Cloning And Functional Analysis Of A Rice (Oryza Sativa L.) Gene Encoding A Endoplasmic Reticulum ω-6 Fatty Acid Desaturase

Rice(Oryza sativa L.) is one of the most important grain crops and originates in tropical and subtropical climates and very sensitive to low temperatures. Polyunsaturated phospholipids in membranes are essential formaintaining cellular function and plant viability at lowered temperatures.Therefore, rice cold-resistant breeding is important and urgent according to genetic engineering method to increase the unsaturase fatty acid content.Open reading frame (ORF) of the riceω-6 fatty acid desaturase gene was attained by homology cloning. The ORF is a 1167bp fragment, and encoding 388 amino acids, and was designated as OsFAD2 (GeneBank accession number:No. FJ768953). The deduced amino acid sequence was aligned with other representative FAD2 and demonstrated significant homology to FAD2 of other plants. Three conserved histidine-rich motifs are contained in the putative protein. These conserved motifs are found in all membrane bound desaturases. here was an aromatic amino acid enriched signal at the C-terminus of the protein (-YNNKL), which has been reported to be necessary and sufficient for retaining enzymes in the ER. Phylogenetic analysis revealed OsFAD2 to be a house-keeping type FAD2.RT-PCR revealed that OsFAD2 is a constitutive expressing gene, and is highly expressed in leaves. In situ hybridization demonstrated the accumulation of OsFAD2 mRNA increased in mesophyll cells when the seedlings were exposed to 15℃for 4 days. However, in the roots, OsFAD2 mRNA was no obvious difference under the same conditions. In addition,6-BA also induced the expression of OsFAD2.Recombinant OsFAD2 was overexpressed in Xenopus oocyte using pT7TS, and also in yeast (InvScI) by the vector of pYES2/NT (C) to study its possible desaturase function. The recombinant Xenopus oocyte and yeast cells that contained OsFAD2 showed fatty caid desaturase ability and improved yeast cells viability under low temperature, as compared with control cultures. Transgenic Arabidopsis thaliana that constitutively expressed OsFAD2 could complement for the absence of the FAD2 enzyme in the Arabidopsis fad2 mutant.The recombinant OsFAD2 was introduced into indica rice Kasalath by Agrobacterium-mediated transformation. Using the germination rate calculation, electrolyte leakage measurement and Fv/Fm assay, we established that, in comparison with seedlings or germinating seeds, transgenic rice plants have a higher level of basal tolerance to low temperature. More importantly, the 35S::FAD2 plants showed significantly enhanced cold tolerance at the reproductive stage, increasing grain yield by 46%over controls in the field under cold conditions. It demonstrated OsFAD2 could convert oleic acid to linoleic acid under low temperature condition and increased membrane fluidity,finally enhanced the cold tolerance of transgenic rice.These results suggest that expression of OsFAD2 enhances the cold tolerance resistance in both yeast and rice. Besides, the linoleic acid accumulation in rice is parallel with the expressing of OsFAD2.It demonstrated OsFAD2 gene could apply on cold-resistant rice breeding.