Gene Cloning, Vector Construction And Genetic Transformation Of FAD2in Flax (Linum Usitatissimum L.)

The plant fatty acids are known to possess highly edible value. Fatty acid content andcomposition have significant impact on quality of vegetable oils.△12-fatty aciddehydrogenase is a limiting velocity enzyme of metabolize to pathway ω-3and ω-6. Itcatalyzes conversion of oleic acid to linoleic acid, leading to synthesis of polyunsaturatedfatty acids of the plants. Improvement in the quality of oil flax is one of the breeding aims ofthe flax. Using genetic engineering to change fatty acid compositions is an important way toimprove the quality of flax.In this study, the FAD2gene was cloned by the method of molecular biology from oilflax. Primers were designed according to the published sequence DQ222824.1in GenBank.The full-length cDNA fragment of FAD2(LuFAD2) was amplified from oil flax byRT-PCR.The FAD2were Constructed into over expression vectors and antisense expressionvector by Gateway technology. The transgenic oil flaxes were obtained byagrobacterium-mediated method. The main results as follows:1. The LuFAD2gene was connected into the TOPO vector pENTRTM/D-TOPO.Sequencing analysis indicated that the full-length of LuFAD2was1149bp and code382amino acids, which share99.04%and95.65%homology in nucleotide level to the reportedsequence FAD2-1(KC469054.1) and FAD2-2(EU660501.1), amino acid sequence wasshowing99.74%and99.21%.2. After LR reaction, LuFAD2were connected to the expression vector pMDC139、pEarleyGate100and interference vector pBIB-BASTA-35S. Therefore, two over expressionvectors pMDC139-LuFAD2、pEarleyGate100-LuFAD2and one antisense expression vectorpBIB-BASTA-35S-LuFAD2were constructed successfully.3. Using hypocotyl of aseptic seedlings of Longya10of growed7days as explants, theexpression vector pEarleyGate100-LuFAD2and pBIB-BASTA-35S-LuFAD2with markergene PPT, the expression vector pMDC139-LuFAD2with marker gene Hyp and gene GUSwere used genetic transformation by agrobacterium-mediated method.42over expressionvector transformed plants and12antisense expression vector transformed plants wereobtained through antibiotics screening,13plants were positive verified by PCR analysis fromDNA of young leaves of resistant shoots of transformed, results have showed11overexpression vectors transformed plants and2antisense expression vectors are transformedplants.