基因枪法获得逆境诱导转录因子DREB1A转基因小麦的研究

Study on Production of Transgenic Wheat with a Stress-inducible Transcription Factor Gene DREB1A by Microprojectile Bombardment

以小麦品种H675 6和藁城 890 1作为基因枪转化的靶材料 ,取其护颖至雌雄蕊原基形成期的幼穗 ,用含逆境诱导转录因子DREB1A和bar基因的质粒pAHC2 5轰击胚性愈伤组织 ,在分别含有 5mg L和 1 0mg LBasta溶液的培养基上进行筛选。得到的抗性愈伤组织在不含Basta溶液的培养基上再生培养 ,获得 2 1 8棵再生植株。田间涂抹浓度为 1 0 0mg L的Basta溶液检测后 ,对抗性植株作PCR检测 ,获得 5 4棵再生植株。通过对其中 2 0株T1代的PCR和Southern杂交分析 ,已获得 1 4株含DREB1A和bar基因的转基因小麦植株 ,其中H675 61 3株 ,藁城 890 1 1株。

Young spikes at the stage from the formation of protective glume primordium to the formation of pistil and stamen primordium of wheat cv. H6756 and Gaocheng 8901 were taken and cultured on MS medium for callus induction. As the receptor of target genes of stress inducible transcription factor gene DREB1A and bar, the calli were bombarded by microprojectile coated with the DNA of plasmid pAHC25 218 plantnets were finaly regenerated from the bombarded calli induced and screened on the selection medium containing 5mg/L and 10mg/L Basta, respectively. According to the resistance tests by daubing Basta solution of 100 mg/L on the wheat leaves in the field, further with the molecular analysis of PCR, 54 transplants were obtained. By PCR and Southern analysis of the genomic DNA extracting from the 20 T 1 plants, 14 plants, in which 13 from H6756 and 1 from Gaocheng 8901 were identified to be the transgenic ones contaning the target genes of stress inducible transcription factor gene DREB1A.