Production Of Marker-Free Transgenic Soybeans With γ-linolenic Acid

γ -linolenic acid(C18:3 Δ ^6,9,12,GLA), one of essential fatty acid, which is a nutritional and important polyunsaturated fatty acid in human and animal diet, plays an important role in hormone regulation and fatty acid metabolization. Futhermore, it is also the biological precursor of a group of molecules, including prostaglandins, leukotrienes and thromboxanes. Δ⁶ —fatty acid desaturase (D6D) is the rate key enzyme in the production of GLA. Linoleic acid can be converted to GLA by D6D. Soybean(Glycin max L.) is one of the major oilseed crops,which has rich content of linoleic acid, accounting for 40%-60% in gross content of fatty acid, while has no or very low content of GLA. Currently transformation systems require selectable marker genes encoding antibiotic or herbicide resistance, along with the interesting, to select transformed cells from a large population of untransformed cells. The continued presence of these selectable markers, especially in food crops such as soybean, is of increasing public concern. Thus the demand for selectable marker-free(SMF) transgenic plants is one of the most popular tendency.In this paper, RT—PCR was employed for the amplification of D6D from Borage in Hainan, China. DNA sequence results showed that D6D was composed of 1344 base pairs and encoded 448 amino acid residues. Sequence alignments with BLAST revealed that the nucleotide sequence and its deduced amino acid sequence of D6D from Chinese Borage were complete homology to those from American Borage. D6D was cloned into T—easy vector as pGΔ⁶—DSA. The fragment containing D6D on pGΔ⁶—DSA was sub-cloned into pGIN22 containing T—DNA and 35S promoter as expression vector pGIN23. And pGIN23 was further sub-cloned with another expression vector pLPN28 containing another T — DNA, promoter and selectable marker gene. Then a co-transformation expression vector, designated as pLIN61, was constructed with two separate T—DNAs and expression cassettes, on which one contained D6D and another contained selectable marker gene. Two types of T-DNA inserts, one containing the selectable gene (bar), another containing interesting gene(D6D), were expected to be produced and integrated into the genome. In the subsequent generation, these inserts could segregate away each other, allowing the selection of the progeny plants with only the D6D.