摘要
用小细胞团和分散细胞混合物与两种带不同质粒的农杆菌共培养的方法,将质粒PGV3850:1103neo T-DNA上的km抗性基因和Nos/Npt基因、质粒PGV517的Km抗性基因、NPTⅡ基因和毒蛋白基因转入贡柑细胞,在含有卡那霉素的选择培养基上选择出抗性愈伤组织和幼胚。通过Nopalim和NPTⅡ酶性检测、DNA/DNA分子杂交鉴定,证明了Nos基因、NPTⅡ基因已整合到贡柑体胚的细胞基因组中并表达,苏云金杆菌的毒蛋白基因整合到贡柑细胞基因组中,第一次在柑桔类果树实现了外源基因转移。用3％海藻酸钠包埋转化体胚制备人工种子,2粒转基因人工种子已萌发。
A transformation system was first established by cocultivation of Citrus embryonic calli with two kinds of Ajrobacterium turnefaciens harboring the recon-structed plasmids, PGV3850: 1103neo and PGV517, respectively. On the MS based medium containing 4mg/L GA_3, 500mg/L Cef (Cefazolium natricum) and 50mg/L Km(kanamycin), Kanamycin-rosistant transgenic embryonic calli and somatic embryos were selected. Through DNA/DNA molecular hybridization, Nopaline as-say and NPT Ⅱ assay, it is certain that the Nos/Npt gene in T-DNA was transfer-red into the somatic embryos of Citrus reticulata Blano cv. yu dong, and expressed in the transformed embryos, and the toxic protein gene of Bacillus thuringiensis was transferred into the somatic embryos. It is the first example of introducing foreign gene into Citrus. Artificial seeds were formed by encapsulating the trans-genic embeyos resistant to Km(100μg/ml) with 3% (w/v) sodium alginate. Up today,2 of the artificial seeds have already germinated.
出处
《中山大学学报（自然科学版）》
CAS
CSCD
1992年第1期79-85,共7页
Acta Scientiarum Naturalium Universitatis Sunyatseni
基金
国家"863"高技术计划资助项目
关键词
外源基因
四会贡柑
人工种子
柑桔
transformation
foreign gene
embryonic calli
artificial seeds
Citrus reticulata Blano cv. yu dong
