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Transcriptome Analysis and Identification of Lipid Genes in Physaria lindheimeri, a Genetic Resource for Hydroxy Fatty Acids in Seed Oil.

ABSTRACT: Hydroxy fatty acids (HFAs) have numerous industrial applications but are absent in most vegetable oils. Physaria lindheimeri accumulating 85% HFA in its seed oil makes it a valuable resource for engineering oilseed crops for HFA production. To discover lipid genes involved in HFA synthesis in P. lindheimeri, transcripts from developing seeds at various stages, as well as leaf and flower buds, were sequenced. Ninety-seven percent clean reads from 552,614,582 raw reads were assembled to 129,633 contigs (or transcripts) which represented 85,948 unique genes. Gene Ontology analysis indicated that 60% of the contigs matched proteins involved in biological process, cellular component or molecular function, while the remaining matched unknown proteins. We identified 42 P. lindheimeri genes involved in fatty acid and seed oil biosynthesis, and 39 of them shared 78-100% nucleotide identity with Arabidopsis orthologs. We manually annotated 16 key genes and 14 of them contained full-length protein sequences, indicating high coverage of clean reads to the assembled contigs. A detailed profiling of the 16 genes revealed various spatial and temporal expression patterns. The further comparison of their protein sequences uncovered amino acids conserved among HFA-producing species, but these varied among non-HFA-producing species. Our findings provide essential information for basic and applied research on HFA biosynthesis.

SUBMITTER: Chen GQ

PROVIDER: S-EPMC7825617 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Castor oil is the only commercial source of hydroxy fatty acid that has industrial value. The production of castor oil is hampered by the presence of the toxin ricin in its seed. Lesquerella seed also accumulates hydroxy fatty acid and is free of ricin, and thus it is being developed as a new crop for hydroxy fatty acid production. A high-throughput, large-scale sequencing of transcripts from developing lesquerella seeds was carried out by 454 pyrosequencing to generate a database for quality improvement of seed oil and other agronomic traits. Deep mining and characterization of acyl-lipid genes were conducted to uncover candidate genes for further studies of mechanisms underlying hydroxy fatty acid and seed oil synthesis.A total of 651 megabases of raw sequences from an mRNA sample of developing seeds was acquired. Bioinformatic analysis of these sequences revealed 59,914 transcripts representing 26,995 unique genes that include nearly all known seed expressed genes. Based on sequence similarity with known plant proteins, about 74% (19,861) genes matched with annotated coding genes. Among them, 95% (18,868) showed highest sequence homology with Arabidopsis genes, which will allow translation of genomics and genetics findings from Arabidopsis to lesquerella. Using Arabidopsis acyl-lipid genes as queries, we searched the transcriptome assembly and identified 615 lesquerella genes involved in all known pathways of acyl-lipid metabolism. Further deep mining the transcriptome assembly led to identification of almost all lesquerella genes involved in fatty acid and triacylglycerol synthesis. Moreover, we characterized the spatial and temporal expression profiles of 15 key genes using the quantitative PCR assay.We have built a lesquerella seed transcriptome that provides a valuable reference in addition to the castor database for discovering genes involved in the synthesis of triacylglycerols enriched with hydroxy fatty acids. The information obtained from data mining and gene expression profiling will provide a resource not only for the study of hydroxy fatty acid metabolism, but also for the biotechnological production of hydroxy fatty acids in existing oilseed crops.

Project description:Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are an important family of endogenous lipids, possessing antidiabetic and anti-inflammatory functions. Therefore, analysis of FAHFAs in biological samples obtained under healthy and disease states can uncover underlying mechanisms of various relevant disorders (e.g., diabetes and autoimmune diseases). Up to now, due to their extremely low abundance, the determination of the changed levels of these species is still a huge challenge, even though great efforts have been made by utilizing liquid chromatography-tandem mass spectrometry with or without derivatization. Herein, we described a novel method for analysis of FAHFAs present in lipid extracts of biological examples after solid-phase extraction and chemical derivatization with one authentic FAHFA specie as an internal standard based on the principles of multi-dimensional mass spectrometry-based shotgun lipidomics. The approach possessed marked sensitivity, high specificity, and broad linear dynamic range of over 3 orders without obvious matrix effects. Moreover, after chemical derivatization, the molecular masses of FAHFAs shift from an overlapped region with ceramide species to a new region without overlaps, removing these contaminating signals from ceramides, and thereby reducing the false results of FAHFAs. Finally, this novel method was successfully applied for determining FAHFAs levels in varieties of representative biological samples, including plasma from lean and overweight/obese individuals of normoglycemia, and tissue samples (such as liver and white adipose tissue from diabetic (db/db) mice). We revealed significant alterations of FAHFAs in samples under patho(physio)logical conditions compared to their respective controls. Taken together, the developed method could greatly contribute to studying altered FAHFA levels under a variety of biological/biomedical conditions, and facilitate the understanding of these lipid species in the patho(physio)logical process.

Dataset Information

Transcriptome Analysis and Identification of Lipid Genes in Physaria lindheimeri, a Genetic Resource for Hydroxy Fatty Acids in Seed Oil.

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