苹果MdRGL1a基因对烟草遗传转化研究  

Transformation analysis of the apple MdRGL1a gene in transgenic tobacco

作　　者：翟晓霏[1] 张文[1] 赵旭勉[1] 王琼[1] 朱元娣[1] 

机构地区：[1]中国农业大学农学与生物技术学院/果树逆境生理与分子生物学北京市重点实验室,北京100193

出　　处：《中国农业大学学报》2011年第1期36-41,共6页Journal of China Agricultural University

基　　金：国家支撑计划新疆特色果品有机栽培关键技术与示范项目(2007BAD26B02)

摘　　要：植物DELLA蛋白是赤霉素信号传导途径的反向调节因子,DELLA蛋白突变体导致植株矮化。苹果MdRGL1a是编码苹果DELLA蛋白家族的基因之一,为了研究该基因的生理功能,用从‘威赛克’苹果中克隆的MdRGL1a基因,分别构建GFP瞬时表达载体和植物表达载体。通过基因枪法轰击洋葱表皮细胞,激光共聚焦显微镜下观察MdRGL1a基因的亚细胞定位。利用农杆菌EHA105介导的叶盘法转化烟草(Nicotianna tabacumL.),对转基因烟草进行PCR扩增和GUS染色法检测,观察转基因烟草的形态学性状,利用ELISA方法测定烟草的内源激素水平。结果表明,MdRGL1a-GFP融合蛋白定位在细胞核内。转MdRGL1a基因的烟草组培苗根短粗、根系小,田间生长表现为植株矮化,75%转基因烟草提早开花。50%以上转基因植株的內源生长素和细胞分裂素水平显著低于对照非转基因烟草,而內源ABA水平显著高于对照。过量表达MdRGL1a导致转基因烟草植株的矮化和花期改变与內源激素生长素、细胞分裂素水平降低和ABA水平提高相关。DELLA proteins in plants are transcriptional regulators involved in GA signal pathway.Mutation of the DELLA proteins induces dwarf phenotypes.MdRGL1a gene is a member of the apple MdDELLAs.In order to elucidate its physiological functions,the full length of MdRGL1a gene isolated from ＇Wijcik＇ apple was cloned to construct an MdRGL1a-GFP transient expression vector and a 35S：MdRGL1a expression vector.The MdRGL1a subcellular localization in the particle bombardment transformed onion epidermal cells was observed under a confocal laser scanning microscope.The Agrobacterium-mediated transgenic method was used to introduce the MdRGL1a gene into Nicotianna tabacum.Transgenic plants were identified by PCR amplification and GUS histochemical staining.Transgenic tobacco plants were characterized.The endogenous hormones of tobacco were quantitatively analyzed by ELISA.Our results showed that MdRGL1a-GFP fusion protein was localized in the cell nucleus.The root system of in vitro transgenic plantlets carrying 35S：MdRGL1a was generally smaller and shorter than those of control plantlets.The transgenic tobacco displayed dwarf phenotypes when transplanted into the greenhouse and 75% flowered earlier than non transgenic control.Compared to control,the concentrations of endo-auxins and cytokinins in most transgenic plants were lower while the ABA level was higher.The dwarf phenotypes and the change in blooming time may probably result from the MdRGL1a overexpression induced changes in the concentrations of the endogenous IAA,ZR,and ABA in the transgenic plants.

关 键 词：苹果 MdRGL1a基因 DELLA蛋白 赤霉素 遗传转化 

分 类 号：S661.1[农业科学—果树学]