Transformation of tobacco (Nicotiana tabacum) with antisense soybean (Glycine max) oleic acid desaturase gene

Abstract

Soybean (Glycine max L.) antisense oleic acid desaturase gene FAD2-1 was cloned from soybean genomic DNA by
PCR for its funtions to be determined. The amplicon was linked to pMD18-T vector and transformed into E. coli JM109.
After sequencing, it was reversely inserted in pbt expression vector in order to construct plant antisense expression vector,
which was introduced into Agrobacterium tumefaciens strains LBA4404 by freeze-thawing method. The modified strain
LBA4404 was confirmed by double enzyme digestion and PCR detection. The antisense FAD2-1 was transferred into
tobacco (Nicotiana tabacum) by Agrobacterium tumefaciens-mediated leaf disc transformation, and 75 kanamycin-resistant tobacco plantplets were regenerated. PCR, PCR-Southern blot, RT-PCR methods and GC/MS analysis were used to detect npt-II gene, antisense FAD2-1, its transcript and oleic acid content to get the positive transgenic plants. The results indicated that the gene FAD2-1 was 1196bp in length, bearing 96.7% identity with the published data in NCBI database,that antisense FAD2-1 expression vector was successfully constructed and transformed into Agrobacterium tumefacien strains LBA4404 and tobacco cells, and that antisense gene FAD2-1 was successfully integrated into the genomes of tobacco cells, expressed in transgenic tobacco plant cells and improved tobacco fatty acids.