【摘要】 建立美洲黑杨G2组培再生体系,确立美洲黑杨G2分化最适宜培养基和生根培养基。然后分别进行卡那霉素和潮霉素敏感性试验,利用叶盘法依次将反义磷脂酶Dγ(Anti_PLDγ)基因和几丁质酶(CH5B)基因转入美洲黑杨G2中。首先转入反义磷脂酶Dγ基因,经诱导不定芽及生根阶段卡那霉素(选择性抗生素)连续筛选,获得了21株卡那霉素抗性植株。抗性植株经PCR及PCR_Southern杂交检测,有13株均呈阳性,证明Anti_PLDγ基因成功整合到美洲黑杨G2基因组中。耐盐性试验表明,4株转基因植株抗NaCl能力比对照都有不同程度提高。选择4号株系继续转入几丁质酶基因,通过潮霉素(选择性抗生素)筛选不定芽及诱导生根获6株潮霉素抗性植株,经PCR及PCR_Southern杂交检测,6株均呈阳性,证明CH5B基因成功整合到美洲黑杨G2基因组中。由于CH5B基因与报告基因GUS处于同一开放阅读框(ORF)内,因此通过GUS基因的组织化学染色分析,证明了6株转化植株几丁质酶基因均获得正常表达。更多还原

【Abstract】 Via sequential single-gene transformation strategy, antisense phospholipase Dγ(Anti-PLDγ)gene and chitinase (CH5B) gene were introduced into Populus deltoides (G2) in proper order by Agrobacterium tumefacien mediated so as to enhance salt-tolerance and disease resistance. Firstly, the optimal media of P. deltoides (G2) for bud differentiation and rooting were established. The sensitivity of explants to kanamycin and hygromycin were tested separately in order to decide a reasonable selective pressure for transformation. Anti-PLDγ gene was introduced firstly. 21 kanamycin-resistant rooted plants were obtained through successive selection in shoot and root induction stage under high level kanamycin pressure. PCR and PCR-Southern analysis showed that 13 of 21 kanamycin-resistant rooted plants were positive. This meant that Anti-PLDγ gene was successfully integrated into the genome of these 13 plants. Salt-tolerance test demonstrated that 4/13 plants were enhanced to some extent. The No.4 plant was selected again to introduce chitinase (CH5B) gene. After successive selection in shoot and root induction stage under high level hygromycin pressure, 6 hygromycin_resistant rooted plants were gained. PCR and PCR-Southern analysis showed that 6 of these rooted plants were positive. This meant that CH5B gene was also successfully integrated into the genome of P. deltoides (G2). The CH5B gene and reporter gene GUS are in the same open reading frame (ORF), so via histochemical staining of GUS gene test, demonstrated that 6 plants can express CH5B gene normally.更多还原