摘要
将水稻黄单胞细菌白叶枯致病变种(Xanthomonas oryzaepv.oryzae)编码HarpinXoo的hrfA基因及其N-末端缺失突变基因AB分别连接到双元载体pBI121上,构建成转基因载体,并经冻融法转化土壤杆菌EHA105。采用叶盘法转化烟草,用卡那霉素抗性筛选再生植株。通过PCR扩增检测了转化烟草中的靶基因序列及35S启动子序列,对T1代PCR阳性植株进行了PCR-Southern杂交和RT-PCR分析。结果显示,外源基因已经整合到转基因烟草基因组中,并在转录水平正常表达。用从转hrfA基因和AB片段的烟草中提取的可溶性蛋白注射烟草都能引起过敏反应,说明目的基因在转基因烟草中能表达出有活性的蛋白。抗病性鉴定结果表明,转N-末端AB片段的烟草和转hrfA全长基因的烟草一样表现出对烟草花叶病毒(TMV)的抗性。
The hrfA gene （420 bp）, which encodes Harpinxoo of Xanthomonas oryzae pv. oryzae, and its N-terminal deletion derivative AB （177 bp）, were inserted into the binary vector pBI121, respectively. The resulting two transgenic constructs were transferred into Agribacterium turnefaciens EHA105 by the freeze-thaw method. Transformation was conducted by infection of tobacco leaf discs with recombinant A. turnefaciens, and transgenic tobacco plants were obtained by screening with kanamycin. Genomic integration and expression of the transferred genes were determined by PCR, PCR-Southern blot hybridization and reverse transcriptase PCR. Soluble proteins extracted from plants transformed with hrfA and AB could elicit HR on tobacco leaves, indicating that the proteins expressed in transgenic tobacco plants are bioactive. Disease bioassays showed that, the tobacco plants transformed with N- terminal sequence AB enhanced resistance to TMV similarly as those transformed with the whole hrfA gene.
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2007年第3期47-52,共6页
Journal of Nanjing Agricultural University
基金
国家自然科学基金项目(30230240)
国家重点基础研究发展规划资助项目(G2000016201)
