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Isolation and functional analysis of fatty acid desaturase genes from peanut (Arachis hypogaea L.).

ABSTRACT: Fatty acid desaturases are enzymes that introduce double bonds into fatty acyl chains. Extensive studies of fatty acid desaturases have been done in many plants. However, less is known about the diversity of this gene family in peanut (Arachis hypogaea L.), an important oilseed crop that is cultivated worldwide.In this study, twelve novel AhFADs genes were identified and isolated from peanut. Quantitative real-time PCR analysis indicated that the transcript abundances of AhFAB2-2 and AhFAD3-1 were higher in seeds than in other tissues examined, whereas the AhADS and AhFAD7-1 transcripts were more abundant in leaves. AhFAB2-3, AhFAD3-2, AhFAD4, AhSLD-4, and AhDES genes were highly expressed in flowers, whereas AhFAD7-2, AhSLD-2, and AhSLD-3 were expressed most strongly in stems. During seed development, the expressions of AhFAB2-2, AhFAD3-1, AhFAD7-1, and AhSLD-3 gradually increased in abundance, reached a maximum expression level, and then decreased. The AhFAB2-3, AhFAD3-2, AhFAD4, AhADS, and AhDES transcript levels remained relatively high at the initial stage of seed development, but decreased thereafter. The AhSLD-4 transcript level remained relatively low at the initial stage of seed development, but showed a dramatic increase in abundance at the final stage. The AhFAD7-2 and AhSLD-2 transcript levels remained relatively high at the initial stage of seed development, but then decreased, and finally increased again. The AhFAD transcripts were differentially expressed following exposure to abiotic stresses or abscisic acid. Moreover, the functions of one AhFAD6 and four AhSLD genes were confirmed by heterologous expression in Synechococcus elongates or Saccharomyces cerevisiae.The present study provides valuable information that improves understanding of the biological roles of FAD genes in fatty acid synthesis, and will help peanut breeders improve the quality of peanut oil via molecular design breeding.

SUBMITTER: Chi X

PROVIDER: S-EPMC5731756 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Isolation and functional analysis of fatty acid desaturase genes from peanut (Arachis hypogaea L.).

Chi Xiaoyuan X Zhang Zhimeng Z Chen Na N Zhang Xiaowen X Wang Mian M Chen Mingna M Wang Tong T Pan Lijuan L Chen Jing J Yang Zhen Z Guan Xiangyu X Yu Shanlin S

PloS one 20171215 12

<h4>Background</h4>Fatty acid desaturases are enzymes that introduce double bonds into fatty acyl chains. Extensive studies of fatty acid desaturases have been done in many plants. However, less is known about the diversity of this gene family in peanut (Arachis hypogaea L.), an important oilseed crop that is cultivated worldwide.<h4>Results</h4>In this study, twelve novel AhFADs genes were identified and isolated from peanut. Quantitative real-time PCR analysis indicated that the transcript abu ...[more]

PMID: 29244878

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Project description:BackgroundFatty acid composition of oil extracted from peanut (Arachis hypogaea L.) seed is an important quality trait because it may affect the flavor and shelf life of resulting food products. In particular, a high ratio of oleic (C18:1) relative to linoleic (C18:2) fatty acid (O/L ≥ 10) results in a longer shelf life. Previous reports suggest that the high oleic (~80%) trait was controlled by recessive alleles of ahFAD2A and ahFAD2B, the former of which is thought to have a high frequency in US runner- and virginia-type cultivars. Functional mutations, G448A in ahFAD2A and 442insA in ahFAD2B eliminate or knock down desaturase activity and have been demonstrated to produce peanut oil with high O/L ratios. In order to employ marker assisted selection (MAS) to select a high oleic disease resistant peanut and to evaluate genotypic and phenotypic variation, crosses were made between high oleic (~80%) and normal oleic (~50%) peanuts to produce segregating populations.ResultsA total of 539 F2 progenies were randomly selected to empirically determine each ahFAD2 genotype and the resulting fatty acid composition. Five of the six crosses segregated for the high oleic trait in a digenic fashion. The remaining cross was consistent with monogenic segregation because both parental genotypes were fixed for the ahFAD2A mutation. Segregation distortion was significant in ahFAD2A in one cross; however, the remaining crosses showed no distortion. Quantitative analyses revealed that dominance was incomplete for the wild type allele of ahFAD2, and both loci showed significant additive effects. Oleic and linoleic acid displayed five unique phenotypes, based on the number of ahFAD2 mutant alleles. Further, the ahFAD2 loci did exhibit pleiotropic interactions with palmitic (C16:0), oleic (C18:1), linoleic (C18:2) acids and the O/L ratio. Fatty acid levels in these progeny were affected by the parental genotype suggesting that other genes also influence fatty acid composition in peanut. As far as the authors are aware, this is the first study in which all of the nine possible ahFAD2 genotypes were quantitatively measured.ConclusionsThe inheritance of the high oleic trait initially was suggested to be controlled by dominant gene action from two homoeologous genes (ahFAD2A and ahFAD2B) exhibiting complete recessivity. Analyzing the ahFAD2 genotypes and fatty acid compositions of these segregating peanut populations clearly demonstrated that the fatty acid contents are quantitative in nature although much of the variability in the predominant fatty acids (oleic, linoleic, and palmitic) is controlled by only two loci.

Project description:Purpose: to screen the candidate genes involved in the peanut drought stress response, we conducted global transcriptome analysis of peanut plants challenged with water deficit and ABA, using the Illumina HiSeq2000 sequencing platform. Methods: a sequences library of Yueyou7 were constructed at first. Then the profile of diffentialy expressed genes (DEGs) under three different treatments (control, water deficit without ABA, and water deficit with ABA) were conducted based on above sequence library. For sequencing library construction, plants were grown under normal conditions, as described previously , Seeds were planted in soil and kep in the greenhouse at temperature of 25-30℃ and water well. Three tissues (leaves, roots, and stems) were collected at three development stages (four-leaf, flowering and podding stages), respectively. Then all of these tissues were mixed to extract the total RNA for sequence library construction. For DEGs study, two-week-old plants were divided into three groups with three independent replication: (1). Water deficit without ABA groups. Plants directly steeped in water containing 30% PEG600 for 30 min in this groups. (2) Water deficit with ABA groups. Plant was subjected to 100 µmol/L ABA for 30 min and then steeped in water containing 30% PEG6000 for 30 min in this groups, (3) Control. Plants steeped in H2O. All treatments were conducted in parallel. After treatments, Total RNA was extracted from 100 mg of plant material, and RNA integrity was checked by gel electrophoresis. Also RNA quality was checked and RNA was quantified using the Agilent 2100 Bioanalyzer (Agilent technologies, Santa Clara, CA) and Nanodrop ND-1000 (Thermo Scientific, Waltham, USA). Results: we generated 4.96×107 raw sequence reads and assembled the high quality reads into 92,390 unique genes. Compared with the control, we found that 621 genes (≥1.5 fold change) responded rapidly to water deficit and 2665 genes (≥1.5 fold change) responded rapidly to ABA. We found 279 genes that overlapped between water deficit and ABA responses, 264 genes that showed the same trend in expression while 15 genes expression that showed opposite trend. Among the identified genes, 257 showed high induction by ABA (>5 fold), and 19 showed high induction by drought (>5 fold). In addition, we identified 100 transcription factor genes among the ABA-inducible genes but only 22 transcription factor genes among the water deficit-inducible genes. Conclusions: we identified genes differentially expressed in the early response to water deficit or ABA. These genes were annotated with GO functional categories for water deficit (33 categories) or ABA (31 categories). We found that only 19 genes were highly induced by water deficit, but 257 genes were highly induced by ABA. Our previous work has examined many of these genes and our future work will reveal their functions and relationships. These data will facilitate functional genomic studies and have established a biotechnological platform for examination of the early drought- and ABA-responsive transcriptome regulatory network in peanut.

Project description:Peanut (Arachis hypogaea L.) is an important source of edible oil and protein for human nutrition. The quality of peanut seed oil is mainly determined by the composition of fatty acids, especially the contents of oleic acid and linoleic acid. Improving the composition of fatty acids in the seed oil is one of the main objectives for peanut breeding globally. To uncover the genetic basis of fatty acids and broaden the genetic variation in future peanut breeding programs, this study used genome-wide association studies (GWAS) to identify loci associated with target traits and developed diagnostic marker. The contents of eight fatty acid components of the Chinese peanut mini-core collection were measured under four environments. Using the phenotypic information and over one hundred thousand single nucleotide polymorphisms (SNPs), GWAS were conducted to investigate the genetics basis of fatty acids under multi-environments. Overall, 75 SNPs were identified significant trait associations with fatty acid components. Nineteen associations were repeatedly identified in multiple environments, and 13 loci were co-associated with two or three traits. Three stable major associated loci were identified, including two loci for oleic acid and linoleic acid on chromosome A09 [mean phenotypic variation explained (PVE): 38.5%, 10.35%] and one for stearic acid on B06 (mean PVE: 23%). According to functional annotations, 21 putative candidate genes related to fatty acid biosynthesis were found underlying the three associations. The allelic effect of SNP A09-114690064 showed that the base variation was highly correlated with the phenotypic variation of oleic acid and linoleic acid contents, and a cost-effective Kompetitive allele-Specific PCR (KASP) diagnostic marker was developed. Furthermore, the SNP A09-114690064 was found to change the cis-element CAAT (-) in the promoter of ahFAD2A to YACT (+), leading dozens of times higher expression level. The enhancer-like activity of ahFAD2A promoter was identified that was valuable for enriching the regulation mechanism of ahFAD2A. This study improved our understanding on the genetic architecture of fatty acid components in peanut, and the new effective diagnostic marker would be useful for marker-assisted selection of high-oleic peanut breeding.

Project description:Purpose: to screen the candidate genes involved in the peanut drought stress response, we conducted global transcriptome analysis of peanut plants challenged with water deficit and ABA, using the Illumina HiSeq2000 sequencing platform. Methods: a sequences library of Yueyou7 were constructed at first. Then the profile of diffentialy expressed genes (DEGs) under three different treatments (control, water deficit without ABA, and water deficit with ABA) were conducted based on above sequence library. For sequencing library construction, plants were grown under normal conditions, as described previously , Seeds were planted in soil and kep in the greenhouse at temperature of 25-30M-bM-^DM-^C and water well. Three tissues (leaves, roots, and stems) were collected at three development stages (four-leaf, flowering and podding stages), respectively. Then all of these tissues were mixed to extract the total RNA for sequence library construction. For DEGs study, two-week-old plants were divided into three groups with three independent replication: (1). Water deficit without ABA groups. Plants directly steeped in water containing 30% PEG600 for 30 min in this groups. (2) Water deficit with ABA groups. Plant was subjected to 100 M-BM-5mol/L ABA for 30 min and then steeped in water containing 30% PEG6000 for 30 min in this groups, (3) Control. Plants steeped in H2O. All treatments were conducted in parallel. After treatments, Total RNA was extracted from 100 mg of plant material, and RNA integrity was checked by gel electrophoresis. Also RNA quality was checked and RNA was quantified using the Agilent 2100 Bioanalyzer (Agilent technologies, Santa Clara, CA) and Nanodrop ND-1000 (Thermo Scientific, Waltham, USA). Results: we generated 4.96M-CM-^W107 raw sequence reads and assembled the high quality reads into 92,390 unique genes. Compared with the control, we found that 621 genes (M-bM-^IM-%1.5 fold change) responded rapidly to water deficit and 2665 genes (M-bM-^IM-%1.5 fold change) responded rapidly to ABA. We found 279 genes that overlapped between water deficit and ABA responses, 264 genes that showed the same trend in expression while 15 genes expression that showed opposite trend. Among the identified genes, 257 showed high induction by ABA (>5 fold), and 19 showed high induction by drought (>5 fold). In addition, we identified 100 transcription factor genes among the ABA-inducible genes but only 22 transcription factor genes among the water deficit-inducible genes. Conclusions: we identified genes differentially expressed in the early response to water deficit or ABA. These genes were annotated with GO functional categories for water deficit (33 categories) or ABA (31 categories). We found that only 19 genes were highly induced by water deficit, but 257 genes were highly induced by ABA. Our previous work has examined many of these genes and our future work will reveal their functions and relationships. These data will facilitate functional genomic studies and have established a biotechnological platform for examination of the early drought- and ABA-responsive transcriptome regulatory network in peanut. Two-week-old plants were divided into three groups with three independent replication: (1). Water deficit without ABA groups. Plants directly steeped in water containing 30% PEG600 for 30 min in this groups. (2) Water deficit with ABA groups. Plant was subjected to 100 M-BM-5mol/L ABA for 30 min and then steeped in water containing 30% PEG6000 for 30 min in this groups, (3) Control. Plants steeped in H2O. All treatments were conducted in parallel.

Dataset Information

Isolation and functional analysis of fatty acid desaturase genes from peanut (Arachis hypogaea L.).

Publications

Isolation and functional analysis of fatty acid desaturase genes from peanut (Arachis hypogaea L.).

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