摘要
以成熟苹果果实的RNA为模板,经RT-PCR扩增并克隆苹果多酚氧化酶(APPO)长度为710bp的反义、正义基因片段。以副球菌中类胡罗卜素合成有关的(crtW+crtY)融合基因片段YYT为间隔区,将APPO反义基因片段、YYT和APPO正义基因片段串联,构成全长为2446bp的DNA并插入到植物双元载体pYPX145中,构成可表达苹果多酚氧化酶双链RNA的植物双元载体pYF7704。以根癌农杆菌介导的叶盘转化法转化苹果栽培品种红富士,通过50mg/L卡那霉素筛选和CUS检测,获得了转基因苹果抗性芽。荧光定量RT-PCR检测结果显示,转基因苹果抗性芽内多酚氧化酶基因的干扰效果达91.69%以上,研究结果证实多酚氧化酶双链RNA干扰在转基因苹果上是可行的。
Antisense and sense gene fragments(710 base pairs)of apple polyphenol oxidase (APPO)gene were obtained by RT-PCR amplification,using the total RNAs isolated from ripen ap- ple fruit as the template.These two fragments were ligated with a 1000bp spacer,YYT(crtW+crtY fusion)gene,which is relative to carotenoid synthesization in subcocci.The full-length 2446bp-tar- get gene was then inserted into plant binary vector pYPX145 to generate the recombinant plasmid pYF7704,which carried the expression unit of A PPO dsRNA,pYF7704 was transformed to apple (Malus×domestica)var.Red Fuji via agrobacterium tumefaciens mediated leaf disc transformation. With the selection of Karamycin and GUS detection assays,transgenic shoots of A PPO dsRNA were obtained.The results of FQ-RT-PCR indicated that A PPO mRNA level was suppressed to 91.69% in transgenic shoots compared to wide shoots.The data suggested that dsRNAi technology on apple polyphenol oxidase is feasible to be utilized in transgenic shoots.
出处
《实验生物学报》
CSCD
北大核心
2004年第6期487-493,共7页
Acta Biologiae Experimentalis Sinica
