【摘要】 【目的】筛选大豆RACK1基因的RNAi突变体,为研究RACK1基因在大豆生长发育过程的调控作用提供依据。【方法】采用RT-PCR克隆大豆叶片RACK1基因核心保守序列片段,以植物表达载体pCAMBIA1301为基本载体,构建抑制大豆RACKl基因表达的RNAi载体。通过农杆菌介导转入大豆子叶节,经潮霉素筛选转基因植株,利用PCR、Southern blot及RT-qPCR进行转基因植株检测。【结果】克隆获得大豆RACK1基因核心保守序列片段432 bp;将该基因片段连接到pCAMBIAl301表达载体内含子两侧,通过酶切分析,RNAi载体构建正确。通过农杆菌介导,将该载体转入大豆中黄13号,获得23个转基因大豆株系;经PCR和Southern blot检测,确定大豆RACK1基因RNAi片段已融合到大豆基因组中。经定量RT-qPCR分析,不同转基因大豆株系RACK1基因mRNA的表达量具有明显差异,其在株系5的表达量最高,为对照的68.5%;株系7最低,降至对照的19.9%。【结论】成功构建了大豆RACK1 RNAi表达载体并导入大豆基因组中,获得23个农杆菌介导的RACK1 RNA干扰表达的大豆转基因植株,为研究RACK1基因在大豆生长发育过程中的功能和作用奠定了基础。更多还原

【Abstract】 【Objective】The RNAi mutant of soybean RACK1 gene was screened in order to provide references for studying regulation role of RACK1 gene in the process of soybean growth and development. 【Method】The conserved sequence of RACK1 in soybean leaf was cloned by RT-PCR. A RNAi expression vector to inhibit RACK1 expression was constructed by using plant expression vector pCAMBIAl301. RACK1 gene was transferred into cotyledonary node of soybean Zhonghuang 13 via Agrobacterium-mediated transformation method. Transgenic seedlings was screened by hygromycin and was detected by PCR, Southern blot and RT-PCR. 【Result】The cloned soybean RACK1 gene fragment was 432 bp.The gene fragment was connected to both sides of intron in expression vector pCAMBIAl301. Through restriction enzyme digestion analysis and DNA sequencing, it showed that all the recombinant plasmids were according with the design and RNAi vector was constructed correctly. By Agrobacterium-mediated, the constructed RNAi vector was transformated into soybean Zhonghuang 13 and 23 transgenic lines were obtained. By detection of PCR and Southern blot, RNAi fragment of RACK1 gene was confirmed to have transferred into soybean genome. The quantitative PCR analysis results showed that mRNA expression level in RACK1 gene significantly differed in different soybean transgenic lines. The mRNA expression level in line 5 was the highest being accounting for 68.5% of control, and that of line 7 was the lowest accounting for19.9% of control. 【Conclusion】The RNAi expression vector of soybean RACK1 gene was successfully constructed and transformated into soybean genome, and 23 transgenic plants with RACK1 RNAi expression are obtained to lay the foundation for studying the function and role of RACK1 in the process of soybean growth and development.更多还原