Yumei Jiang

De novo Sequence Assembly and Characterization of Lycoris aurea Transcriptome Using GS FLX Titanium Platform of 454 Pyrosequencing

Background Lycoris aurea, also called Golden Magic Lily, is an ornamentally and medicinally important species of the Amaryllidaceae family. To date, the sequencing of its whole genome is unavailable as a non-model organism. Transcriptomic information is also scarce for this species. In this study, we performed de novo transcriptome sequencing to produce the first comprehensive expressed sequence tag (EST) dataset for L. aurea using high-throughput sequencing technology. Methodology and Principal Findings Total RNA was isolated from leaves with sodium nitroprusside (SNP), salicylic acid (SA), or methyl jasmonate (MeJA) treatment, stems, and flowers at the bud, blooming, and wilting stages. Equal quantities of RNA from each tissue and stage were pooled to construct a cDNA library. Using 454 pyrosequencing technology, a total of 937,990 high quality reads (308.63 Mb) with an average read length of 329 bp were generated. Clustering and assembly of these reads produced a non-redundant set of 141,111 unique sequences, comprising 24,604 contigs and 116,507 singletons. All of the unique sequences were involved in the biological process, cellular component and molecular function categories by GO analysis. Potential genes and their functions were predicted by KEGG pathway mapping and COG analysis. Based on our sequence analysis and published literatures, many putative genes involved in Amaryllidaceae alkaloids synthesis, including PAL, TYDC OMT, NMT, P450, and other potentially important candidate genes, were identified for the first time in this Lycoris. Furthermore, 6,386 SSRs and 18,107 high-confidence SNPs were identified in this EST dataset. Conclusions The transcriptome provides an invaluable new data for a functional genomics resource and future biological research in L. aurea. The molecular markers identified in this study will provide a material basis for future genetic linkage and quantitative trait loci analyses, and will provide useful information for functional genomic research in future.

Effect of abiotic and biotic elicitors on growth and alkaloid accumulation of Lycoris chinensis seedlings.

Three-month-old seedlings of Lycoris chinensis were treated with biotic and abiotic elicitors: yeast elicitor (YE), methyl jasmonate (MJ), salicylic acid (SA), and sodium nitroprusside as NO donator (NO). We have shown that the addition of MJ and NO promotes the accumulation of galanthamine in these seedlings. The effect of these elicitors on the growth of the seedlings, as well as on the amount of the alkaloids accumulated in the seedlings was studied. The results showed that, in general, high doses of MJ and SA had a negative effect on the growth of the seedlings, while appropriate doses of NO and YE had a positive effect on the growth of the seedlings. It was remarkable that the addition of MJ, NO, and YE can promote galanthamine accumulation in seedlings. The accumulation was higher in treatments at higher concentrations of NO (100 μM), where the release of galanthamine was 1.72-fold higher than that of the control at the 10th day of culture. The highest values of lycorine were obtained in seedlings treated with YE at a concentration of 0.01 g/l and by the 10th day of culture; the level was 1.38 times of the control.

Transcriptome Analysis of Secondary Metabolism Pathway, Transcription Factors, and Transporters in Response to Methyl Jasmonate in Lycoris aurea.

Lycoris aurea, a medicinal species of the Amaryllidaceae family, is used in the practice of traditional Chinese medicine (TCM) because of its broad pharmacological activities of Amaryllidaceae alkaloids. Despite the officinal and economic importance of Lycoris species, the secondary mechanism for this species is relatively deficient. In this study, we attempted to characterize the transcriptome profiling of L. aurea seedlings with the methyl jasmonate (MeJA) treatment to uncover the molecular mechanisms regulating plant secondary metabolite pathway. By using short reads sequencing technology (Illumina), two sequencing cDNA libraries prepared from control (Con) and 100 μM MeJA-treated (MJ100) samples were sequenced. A total of 26,809,842 and 25,874,478 clean reads in the Con and MJ100 libraries, respectively, were obtained and assembled into 59,643 unigenes. Among them, 41,585 (69.72%) unigenes were annotated by basic local alignment search tool similarity searches against public sequence databases. These included 55 Gene Ontology (GO) terms, 128 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and 25 Clusters of Orthologous Groups (COG) families. Additionally, 4,175 differentially expressed genes (DEGs; false discovery rate ≤ 0.001 and |log2 Ratio| ≥ 1) with 2,291 up-regulated and 1,884 down-regulated, were found to be affected significantly under MeJA treatment. Subsequently, the DEGs encoding key enzymes involving in the secondary metabolite biosynthetic pathways, transcription factors, and transporter proteins were also analyzed and summarized. Meanwhile, we confirmed the altered expression levels of the unigenes that encode transporters and transcription factors using quantitative real-time PCR (qRT-PCR). With this transcriptome sequencing, future genetic and genomics studies related to the molecular mechanisms associated with the chemical composition of L. aurea may be improved. Additionally, the genes involved in the enrichment of secondary metabolite biosynthesis-related pathways could enhance the potential applications of L. aurea.

Identification and differential regulation of microRNAs in response to methyl jasmonate treatment in Lycoris aurea by deep sequencing

BackgroundLycoris aurea is a medicine-valuable and ornamental herb widely distributed in China. Former studied have showed that methyl jasmonate (MJ) treatment could increase the content of glanthamine-a worldwide medicine for symptomatic treatment of Alzheimer’s disease in genus Lycoris plants. To explore the possible role of miRNAs in the regulation of jasmonic acid signaling pathway and uncover their potential correlations, we investigated the expression profiles of small RNAs (sRNAs) and their targets in Lycoris aurea, with MJ treatment by using next-generation deep sequencing.ResultsA total of 365 miRNAs were identified, comprising 342 known miRNAs (representing 60 miRNA families) and 23 novel miRNAs. Among them, 143 known and 11 novel miRNAs were expressed differently under MJ treatment. Quantitative real-time PCR of eight selected miRNAs validated the expression pattern of these loci in response to MJ treatment. In addition, degradome sequencing analysis showed that 32 target genes were validated to be targeted by the 49 miRNAs, respectively. Gene function and pathway analyses showed that these targets such as auxin response factors (ARFs), squamosa promoter-binding like (SPL) proteins, basic helix-loop-helix (bHLH) proteins, and ubiquitin-conjugating enzyme E2 are involved in different plant processes, indicating miRNAs mediated regulation might play important roles in L. aurea response to MJ treatment. Furthermore, several L. aurea miRNAs associated with their target genes that might be involved in Amaryllidaceae alkloids biosynthehsis were also analyzed.ConclusionsA number of miRNAs with diverse expression patterns, and complex relationships between expression of miRNAs and targets were identified. This study represents the first transcriptome-based analysis of miRNAs in Lycoris and will contribute to understanding the potential roles of miRNAs involved in regulation of MJ response.

Alkaloid accumulation in different parts and ages of Lycoris chinensis.

The galanthamine, lycorine, and lycoramine content of Lycoris chinensis was researched during development from young to old plants, i.e. in seeds, ten-day-old seedlings, threemonth- old seedlings, one-year-old seedlings, and perennial seedlings. Notably the alkaloid level reduced to its lowest content 10 days after seed germinating. Then the accumulation of galanthamine tended to increase with age, reaching a higher value in perennial seedlings. The production pattern of lycorine and lycoramine was found similar to that of galanthamine. Different plant organs were also evaluated for their galanthamine, lycorine, and lycoramine contents. Mature seeds had the highest content of galanthamine (671.33 μg/g DW). Kernels, seed capsules, and root-hairs were the main repository sites for galanthamine, lycorine, and lycoramine. The leaves were the least productive organs.

Cloning and heterologous expression of a new 3'-hydroxylase gene from Lycoris radiata.

A full-length cDNA (LC3ʹH) was obtained from a cDNA library of Lycoris radiata by DOP-PCR (degenerate oligonucleotide primer PCR), 3ʹrace and 5ʹrace methods. Compared with the other reported enzymes from different plants, the deduced amino acid sequence of LC3ʹH exhibits significant homologies to 3ʹ-hydroxylases that are involved in the caffeic acid biosynthesis. These findings suggest that the new gene is closely related to the biosynthesis of caffeic acid, which is also an important step of the galanthamine biosynthesis in Amaryllidaceae plants.