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为了获得小麦wx-B1a基因的特异RNAi表达载体,以小麦品种‘京花1号’开花12天的籽粒为材料,用天根公司植物总RNA提取试剂盒提取总RNA,以wx-B1a基因(GenBank NO:AB019623)的cDNA序列设计一对特异性引物,利用RT-PCR克隆了wx-B1a基因部分cDNA片段。Blastn结果显示,它与GenBank上报道的Triticum aestivum wx-1 gene(GenBank NO:EU719611.1)同源。通过酶切连接将此片段分别置于拟南芥FAD2的Intron1(GenBank NO:AJ271841)的上、下游,然后将此发夹结构置于小麦HMW-GS 1Dx5启动子的下游,从而构建了小麦wx-B1a基因的特异RNAi表达载体pBAC47p-wx-B1aIR。从而为下一步转化高产强筋小麦品种培育优质面条专用小麦奠定基础。
In order to obtain the specific RNAi expression vector of wheat wx-B1a gene, the total RNA was extracted from the wheat genotype “Jinghua 1” with 12 days of flowering. Total RNA was extracted from genomic DNA of wx-B1a GenBank NO: AB019623) cDNA sequence designed a pair of specific primers, using RT-PCR cloned wx-B1a gene partial cDNA fragments. Blastn results showed that it is homologous to the Triticum aestivum wx-1 gene (GenBank NO: EU719611.1) reported in GenBank. This fragment was placed on the upstream and downstream of Intron1 (GenBank NO: AJ271841) of Arabidopsis FAD2 by restriction enzyme ligation, and then the hairpin structure was placed downstream of the wheat HMW-GS 1Dx5 promoter to construct a wheat wx -B1a gene specific RNAi expression vector pBAC47p-wx-B1aIR. Which laid the foundation for the next step to transform high yield and strong gluten wheat varieties to cultivate high quality wheat for noodles.