The Functional Analysis Of The AAD5 Gene In Regulating Fatty Acid Accumulation In Brassica Napus And Arabidopsis Thaliana

ACYL-ACYL CARRYIER PROTEIN DESATURASEs?AADs?as a protein family in plants play an important role in converting stearic acid?C18:0?into oleic acid?C18:1?.There are 7 AAD members in total in Arabidopsis thaliana genome,including AtSSI2,AtAAD1,AtAAD2,AtAAD3,AtAAD4,AtAAD5,and AtAAD6.Except for AtAAD5,other members of AADs have already been studied in fatty acid accumulation,stress resistance,and other aspects of plant growth and development.In addition,the function of the homologous genes of AtAAD5 in Brassica napus is not clear.In this study,we studied the expression pattern of AtAAD5 in A.thaliana and its role in seed fatty acid accumulation.We used the bioinformatic method to analyze the genetic relationship of AAD5 in cruciferous plants and major oil-producing crops.We cloned the full-length coding domain sequence of the BnAAD5-1,closest to AtAAD5,from the B.napus genotype L111,and then we constructed its overexpression vector,transformed into the mutant line aad5-2,and successfully obtained the T3 homozygous transgenic plants of aad5-2 35S:BnAAD5-1.Based on these abovementioned,we analyzed the function of AtAAD5 and BnAAD5-1 on seed fatty acid accumulation.The main results are listed as follows below:?1?The pAtAAD5:GUS vector was constructed and then transformed into the A.thaliana wild type?Col-0?.The GUS staining results showed that AtAAD5 is mainly expressed in developing embryos and several other tissues,including hypocotyl vascular bundles,root tips,cotyledons,and true leaves.It can be preliminarily speculated that AtAAD5 may affect some processes in seed development.?2?Molecular identification results showed that we successfully obtained two T-DNA insertion homozygous mutants,SALK₁29779c?aad5-1?and SALK₀35968C?aad5-2?,which are loss-of-function mutants.?3?The results of seed fatty acid analysis by GC indicated that both total fatty acid and oleic acid contents in aad5-1 and aad5-2 seeds were significantly decreased,whereas the stearic acid content is greatly increased,compared with that of the wild-type.This suggested that AtAAD5 promotes the accumulation of seed fatty acids and the convertion of fromstearic acid into oleic acid in A.thaliana.?4?The phylogenetic tree analysis of AAD5 proteins from different species was carried out by bioinformatics.The result represented that AAD5 is always present in monocotyledons and dicotyledons and the sequence structure is highly conserved.It is worthy to note that AtAAD5 has clost relationship with BnAAD5-1?XP₀13735719.1?,BrAAD5-1?XP₀09134697.1?,and BoAAD5-1?XP₀13630756.1?.These suggested that the AAD5 proteins are evolutionally conserved and may have similar functions.?5?There are seven copies of AtAAD5 in B.napus genome.We cloned and constructed the overexpression vector of one copy of BnAAD5,which is named BnAAD5-1 and located on A3 chromosome,and transformed it into the A.thaliana mutant aad5-2.The highly expressed transgenic lines were confirmed by PCR and quantitative fluorescence PCR.In addition,T3 homozygous transgenic lines were obtained through multi-generation selection.?6?We determined the contents of major fatty aicd compositions of T3 homozygous transgenic lines,the results showed that BnAAD5-1 completely restored the phenotypes of the A.thaliana aad5-2 mutant with lower oil content,lower oleic acid content,and higher stearic acid content.It suggested that BnAAD5-1 and AtAAD5 had similar functions in regulating seed fatty acid accumulation.Meanwhile.We found that the effect of BnAAD5-1on seed fatty acid accumulation in A.thaliana was independent of its dosage.In summary,in cruciferous plants A.thaliana and B.napus,AAD5 significantly promotes seed oil accumulation and the convertion of stearic acid to oleic acid,and has similar physiological and biochemical functions.This study not only has good theoretical value,helping us to understand the fatty acid biosynthesis in cruciferous seeds,but also has certain application value,developing it to be a useful molecular marker or manipulating it to modify the contents of fatty acid compositions in oil-producing crops by genetic engineering technology.