根癌农杆菌介导的百脉根遗传转化体系的优化研究

Optimization of A.tumefaciens-mediated Transformation Procedure for Lotus Corniculatus

百脉根(L otus corn icu la tus L.)是世界著名的多年生优良豆科牧草之一,也是常见的庭院观赏植物.本文通过比较不同根癌农杆菌转化百脉根的效率和优化百脉根子叶遗传转化的条件,建立了一套有效的遗传转化体系.实验结果表明,EHA 105作为宿主菌对百脉根进行转化较LBA 4404和GV 3101具有更高的转化率;5 d苗龄的子叶最适合作为转化的外植体;浸染过程中0.05 M Pa负压处理5 m im以及在共培养培养基中添加20 m g/L的乙酰丁香酮均可提高转化效率.卡那霉素抗性植株经3次选择继代培养后,PCR检测全部为阳性.对部分PCR阳性植株进行PCR-Sou thern杂交,证实PCR产物真实可靠,表明外源基因已整合进入百脉根基因组中.

Lotus corniculatus L. is one of well-known leguminous forages around the world and also often used as ornamentals to decorate houses. In this study, a reproducible and efficient Lotus corniculatus transgenie system was established through comparing transformation efficiency of different Agrobacterium strains and optimizing factors affecting the transformation. The results show that strain EHA105 is more efficient than strans LBA4404 and GV3101 in performing transfer of foreign genes into Lotus corniculatus. Cotyledons of 5-day-old seedlings were found to be most suitable for infecting L. corniculatus with A. turnefaciens. And transformation efficiency was improved either by subjecting cotyledons to -0.05 MPa pressure for 5 min while they were being infected or by adding AS at 20 mg/L into co-culture medium. After a 3-round selection of kan-resistance, selected kan-resistant plants were further identified by PCR and PCR based Southern blot analysis. The results demonstrate that all the kan-resistant plants are transgenic ones and that the exogenous target gene, the AVP1 gene, has been integrated into the genomic DNA of all transformants obtained.