小麦wx-B1a基因片段的克隆及其RNAi载体构建

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。

Cloning of wx-B1a Gene Partial Sequence and Construction of its RNAi Expression Vectors

In order to obtain wheat wx-Bla gene-specific RNAi expression vector, the total RNA was isolated from wheat grain of 12-day post anthering, a pair of special primers was designed according to the wx-Bla gene （GenBank No. AB019623）, wx-Bla partial cDNA sequences （461 bp） was amplified by RT-PCR. The results demonstrated that, the cloned wx-Bla gene sequences were high homology with the reported waxy genes in GenBank previously. Then the fragment ligated to the FAD2 Intronl （Arabidopsis FAD2 Intron） in sense and antisense orientation to produce a hairpin fragment. The hairpin fragment was then inserted downstream of the endosperm specific expression promoter of high moleculer weight glutein gene IDx5. And a RNAi vector was obtained, which was drove by HMW-GS 1Dx5 promoter in pBAC47p. This RNAi vector willbe used to breed good-quality noodle lines with high yield-strong gluten wheat.