基因枪法获得转cry1Ac基因甘蔗的研究

Studies on cry1Ac Gene Transgenic Plants of Saccharum Complex by Particle Bombardment

摘要:

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8602 bp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。

Abstract:

Plant expression vectors pUBCG0229-cry1Ac 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-cry1Ac was completely correct. The embryonic callus of Saccharum Complex ‘FN95-1702’ and ‘GT94-119’ were transformed with the plasmid of pUBCG0229-cry1Ac 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 cry1Ac gene had been integrated into sugarcane genome.