Genome-Wide Identification of Peanut B-Boxs and Functional Characterization of AhBBX6 in Salt and Drought Stresses.

The B-box (BBX) gene family includes zinc finger protein transcription factors that regulate a multitude of physiological and developmental processes in plants. While BBX gene families have been previously determined in various plants, the members and roles of peanut BBXs are largely unknown. In this research, on the basis of the genome-wide identification of BBXs in three peanut species (Arachis hypogaea, A. duranensis, and A. ipaensis), we investigated the expression profile of the BBXs in various tissues and in response to salt and drought stresses and selected AhBBX6 for functional characterization. We identified a total of 77 BBXs in peanuts, which could be grouped into five subfamilies, with the genes from the same branch of the same subgroup having comparable exon-intron structures. In addition, a significant number of cis-regulatory elements involved in the regulation of responses to light and hormones and abiotic stresses were found in the promoter region of peanut BBXs. Based on the analysis of transcriptome data and qRT-PCR, we identified AhBBX6, AhBBX11, AhBBX13, and AhBBX38 as potential genes associated with tolerance to salt and drought. Silencing AhBBX6 using virus-induced gene silencing compromised the tolerance of peanut plants to salt and drought stresses. The results of this study provide knowledge on peanut BBXs and establish a foundation for future research into their functional roles in peanut development and stress response.

Genome-wide identification of the LRR-RLK gene family in peanut and functional characterization of AhLRR-RLK265 in salt and drought stresses.

Leucine-rich repeat receptor-like kinases (LRR-RLKs) play important roles in plant developmental regulations and various stress responses. Peanut (Arachis hypogaea L.) is a worldwide important oil crop; however, no systematic identification or analysis of the peanut LRR-RLK gene family has been reported. In present study, 495 LRR-RLK genes in peanut were identified and analyzed. The 495 AhLRR-RLK genes were classed into 14 groups and 10 subgroups together with their Arabidopsis homologs according to phylogenetic analyses, and 491 of 495 AhLRR-RLK genes unequally located on 20 chromosomes. Analyses of gene structure and protein motif organization revealed similarity in exon/intron and motif organization among members of the same subgroup, further supporting the phylogenetic results. Gene duplication events were found in peanut LRR-RLK gene family via syntenic analysis, which were important in LRR-RLK gene family expansion in peanut. We found that the expression of AhLRR-RLK genes was detected in different tissues using RNA-seq data, implying that AhLRR-RLK genes may differ in function. In addition, Arabidopsis plants overexpressing stress-induced AhLRR-RLK265 displayed lower seed germination rates and root lengths compared to wild-type under exogenous ABA treatment. Notably, overexpression of AhLRR-RLK265 enhanced tolerance to salt and drought stresses in transgenic Arabidopsis. Moreover, the AhLRR-RLK265-OE lines were found to have higher activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) under salt and drought stress treatments. We believe these results may provide valuable information about the function of peanut LRR-RLK genes for further analysis.

The bHLH transcription factor AhbHLH121 improves salt tolerance in peanut.

Plants have developed a number of protective mechanisms to respond to salt and other stresses. Previous studies have shown that the basic helix-loop-helix (bHLH) transcription factor AhbHLH121 plays a crucial role in the response to abiotic stresses in peanut, but the mechanisms and functions related to AhbHLH121 remain unclear. In the current research, AhbHLH121 was induced by salt treatment. Overexpression of AhbHLH121 improved salt resistance, whereas silencing AhbHLH121 resulted in the inverse correlation. Our results also demonstrated that overexpression of AhbHLH121 results in greater activity of antioxidant enzymes under stress condition by promoting the expression of the genes for peroxidase, catalase and superoxide dismutase (AhPOD, AhCAT and AhSOD), indicating enhanced scavenging of reactive oxygen species. Further analysis including Yeast one-hybrid (Y1H) assays and electrophoretic mobility shift assays (EMSAs), suggested that AhbHLH121 can bind directly to the G/E-box regions of the AhPOD, AhCAT and AhSOD promoters, thereby promoting their expression and leading to improved antioxidant enzyme activity. Our research improves the understanding of the mechanisms that allow this peanut bHLH transcription factor to improve abiotic tolerance, and provides valuable gene resources for breeding programs to promote salt stress resistance.

Hsf transcription factor gene family in peanut (Arachis hypogaea L.): genome-wide characterization and expression analysis under drought and salt stresses.

Heat shock transcription factors (Hsfs) play important roles in plant developmental regulations and various stress responses. In present study, 46 Hsf genes in peanut (AhHsf) were identified and analyzed. The 46 AhHsf genes were classed into three groups (A, B, and C) and 14 subgroups (A1-A9, B1-B4, and C1) together with their Arabidopsis homologs according to phylogenetic analyses, and 46 AhHsf genes unequally located on 17 chromosomes. Gene structure and protein motif analysis revealed that members from the same subgroup possessed similar exon/intron and motif organization, further supporting the results of phylogenetic analyses. Gene duplication events were found in peanut Hsf gene family via syntenic analysis, which were important in Hsf gene family expansion in peanut. The expression of AhHsf genes were detected in different tissues using published data, implying that AhHsf genes may differ in function. In addition, several AhHsf genes (AhHsf5, AhHsf11, AhHsf20, AhHsf24, AhHsf30, AhHsf35) were induced by drought and salt stresses. Furthermore, the stress-induced member AhHsf20 was found to be located in nucleus. Notably, overexpression of AhHsf20 was able to enhance salt tolerance. These results from this study may provide valuable information for further functional analysis of peanut Hsf genes.

Identification of Quantitative Trait Nucleotides and Development of Diagnostic Markers for Nine Fatty Acids in the Peanut.

The cultivated peanut (Arachis hypogaea L.) is an important oilseed crop worldwide, and fatty acid composition is a major determinant of peanut oil quality. In the present study, we conducted a genome-wide association study (GWAS) for nine fatty acid traits using the whole genome sequences of 160 representative Chinese peanut landraces and identified 6-1195 significant SNPs for different fatty acid contents. Particularly for oleic acid and linoleic acid, two peak SNP clusters on Arahy.09 and Arahy.19 were found to contain the majority of the significant SNPs associated with these two fatty acids. Additionally, a significant proportion of the candidate genes identified on Arahy.09 overlap with those identified in early studies, among which three candidate genes are of special interest. One possesses a significant missense SNP and encodes a known candidate gene FAD2A. The second gene is the gene closest to the most significant SNP for linoleic acid. It codes for an MYB protein that has been demonstrated to impact fatty acid biosynthesis in Arabidopsis. The third gene harbors a missense SNP and encodes a JmjC domain-containing protein. The significant phenotypic difference in the oleic acid/linoleic acid between the genotypes at the first and third candidate genes was further confirmed with PARMS analysis. In addition, we have also identified different candidate genes (i.e., Arahy.ZV39IJ, Arahy.F9E3EA, Arahy.X9ZZC1, and Arahy.Z0ELT9) for the remaining fatty acids. Our findings can help us gain a better understanding of the genetic foundation of peanut fatty acid contents and may hold great potential for enhancing peanut quality in the future.

MIKC-type MADS-box transcription factor gene family in peanut: Genome-wide characterization and expression analysis under abiotic stress.

Peanut (Arachis hypogaea) is one of the most important economic crops around the world, especially since it provides vegetable oil and high-quality protein for humans. Proteins encoded by MADS-box transcription factors are widely involved in regulating plant growth and development as well as responses to abiotic stresses. However, the MIKC-type MADS-box TFs in peanut remains currently unclear. Hence, in this study, 166 MIKC-type MADS-box genes were identified in both cultivated and wild-type peanut genomes, which were divided into 12 subfamilies. We found a variety of development-, hormone-, and stress-related cis-acting elements in the promoter region of peanut MIKC-type MADS-box genes. The chromosomal distribution of peanut MADS-box genes was not random, and gene duplication contributed to the expansion of the MADS-box gene family. The interaction network of the peanut AhMADS proteins was established. Expression pattern analysis showed that AhMADS genes were specifically expressed in tissues and under abiotic stresses. It was further confirmed via the qRT-PCR technique that five selected AhMADS genes could be induced by abiotic and hormone treatments and presented different expressive profiles under various stresses. Taken together, these findings provide valuable information for the exploration of candidate genes in molecular breeding and further study of AhMADS gene functions.

Identification of the LOX Gene Family in Peanut and Functional Characterization of AhLOX29 in Drought Tolerance.

Lipoxygenases (LOXs) are a gene family of nonheme iron-containing dioxygenases that play important roles in plant development and defense responses. To date, a comprehensive analysis of LOX genes and their biological functions in response to abiotic stresses in peanut has not been performed. In this study, a total of 72 putative LOX genes were identified in cultivated (Arachis hypogaea) and wild-type peanut (Arachis duranensis and Arachis ipaensis) and classified into three subfamilies: 9-LOX, type I 13-LOX and type II 13-LOX. The gene structures and protein motifs of these peanut LOX genes were highly conserved among most LOXs. We found that the chromosomal distribution of peanut LOXs was not random and that gene duplication played a crucial role in the expansion of the LOX gene family. Cis-acting elements related to development, hormones, and biotic and abiotic stresses were identified in the promoters of peanut LOX genes. The expression patterns of peanut LOX genes were tissue-specific and stress-inducible. Quantitative real-time PCR results further confirmed that peanut LOX gene expression could be induced by drought, salt, methyl jasmonate and abscisic acid treatments, and these genes exhibited diverse expression patterns. Furthermore, overexpression of AhLOX29 in Arabidopsis enhanced the resistance to drought stress. Compared with wide-type, AhLOX29-overexpressing plants showed significantly decreased malondialdehyde contents, as well as increased chlorophyll degradation, proline accumulation and superoxide dismutase activity, suggesting that the transgenic plants exhibit strengthened capacity to scavenge reactive oxygen species and prevent membrane damage. This systematic study provides valuable information about the functional characteristics of AhLOXs in the regulation of abiotic stress responses of peanut.

The bHLH transcription factor AhbHLH112 improves the drought tolerance of peanut.

BACKGROUND: Basic helix-loop-helix (bHLH) transcription factors (TFs) are one of the largest gene families in plants. They regulate gene expression through interactions with specific motifs in target genes. bHLH TFs are not only universally involved in plant growth but also play an important role in plant responses to abiotic stress. However, most members of this family have not been functionally characterized. RESULTS: Here, we characterized the function of a bHLH TF in the peanut, AhHLH112, in response to drought stress. AhHLH112 is localized in the nucleus and it was induced by drought stress. The overexpression of this gene improves the drought tolerance of transgenic plants both in seedling and adult stages. Compared to wild-type plants, the transgenic plants accumulated less reactive oxygen species (ROS), accompanied by increased activity and transcript levels of antioxidant enzymes (superoxide dismutase, peroxidase and catalase). In addition, the WT plants demonstrated higher MDA concentration levels and higher water loss rate than the transgenic plants under drought treatment. The Yeast one-hybrid result also demonstrates that AhbHLH112 directly and specifically binds to and activates the promoter of the peroxidase (POD) gene. Besides, overexpression of AhHLH112 improved ABA level under drought condition, and elevated the expression of genes associated with ABA biosynthesis and ABA responding, including AtNCED3 and AtRD29A. CONCLUSIONS: Drawing on the results of our experiments, we propose that, by improving ROS-scavenging ability, at least in part through the regulation of POD -mediated H2O2 homeostasis, and possibly participates in ABA-dependent stress-responding pathway, AhbHLH112 acts as a positive factor in drought stress tolerance.

Genome-Wide Identification and Characterization of HSP90-RAR1-SGT1-Complex Members From Arachis Genomes and Their Responses to Biotic and Abiotic Stresses.

The molecular chaperone complex HSP90-RAR1-SGT1 (HRS) plays important roles in both biotic and abiotic stress responses in plants. A previous study showed that wild peanut Arachis diogoi SGT1 (AdSGT1) could enhance disease resistance in transgenic tobacco and peanut. However, no systematic analysis of the HRS complex in Arachis has been conducted to date. In this study, a comprehensive analysis of the HRS complex were performed in Arachis. Nineteen HSP90, two RAR1 and six SGT1 genes were identified from the allotetraploid peanut Arachis hypogaea, a number close to the sum of those from the two wild diploid peanut species Arachis duranensis and Arachis ipaensis. According to phylogenetic and chromosomal location analyses, thirteen orthologous gene pairs from Arachis were identified, all of which except AhHSP90-A8, AhHSP90-B9, AdHSP90-9, and AiHSP90-9 were localized on the syntenic locus, and they shared similar exon-intron structures, conserved motifs and expression patterns. Phylogenetic analysis showed that HSP90 and RAR1 from dicot and monocot plants diverged into different clusters throughout their evolution. Chromosomal location analysis indicated that AdSGT1 (the orthologous gene of AhSGT1-B3 in this study) might provide resistance to leaf late spot disease dependent on the orthologous genes of AhHSP90-B10 and AhRAR1-B in the wild peanut A. diogoi. Several HRS genes exhibited tissue-specific expression patterns, which may reflect the sites where they perform functions. By exploring published RNA-seq data, we found that several HSP90 genes play major roles in both biotic and abiotic stress responses, especially salt and drought responses. Autoactivation assays showed that AhSGT1-B1 could not be used as bait for yeast two-hybrid (Y2H) library screening. AhRAR1 and AhSGT1 could strongly interact with each other and interact with AhHSP90-B8. The present study represents the first systematic analysis of HRS complex genes in Arachis and provides valuable information for functional analyses of HRS complex genes. This study also offers potential stress-resistant genes for peanut improvement.

The Genetic Base for Peanut Height-Related Traits Revealed by a Meta-Analysis.

Peanut (Arachis hypogaea L.) is an important oilseed crop worldwide, and peanut height has been shown to be closely related to yield, therefore a better understanding of the genetic base of plant height-related traits may allow us to have better control of crop yield. Plant height-related traits are quantitative traits that are genetically controlled by many genes, and distinct quantitive trait loci (QTLs) may be identified for different peanut accessions/genotypes. In the present study, in order to gain a more complete picture of the genetic base for peanut height-related traits, we first make use of the high quality NGS sequence data for 159 peanut accessions that are available within our research groups, to carry out a GWAS study for searching plant height-related regions. We then perform a literature survey and collect QTLs for two plant height-related traits (Ph: peanut main stem height, and Fbl: the first branch length) from earlier related QTL/GWAS studies in peanut. In total, we find 74 and 21 genomic regions that are, associated with traits Ph and Fbl, respectively. Annotation of these regions found a total of 692 and 229 genes for, respectively, Ph and Fbl, and among those genes, 158 genes are shared. KEGG and GO enrichment analyses of those candidate genes reveal that Ph- and Fbl-associated genes are both enriched in the biosynthesis of secondary metabolites, some basic processes, pathways, or complexes that are supposed to be crucial for plant development and growth.

Alternative splicing profiling provides insights into the molecular mechanisms of peanut peg development.

BACKGROUND: The cultivated peanut (Arachis hypogaea) is one of the most important oilseed crops worldwide, and the generation of pegs and formation of subterranean pods are essential processes in peanut reproductive development. However, little information has been reported about alternative splicing (AS) in peanut peg formation and development. RESULTS: Herein, we presented a comprehensive full-length (FL) transcriptome profiling of AS isoforms during peanut peg and early pod development. We identified 1448, 1102, 832, and 902 specific spliced transcripts in aerial pegs, subterranean pegs, subterranean unswollen pegs, and early swelling pods, respectively. A total of 184 spliced transcripts related to gravity stimulation, light and mechanical response, hormone mediated signaling pathways, and calcium-dependent proteins were identified as possibly involved in peanut peg development. For aerial pegs, spliced transcripts we got were mainly involved in gravity stimulation and cell wall morphogenetic processes. The genes undergoing AS in subterranean peg were possibly involved in gravity stimulation, cell wall morphogenetic processes, and abiotic response. For subterranean unswollen pegs, spliced transcripts were predominantly related to the embryo development and root formation. The genes undergoing splice in early swelling pods were mainly related to ovule development, root hair cells enlargement, root apex division, and seed germination. CONCLUSION: This study provides evidence that multiple genes are related to gravity stimulation, light and mechanical response, hormone mediated signaling pathways, and calcium-dependent proteins undergoing AS express development-specific spliced isoforms or exhibit an obvious isoform switch during the peanut peg development. AS isoforms in subterranean pegs and pods provides valuable sources to further understand post-transcriptional regulatory mechanisms of AS in the generation of pegs and formation of subterranean pods.

Comprehensive genomic characterization of NAC transcription factor family and their response to salt and drought stress in peanut.

BACKGROUND: Peanut is one of the most important oil crop species worldwide. NAC transcription factor (TF) genes play important roles in the salt and drought stress responses of plants by activating or repressing target gene expression. However, little is known about NAC genes in peanut. RESULTS: We performed a genome-wide characterization of NAC genes from the diploid wild peanut species Arachis duranensis and Arachis ipaensis, which included analyses of chromosomal locations, gene structures, conserved motifs, expression patterns, and cis-acting elements within their promoter regions. In total, 81 and 79 NAC genes were identified from A. duranensis and A. ipaensis genomes. Phylogenetic analysis of peanut NACs along with their Arabidopsis and rice counterparts categorized these proteins into 18 distinct subgroups. Fifty-one orthologous gene pairs were identified, and 46 orthologues were found to be highly syntenic on the chromosomes of both A. duranensis and A. ipaensis. Comparative RNA sequencing (RNA-seq)-based analysis revealed that the expression of 43 NAC genes was up- or downregulated under salt stress and under drought stress. Among these genes, the expression of 17 genes in cultivated peanut (Arachis hypogaea) was up- or downregulated under both stresses. Moreover, quantitative reverse transcription PCR (RT-qPCR)-based analysis revealed that the expression of most of the randomly selected NAC genes tended to be consistent with the comparative RNA-seq results. CONCLUSION: Our results facilitated the functional characterization of peanut NAC genes, and the genes involved in salt and drought stress responses identified in this study could be potential genes for peanut improvement.

Identification of AflR Binding Sites in the Genome of Aspergillus flavus by ChIP-Seq.

We report here the AflR binding motif of Aspergillus flavus for the first time with the aid of ChIP-seq analysis. Of the 540 peak sequences associated with AflR binding events, 66.8% were located within 2 kb upstream (promoter region) of translational start sites. The identified 18-bp binding motif was a perfect palindromic sequence, 5'-CSSGGGWTCGAWCCCSSG'3' with S representing G or C and W representing A or T. On closer examination, we hypothesized that the 18-bp motif sequence identified contained two identical parts (here called motif A and motif B). Motif A was in positions 8-18 on the upper strand, while motif B was in positions 11-1 on the bottom strand. The inferred length and sequence of the putative motif identified in A. flavus were similar to previous findings in A. parasiticus and A. nidulans. Gene ontology analysis indicated that AflR bound to other genes outside the aflatoxin biosynthetic gene cluster.

Comparative Transcriptome Analysis Reveals Molecular Defensive Mechanism of Arachis hypogaea in Response to Salt Stress.

Abiotic stresses comprise all nonliving factors, such as soil salinity, drought, extreme temperatures, and metal toxicity, posing a serious threat to agriculture and affecting the plant production around the world. Peanut (Arachis hypogaea L.) is one of the most important crops for vegetable oil, proteins, minerals, and vitamins in the world. Therefore, it is of importance to understand the molecular mechanism of peanut against salt stress. Six transcriptome sequencing libraries including 24-hour salt treatments and control samples were constructed from the young leaves of peanut. A comprehensive analysis between two groups detected 3,425 differentially expressed genes (DEGs) including 2,013 upregulated genes and 1,412 downregulated genes. Of these DEGs, 141 transcription factors (TFs) mainly consisting of MYB, AP2/ERF, WRKY, bHLH, and HSF were identified in response to salinity stress. Further, GO categories of the DEGs highly related to regulation of cell growth, cell periphery, sustained external encapsulating structure, cell wall organization or biogenesis, antioxidant activity, and peroxidase activity were significantly enriched for upregulated DEGs. The function of downregulated DEGs was mainly enriched in regulation of metabolic processes, oxidoreductase activity, and catalytic activity. Fourteen DEGs with response to salt tolerance were validated by real-time PCR. Taken together, the identification of DEGs' response to salt tolerance of cultivated peanut will provide a solid foundation for improving salt-tolerant peanut genetic manipulation in the future.

Twelve complete chloroplast genomes of wild peanuts: great genetic resources and a better understanding of Arachis phylogeny.

BACKGROUND: The cultivated peanut (Arachis hypogaea) is one of the most important oilseed crops worldwide, however, its improvement is restricted by its narrow genetic base. The highly variable wild peanut species, especially within Sect. Arachis, may serve as a rich genetic source of favorable alleles to peanut improvement; Sect. Arachis is the biggest taxonomic section within genus Arachis and its members also include the cultivated peanut. In order to make good use of these wild resources, the genetic bases and the relationships of the Arachis species need first to be better understood. RESULTS: Here, in this study, we have sequenced and/or assembled twelve Arachis complete chloroplast (cp) genomes (eleven from Sect. Arachis). These cp genome sequences enriched the published Arachis cp genome data. From the twelve acquired cp genomes, substantial genetic variation (1368 SNDs, 311 indels) has been identified, which, together with 69 SSR loci that have been identified from the same data set, will provide powerful tools for future explorations. Phylogenetic analyses in our study have grouped the Sect. Arachis species into two major lineages (I & II), this result together with reports from many earlier studies show that lineage II is dominated by AA genome species that are mostly perennial, while lineage I includes species that have more diverse genome types and are mostly annual/biennial. Moreover, the cultivated peanuts and A. monticola that are the only tetraploid (AABB) species within Arachis are nested within the AA genome species-dominated lineage, this result together with the maternal inheritance of chloroplast indicate a maternal origin of the two tetraploid species from an AA genome species. CONCLUSION: In summary, we have acquired sequences of twelve complete Arachis cp genomes, which have not only helped us better understand how the cultivated peanut and its close wild relatives are related, but also provided us with rich genetic resources that may hold great potentials for future peanut breeding.

GWAS Discovery Of Candidate Genes for Yield-Related Traits in Peanut and Support from Earlier QTL Mapping Studies.

Peanut (Arachishypogaea L.) is one of the most important oil crops worldwide, and its yet increasing market demand may be met by genetic improvement of yield related traits, which may be facilitated by a good understanding of the underlying genetic base of these traits. Here, we have carried out a genome-wide association study (GWAS) with the aim to identify genomic regions and the candidate genes within these regions that may be involved in determining the phenotypic variation at seven yield-related traits in peanut. For the GWAS analyses, 195 peanut accessions were phenotyped and/or genotyped; the latter was done using a genotyping-by-sequencing approach, which produced a total of 13,435 high-quality single nucleotide polymorphisms (SNPs). Analyses of these SNPs show that the analyzed peanut accessions can be approximately grouped into two big groups that, to some extent, agree with the botanical classification of peanut at the subspecies level. By taking this genetic structure as well as the relationships between the analyzed accessions into consideration, our GWAS analyses have identified 93 non-overlapping peak SNPs that are significantly associated with four of the studied traits. Gene annotation of the genome regions surrounding these peak SNPs have found a total of 311 unique candidate genes. Among the 93 yield-related-trait-associated SNP peaks, 12 are found to be co-localized with the quantitative trait loci (QTLs) that were identified by earlier related QTL mapping studies, and these 12 SNP peaks are only related to three traits and are almost all located on chromosomes Arahy.05 and Arahy.16. Gene annotation of these 12 co-localized SNP peaks have found 36 candidates genes, and a close examination of these candidate genes found one very interesting gene (arahy.RI9HIF), the rice homolog of which produces a protein that has been shown to improve rice yield when over-expressed. Further tests of the arahy.RI9HIF gene, as well as other candidate genes especially those within the more confident co-localized genomic regions, may hold the potential for significantly improving peanut yield.

Genome-Wide Identification and Characterization of Long Non-Coding RNAs in Peanut.

Long non-coding RNAs (lncRNAs) are involved in various regulatory processes although they do not encode protein. Presently, there is little information regarding the identification of lncRNAs in peanut (Arachis hypogaea Linn.). In this study, 50,873 lncRNAs of peanut were identified from large-scale published RNA sequencing data that belonged to 124 samples involving 15 different tissues. The average lengths of lncRNA and mRNA were 4335 bp and 954 bp, respectively. Compared to the mRNAs, the lncRNAs were shorter, with fewer exons and lower expression levels. The 4713 co-expression lncRNAs (expressed in all samples) were used to construct co-expression networks by using the weighted correlation network analysis (WGCNA). LncRNAs correlating with the growth and development of different peanut tissues were obtained, and target genes for 386 hub lncRNAs of all lncRNAs co-expressions were predicted. Taken together, these findings can provide a comprehensive identification of lncRNAs in peanut.

Isolation and characterization of a novel seed-specific promoter from peanut (Arachis hypogaea L.).

Peanut, whose seeds are ideal bioreactors for the production of recombinant proteins and/or nutrient metabolites, is one of the most important crop species worldwide. As important molecular tools, seed-specific promoters (SSPs) can direct the expression of foreign proteins specifically in seeds to avoid constitutive expression that can damage plants. However, few SSPs have been identified from this species. In this study, we isolated a novel SSP (we named it AHSSP2) from peanut. Several cis-acting elements commonly found in SSPs, including 3 copies of RYREPEAT elements, were dispersed throughout the 1970-bp sequence of AHSSP2. The sequence was then substituted in place of the 35S promoter sequence in a pBI121 plasmid, which was subsequently transformed into Arabidopsis. Beta-glucuronidase (GUS) staining showed that AHSSP2 can drive GUS gene expression in the mature seeds of transgenic Arabidopsis, excluding within the testa. The cotyledons and hypocotyls of the germinating seeds of transgenic Arabidopsis seedlings also exhibited GUS activity, even after the seedlings became adult plants. No GUS activity was detected in nontransformed Arabidopsis at any stage. These results strongly suggested that AHSSP2 could drive the expression of foreign genes in a seed-specific manner. This study enriched SSP resources, and the results showed that AHSSP2 could be potentially utilized in peanut and other crop species to improve seed quality, such as modifications to seed oil content.

Genome-wide mining seed-specific candidate genes from peanut for promoter cloning.

Peanut seeds are ideal bioreactors for the production of foreign recombinant proteins and/or nutrient metabolites. Seed-Specific Promoters (SSPs) are important molecular tools for bioreactor research. However, few SSPs have been characterized in peanut seeds. The mining of Seed-Specific Candidate Genes (SSCGs) is a prerequisite for promoter cloning. Here, we described an approach for the genome-wide mining of SSCGs via comparative gene expression between seed and nonseed tissues. Three hundred thirty-seven SSCGs were ultimately identified, and the top 108 SSCGs were characterized. Gene Ontology (GO) analysis revealed that some SSCGs were involved in seed development, allergens, seed storage and fatty acid metabolism. RY REPEAT and GCN4 motifs, which are commonly found in SSPs, were dispersed throughout most of the promoters of SSCGs. Expression pattern analysis revealed that all 108 SSCGs were expressed specifically or preferentially in the seed. These results indicated that the promoters of the 108 SSCGs may perform functions in a seed-specific and/or seed-preferential manner. Moreover, a novel SSP was cloned and characterized from a paralogous gene of SSCG29 from cultivated peanut. Together with the previously characterized SSP of the SSCG5 paralogous gene in cultivated peanut, these results implied that the method for SSCG identification in this study was feasible and accurate. The SSCGs identified in this work could be widely applied to SSP cloning by other researchers. Additionally, this study identified a low-cost, high-throughput approach for exploring tissue-specific genes in other crop species.

Production of eicosapentaenoic acid (EPA, 20:5n-3) in transgenic peanut (Arachis hypogaea L.) through the alternative Δ8-desaturase pathway.

An important alternative source of fish oil is its production by plants through metabolic engineering. To produce eicosapentaenoic acid (EPA, 20:5n-3) in peanut through the alternative Δ8-pathway, a plant expression vector containing five heterologous genes driven by the constitutive 35S promoter respectively, namely, ∆9-elongase (Isochrysis galbana), ∆8-desaturase (Euglena gracilis), ∆5-desaturase (Mortierella alpina), ∆15-desaturase (Arabidopsis thaliana) and ∆17-desaturase (Phytophthora infestans) were transferred into peanut through Agrobacterium-mediated transformation method. The gas chromatography results indicated that the average content of EPA in the leaves of the transgenic lines was 0.68%, and the highest accumulation of EPA in an individual line reached 0.84%. This finding indicates that it is feasible to synthesize EPA in peanut through metabolic engineering and lays the foundations for the production of very-long-chain polyunsaturated fatty acids (VLCPUFAs) in peanut seeds.