| Fatty acid desaturase(FAD)is a key enzyme in the synthesis of unsaturated fatty acids(UFA),and it can introduce one or more unsaturated fatty acids into saturated fatty acid(SFA)to form monounsaturated fatty acids(MFA)and polyunsaturated fatty acids(PUFA).PUFA is a necessary fatty acid in mammals,especially inhuman being,they all need to maintain the normal operation of the body by ingesting exogenous PUFA.In plants,PUFA can maintain the relative fluidity of cell membrane to ensure the normal physiological function of cells.In addition,PUFA plays an important role in plant response to biotic and abiotic stress.The derivatives of UFA can be also used in industrial production.PUFA can be divided into ?-3 and ?-6 PUFA according to the position of the first double bond from the methyl end,where ?-3 PUFA,like vitamins and minerals,are essential nutrients for animals,and inadequate intake can easily lead to metabolic disorders in vital organs such as the heart and brain.Peanut is one of the most important oil crops and cash crops in the world,the oil content of seed is about 50%,and the content of oleic acid and linoleic acid is about 80%of the total oil content.UFA content is abundant,but the content of ?-3 PUFA is low.As the demand for peanuts is increasing year by year in the world,increasing the production of peanut and improving the quality of oil have attracted more and more attention.The previous studies on peanut were mainly focused on high yield and high oleic acid breeding,but less on raising ?-3 PUFA.In this study,several ?-3 peanut FAD(?-3 AhFAD)and their promoters were cloned from FH-1,a peanut cultivar,and their functions were verified.The main results are as follows:1:Genome-wide identification and expression pattern analysis of peanut FAD gene family.There were 31 AhFADs in peanut genome,which were divided into 8 subfamilies,12 members in AhSAD subfamily and 6 in AhFAD2,4 in AhFAD3,3 in AhFAD4/5,2 in AhFAD6 and 4 in AhFAD7/8.There are 15 AhFADs unevenly distributed on 8 chromosomes in A genome,and 16 AhFADs s in B genome are distributed on 7 chromosomes.Clustering analysis showed that there were two major branches of AhFAD:free AhSAD branch and membrane-bound AhFAD branch,the origin of AhFAD4/5 in membrane-bound AhFAD branches is the earliest,co-3 AhFAD evolved from co-6 AhFAD,and mono-dicotyledonous AhFAD branch was clustered in different branches.Subcellular localization analysis showed that AhFAD2 and AhFAD3 were located in endoplasmic reticulum and others were located in chloroplasts.The analysis of alternative splicing shows that 12 AhFADs have alternative splicing,TTS and TSS are the most frequent alternative splicing events,followed by IR,the fewest ones are AE and ES.2:Gene cloning and sequence analysis of ?-3 AhFADs.According to the sequence information of the wild peanut genome(https://www.peanutbase.org/),seven ?-3 AhFAD genes were cloned.Among them,four AhFAD8 genes(AhFAD8-1,AhFAD8-2,AhFAD8-3,AhFAD8-4)and three AhFAD3 genes(AhFAD3-1,AhFAD3-2,AhFAD3-3).The three pairs of homologous genes are AhFAD8-1 and AhFAD8-2,AhFAD8-3 and AhFAD8-4,AhFAD3-2 and AhFAD3-3.AhFAD8-1 has two alternative splicing isoforms AhFAD8-1.1 and AhFAD8-1.2.The sequences of the gene CDSs varies from 1100 bp to 1400 bp,with 8 exons and 7 introns.The number of amino acids of the encoded proteins is about 400.Compared with other plant ?-3 FADs,7 sequences have conserved domain of ?-3 FAD and 3 histidine rich regions except AhFAD8-1.1.AhFAD8-1.1 has conserved domain,but alternative splicing results in partial deletion of the third histidine rich region.3:Expression pattern analysis of ?-3 AhFADs.Taqman-PCR was used to detect the expression level of each gene in 8 tissues of peanut.Results showed that the expression patterns of the six detected genes had different characteristics.In the four tissues of peanut root,stem,leaf and flower,AhFAD8-1 and AhFAD8-2 expressed at higher level in leaves and flowers than those in roots and stems,the highest in leaves whereas the lowest in roots;the expression level of AhFAD8-3 and AhFAD8-4 was the highest in root,followed by flower,stem and leaf;the expression level of AhFAD3-1 was low in four tissues;the AhFAD3-3 expression level was highest in flowers,followed by leaves,stems and roots.The expression patterns of co-3 AhFADs in different stages of peanut seed development(15 d,30 d,45 d and 60 d)were detected,respectively.Result showed that the expression levels of 6 ?-3 AhFADs were high at 15 d and 30 d stages.The expression level of AhFAD8-1,AhFAD8-2 and AhFAD8-3 was significantly higher at 15 d than that at 30 d,while the expression level of AhFAD3-1 and AhFAD3-3was higher at 30 d than that at 15 d;The expression level of AhFAD8-4 was higher at 15 d and 30 d,but there was no significant difference between them.4:Subcellular localization analysis of ?-3 AhFADs.Subcellular localizations of AhFADs were detected by transient expression in Arabidopsis thaliana protoplasts.Results showed that 5 AhFAD8s were located in the chloroplast membrane,and the 3 AhFAD3s were located in the endoplasmic reticulum.5:Obtaining of ?-3 AhFAD transgenic yeast.The eukaryotic yeast surface vector pESC-URA fusion plasmid was successfully constructed and transformed into Saccharomyces cerevisiae W303 receptive cells,and the transgenic yeast positive strain was obtained.6:Function validation of ?-3 AhFADs intransgenic A.thaliana.The plant expression vector was constructed and over expressed in Arabidopsis for functional verification.Fatty acids from T2 transgenic Arabidopsis seeds were extracted and analyzed by GC method.Result showed that the contents of several kinds of fatty acids increased in transgenic Arabidopsis seeds,the total fatty acids content increased 8%-28%,and linolenic acid content increased 2.4%.These results proved that ?-3 AhFADs could promote the synthesis of ALA.Stress resistance experiment of the T2 transgenic Arabidopsis strains showed that the survival rate of the transgenic Arabidopsis seedlings at different salt concentrations was 1%-13%,higher than that of wild type,and the survival rate of AhFAD8-3 and AhFAD8-4 transgenic Arabidopsis lines was the highest.7:Promoter function verification of co-3 AhFADs.The corresponding promoter sequences of the seven genes were cloned,the length of which varied from 1000 bp to 1500 bp.GUS-plant expression vector was constructed to transform Arabidopsis and screen the positive plants.GUS staining was performed on different tissues of the positive plants.Result showed that there was a strong GUS staining reaction in the roots,stems,leaves,flowers,fruits and seeds,which showed that all the 7 promoters were active. |
PDF Full Text Request