水稻OsNCED3基因的RNAi载体构建

摘要

OsNCED3 gene is an important gene during stress tolerance. The genomic DNA was isolated from the seedings of Oryza sati-υa "zhonghua NO. 10. " The specific genomic DNA segment was chosen, the restriction site was inserted, the genomic DNA was used as a template for KNAi-0sNCED3 cis-and trans fragments of PCR amplification. The PCR amplification fragments was connected to the pMD19 - T vectors and sequenced after restriction enzyme digestion and PCR. The sequencing results showed that RNAi-OsNCED3 cis-and trans fragments were correctly connected to the pMD19 - T vectors. The RNAi-OsNCED3 cis-and trans-fragment was digested by restriction enzyme and connected to the plasmid pFGC5941with a hairpin structure. PCR and double digestion verification showed that constructed pFGC5941-OsNCED3 vector (KNAi-0sNCED3) structural integrity.%水稻OsNCED3基因是水稻抗逆过程中重要的基因之一.以水稻中花10号幼苗为材料,提取基因组DNA.设计引物扩增区段cDNA并引入相应的酶切位点,以基因组DNA作为模板,进行RNAi-OsNCED3顺式和反式目的片段的PCR扩增.将PCR产物连接到pMD19-T载体上,经酶切和PCR检测后进行测序.测序结果表明:RNAi-OsNCED3顺式和反式目的片段均已正确的连接到pMD19-T载体上.然后将RNAi-OsNCED3顺式和反式目的片段通过酶切和连接,连接到含有发夹结构的质粒pFGC5941上.PCR及双酶切结果显示,构建的pFGC5941-OsNCED3即RNAi-OsNCED3载体结构完整.