【摘要】 大片段克隆在农杆菌中的稳定性是利用可转化大片段载体进行农杆菌介导遗传转化的关键问题.选用插入片段分子质量分别为150kb和50kb的BIBAC克隆,测定了在3种农杆菌AGL-1,EHA105和LBA4404中的遗传稳定性.从第1、3、5次继代培养的农杆菌中抽提的质粒及酶切结果显示,由于农杆菌背景质粒的干扰,难以判断质粒的降解与否.将农杆菌质粒再转化到大肠杆菌宿主中,发现来自农杆菌AGL-1和EHA105的质粒出现了明显的降解,片段变小,而来自农杆菌LBA4404的质粒没有变化.结果表明,大片段BIBAC质粒在不同农杆菌菌株中的稳定性不同,在农杆菌LBA4404中比较稳定,适合用于遗传转化.更多还原

【Abstract】 Genetic stability of BIBAC clones containing large DNA fragments in agrobacterium is the key factor to influence Agrobacterium-mediated transformation with transformation-competent artificial chromosome.2 BIBAC clones,150 kb and 50 kb were used respectively to test their stability in agrobacterium AGL-1,EHA105 and LBA4404.Electrophoresis of plasmid and its digestion,which was extracted from first culture,third culture and fifth culture of Agrobacterium showed uncertainty of degradation because of background of agrobacterium plasmid.Then plasmid from agrobacterium was transferred into E.coli DH10B by electroporation again to avoid affection of agrobacterium background and extracted to perform electrophoresis.Results showed that BIBAC clones were unstable in AGL-1 and EHA105 because significant degradation was discovered,but BIBAC clones was stable in LBA4404,so it is suitable to be used in transformation.更多还原