HbCBL1::GFP在质膜上可见,同时在核中也有信号;HbCBL1的N端豆蔻酰化位点参与了该基因的定位。稳定表达结果表明,HbCBL2主要定位于液泡膜上;HbCBL1主要定位于质膜和细胞核;同时这两个蛋白在保卫细胞中也有表达。研究表明,HbCBL1和Hb-CBL2的结构决定了其亚细胞水平的表达部位,为进一步分析其生物学功能奠定了基础。
RT-PCR was used to obtain two genes named as HbCBL1 and HbCBL2 from Hordeum brevisubulatum.Protein structure analysis showed that the proteins encoded by these two genes have the typical EF-hands,and HbCBL1 has N-myristoylation motif.In order to clarify whether HbCBL1 and HbCBL2 protein structures contribute to their subcellular localization,several plant expression vectors were constructed and transformed into onion epidermal cells by particle bombardment,respectively.These vectors were also transformed into Arabidopsis by floral dip.The subcellular localization of fusion proteins was determined by Confocal laser scanning microscope.The results of transient expression showed that the fusion protein HbCBL2-GFP was accumulated in tonoplast,but the HbCBL1-GFP fussion protein was accumulated in plasma membrane as well as in nucleus;N-myristoylation motif of HbCBL1 involves in its localization at cell level.The results of stable expression showed that HbCBL2 was located at tonoplast and HbCBL1 was located at plasma memebrane,additionally,both HbCBL2 and HbCBL1 are located at the guard cells.It was suggested that HbCBL1 and HbCBL2 protein structures involved in their subcellular localization,which play roles for further analyses of gene functions.
出处
《华北农学报》
CSCD
北大核心
2011年第4期1-7,共7页
Acta Agriculturae Boreali-Sinica
基金
国家重大转基因专项(2009ZX08009-060B)
国家自然科学基金项目(30971850)
北京自然科学基金项目(5102017)
北京市农林科学院科技项目(2010A003)
作者简介
吴广宇（1983-），女，蒙古族，内蒙古呼伦贝尔人，在读硕士，主要从事植物抗逆基因资源挖掘与利用研究。
李瑞芬（1970-），女，内蒙古人，研究员，博士，主要从事植物抗逆基因资源挖掘与利用研究。
通讯作者：魏建华（1971-），男，内蒙古人，研究员，博士，主要从事植物基因工程研究。
