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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。
Fragrance is one of the main traits of rice eating quality. To develop a new line of high-quality fragrant rice with selectable marker-resistant genes, Agrobacterium tumefaciens strain EHA105 carrying the super-binary vector OSBADH2-RNAi was used to mediate the interference of OSBADH2 RNA interference The structure and hygromycin resistant marker (HPT) were introduced into callus derived from mature embryos of high quality japonica rice Wuyujing 3 simultaneously. After selection and differentiation on hygromycin containing medium, 54 independent Among them, 36 transformants contained the target gene and HPT gene at the same time, and the frequency of co-transformation was 66.7%. From the progenies of these transformants, 25 transformants were screened for removing HPT gene. The frequency of de-labeling was 69.4% France and other four methods for the identification of fragrance, access to 15 scented strains. Real-time quantitative PCR analysis showed that the expression of OSBADH2 gene in the scented strains was significantly inhibited, which further proves that the production of the fragrance is caused by the interference of the OSBADH2 gene. Agronomic traits investigation showed that inhibiting the expression of OSBADH2 gene had no obvious effect on the yield-related traits of rice, including plant height, grain weight, grains per spike and seed setting rate.