摘要
旨在通过转基因途径诱导油菜种子中fad2基因发生转录后基因沉默,本文构建了fad2基因的ihpRNA植物表达载体。通过PCR扩增分离到甘蓝型油菜种子特异性表达的Nap in启动子序列(1147bp)以及油酸脱饱和酶基因(fad2)的一个537bp的片段,并将它们分别克隆到pGEM-T easy载体中。利用中间载体pHurrican构建fad2基因的反向重复框。将fad2基因片段以正向的方式连接在一个可剪切的内含子的5’末端,而以反向的方式连接到该内含子的3’末端;然后将Nap in启动子序列连接到该反向重复框的5’端,而在其3’端接有一个nos终止子序列;最后将fad2基因的反向重复表达框分两步克隆到植物双元载体pCAMB IA2300的pUC18多克隆位点,构建具有种子特异性表达的fad2基因的ihpRNA表达载体pCNFIRnos。限制性内切酶酶切对载体作了鉴定分析。
The full- length napin promoter sequence （1147bp） and a conserved region （537bp） of microsomal △ - 12 desaturase gene （fad2） were amplified from genomic DNA of Brassica napus L. and cloned into the pGEM - T easy vector respectively. To develop an iphRNA vector for seed - specific expression targeting the fad2 gene in Brassica napus L. ,an inverted repeated unit of the 537bp segment of the fad2 gene was first cloned into a medium vector,the pHurrican Vector,with a spliceable fad2 intron sequence in between. Then the seed -specific napin promoter was placed to the 5 ＇ - end of the inverted repeat cassette, and a nos polyA to the 3 ＇ - end of it. Finally,an ihpRNA expression vector pCNFIRnos was constructed by inserting the whole inverted repeat cassette into the multiple clone sites in the binary vector pCAMBIA2300. The pCNFIRnos was confirmed by the digestion of restriction enzymes. The vector is aim to transform the Brassica napus L and silence the post - transcription expression of fad2 gene during the seed development.
出处
《中国油料作物学报》
CAS
CSCD
北大核心
2006年第3期251-256,共6页
Chinese Journal of Oil Crop Sciences
基金
农业部"948"项目(2003-Q4)
江苏省农业科学院基金项目(6110508)
