Cloning MEX Gene In Malania Oleifera Chun Et Lee And Plant Transformation

Nervonic acid(NA), also known as shark acid (Selacholeic Acid), scientific name:cis-15-Tetracosenic Acid, is a kind of super-long chain fatty acid. Nervonic acid is an important composition of the brain white matter and an nutrient required for brain development and function maintenance. Nervonic acid can play a key role in enhancing immune function, prevention and treatment of AIDS, promoting neural development, lowering blood glucose and lipid, improving brain function, improving memory and preventing cardiovascular disease. At present, it has been developed and untilized as an medical lipid acid. Although nervonic acid has great effect to the body, it is difficult to synthesize the body’s own nerve acid and the body needs to rely on in vitro uptake of nerve acid. In the past, the major way to gain nervonic acid was from hunted sharks and nowdays due to the opposition of shark fishing by the international community, the sources of nervonic acid face crisis. Large amount production of NA in a cheaper way can be fulfilled on condition that crops producing NA are widely cultivated.The major rate-limiting enzyme in nervonic acid synthesis is β-ketoacyl-coenzyme A synthase (KCS), the study designed a pair of primers based on the highly conservation of β-ketoacyl-coenzyme A synthase gene, use Malania oleifera Chun et Lee total DNA as template, we cloned Malania oleifera Chun et Lee β-ketoacyl-coenzyme A synthase gene, named MEX. Construct a seed-specific expression vector pNapinMEX.Transformed wild-type Arabidopsis thaliana (Col-0) with the use of Floral dip method, and measured the fatty acid composition of T1transgenic Arabidopsis thaliana with GC analysis; using Agrobacterium infection as the method,cotyledon petioles as explant, transformed a high erucic acid content Brassica napus L.:GX-272to study whether MEX gene can get expressed in Brassica napus L.,thereby producing nervonic acid; results are as follows:1.Primers were generated based on the high similarity of KCS genes in different species and MEX CDS encoding β-ketoacyl-coenzyme A synthase was cloned in Malania oleifera Chun et Lee for the first time. In silico analysis revealed the product AA shares the same functional actives with that of Lunaria annua KCS. 2.Seeds-specific expressing recombinant plasmid pNapinMEX was constructed。3.The recombinant plasmid was transformed into Col-0ecotype via floral-dipped method and8transgenic lines were obtained。4.Lipid acid ingredients of T1transgenic Arabidopsis seeds were measured by GC。5.Via agrobacterium-mediated method and cotyledon petioles were used as explant, we conducted genentic transformation in GX-272and obtained6tranformants, among which1genetic-stable line was indentified。...