摘要
根据烟草(Nicotiana tabacum)GA 20-氧化酶基因序列,设计2对分别含有特定酶切位点的特异引物,以烟草基因组DNA为模板,扩增目的基因(约250bp)片段.将正、反向目的片段分别插入中间载体的内含子两侧,再经BamHI和SacI双酶切回收约700bp的目的片段,插入到双元载体质粒p2355中,成功构建了含GA20-氧化酶基因片段的反向重复序列植物表达载体p23700,其转录产物能形成发夹RNA(hpRNA),产生小分子干扰RNA,干扰目的基因的表达.将p23700质粒导入根癌农杆菌EHA105中并转化烟草叶片细胞,经选择分化培养,获得表型矮化的转基因烟草.
According to GA 20-oxidase gene sequence of tobacco （Nicotiana tabacum） , two pairs of specific primers containing special restriction enzyme sites were designed. The target fragments forward and reverse,were amplified and inserted into both sides of intron in intermediated vector pSK-int, respectively. Then the target fragment about 700 bp was cut by BamHI and Sacl from recombinant-intermediated vector, and was cloned into binary plasmid p2355. Plant expression plasmid p23700, with inverted repeat DNA fragment of GA 20-oxidase gene, was successfully constructed. And p23700 was introduced into Agrobacterium tumefaciens EHA105 and then transformed tobacco leaves. The transcriptional products of the foreign fragment probably form hairpin RNA （hpRNA） to interfere with target gene＇s expression. The transgenic plants of tobacco with dwarf phenotype were obtained. Fig 4, Ref 14
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2008年第1期48-52,共5页
Chinese Journal of Applied and Environmental Biology
