农杆菌介导雪莲PBP基因对棉花的遗传转化研究

Studies on Genetic Transformation of Transferring the XLPBP Gene to Cotton Mediated by Agrobacterium tumefaciens

雪莲磷脂酰乙醇胺结合蛋白(Phosphatidyl ethanolamine-binding family protein,PBP)基因表达产物与细胞膜的冷稳定性有关,可能在植物抗寒方面起一定作用。本研究将XLPBP基因以农杆菌介导的方法转化新陆早17号、新陆早13号、新陆早1号3个品种陆地棉,通过对不同激素浓度、预培养时间、菌液浓度和浸菌时间等因素对转化培养物的影响以及茎段外植体对卡那霉素(Kan)的敏感性等方面的探讨,确定了新陆早13号外植体材料(茎段)的最佳转化条件,即:在OD600为0.4的农杆菌菌液中侵染10 min,在茎段最适培养基(含0.1 mg/L KT和0.1 mg/L 2,4-D,pH5.8)上进行预培养2 d,共培养2 d,转至不含选择压的培养基(含Cef 500 mg/L)上进行延迟培养7 d,再进行选择培养,茎段愈伤分化达25%(诱导出愈伤组织的外植体数占接种的外植体数)以上;茎段转化适宜的选择压力为Kan 50 mg/L;在获得的抗性愈伤中8%左右经检测为GUS阳性。

The phosphatidyl ethanolamine-binding family protein gene from Saussurea involucrata Kar. et belongs to cold-resistant revelant gene. It is revelant to the stability of cellular membrane, and may play an important role in the cold resistance of plant. This study established a highly efficient regeneration system of cotton from genes engineering and then transfer red the XLPBP gene to cotton, which was mediated by Agrobacterium tumefaciens. The main factors influencing regeneration and transformation rate were discussed and the best conduction of regeneration and transformation were determined. The positive regeneration was obtained.