小麦农杆菌转化体系的优化及HMW-GS 1Bx14基因转化

Optimization of Agrobacterium-mediated genetic transformation of wheat and transformation of HMW-GS 1Bx14 gene

以小麦绵阳19和洛阳8716幼胚愈伤组织为材料,利用gus基因的瞬时表达,探讨了农杆菌菌株和侵染条件对转化效率的影响。结果发现,对于农杆菌菌株EHA105,小麦绵阳19和洛阳8716幼胚愈伤组织侵染的最佳条件均为OD6000.8,侵染30min。对于农杆菌菌株LBA4404,绵阳19和洛阳8716幼胚愈伤组织侵染的最佳条件分别为OD6001.0,侵染30min和OD6000.8,侵染60min。利用建立的农杆菌转化体系对小麦高分子量麦谷蛋白亚基1Bx14基因进行了转化,经潮霉素抗性筛选、PCR和PCR-Southern杂交检测,平均转化率为0.61%。

The genetic transformation of wheat Mianyang 19 and Luoyang 8716 immature embryo callus mediated by Agrobacterium tumefaciens was studied.The factors that influence the efficiency of transformation were investigated by using the transient expression of gus gene.The result showed that for both varieties,the best transformation procedure of EHA105 was Agrobacterium cell density of OD600≈0.8,incubation for 30 min.When using Agrobacterium LBA4404,the transformation efficiency of Mianyang 19 and Luoyang 8716 immature embryo callus was highest when cell density was OD600≈1.0,incubation for 30 min and cell density of OD600≈0.8,incubation for 1h,respectively.Using the optimized conditions,the high molecular weight glutenin subunit 1Bx14 gene was transformed.After selected with hygromycin,tested by PCR and PCR-Southern,the positive progenies were obtained,yielding an average transformation frequency of 0.61%