Functional Study On LncRNAs And Key Genes Related To Oil Synthesis Pathway In Peanut

Triacylglycerol(TAG)is the main component of vegetable oil and can be directly catalyzed by diacylglycerol acyltransferase(DGAT)and phospholipids:diacylglycerol acyltransferase(PDAT).Long non-coding RNAs(lncRNAs),as key regulators of cell processes,can regulate gene expression at multiple levels including epigenetics,transcriptional regulation and post-transcriptional regulation in the form of RNA molecules,but its role in peanut oil synthesis has not been fully studied.Here,we use peanut as experimental materials,and combine with the expression profiles of mRNAs and lncRNAs to identify the key genes,such as IncRNAs,AhDGAT and AhPDAT that involved in oil synthesis in the peanut genome.This will provide a theoretical basis for high-oil breeding and quality improvement of peanut.1.Genome-wide identification and characterization of lncRNAs in peanutPeanut lncRNAs were identified by transcriptome sequencing combined with bioinformatics and validated by RT-qPCR.A few of lncRNAs as well as their target genes that may be involved in peanut oil synthesis were identified.The result are as follows:(1)A total of 1,442 peanut lncRNAs were identified in the whole genome,and 189 lncRNAs were differentially expressed in roots,leaves or seeds.(2)The expression of lncRNAs in peanut was tissue-specific and spatiotemporally specific,and their expression levels and alternative splicing rate were lower than those of mRNAs.Moreover,it was found that some protein-coding genes could produce the transcripts that cannot be encoded during the splicing process,which can be regarded as a new source of lncRNAs.(3)The candidate target genes of AhlncR1,AhlncR2 and AhlncR3 were mostly annotated as AhPDAT1,suggesting that these lncRNAs might be involved in peanut oil metabolism as trans regulators of AhPDAT1.Two splicing isoforms of AhlncR1,named AhlncR1.1 and AhlncR1.2 respectively,were identified;and the interaction of AhlncRl and AhPDAT1 in lipid synthesis was verified subsequently.2.Identification and functional analysis of AhPDAT gene family in peanut(1)Genomic identification of AhPDAT gene family.We performed bioinformatics analysis of AhPDAT gene family using genome and transcriptome data.In peanut genome,there were 17 members distributed unequally in nine chromosomes.Phylogenetic analysis showed that AhPDATs were closely related to soybean,Medicago truncatula and Phaseolus vulgaris.Gene structure analysis showed that the exon number of these genes ranged from 2 to 8,and the protein sequence length varied from 88 to 742 amino acids.Expression pattern analysis indicated that AhPDAT genes showed diverse expression patterns,suggesting a significant divergence in their function.Meanwhile,there were rich alternative splicing types of these genes in different organs or tissues,among which Aradu.S9XBY owned the most alternative splicing isoforms.(2)The cloning and expression pattern analysis of AhPDATs.Six transcripts were cloned from peanut and named AhPDAT1A、AhPDAT1B-1、AhPDAT1B-2 and AhPDAT2A-1、AhPDAT2A-2、AhPDAT2B.Taqman fluorescence probe was designed to verify the expression pattern of each splice isomer.Because of no specific probe of AhPDAT1B-2 was found,its expression pattern was not analyzed.The results showed that AhPDAT1 and AhPDAT2 were expressed at the highest transcript levels in 15 d immature seeds,followed by 30 d,root and flower,and the expression was lower at 45 d and 60 d.The difference was that the expression of AhPDAT1A was significantly higher than that of AhPDAT1B-1 in flowers,and significantly lower than that of AhPDAT1B-1 in 60 d immature seeds.(3)The function of AhPDAT1 and AhPDAT2 was verified by TAG synthesis mutant H1246.AhPDAT1A and AhPDAT2A-1,AhPDAT2A-2,AhPDAT2B can encode a complete protein and restore the TAG synthesis function of H1246,while AhPDAT1B-1 and AhPDAT1B-2 cannot encode proteins and lose the TAG synthesis function of H1246 due to the premature termination codon.To explore the interaction between AhPDAT1B-1,AhPDAT1B-2 and AhPDAT1A,the co-expression vectors of AhPDAT1A+1B-1 and AhPDAT1A+1B-2 were constructed and both the transgenic yeasts could restore the TAG synthesis function of H1246.Compared with AhPDAT1A transformed yeast,the lipid content of transgenic yeasts with double genes was slightly reduced.3.The interaction verification between AhlncRl and AhPDAT1.(1)Western blot results showed that AhlncR1.1 and AhlncR1.2 could not encode any protein products,and had little effect on the oil synthesis of H1246.(2)The co-expression vector of AhPDAT1 A+AhlncR1.l and AhPDAT1A+AhlncR1.2 genes was constructed and transformed into yeast H1246 to verify whether AhlncR1.l or AhlncR1.2 can affect the function of AhPDAT1A.We found that both of them could restore the TAG synthesis function of H1246,but the total oil content of AhPDAT1A+AhlncR1.1 and AhPDAT1A+AhlncR1.2 transformed yeast was significantly lower than that of AhPDAT1A.4.Regulation of AhDGAT2-Like by alternative splicing.Our previous studies have found that some lncRNAs were derived from coding genes,so the function of such lncRNAs were studied.In the AhDGAT2 subfamily,the similarity between Aradu.Z4DIZ and other AhDGAT2 members was only 64.67%,so it was named as AhDGAT2-Like,and its five splicing isomers were named as AhDGAT2-L1.1-1.5.Only AhDGAT2-L1.1 can encode the complete protein.We speculated that the other four might play functions by regulating the expression of AhDGAT2-L1.1 as lncRNAs and further analyzed their interaction from the following four aspects.:(1)Identification and analysis of AhDGAT genefamily in peanut.A total of 29 DGAT genes were identified in peanut genome and clustered into four independent branches,DGAT1,DGAT2,DGAT3 and WSD subfamilies.Among them,DGAT1 has the least members and the closest genetic relationship with DGAT2,and the farthest genetic relationship with WSD.At the same time,each subfamily has its own unique conserved domain.There are some differences in gene structure,intron number,transmembrane region number,expression pattern,subcellular localization and alternative splicing.(2)Expression pattern analysis of AhDGAT2-Like.In developing seeds,AhDGAT2-L1.2-1.5 was highest expressed in 15 and 30 d immature seeds,while AhDGAT2-L1.3 did not express in 60 d.Among the four organs of root,stem,leaf and flower,AhDGAT2-L1.2 was expressed highest in flowers and lowest in leaves.AhDGAT2-L1.3 was highest expressed in stems,but not in roots and flowers;AhDGAT2-L1.4 was expressed only in roots;the expression of AhDGAT2-L 1.5 was highest in roots and lowest in leaves and flowers.(3)The function verification of the splicing isoforms of AhDGAT2-Like.Five isoforms were overexpressed in H1246,respectively.Only AhDGAT2-L1.1 could restore the ability of H1246 to synthesize TAG,and the total lipid and fatty acid contents of the transgenic yeast were significantly higher than those of control,indicating that AhDGAT2-L1.1 had acyltransferase activity.AhDGAT2-L1.2-1.5 could not restore the ability of mutant to synthesize TAG,because these four splicing isoforms could not encode proteins.(4)Verification of the interaction between AhDGAT2-L1.1 and AhDGAT2-L1.2-1.5.In order to explore whether AhDGAT2-L1.2-1.5 is involved in regulating the expression of AhDGAT2-L1.1 as lncRNAs,AhDGAT2-L1.2/1.3/1.4/1.5+1.1 co-expression vectors were constructed and transformed into H1246.The results showed that all the four transgenic yeasts restored the TAG synthesis ability.Compared with AhDGAT2-L1.1,the oil content and fatty acid composition of the four yeasts changed in different degrees.In terms of oil content,the total oil content of AhDGAT2-L1.2+1.1 and AhDGAT-L1.3+1.1 transgenic lines was significantly lower than that of AhDGAT2L1.1,and AhDGAT2-L1.4+1.1 was basically the same as that of AhDGAT2-L1.1,while AhDGAT2-L1.5+1.1 was significantly higher than that of AhDGAT2-L1.1.Compared with the AhDGAT2-L1.1transgenic yeast,the fatty acid composition was changed and the proportion of four major fatty acids was decreased,indicating that the substrate specificity of the transgenic yeast was changed.Western blot results showed that AhDGAT2-L1.2-1.5 did not encode protein,but they could affect the function of AhDGAT2-L1.1 in the form of RNA molecules,thereby affecting the biosynthesis of TAG.In summary,this study showed that AhlncR1 could regulate the expression of the key gene AhPDAT1A for oil synthesis in the form of RNA molecules,affecting oil accumulation and then participating in oil metabolism.While the four splicing isoforms of AhDGAT2-Like that could not encode proteins,they could effect the function of AhDGAT2-L1.1,suggesting that they might have similar action modes with lncRNA to regulate the expression of coding genes.The regulatory mechanism between lncRNA and functional genes is still unclear,and needs further study.