农杆菌介导的二氢黄酮醇3’5’羟化酶cDNA对非洲菊的转化及对其花色的影响(英文)

Agrobacterium -mediated Transfer of Flavonoid 3' 5'-Hydroxylase cDNA to Gerbera hybrida Modifies Flower Colour

通过非洲菊(Gerbera hybrida)组织培养建立起适合于农杆菌介导的基因转化受体系统.非洲菊叶柄外植体在MS+3 mg/L BA+0.01 mg/L NAA培养基中直接诱导芽生长,在MS+0.1 mg/L NAA培养基中生根培养,获得再生植株,诱导率达57％.带有云南矮牵牛(Petunia hybrida)二氢黄酮醇3’5’羟 化酶cDNA的pEH表达载体与农杆菌(Agrobacterium tumefaciens)LBA4404融合后,与非洲菊叶柄外植体共培养3 d,通过含有卡那霉素25 mg/L和羧苄青霉素500 mg/L的选择培养,诱导出转基因非洲菊.经PCR和Southern分子杂交检测,证明目的基因已整合入非洲菊的基因组中,转基因非洲菊花色、花型及叶型都有显著变化.

A protocol for regeneration and genetic transformation has been established for gerbera (Gerbera hybrida). High frequency regeneration of shoot buds from petioles was achieved with Murashigeand Skoog's (MS) medium supplemented with 3 mg/L 6-benzylaminopurine (BA) and 0.01 mg/L α-naphthalene acetic acid (NAA). Elongation of shoot buds and subsequent rooting were obtained on MS medium with 0.1 mg/L NAA. The Agrobacterium twnefaciens strain LBA 4404 carrying a binary vector plasmid pEH subcloned with cDNA for flavonoid 3' 5' -hydroxylase has been used for transformation. The petiole explants from in vitro grown shoots were cocultivated with A. tumefaciens for 3 days. Shoot buds were produced on the regeneration medium containing kanamycin (25 mg/L) and carbenicillin (500 mg/L). The transgenic nature of the regenerated plants was confirmed by polymerase chain reaction and Southern hybridization analyses. Transgenic gerbera plants had obvious changes in flower colour and some alterations in the form of flowers and leaves.