Clon ing of fad2 Gene Fragments from Ca ragana in te rm edia, Constructing ofAn isence fad2 Expression Vector and Its Genetic Transforma tion in Tobacco
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1.
Research Institute of Forestry, CAF
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2.
Key Laboratory of Tree Breeding and Cultivation, state Forestry Administration, Beijing 100091, China
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Received Date:
2006-10-30
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Abstract
△-12 fatty acid desaturase is an important enzyme in the route of p roduction of polyunsaturated lip ids. Control the gene exp ression of △-12 fatty acid desaturase can change p lant fatty acid for good p lant biodiesel. Two cDNAfragments of fad2 gene were cloned using degenerate primers from Caragana interm edia, an important biodiesel plant.The DNA sequence analysis indicated that the fragments contained 452 and 480 bp respectively, and shared 88% ofhomology with Glycine max Gmfad2-2a in GenBank. Both of the gene fragments were registered in GenBank(AY957393). The fragment AY957394 (452 bp)was digested with the enzyme BamH I and Sac I, and ligated to thecorresponding sites of the pB I121 in the antisense orientation. The pB I121fad2 vectorwas introduced into Agrobacterium tum efaciens strain LB4404 by electroporation and transformed to leaves of tobacco via Agrobacterium tum efacienssystem. Plantletswere regenerated in vitro by resistance selection on medium containing Amp icillin and Kanamycin.PCR amp lification with p rimer designed according to the fad2 gene fragment and NPTII gene in pB I121 demonstratedthat antisense fad2 was integrated into tobacco genomes. The analysis of fatty acids of transgenic tobacco p lants withantisense fad2 showed that the content of linoleic acid was 10. 3% less than that of the un2transgenic tobacco plants.
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