Cloning And Express Analysis Of Fatty Acid Desaturase Gene CmFAD7’m Chrysanthemum

Temperature is a major environmental factor limiting plant growth，developmentand outputs in the process of plant growth and development. The biological membrane wasregarded as the position which is firstly suffered damage under temperature stresses.The contentof unsaturated fatty acid in plant membrane lipids was higher, the temperature of transition ofmembrane lipids was lower and the chilling tolerance of plans was stronger. Contrarily, thetemperature of transition of membrane lipids was higher and the thermal tolerance was stronger.Omega-3fatty acid desaturase was the key enzyme which catalyzed dienoic fatty acids toform trienoic fatty acids. The change of its content can influence the composition of membranelipid, especially the level of TAs. Then it can affect the temperature of transition of membranelipids and change the tolerance under temperature stresses.Chrysanthemum （Chrysanthemum×morifolium Ramat.） cultivar ‘Xingguangcanlan’ withcold resistance was used as experimental materials in this study, ω-3fatty acid desaturase genewere separated and cloned from the leaf by RT-PCR and RACE, and analysis sequence.Expression of CmFAD7gene and fatty acid content in leaf and root under low temperature stresswere determined with real-time fluorescent quantitative PCR （RT-qPCR） and gaschromatography technology, explain its expression patterns under low temperature stress. Themain results are as follows:1. Two degenerate primers were designed to amplify specific DNA fragment using cDNAprepared from chrysanthemum leaves according to the homologous sequences from other plants.The middle fragment of interested cDNA was obtained by RT-PCR. Then the full length of thecDNA was isolated by5’RACE and3’RACE. The clone contains an open reading frame（ORE）of1284bp comprising428amino acid residues with the predicted molecular mass of48.98kD．Then these sequence data have been submitted to the GenBank databases under accessionnumber KC567246.2. The identities of putative amino acid sequence of chrysanthemum chloroplast omega-3fatty acids desaturase with that of plastidial ω-3fatty acid desaturase from Crepis alpine andCarthamus tinctorius are84％and83％, respectively. With other species of omega-3fatty acidsdesaturated amino acid sequence of enzyme family has high similarity （figure15）, such asPfFAD7（78%）, EgFAD7（77%）, AtFAD8（71%）, ZmFAD8（67%）, PoFAD8（54%）,AtFAD3（58%）, BnFAD3（58%）, CmFAD7protein contains a delta12-FADs conservativedomain, three transmembrane helical structure, and a chloroplast transit peptide at N-terminal,which is belonging to the Membrane-FADs-like superfamily, and there are four conservativestructure domain, the last three conservative area is rich in histidine and necessary for maintainingthe desaturase enzymatic activity. Phylogenetic tree analysis showed that omega-3fatty acidsdesaturase gene and the plastidial FAD7of CaFAD7CtFAD7/8is the closest, plastidial FAD8iscloser and endoplasmic reticulum FAD3is farther, so named CmFAD7. 3. Leaf and root of CmFAD7gene expression analysis showed that CmFAD7gene expressedin root and leaf under different temperatures, the level of expression in leaves was higher than thatin roots. CmFAD7expression in the leaves with lower temperature, the amount at first andlowered later, in-4℃isthe highest,16、5、8℃of13.5,2.6,7.6times,respectively; the expressionlevel of root in5℃reaches the highest, then gradually decline. Change of fatty acid content showunsaturated fatty acid in the leaves with C18:3content is highest, from16to-8℃, C18:2contentdeclined slightly, C18:3content showed a rising trend, the highest content-8℃（66.09%）,increase of32.68%. Unsaturated fatty acid in the root with C18:2the highest levels, in total fattyacids content was35.49%~35.49%; With the decreasing temperature, C18:2content has a risingtrend, C18:3levels show the first trend, lowered after but no obvious difference; At5℃, C18:3content is the highest （18.26%）, which chrysanthemum CmFAD7gene expression is consistentin root at5℃.