【摘要】 香味是稻米食味品质的主要性状之一,为培育无抗性选择标记基因的优质香稻新品系,以携带有超双元载体OSBADH2-RNAi的农杆菌菌株EHA105介导,将OSBADH2基因的RNA干扰结构和潮霉素抗性选择标记(HPT)基因同时导入优质粳稻武育粳3号成熟胚来源的愈伤组织中,经过在含有潮霉素的培养基上筛选和分化,获得了54个独立转化子,其中36个转化子同时含目的基因和HPT基因,共转化频率为66.7%;从这些转化子的后代中共筛选获得25个去除HPT基因的转化子,去标记频率为69.4%;通过KOH法等4种方法进行香味的鉴定,获得15个具有香味的株系。实时定量PCR分析结果表明,具香味株系中的OSBADH2基因的表达受到了明显抑制,进一步证明了香味的产生是由于OSBADH2基因被干扰而引起。农艺性状调查表明,抑制OSBADH2基因的表达对水稻产量相关性状,包括株高、千粒质量、穗粒数和结实率影响不明显。更多还原

【Abstract】 In order to generate marker-free fragrant transgenic rice,OSBADH2-RNAi and hygromycin phosphotransferase gene( HPT) were transferred to calli derived from a japonica rice cultivar,Wuyujing 3,mediated with a Agrobacterium strain EHA105 harboring a single twin T-DNA binary vector. After selected and regenerated on the media with hygromycin,54 independent transgenic lines were generated. According to PCR results,we got 36 cotransformation lines harboring both HPT gene and OSBADH2 gene RNAi structure. The frequency of co-transformation was 66. 7%. After two generation selections,we got 25 HPT gene free T2 lines harboring OSBADH2 gene RNAi structures. The frequency was 69. 4%. 15 fragrance lines were screened from these transgenic lines using four kind methods detecting fragrance. Real-time quantitative PCR results showed that OSBADH2 gene expression level was reduced significantly in these transgenic fragrance lines. We found that there was no obvious difference between the transgenic lines and the wild type,in the main agronomy characteristics,including plant height,1000-grain weight,grain number of per spike,and seed setting rate.更多还原