Abstract: 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.

Key words: Sulphur-amino acid genes, Green fluorescent protein, Allium cepa L., Nicotiana tabacum, Gene expression

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(Allium cepa 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(Agrobacterium tumefaciens)菌株LBA4404,利用该菌株转化烟草(Nicotiana tabacum);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.

关键词: 含硫氨基酸基因, 绿色荧光蛋白, 洋葱, 烟草, 基因表达