Function And Regulation Research Of Peanut Diacylgycerol Acyltransferase (AhDGAT) Gene Family

The increasing consumption of plant oil leads to its serious shortage.In 2016,more than 80 million ten soybean was imported into China.In a period of time,our country will continue to face the serious situation of edible plant oil.Peanut oil is one of the main cooking oil kinds.Because of good proportion of fatty acid and high smoke point of peanut oil,its proportion is getting bigger and bigger in our edible plant oil.The huge demands of peanut oil attract people’s more attention to oil content and quality of peanut.According to statistics,the economic benefits generated by increasing 1%oil content of peanut is equal to that of increasing 2%peanut yield or increasing7%in benefit of processing enterprise.Oil content in common peanut varieties is about 50%,while it can be more than 55%in some high oil varieties,indicating higher oil content can be explored.For a long time,people mainly focused on yield in peanut breeding,resulting in less progress in oil content and quality.Studying the genes related to lipid synthesis and the its regulatory mechanism in peanut,will be helpful for breeding new peanut varieties with high oil content,and also be of great significance for resolving the oil crisis in our country.In oil crops,triacylglycerols(TAGs)as main lipid component stores in seeds,TAG synthesis can be catalyzed by diacylgycerol acyltransferase(DGAT).The DGATs were classified into four types:DGAT1,DGAT2,DGAT3 and WSD/DGAT.DGAT1 and DGAT2 are key genes which are responsible for major lipid synthesis.Although both of them can catalyze TAG synthesis,they have distinct subcellular localization and expression pattern,suggesting no functional redundancy between them.The studies about DGAT1 and DGAT2 mainly focused function in increasing lipid content by over-expression them in transgenic plants.However,the study of DGAT evolution and their regulation mechanism is scarce,especially in peanut.Therefore,we analyzed the evolution,function and regulatory mechanism of DGAT1 and DGAT2 in detail,which will lay good foundation for higher oil quality and quantity peanut breeding.1.Genetic variation and phylogenetic analysis of plant DGATsLipid synthesis is a complex biological process in plant,involving many genes.In order to understand the evolution of plant DGAT genes,sequences of DGAT homologous genes were aligned and analyzed in 30 species.(1)Phylogenetic tree analysis.DGAT homologous sequences could be identified in all 30 plant species,indicating their ubiquitous and ancient.DGAT had four clades indicating different DGAT types.Among them,the genetic relationship of DGAT1,DGAT2 and DGAT3 is closer than that of WSD/DGAT.The diversity of DGAT may occur before plant evolved.For WSD/DGAT clade,there were several subbranches,two of which only contained eudicot plants.The pattern of gene duplication was obvious within DGAT,which might cause two special subbranches existence.(2)The analysis of gene structures,expression patterns,transmembrane and conserved domains of DGATs.A total of 18 of 30 species were selected for DGATs analysis.Results showed significant differences among the four DGAT subfamilies.A:Differences in gene structure.There were 15-16 exons in DGAT1,while 8-9 in DGAT2,2 in DGAT3 and 5-7 in WSD/DGAT.B:Differences in transmembrane domains.There were 8-9 transmembrane domains in DGAT1,whereas 1-4 in DGAT2,no transmembrane domains in DGAT3,and 0-1 in WSD/DGAT.C:Differences in conservative domains.Each subfamily has its own unique conservative domains.D:Differences in expression pattern.In A.thaliana and G.max,DGAT1、DGAT2 and DGAT3 expressed almost in all the tissues,especially higher in seeds,whereas WSD/DGAT expressed relatively higher in flower,stem and leaf,and lower in seed.However,in Z.mays,all types of DGAT genes expressed in all the tissues.(3)Analysis of alternative splicing(AS)in plant DGAT.AS is an important regulation mechanism in eukaryotes.Intron retention(IR),alternative splice sites(5’-SS,3’-SS)are the predominant AS types in DGAT genes of the 18 selected species.Splice sites and AS types of DGAT1 are relatively consered,while DGAT2 not.The AS sites of WSD/DGAT in G raimondi,thaliana and G.max were distinctly different from that in B.distachyon.2.AS regulate the function of AhDGATlSeven distinct AS isoforms of AhDGATl(AhDGATl.1-AhDGATl.7)were identified from Fenghua 1.It’s the first time to find AS in plant DGAT genes.In order to verify the function of AhDGAT 1.1-AhDGAT1.7 and whether the AS could regulate the function of AhDGATl,we conducted experiments from the following aspects:(1)The expression profiles analysis of AhDGATl.AhDGATl.1-AhDGAT 1.7 were divided into two types.AhGATl.1-1.3 had 98.4%homologous identity,while AhDGAT1.4-1.7 had 97.2%identity.AhDGATl.2 and AhDGAT1.4 have premature termination codons.Taqman PCR assays were employed to analyse the expression levels of the each AhDGATl isoform.The patterns of each isoform were similar,with high transcript levels in 15-30 d immature seeds.AhDGAT1.1 expressed higher than AhDGAT 1.2 and AhDGATl.3 in all tissues.In root,stem,leaf and flower,AhDGAT1.1 expressed higher in root,but AhDGATl.2 in flower.AhDGATl.4 was detected only in 15 d and leaves,but AhDGAT1.5-1.7 was detected in all tissues.(2)Function anlysis of AhDGAT1.1-AhDGATl.7 in S.cerevisiae stain H1246.H1246 was a mutant yeast strain that can not synthesize TAGs.AhDGAT1.1-AhDGATl.7 were transformed into H1246 respectively.AhDGAT1.2 and AhDGAT1.4 did not produce TAG,whereas the other isoforms significantly increased TAG content about 3-5 times than pESC-URA.AhDGAT 1.5 presented the highest lipid content.The content of C16:0,C16:1,C18:0 and C18:1 in AhDGAT1.1 and AhDGAT1.5-1.7 transformed yeasts also increased significantly.(3)Regulation of AS on the function of AhDGATl.AhDGAT1.2 and AhDGAT1.4 didn’t produce protein because of the premature termination codon.Double gene co-expression yeast vectors were constructed to explore the function of AhDGAT1.2 and AhDGAT 1.4.Five double-gene expression vectors(AhDGAT1.1+1.2,AhDGATl.2+1.3,AhDGATl.4+1.5/1.6/1.7)were transformed into H1246 respectively.All double-gene transformed yeasts could produce TAG,but the content of lipid and FA changed.The lipid and FA content of AhDGAT1.2+1.3 were higher than AhDGAT1.3,whereas AhDGAT1.4+1.5 and AhDGAT1.4+1.6 were lower than AhDGAT1.5 and AhDGAT1.6.The percentage of C16:0 in AhDGAT1.1+1.2 decreased from 17.5%to 7.6%and C16:1 in AhDGAT14+1.5 from 26.7%to 0.8%,accompanying C18:0 increased from 10.9%to 22.9%in AhDGAT1.4+1.5.These results suggested that substrate specificity of double-gene transformed yeasts altered.AhDGAT1.2 and AhDGAT1.4 could regulate other isoforms’ function.(4)Over-expression of AhDGATl.l in tobacco*Transforming AhDGAT1.1 into tobacco,the FA content in transgenic tobacco seeds increased 16.1%-23.5%,but the ratio of oleic acid/linoleic acid declined.3.The key enzyme activity sites identification of AhDGAT2Many researches have shown that AhDGAT2 is associated with uncommon fatty acid accumulation insome plants,such as castor acid and palmitic acid,but little is known about key enzyme activity sites affecting the DGAT function and the substrate specificity.We cloned nine AhDGAT2 sequences from 11 the peanut varieties(AhDGAT2a-i),and found 9 amino acid differences among them.We made nine single-point-mutations to verify the relationship of these sites with enzyme activity.The results are as follows:(1)Single-point-mutants and their function verfication.Sequence alignments of AhDGAT2 revealed 9 differences in predicted amino acid sequences(D3V、N6D、A9V、A26P、T37M、T107M、S118P、K251R、L316P).AhDGAT2a and its 9 mutant sequences were transformed into H1246.The lipid content of N6D and A26P mutants raised 16.4%and 17.1%,and the FA content raised 35.6%and 26.8%,respectively.FA content of L316P mutant was the same as pESC-URA transformed yeast indicating its disability in TAG synthesis.The pertentages of various FAs also changed in mutants.D3V and T107M mutants mainly reduced the percentage of C18:0,about 24.8%and 37.3%.N6D mutant mainly increased the percentage of C16:1,about 46.8%.A26P mutant mainly increased the percentage of C16:0.In short,the enzyme activity of N6D and A26P mutants were increased and L316P mutant was lost.Single-point-mutation could affect the substrate specificity of AhDGAT2a.(2)Heterogenous expression of AhDGAT2a in tobacco.Expressing AhDGAT2a in tobacco significantly increased the FA content of seeds,about 21.1%and 26.9%,meanwhile increased the FA content of leaves.The ratio of oleic acid/linoleic acid in toacco seed increased.Lipid content is a quantitative character,and regulated by a series of genes which coordinate with each other in some way and determine the oil content.In this study,we studied the function and regulation way of AhDGATl and AhDGAT2,and obtained some new discoveries.1.AS plays some roles in function regulation of AhDGAT.These results showed that function of DGAT1 could be controlled by splicing isoforms produced by itself.2.Several enzyme activity sites of AhDGAT2 were detected.N6D and A26P mutants could increase the enzyme activity and oil content in transformed yeasts,whereas L316P mutant lost the enzyme activity.This paper revealed the complexity of peanut oil synthesis and regulation from a new angle.These results will supplement the regulation mechanism of plant oil synthesis and provide theoretical basis for higher quality oil peanut breeding.