无选择标记和载体骨干序列的Xa21转基因水稻的获得  

Generation of Selectable Marker-free and Vector Backbone Sequence-free Xa21 Transgenic Rice

作　　者：夏志辉[1] 李晓兵[1] 陈彩艳[1] 范海阔[1] 江光怀[1] 朱立煌[1] 翟文学[1] 

机构地区：[1]中国科学院遗传与发育生物学研究所,北京100101

出　　处：《生物工程学报》2006年第2期204-210,共7页Chinese Journal of Biotechnology

基　　金：国家高技术研究发展计划项目(No.2002AA212131);国家自然科学基金项目(No.30270857)资助。~~

摘　　要：利用双右边界T-DNA载体通过根癌农杆菌介导法将水稻白叶枯病广谱抗性基因Xa21导入杂交稻重要恢复系C418中。T0代共获得27个独立转基因株系,通过田间抗性鉴定与PCR分析,有17个株系的Xa21基因分子鉴定为阳性,且对白叶枯病原菌P6生理小种具有抗性。通过对17个株系的后代植株进行田间抗性鉴定,分子标记辅助选择及Southern杂交分析,结果显示4个株系的T1代植株中能分离出无潮霉素标记基因的Xa21转基因植株。无选择标记Xa21转基因株系的获得率为15%。PCR检测还表明,这些无选择标记的Xa21转基因植株不带有载体骨架序列。通过对转基因后代进一步的抗性鉴定与PCR辅助选择,获得了无选择标记和载体骨架序列的转基因Xa21纯合的抗白叶枯病水稻。The dominant gene Xa21 with broad-spectrum and high resistance to Xanthomonas oryzae pv. oryzae （Xoo） was transferred into C418, an important restorer line of japonica hybrid rice in China using double right-border （DRB） T-DNA binary vector through Agrobacterium-mediated transformation. 17 transgenic lines were Xa21-pesitive with high resistance to the race P6 of Xoo through PCR analysis and resistance identification, among the total 27 independent primary transformants （To ） obtained. The subsequent analysis of the T1 progenies of these 17 T0 lines through PCR-assisted selection and resistance investigation showed that four Xa21 transgenic To lines could produce selectable marker-free （SMF） progenies. The frequency of primary transformants producing SMF progenies was 15 %. In addition, PCR analysis also revealed these SMF progenies did not contain vector backbone sequence, and they were named as SMF and vector backbone sequence-free （SMF-VBSF） Xa21 transgenic plants. The further molecular and phenotypic analysis of the T2 and T3 progenies testified the homozygous SMF-VBSF Xa21 transgenic plants were obtained with high resistance to Xoo.

关 键 词：转基因水稻 XA21 白叶枯病抗性 无选择标记 无载体骨干 双右边界T-DNA载体 

分 类 号：S511[农业科学—作物学]