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根据GenBank和MaizeSeq等数据库中的玉米矮花叶病毒和粗缩病毒外壳蛋白基因(CP)全序列设计特异性引物,RT-PCR扩增玉米矮花叶病毒及粗缩病毒的CP基因特异性RNA干扰片段,分别构建反向重复片段并串联成pBluscript+SM;Hind III-EcoR I双酶切2T-DNA真核表达载体pDTB,插入Ubiquitin启动子及Nos终止子,构建pDTBU;串联的反向重复片段插入pDTBU,构建兼抗玉米矮花叶病毒和粗缩病毒的siRNA复合表达载体pDTBUSM。酶切结果显示,目的片段均正确插入到相应的载体中。本研究对开展抗病毒RNA干扰调控技术研究,创建高抗病毒玉米新种质具有重要的理论和实践意义。
Specific primers were designed based on the full-length cDNA sequences of maize dwarf mosaic virus and cull coat protein gene (CP) in GenBank and MaizeSeq databases. The CP gene-specific RNAs of maize dwarf mosaic virus and cull virus were amplified by RT-PCR The reverse fragment was constructed and inserted into pBluscript + SM in series respectively. The recombinant plasmid pDTB was digested with Hind III-EcoR I and inserted into Ubiquitin promoter and Nos terminator to construct pDTBU. Inverted tandem repeats The fragment was inserted into pDTBU to construct a pDTBUSM siRNA expression vector against maize dwarf virus and cull virus. The results of digestion showed that the target fragments were correctly inserted into the corresponding vectors. This study has important theoretical and practical significance for the research on the regulation of antiviral RNA interference and for the creation of new high resistant antiviral maize germplasm.