| Linoleic acid(C18:2)is an important polyunsaturated fatty acid necessary for the normal growth of the eukaryotes.Fatty acid desaturase 2(FAD2)is a key enzyme in the synthesis of linoleic acid from oleic acid.Artemisia sphaerocephala Kraschen is rich in linoleic acid,the content up to 83.45% in seeds,and has the largest FAD2 gene family in plants with 26 members.In this study,based on the transcriptome sequencing of A.sphaerocephala,the AsFAD2 gene family was cloned and its function was studied.Transgenic alfalfa lines with high fat and linoleic acid were obtained,and the quality and resistance of the transgenic alfalfa were evaluated.The main results were as follows:1.The full-length c DNA sequences of 21 AsFAD2 genes were cloned from A.sphaerocephala,of which 16 AsFAD2 had different amino acid sequences,and they were highly homologous and relatively conservative in the process of evolution.AsFAD2-1 and AsFAD2-10 genes belong to seed-specific and constitutive expression genes,respectively,and their high expression may play an major role in the formation of high linoleic acid traits in A.sphaerocephala seeds.Although the responses of AsFAD2 family members to salt stress were different,the total expression level was relatively stable,which may play an important role in the adaptation to salt stress in A.sphaerocephala.2.The functional verification of 16 AsFAD2 genes in Saccharomyces cerevisiae and Arabidopsis thaliana fad2 mutants showed that five AsFAD2 enzymes,namely AsFAD2-1,-10,-15,-20 and-23,had oleate desaturase function.3.46 and 70 transgenic plants of "Zhongmu No.1" alfalfa were obtained by transferring AsFAD2-1 and AsFAD2-10 genes,respectively.The expression of target gene was 12.8?676.6 times and 1.27?1392.7 times higher than that of the control,respectively and the total fatty acids,unsaturated fatty acids and linoleic acid increased by 0.19%?70.52% and 1.17%?34.49%,1.58%?79.60% and 4.92%?39.13%,14.18%?92.66% and 20.51%?56.29% respectively compared with wild type.Among them,AsFAD2-1-10 and AsFAD2-10-39 strains had the highest contents of total fatty acids,unsaturated fatty acids,oleic acid,linoleic acid and linolenic acid.4.Transformation of AsFAD2-1 and AsFAD2-10 significantly affected agronomic and quality characters of alfalfa.The aboveground biomass,leaf-stem ratio,plant height and branch number of AsFAD2-1 and AsFAD2-10 transgenic alfalfa were increased by 2.67%?69.77% and 0.76%?171.83%,2.78%?55.90% and 1.19%?49.83%,2.03%?39.62% and 0.69%?39.05%,2.86%?80.95% and 2.86%?42.86% compared with the control,respectively.Crude fat,crude protein and RFV increased by 5.02%?54.65% and 1.18%?106.83%,0.90%?29.68% and 0.05%?26.14%,0.65%?29.15% and 0.59%?22.56% compared with the control,respectively.5.Based on the comprehensive evaluation of 96 transgenic alfalfa,8 excellent materials were obtained: 1 material was high in fat,linoleic acid,protein and high yield;2 materials were high in fat,linoleic acid and protein;1 material was high in fat,linoleic acid and high yield;2 materials had high protein and high yield;1 material had high protein,high relative feeding value and high yield;1 material had high protein and high relative feeding value.In addition,due to the excellent performance of individual characters,12 reserve materials for quality breeding were obtained.6.Under salt stress,the growth and photosynthetic capacity of AsFAD2-1 and AsFAD2-10 transgenic alfalfa were stronger than those of the non-transgenic control plants,and the damage of cell membrane was lighter than the latter.With the increase of salt concentration,linoleic acid content increased,linolenic acid and membrane lipid unsaturation decreased,but the decrease of mesangial lipid unsaturation of transgenic plants was slower than that of control plants.The above results indicated that the alfalfa transformed with AsFAD2 gene had stronger salt tolerance,and the salt tolerance of AsFAD2-1-10 strains was stronger than that of AsFAD2-10-39 strains,indicating there were differences in the contribution of gene family members to salt tolerance. |
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