摘要
双元细菌人工染色体（BIBAC）栽体具有克隆大片段DNA构建文库和农杆菌介导转化植物的双重功能．利用BIBAC载体，在双子叶植物烟草和番茄中已实现了大片段DNA的转移，但在单子叶植物中的应用还未见报道．本研究利用不同水稻基因型、不同农杆菌菌株，对水稻进行了BIBAC2栽体的转化研究．水稻成熟胚诱导愈伤组织经过1～2次继代培养，在含乙酰丁香酮的N6培养基上预培养3d，在含BIBAC2栽体的农杆菌中侵染10min，26℃下共培养3d．感染后的愈伤组织在含25～50mg／L潮霉素培养基上经过4～8周的筛选，共得到了66株抗性再生植株．通过对抗性植株中GUs基因的组织化学染色检测，NPTⅡ基因的PCR检测和ELISA检测，HYG基因的PCR检测，确认2株阳性植株来自EHA105菌系侵染的中花15号愈伤组织，1株阳性植株来自EHA105菌系侵染的中花8号愈伤组织，转化率分别为2．9％和0．9％．结果表明，B1BAC2栽体已被成功导入了水稻基因组中，转化效果EHA105菌株优于C58C1菌株，中花15号、中花8号优于中花10号和中花11号．结果还表明，利用三亲交配法可以将大于23．5kb的载体转化到农杆菌中．初步建立了BIBAC2栽体转化水稻的技术体系，为利用BIBAC系统向水稻转入大片段DNA和多个外源功能基因奠定了基础．
An Agrobacterium mediated transformation protocol in rice using binary bacterial artificial chromosome （BIBAC） vector system was developed in this research. Calli derived from mature embryos of different rice genotypes were used as target tissues to be infected by different Agrobacteriurn strains. After cultured on the selection medium containing 25-50 mg/l hygromycin for 4-8 weeks, 66 resistant plants to the antibiotics were obtained. Tested by histochemical staining for GUS gene, PCR and ELISA assay for NPTII gene or HYG gene, three regeneration plants were identified to be positive, among which two plants derived from Zhonghua 15 mediated with EHA105, one plant from Zhonghua 8 of the same strain. The results indicated that the report gene, marker genes on the insert fragment carried by BIBAC2 were stably integrated into the rice genome. No transgenic plant was got from other rice genotypes or C58Cl infected rice calli. It seemed that Zhonghua 15 or Zhonghua 8 was better than other tested varieties, EHA105 better than C58Cl for the transformation efficiency. The results demonstrated that it is possible to transfer larger DNA fragment and a few functional genes into rice by using BIBAC2 as plasmid vector. Tri parents mating method can be used to transform a plasmid larger than 23. 5kb from E. coli into Agrobacterium.
出处
《农业科学研究》
2006年第1期6-9,12,共5页
Journal of Agricultural Sciences
关键词
水稻
双元细菌人工染色体
农杆菌
遗传转化
rice
binary bacterial artificial chromosome （BIBAC）
Agrobacteriurn tumefaciens
genetic transformation
