大豆脂肪酸脱饱和酶反义基因转化根癌农杆菌研究

Study on the Transformation of Antisense Soybean Fat Acid Desaturase to Agrobacterium tumefacien

根据NCBI中大豆fad2-1序列设计引物,用PCR方法从大豆的基因组DNA中扩增大豆脂肪酸脱饱和酶基因,克隆到pMD18-Tvector中,转化大肠杆菌JM109菌株,进行测序与比对。然后,将其反向克隆到表达载体pBt,转化农杆菌菌株LBA4404,经双酶切鉴定和PCR扩增检测,获得具有该基因的农杆菌工程菌。结果表明,克隆的fad2-1基因为1 196 bp,基因序列与NCBI中已发表的fad2-1序列只有4%的差异,相似性大于95%,说明克隆的基因是大豆fad2-1基因;构建了该基因的反向表达载体,转入农杆菌内。这为利用农杆菌介导法把该反义基因转入大豆,改良脂肪酸成分奠定了基础。

In this study,the primers were designed based on the sequences of soybean fat acid desaturase（fad2-1） in NCBI database to amplify fad2-1 gene from the genomic DNA of soybean leaves.The amplicon was cloned into pMD18-T vector to be introduced to E.coli JM109,and then was sequenced and aligned with the sequence of soybean fad2-1 in NCBI database,and reversely inserted in pBt expression vector to construct plant antisense expression vector,which was mobilized into Agrobacterium tumefacien strains LBA4404 by freeze-thawing method to get genetically modified strain LBA4404 confirmed by double enzyme digestion and PCR detection.The results indicated that the size of the isolated gene was 1 196 bp,bearing 95% identity with that in NCBI database showing that it was soybean fad2-1,and that antisense fad2-1 expression vector was successfully constructed and transferred into Agrobacterium tumefacien strains LBA4404.This will build up the foundation for the improvement in soybean faty acids by antisense technique.