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本研究以日龄1~3 d及7 d的小麦幼胚为受体,利用携带GUS基因的农杆菌LBA4404侵染,恢复培养至3~5叶期时,取第三叶进行GUS组织化学染色分析;并做两个优化对比处理:(1)抽真空处理:在侵染过程中,0.1 MPa抽真空2 min;(2)干燥处理:在共培养阶段,将幼胚转入铺有干燥无菌滤纸的平皿中。结果表明:GUS瞬时表达率随日龄增加而降低,以2~3 d龄的幼胚为最高;抽真空和干燥处理均能显著提高GUS瞬时表达率。最终,在优化处理条件下,获得77株转化苗中,40株第三叶GUS染色显示阳性,高达57.14%。进一步提取GUS阳性小麦叶片基因组DNA,进行PCR扩增,有6株转化苗扩增出目的条带,转化率为7.79%。初步说明以小麦幼胚的生长点作为转化受体是可行的。
In this study, 1 to 3 days and 7 days of wheat immature embryos as recipients, using GUS gene carrying Agrobacterium LBA4404 infection, restore culture to 3 to 5 leaf stage, take the third leaf GUS histochemical staining (2) Drying: During the co-culture stage, the immature embryos were transferred to a dry place without drying Bacteria filter paper in the dish. The results showed that the transient expression rate of GUS decreased with the increase of the age, and reached the highest level at 2 ~ 3 d. Both the evacuation of GUS and the drying treatment could significantly improve the transient expression rate of GUS. Finally, under the optimized conditions, the GUS staining of the 40 third transformed leaves was positive, reaching 57.14% of the 77 transformed seedlings. Genomic DNA of GUS-positive wheat leaves was further extracted for PCR amplification. Six of the transformed plants were expanded to produce the target bands with a conversion rate of 7.79%. Preliminary description of the growth point of wheat immature embryo as a receptor for transformation is feasible.