Cloning And Express Analysis Of Fatty Acid Desaturase Gene CmSAD In Chrysanthemum

△9 Stearoyl-ACP desaturase(SAD)catalyze dehydrogenation between9 and 10 carbon atoms to form the first double bond.SAD participate in the synthesis of cell membrane lipid unsaturated fatty acid.When the content of SAD was changed,it can change the ratio between saturated and unsaturated fatty acid,while it can affect cold resistance of plants.SAD gene expressive patterns under low temperature stress from the plant molecular level was researched in this test,in order to lay the foundation for further cold resistance addition research,cold resistance variety screening and resistance breeding of chrysanthemum,it can be also used for cold resistance seed selection and the ornamental value improvement of chrysanthemum.Chrysanthemum(Chrysanthemum×morifolium Ramat.)variety of cold resistance‘Xingguangcanlan’ was selected as experimental materials.Chrysanthemum’s SAD gene was cloned through improved method of CTAB from Chrysanthemum’s leaf,and its sequence was analyzed.The expression index of chrysanthemum’s SAD was determined using q RT-PCR,and the expressive influence of the gene under different temperatures was analyzed.The main results were as follows:1.Degenerate primers were designed according to homologous sequences of chrysanthemum’s SAD genes.Fragment of chrysanthemum’s SAD genes was cloned from leaf of chrysanthemum ‘Xingguangcanlan’ and the specific primers were designed and a total 1203 bp length of c DNA was obtained through amplification.It can encode the number of 401 amino acids.Its relative molecular weight is 45.57 KD.Registration number is KC529335 in Gene Bank.2.Homologous sequence was compared between chrysanthemum SAD gene and other plants.There is homology of 80.29% between the amino acid encoded by SAD genes fragment of chrysanthemum’s and Xinjiang Snow lotus Herb’s(Saussurea involucrata Kar.Et Kir.Ex Maxim.,ABF66638)of the same family.And there is homology of 79.81% with sunflower(Helianthus annuus,AAB65145.1)of the same family.There is high homology with other plants,such as jatropha(77.62%),paraquat(75.91%),potato(77.86%),Millettia pinnata(77.62%),peanut(77.13%),Excoecaria sebifera(76.89%),cassava(76.89%).It had a higher homology with SAD in otherplants,so the gene was named CmSAD.The protein is soluble and had no transmembrane signal area.There is a transit peptide of chloroplast in N– and a sequence of conservative area of SAD in C– of encoded protein by CmSAD through protein active site analysis.And 3D structure of the protein was predicted.3.The expression quantity of CmSAD gene was decreased in root system with temperature decreasing.When the temperature was 16 ℃,the expression quantity of CmSAD gene is the highest.The expression quantity at 5℃,-4℃ and-8℃ was 0.87,0.11,and 0.03 times than that at 16℃,respectively.With the decrease of temperature,the expression quantity of CmSAD gene in leaves increased first and achieved the highest at 5℃,then it was decreased.The expression quantity at 16℃,-4℃ and-8℃was 1.3,5.6 and 3.5 times than that at 5 ℃.Different temperatures made diffident expression quantity of CmSAD gene.Expression quantity of CmSAD gene in root system was higher than leaves when the temperature were 16℃ and 5℃.Expression quantity of CmSAD gene in leaves was higher than root system when the temperature were 4℃ and-8℃.SAD gene made saturated fatty acid catalyze dehydrogenation to form the first double bond,which changed the content of unsaturated fatty acid.With the decreased of temperature,the expression quantity of CmSAD gene was decreased.Therefore,the expression quantity of CmSAD gene was related to temperature and it also had a close relationship with cold resistance.