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普通双元载体已被广泛应用于农杆菌介导的植物转化,但这类载体通常只能转移5-20 kb的外源DNA片段;而双元细菌人工染色体(BIBAC)载体可以弥补普通双元载体的不足,通过它已在烟草、番茄等双子叶植物中实现了大片段 DNA(150 kb)的转移。BIBAC载体在单子叶植物转化中的应用尚未见报道。由于单、双子叶植物间以及大、小片段转化间的转化体系存在明显差异,常规的农杆菌介导的水稻转化体系不能适应BIBAC系统转化的要求。因此,建立适于BIBAC 系统的水稻转化体系是十分必要的。通过比较不同的受体材料,不同的预培养、共培养条件,不同的去除农杆菌及选择阳性愈伤的方式等对转化效率的影响,建立了适合水稻BIBAC系统的转化体系。该体系的技术要点包括:以水稻品种H1493 为转化受体;以含毒性辅助质粒pCH32的LBA4404菌株(HP4404)为侵染菌株;预培养的培养基pH5．6;以N6A代替AAM 悬浮农杆菌:侵染菌液浓度为OD600=1．0;共培养温度为24℃;采用过渡(Resting)培养除去农杆菌;采用二步法进行选择等。基于PCR检测、Southern印迹分析的结果表明,BIBAC载体所携带的插入片段及标记基因已整合到转化植株的基因组中。这个体系的建立为在水稻中利用BIBAC系统进行大片段DNA转化奠定基础。
Ordinary binary vectors have been widely used in Agrobacterium-mediated plant transformation, but these vectors usually only transfer 5-20 kb exogenous DNA fragments; and Binary Bacterial Artificial Chromosome (BIBAC) vector can make up the ordinary dual Vector deficiency, through which large fragments of DNA (150 kb) have been transferred in dicot plants such as tobacco and tomato. The application of BIBAC vectors in monocot transformation has not been reported yet. Because of the obvious difference between transformation systems of single and double dicot plants and between large and small fragments, the conventional Agrobacterium-mediated rice transformation system can not meet the requirements of BIBAC system transformation. Therefore, it is necessary to establish rice transformation system suitable for BIBAC system. The transformation system suitable for rice BIBAC system was established by comparing different receptor materials, different pre-culture, co-culture conditions, different methods of removing Agrobacterium and selecting positive callus. The main technical points of the system are as follows: the rice variety H1493 is used as the transformation receptor; the LBA4404 strain (HP4404) containing the toxic auxiliary plasmid pCH32 is used as the infection strain; the pre-culture medium is pH5.6; and the N6A is used instead of the AAM suspension Agrobacterium: Inoculum concentration of OD600 = 1.0; co-culture temperature of 24 ℃; using the rest (Resting) to remove Agrobacterium; two-step selection and so on. Based on the PCR test, the results of Southern blot analysis indicated that the insert and marker gene carried by the BIBAC vector had been integrated into the genome of the transformed plant. The establishment of this system lays the foundation for large DNA transformation in rice using BIBAC system.