| Safflower is known as“the king of linoleic acid”.The content of linoleic acid in seed oil safflower is generally more than 70%.Linoleic acid(Linoleic acid,LA,C18:2?9,12)is an important part of cell membranes in plants,which is involved in resisting a variety of external stresses,and it"s also a precursor of many signaling molecules biosynthesis.Linoleic acid is essential fatty acids for human with the functions of reducing blood viscosity and triglyceride content,preventing diseases such as cardiovascular and cerebrovascular diseases.Linoleic acid biosynthesis is catalyzed by multiple enzymes involved in fatty acid synthesis and desaturation,and?-6 fatty acid desaturase genes play a key role in the conversion of oleic acid(oleic acid,OA,C18:1?9)to linoleic acid.Generally,the high content of linoleic acid and very low content of saturated fatty acids in seed oil of safflower are unique characteristics among all oil crops.Therefore,it is important to investigate the function and regulatary mechanism of?-6 fatty acid desaturase genes in safflower.The main results of this study are as follows:1.The external characteristics of seed were surveyed and fatty acid content was measured with the development of safflower seeds,the results found that the contents of total fatty acid and linoleic acid increased rapidly at 14?18 days after flowering(DAF).Transcriptome sequencing was performed at 4 time points(10,14,18 and 22 DAF)and a total of 255 differential unigenes involved in fatty acid biosynthesis and triglyceride assembly were identified.Most of unigenes involved in the biosynthesis of fatty acids expressed highly at 10?14 DAF,and low m RNA accumulation at 18 DAF.The m RNA of Ct SAD and CtFAD2-1 genes highly accumulated at 18 DAF.In addition,13 candidate transcription factors might be involved in regulating the linoleic acid biosynthesis also were identified.Twelve key enzyme genes involved in fatty acid biosynthesis were selected for quantitative PCR.The dynamic expression patterns of these genes at different developmental stages of seeds were consistent with that of transcriptome sequencing.2.According to the transcriptome sequencing analyses,fifteen candidate reference genes involved in multiple metabolic pathways of plant were finally selected.The real-time PCR experiment was executed under a various conditions including different experimental treatments,different seed development stages and different cultivars and tissues for.The suitability evaluation was executed by ge Norm and Norm Finder programs.Overall,EF1,UBCE2,EIF5A,ATPS and 60SRPL10 were the more stable genes,and MBF1 as well as MFC were the most unstable genes by ge Norm and Norm Finder software in all experimental samples.In addition,the multiple stable reference genes were also identified using for different treatment groups,respectively.3.Safflower varieties were commonly divided into high,low and middle linoleic acid types according to their LA relative percentage contents in the seed oil.Fatty acid desaturase 2(FAD2)plays a key role for LA content in seed.At least eleven FAD2 genes exist in safflower and it means that a large number of FAD2 gene copies have been found in one species.Interestingly,the m RNA accumulation of CtFAD2-1,CtFAD2-2,CtFAD2-10 and CtFAD2-11 was detected in safflower developmental seed,and they could convert the OA to LA in yeast.Our research group previously found the change of CtFAD2-1 gene sequence in safflower material with high-OA genotypes which caused the deactivation of delta-12 fatty acid desaturase.In this study,the sequence variations of FAD2(CtFAD2-2,CtFAD2-10,CtFAD2-11)genes which could express in developmental seed of safflower were analyzed in 15 different LA-type materials.The results revealed that the CtFAD2-2 sequences were the same in all materials,and the CtFAD2-10,CtFAD2-11 sequences formed into two haplotypes independent of the LA-type of safflower seed.Yeast expression analysis revealed that two haplotypes of CtFAD2-10 had the function of oleic acid desaturase.The real-time PCR analysis of FAD2 in high and low LA-type materials at different seed developmental stages revealed that the expression pattern of CtFAD2-1 in two different LA-type safflower materials was significantly divergent.For high-LA type,the accumulation of CtFAD2-1 m RNA was extremely low at10 DAF,and its expression level increased sharply between 14 and 18 DAF and decreased slightly after 18 DAF.For low-LA type,the accumulation of CtFAD2-1 m RNA was extremely low during seed development stages.While,CtFAD2-2,CtFAD2-10 and CtFAD2-11 expressed mainly at 10 DAF for two different LA-type materials and the accumulation of few m RNA was detected at 14-22 DAF.In total,CtFAD2-1,CtFAD2-10and CtFAD2-11 may not be major genes controling the high level accumulation of linoleic acid content at mature safflower seeds.4.Cloning and functional analysis of the full-length CtFAD6 gene sequence were done in safflower,and the results showed that CtFAD6 could catalyze not only palmitoleic acid(16:1)but also oleic acid(18:1)to produce 16:2 and 18:2,respectively.In addition,the response of?12 fatty acid desaturase genes to different temperature and salt stress in safflower vegetative tissues(roots,stems and leaves)was systematically investigated.We found that low temperature,high temperature and salt stress could all regulate the expression level of?12 fatty acid desaturase genes at transcriptional level.?12 fatty acid desaturase genes were significantly induced under cold and heat stress in young leaves than young stems and roots.In contrast,CtFAD2-1,CtFAD2-11 and CtFAD2-10 were sensitive to salt stress in all safflower tissues(roots,stem and leaves).5.The 5"-UTR?intron sequence and 1116bp promoter sequence of CtFAD2-1 gene were cloned.Furthermore,the 821bp promoter region was further functionally characterized in Arabidopsis thaliana by using the transgenic approach.Computational analysis affirmed the abiotic stress responsive cis-elements like HSE,LTR,MBS etc,as well as wound,fungus,methyljasmonate responsive motifs,four W-boxes and two E-boxes existing in the promoter.For transformation in Arabidopsis thaliana,different5"-deletion constructs were constructed.The histochemical staining of GUS revealed that the flower,mature seed and silique wall were stained,and the-230?-60 bp was the key region for GUS activity in Arabidopsis thaliana tissues.Further,the transgenic plants were treated with drought,salt,cold,high-temperature,wound and ABA hormone as well as control plants;the results revealed that CtFAD2-1 promoter can be induced by a variety of abiotic stresses and this was relevance with the analysis result of promoter sequence,and the two W-box in P3 region may be the key element responsing to wound.6.Based on transcriptome sequencing,two transcription factors were selected which contained AP2 and HSF domain,named as Ct ERF1B and Ct HSFA6,respectively.Another candidate transcription factor which expressed highly at 10 DAF also was selected,named as Ctb HLH128.The full length ORF of three candidate transcription factors were cloned and subcellular localization amalyses revealed their nuclear localization information.Fuether,three candidate genes were overexpressed in Arabidopsis thaliana,and the fatty acid content of seeds in transgenic lines were measured.Results revealed the content of unsaturated fatty acid in seeds of Arabidopsis thaliana transformed into Ctb HLH128 was significantly higher than wild types.In addition,in agar solid medium containing 15%PEG,T3 homozygous lines transformed into Ctb HLH128 gene had higher germination rate than wild-type.In total,the higher level expression of CtFAD2-1 at seed developmental middle and late stages causes the rapidly and largely accumulation of linoleic acid content,which may be the main reason for higher linoleic acid content in safflower seeds than other oil crops.The promoter of CtFAD2-1 can be regulated by multiple abiotic stresses and CtFAD2 gene can also response to temperature and salt stress in multiple safflower vegetative tissues,which may be partially explain the phenomenon that the better tolerance on multiple stresses appears in safflower. |
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