花生Δ12脂肪酸脱氢酶基因FAD2的克隆及反义表达载体的构建

从花生的幼嫩叶片中提取总DNA,应用PCR方法分离到了两个FAD2基因片段,分别命名为FAD2-1和FAD2-2,将反向的FAD2-1和FAD2-2片段分别与所克隆的大豆油体蛋白基因启动子片段oleosin-a与oleosin-b相连,在中间表达载体pSPROK中构建了三个带T-nos的融合基因,进而把融合基因构建到植物表达载体pCAMBIA1300中,酶切鉴定后进行测序,结果表明,所构建的三个植物表达载体均分别带有Oleosin启动子和反义FAD2基因序列,FAD2-2的长度为593bps,与引用序列的同源性达到97%,包含一个起始密码子;FAD2-1的长度为1175bp,与引用序列的同源性达到99%,包含一个起始密码子与一个终止密码子。将构建好的植物表达载体已经转化农杆菌LBA4404并用于侵染花生外植体。

Cloning of PeanutΔ12 Fatty Acid Desaturase Gene FAD2 and Constructing of Anti-sense Expressing Vector

Genomic DNA was extracted from peanut leaves and two fragments of FAD2,named FAD2-1 and FAD2-2,were isolated by PCR,then the antisense FAD2-1 and FAD2-2 fragments were linked with soybean oleosin gene's promoter oleosin-a and oleosin-b,respectively,and three fusion genes with T-nose were constructed on mediate expressing vector pSPROK.The fussion genes including T-nos from pSPROK were cut out and constructed on plant expressing vector pCAMBIA1300.Digesting,characterization and sequencing was carried,and the sequencing results showed that FAD2-2 is 1175 bps in length and showed 99% identity with cited sequence,containing a start code.FAD2-2 is 593 bps,showing 97% identity with cited sequence,containing a start code and a teminal code.The constructs were then transferred into Agrobacterium LBA4404 via frozen-fusion method and have been used to transformed peanut mediated by agrobacteria.