【作者】 郭健； 陈忠正； 李斌； 文海涛； 赵亮；

【Author】 Guo Jjian Chen Zhongzheng Li Bin Wen Haitao Zhao Liang (College of Food Science,South China Agricultural University Guangzhou 510642,China)

【机构】 华南农业大学食品学院广州；

【摘要】 本研究利用 RNAi 技术,通过 RT-PCR 方法克隆出水稻淀粉分支酶 sbe1及 sbe3基因片断,经两步组装法将 sbe1、sbe3部分基因片断及由此二片段拼接得到的 sbe1+sbe3片段分别正向、反向组装入 pRNAi-ubi,成功构建 RNAi 转化载体 pRNAi-ubi/sbe1、pRNAi-ubi/sbe3 和 pRNAi-ubi/sbe1+sbe3。在农杆菌介导下,使 pRNAi-ubi/sbe3对粳稻中花11未成熟胚诱导的愈伤组织进行转化,通过 PCR、Southern 杂交鉴定获得一批转基因植株。半定量 RT-PCR 鉴定出转化苗 T1代种子中 sbe3基因表达被明显抑制,但只引起转基因水稻胚乳中直链淀粉含量少量的提高,说明采用 RNAi 仅沉默 sbe3基因对水稻胚乳直链淀粉含量没有显著影响。更多还原

【Abstract】 In this study,RNA interference was used to down-regulate the gene of starch branch enzyme in rice endosperm in order to increase the content of amylose. The sbel and sbe3 gene fragment was isolated from rice by RT-PCR.These two fragments and the sbel+sbe3 fragment were constructed into the two multi-cloning site regions of pRNAi-ubi in the sense and antisense orientations respectively.The sbe3 construct was introduced into the rice variety of zhonghua 11 by Agrobacterium-mediated transformation.PCR and Southern blot analysis showed that the sbe3 gene had been integrated into rice genome.The semi-quantity RT-PCR confirmed that the expression of she3 gene was significantly inhibited in the endosperm of T₁ transgenic seeds,while the amylose content in the endosperm of T₁ transgenic seeds was slightly increased compared with the non-transgenic seeds of zhonghua 11.The results indicated that the suppression of sbe3 gene expression alone by RNA interference had no dominant effects on the seeds amylose content of transgenic rice.更多还原