为明确昆虫抗冻蛋白基因转入甘薯(Ipomoea batatas)后是否能提升其抗冻能力, 进而为培育甘薯抗冻育种材料奠定基础, 将黄粉虫(Tenebrio molitor)抗冻蛋白基因TmAFP导入植物基因表达质粒, 经农杆菌介导的遗传转化获得抗冻甘薯新材料。以甘薯品种Huachano为受体材料建立甘薯植株高效再生体系, 并采用不同成分的体细胞胚成熟培养基培养胚性悬浮细胞。胚性愈伤组织对除草剂的敏感性测试结果表明, 转基因阳性植株筛选的最适培养基为MS+0.2 mg·L ^-12,4-D+0.8 mg·L ^-1GAP+100 mg·L ^-1Carb。将表达质粒分别转化Huachano后共获得7个胚性愈伤团并最终获得42株再生抗性植株, 其中转pSUIBEV3-AFP有23个株系, 转pCAMBIA-AFP有19个株系, 经PCR、Southern杂交和RT-PCR检测后证实TmAFP基因已整合至甘薯基因组中并获得表达。将转基因甘薯及对照植株在-1°C下处理15小时后转移至室温, 结果表明, 转基因甘薯植株的抗冻能力显著提升。

To explore whether the gene encoding antifreeze protein from insect can enhance the freezing tolerance of sweet potato through gene transformation, and to prepare freeze-tolerance materials for breeding purposes, we constructed a plant gene expression vector harboring an antifreeze gene TmAFP from yellow mealworms (Tenebrio molitor) and obtained transgenic freeze-tolerance sweet potato lines using Agrobacterium-mediated transformation method. A high-frequency regeneration system of sweet potato was established using the variety Huachano as the recipient material, and the embryogenic suspension cells were cultured in the somatic embryo maturation medium. The sensitivity test of embryogenic cells to herbicides indicated that the combination of MS+0.2 mg·L ^-12,4-D+0.8 mg·L ^-1GAP+100 mg·L ^-1Carb is the most effective medium for screening the transgenic positive plants. Seven embryogenic calli were obtained and 42 resistant seedlings were regenerated, among which 23 harbored pSUIBEV3-AFP and 19 had pCAMBIA-AFP. All resistant seedlings were examined by PCR, Southern hybridization and RT-PCR, and the results showed that the TmAFP gene was integrated into the plant genome and expressed. The transgenic and non-transgenic plants were treated at -1°C for 15 hours, and then transferred to room temperature. The results demonstrated that the freeze-tolerance of the transgenic plants was greatly improved.