基因枪法获得转cry1Ac基因甘蔗的研究

Studies on cry1Ac Transgenic Plants of Saccharum Complex by Particle Bombardment

根据定向克隆原则,以pGreenⅡ0229为载体骨架,cry1Ac为目的基因,bar为筛选标记基因,构建了大小为8602bp的植物表达载体pUBCG0229-cry1Ac。酶切结果表明,构建的载体pUBCG0229-cry1Ac结构完全正确。用基因枪轰击法将pUBCG0229-cry1Ac质粒DNA转化甘蔗(Saccharum Complex)品种福农95-1702和桂糖94-119的胚性愈伤组织,使用PPT(phosphinothricin)对轰击后的材料进行继代、分化和生根筛选,移栽成活后使用Basta溶液喷洒进行初步筛选,共获得86株抗性植株,通过PCR检测、Dot-Southern检测及PCR产物测序,证明已将cry1Ac基因整合到其中22株甘蔗基因组中。

Plant expression vectors pUBCGO229 crylAc with size of 8602 bp, which had anti-insect gene and the bar gene as screening marker gene, was constructed. The result of restriction digestion showed that the construction of the vectors pUBCG0229 crylAc was completely correct. The embryonic callus of Saccharum Complex ＇FN95 1702＇ and ＇GT94 119＇ were transformed with the plasmid of pUBCGO229 crylAc by bombardment. After selection with PPT （Phosphinothricin） or Basta, 86 regenerated plants were obtained. After detected by PCR, Dot Southern blotting and sequencing of PCR products from 22 transformed plants, it proved that crylAc gene had been integrated into sugarcane genome.