Cloning And Expression Vector Construction Of △12-Fatty Acid Desaturase (FAD2) Gene From Artemisia Sphaerocephala

△12-Fatty acid desaturase (FAD2) is a key enzyme that catalyzes the introduction of double bonds at the△12 position into the hydrocarbon chain and thereby converts oleic acid to linoleic acid. The contents of linoleic acid and unsaturated fatty acids and the salt and cold resistance could be regulated and improved by the promoting the activity of FAD2. Artemisia sphaerocephala is an endemic plant in desert and semi-desert areas of China. Seeds of A. sphaerocephala are rich in linoleic acid and their linoleic acid contents are about 80%which are higher than any other vegetable oil. Therefore, cloning, identification and transformation of△12-fatty acid desaturase gene from A. sphaerocephala have important meaning for oil crops quality improvement and genetic improvement of tolerance of pasture.In this study,△12-fatty acid desaturase (FAD2) gene was cloned from Artemisia sphaerocephala, the expression vector PBI121-AsFAD2 was constructed, and PBI121-AsFAD2 was transformed into tobacco NC89. The main results are as follows:1. Degenerate primers were designed based on the conserved sequences of the△12-fatty acid desaturase genes from other plants. A novel△12-fatty acid desaturase gene was isolated from A. sphaerocephala using RT-PCR and RACE methods, and named as AsFAD2 (GenBank No. FJ447484).The AsFAD2 cDNA is 1419bp in length including an open reading frame (ORF) of 1161bp, a 5’untranslated region (5’URT) of 79bp and a 3’untranslated region (3’URT) of 179bp. The AsFAD2 cDNA encodes a protein of 386 amino acids which contains three histidine motifs and six transmembrane regions. Three histidine motifs located at 106-111, 36-146 and 316-320, respectively, in amino acids sequence. The AsFAD2 protein shares 64%, 62%,61%,61%,60%and 59%homology with the six reported other FAD2, namely VgE4D2(AAF04093.1),SiFAD2(AAF80560.1),HaFAD2-3(AAL68983.1), CoFAD2 (AAK26633.1), CaFAD2-2A(ABP49577.1) and AhFAD2(AAB84262.1), respectively.2. AsFAD2 then was combined into the expression vector, PBI121, to generate recombinant plasmid PBI121-AsFAD2.AsFAD2 gene open reading frame (ORF) was inserted behind CaMV35S promoter of vector PBI121 in a sense orientation to generate the binary vector PBI121-AsFAD2 by using traditional enzyme digestion methods. Recombinant plasmid PBI121-AsFAD2 was successfully electroporation into Agrobacterium tumefaciens strain GV3101.3. Plant expression vector PBI121-AsFAD2 was transformed into tobacco NC89 using Agrobacterium-mediated method.