ACC氧化酶反义基因转化青花菜的研究

Studies on the Transformation of Broccoli (Brassica oleracea L.var.italica) with Antisense ACC Oxidase Gene

以青花菜的下胚轴和带 1～ 2mm子叶柄的子叶为转化受体 ,建立了根癌农杆菌介导的转化体系 ,获得了含番茄果实ACC氧化酶反义基因的转基因植株。经筛选获得了 5株抗性植株 ,其中 1株来自下胚轴 ,另外 4株来自带子叶柄的子叶。PCR及Southernblot检测证实 ,外源ACC氧化酶反义基因已整合进其中 2株拟转化青花菜植株的基因组。转化植株移栽到室外均能成活 ,成熟小花蕾的乙烯测定结果表明乙烯的合成受到了不同程度的抑制。

Using hypocotyls and cotyledon with 1～2 mm petiole as receptors, the Agrobacterium tumefaciens mediated transformation of broccoli was studied. The Agrobacterium tumefaciens contained the binary vector pTEW2402 carrying NPT Ⅱ gene, gusA gene and antisense ACC oxidase gene. NPT Ⅱ gene and gusA gene were under the control of CaMV 35S promotor, and antisense ACC oxidase gene was under the control of Act1 promotor. Five putative transgenic plants were selected from the selective regeneration medium with Km 25 mg/L and Car 250 mg/L, and the selective rooting medium with Cef 250 mg/L and Km 15 mg/L. Among them, one was regenerated from hypocotyls, and the other four were regenerated from cotyledon with 1～2 mm petioles. PCR, PCR_Southern blot and Southern blot analysis confirmed that foreign antisense ACC oxidase gene had already integrated into the genome of two plants of the five putative transgenic plants. The two transgenic plants could grow, develop and florescence after they were transplanted into soil. The ethylene production from fully open flowers of the transgenic plants were reduced compared with that released from the nontransgenic plant.