水稻淀粉分支酶SBE3基因RNAi载体构建及愈伤组织转化影响因素

RNAi Vector Construction for Rice Starch Branching Enzyme Gene SBE3 and Influencing Factors of Callus Transformation

为了简便、快捷构建RNAi载体,选择了起始点位置相同两个片段构建了SBE3基因的RNAi干扰载体。选择了长度为496bp和803bp,具有不同的酶切位点,其中互补区域形成dsRNA,而长出形成发夹结构,直接克隆到载体pCAMBIA1301上,不需构建中间载体,从而进一步研究SBE3基因在水稻品质改良中的应用。结果表明：所构建RNA干扰表达载体是可行的,相比与常规方法此方法更简便、快捷。同时研究了激素、外植体、继代培养次数、潮霉素因素对水稻愈伤组织诱导效果的影响,结果表明：水稻愈伤组织诱导效果的最佳因素组合是2,4-D浓度2.0mg·L-1,外植体选用下胚轴,继代培养次数在2~4次,潮霉素浓度为20mg·L-1。

In order to find to a easier and faster method to construct RNAi expression vector, two gene-specific fragment of the same origin positioning constructs RNAi expression vector of SBE3. A 496 bp and a 803 bp gene-specific fragment with different enzyme sites cloned into plant expression vector pCAMBIA1301. The sequence complementary composed double stranded RNA,and sequence remainder composed hairpin structures. RNAi expression vector of rice was constructed in order to find generally way to quality improvement of rice. The results showed that the method was feasiable, easier and faster. The effects of hormone, explant, subculture times and hygromycin on rice callus was studied in order to provide the optimal condition for establishment of efficient and stable rice callus induction effect system. The results showed that the best combination for rice callus induction was that 2,4-D concentration was 2.0 mg· L^-1 ,taking hypocotyl as explants,successive transfer culture times was 2-4 times,hygromycin concentration was 20 mg· L^-1 .