De novo assembly and Characterisation of the Transcriptome during seed development, and generation of genic-SSR markers in Peanut (Arachis hypogaea L.)

<p>Abstract</p> <p>Background</p> <p>The peanut (<it>Arachis hypogaea </it>L.) is an important oilseed crop in tropical and subtropical regions of the world. However, little about the molecular biology of the peanut is currently known. Recently, next-generation sequencing technology, termed RNA-seq, has provided a powerful approach for analysing the transcriptome, and for shedding light on the molecular biology of peanut.</p> <p>Results</p> <p>In this study, we employed RNA-seq to analyse the transcriptomes of the immature seeds of three different peanut varieties with different oil contents. A total of 26.1-27.2 million paired-end reads with lengths of 100 bp were generated from the three varieties and 59,077 unigenes were assembled with N50 of 823 bp. Based on sequence similarity search with known proteins, a total of 40,100 genes were identified. Among these unigenes, only 8,252 unigenes were annotated with 42 gene ontology (GO) functional categories. And 18,028 unigenes mapped to 125 pathways by searching against the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG). In addition, 3,919 microsatellite markers were developed in the unigene library, and 160 PCR primers of SSR loci were used for validation of the amplification and the polymorphism.</p> <p>Conclusion</p> <p>We completed a successful global analysis of the peanut transcriptome using RNA-seq, a large number of unigenes were assembled, and almost four thousand SSR primers were developed. These data will facilitate gene discovery and functional genomic studies of the peanut plant. In addition, this study provides insight into the complex transcriptome of the peanut and established a biotechnological platform for future research.</p

The Peanut (<i>Arachis hypogaea</i> L.) Gene <i>AhLPAT2</i> Increases the Lipid Content of Transgenic Arabidopsis Seeds

<div><p>Lysophosphatidic acid acyltransferase (LPAT), which converts lysophosphatidic acid (LPA) to phosphatidic acid (PA), catalyzes the addition of fatty acyl moieties to the <i>sn</i>-2 position of the LPA glycerol backbone in triacylglycerol (TAG) biosynthesis. We recently reported the cloning and temporal-spatial expression of a peanut (<i>Arachis hypogaea</i>) <i>AhLPAT2</i>gene, showing that an increase in <i>AhLPAT2</i> transcript levels was closely correlated with an increase in seed oil levels. However, the function of the enzyme encoded by the <i>AhLPAT2</i> gene remains unclear. Here, we report that <i>AhLPAT2</i> transcript levels were consistently higher in the seeds of a high-oil cultivar than in those of a low-oil cultivar across different seed developmental stages. Seed-specific overexpression of <i>AhLPAT2</i> in Arabidopsis results in a higher percentage of oil in the seeds and greater-than-average seed weight in the transgenic plants compared with the wild-type plants, leading to a significant increase in total oil yield per plant. The total fatty acid (FA) content and the proportion of unsaturated FAs also increased. In the developing siliques of <i>AhLPAT2</i>-overexpressing plants, the expression levels of genes encoding crucial enzymes involved in <i>de novo</i> FA synthesis, acetyl-CoA subunit (<i>AtBCCP2</i>) and acyl carrier protein 1 (<i>AtACP1</i>) were elevated. <i>AhLPAT2</i> overexpression also promoted the expression of several key genes related to TAG assembly, sucrose metabolism, and glycolysis. These results demonstrate that the expression of <i>AhLPAT2</i> plays an important role in glycerolipid production in peanuts.</p></div

Altered Expression of Genes Involved in the FAs and TAG Biosynthesis Pathway.

<p>Values are means ± SE (<i>n</i> = 3). Total RNA was prepared from developing siliques of transgenic lines. Gene expression levels are shown relative to the expression of <i>AtActin7</i> in each sample. The transcription level of each gene in the wildtype was set as 1.0.Asterisks indicate significant differences between the wild-type and transgenic lines at p < 0.01 (**) and p < 0.05 (*).</p

Effect of <i>AhLPAT2</i> Overexpression on Seed Oil and Protein Contents of Independent Homozygous T3 Lines.

<p>A. Seed oil content as a percentage. B. Oil yield per plant. C. Seed protein content. Values are means ± SE of measurements of individual plants (<i>n</i> = 10). Asterisks indicate a significant difference between the wild-type and transgenic lines at p < 0.01 (**)and p < 0.05 (*).</p

Subcellular Localization of the AhLPAT2–GFP Fusion Protein in Onion Epidermal Cells.

<p>The top panels show the expression vector containing <i>AhLPAT2</i> and the <i>GFP</i> reporter gene. <i>AhLPAT2</i> tagged with <i>GFP</i> in the C-terminus was inserted into the pEGFP vector between the <i>Kpn</i>I and <i>Xma</i>I restriction sites. LB, left border; RB, right border; PNOS and TNOS are the promoter and polyadenylation signal of the nopaline synthase gene, respectively; NptII neomycin phosphotransferase II, CaMV 35S promoter. Bottom panels show the expression of <i>GFP</i> and <i>AhLPAT2</i>-<i>GFP</i>. (A) GFP fluorescence of onion epidermal cells expressing <i>GFP</i>. (B) Bright field image of (A). (C) Cell nuclei counterstained with DAPI. (D) Merged image of (A), (B) and (C). (E) GFP fluorescence of onion epidermal cells expressing AhLPAT2-GFP. (F) Bright field image of (E). (G) Cell nuclei counterstained with DAPI. (H) Merged image of (E), (F) and (G). Scale bars = 50 μm.</p

Effect of <i>AhLPAT2</i> Overexpression on Seed Storage Reserve Content of Independent Homozygous T3 Lines.

<p>A. Seeds per silique. B. Numbers of siliques per plant. C. 100-seed weight. D. Seed yield per plant. Values are means ± SE (<i>n</i> = 10). Asterisks indicate significant differences between the wild-type and transgenic lines at p < 0.01 (**).</p

Effect of <i>AhLPAT2</i> Overexpression on FA Content and Composition in Independent Homozygous Seeds.

<p>A. Total FA content. B. Main FA profiles. C. The ratio of unsaturated to saturated FAs. D. The ratio of 18- to 20-carbon FA. Values are means ± SE (<i>n</i> = 10). Asterisks indicate a significant differences between the wild-type and transgenic lines at p < 0.01 (**) and p < 0.05 (*).</p

Altered Expression of Genes Involved in Sucrose Metabolism and Glycolysis.

<p>Values are means ± SE (<i>n</i> = 3). Total RNA was prepared from developing siliques of transgenic lines. Gene expression levels are shown relative to the expression of <i>AtActin7</i> in each sample. The transcription level of each gene in the wild type was set as 1.0.Asterisks indicate significant differences between the wild-type and transgenic lines at p < 0.01 (**) and p < 0.05 (*).</p

<i>AhLPAT2</i> Gene Expression in Young Siliques of the Selected Transgenic Lines Overexpressing <i>AhLPAT2</i>.

<p>Two transgenic T3 lines, FNH1-21 and FNH2-2, were used. Total RNA was prepared from developing siliques of transgenic lines. Gene expression levels are shown relative to the expression of <i>AtActin7</i> in each sample. Values are means ± SE (<i>n</i> = 3).</p