含硫氨基酸蛋白与GFP融合基因的表达分析  

Expression Analysis of Fusion Gene Sulphur-amino acid Protein with Green Fluorescent Protein

作　　者：关宁[1] 王涌鑫[1] 李聪[1] 苗丽宏[1] 张博[2] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所牧草遗传育种研究室,北京100193 [2]新疆农业大学草地资源与生态自治区重点实验室,乌鲁木齐830052

出　　处：《草地学报》2009年第6期706-712,共7页Acta Agrestia Sinica

基　　金：国家十一五科技支撑计划(2006BAD01A19);新疆农业大学草地资源与生态自治区重点实验室开放课题(XJDX0201-2005-03)

摘　　要：提高饲用植物中的含硫氨基酸水平已成为动物营养学家关注的焦点,含硫氨基酸对奶制品、肉类及羊毛产量有重要影响。γ-zein和zeolin(γ-zein和菜豆球蛋白的融合基因)编码2种含硫氨基酸蛋白基因,利用基因轰击法将质粒pCBzein、pCBzeolin和pCAMBIA1302分别转入洋葱(Alliumcepa L.)表皮细胞,激光共聚焦扫描显微结果表明γ-zein和zeolin在洋葱表皮细胞的细胞核、内质网及细胞壁中均有表达。采用冻融法分别将表达载体pCBzein、pCBzeolin和pCAMBIA1302导入根癌农杆菌(Agrobacteriumtumefaciens)菌株LBA4404,利用该菌株转化烟草(Nicotianatabacum);在Hygromycin选择压力下获得烟草抗性不定芽和再生植株;激光共聚焦扫描显微结果表明γ-zein和zeolin蛋白以颗粒的形式不均匀地分布在烟草原生质体中,并验证表达载体pCBzein和pCBzeolin的选择基因、目的基因和报告基因均得到了表达,为进一步开展豆科牧草营养品质改良奠定了基础。Improvement of the sulfur-containing amino acids level in forage plants has been the focus of attention of the animal nutritionists.Sulphur-containing amino acids play an important role in the efficiency of dairy,meat and wool production.γ-zein and zeolin（fused withγ-zein and phaseolin） are the genes encoding sulphur-containing amino acids proteins.Plasmids pCBzein,pCBzeolin and pCAMBIA1302 were transferred into epidermal cells of onion by gene-gun bombardment technique,respectively.The results of confocal laser scanning microscope（CLSM） showed that γ-zein and zeolin expressed in cell nuclear,endoplasmic reticulum and cell wall of onion epidermal cells transformed γ-zein-GFP and zeolin-GFP.Expression vectors pCBzein,pCBzeolin and pCAMBIA1302 were transferred into Agrobacterium tumefaciens LBA4404 by freeze-thaw method,and then were transformed into Nicotiana tabacum mediated by stain LBA4404.Resistant adventitious shoots and regeneration plants were obtained under the selection pressure of Hygromycin.The results of CLSM confirmed that γ-zein and zeolin accumulated in granular forms were distributed heterogeneously in the protoplast of tobaccoes,and proved that the selection gene,target genes and report gene from the plant expression vectors pCBzein and pCBzeolin were working well detected by PCR and CLSM.

关 键 词：含硫氨基酸基因 绿色荧光蛋白 洋葱 烟草 基因表达 

分 类 号：Q946.1[生物学—植物学]