Digital Gene Expression Library Of Larix Gmelinii Induced By Different Elicitors Between Different Growth Periods

Larix gmelinii is one of the most economically and ecologically important tree species in Northeast China, however, little is understood about its genes involved in growth, development, and resistance of the species. In this study, we performed the de novo assembly of Larix gmelinii transcriptome using a short read sequencing technology (Illumina) and analyzed the transcript differences between Larix gmelinii of different phase induced by jasmonic acid and by methyl jasmonate respectively for6h using a tag-based digital gene expression system. Sample seedlings were treated non in vitro with0.1mM jasmonic acid,0.1mM methyl jasmonate, or0.1%aqueous acetone solution (as control) separately using a handheld sprayer. Tree needles from upper part of the seedling (circa30cm from the top) were sampled for total RNA isolation6h after being sprayed, total RNA isolation following the CTAB method protocol. The following are the results of this study.In a single run,25,977,782short reads were produced, which were assembled into545,211contigs assembled further into92,511scaffolds. After filling gaps in scaffolds by using paired-end reads,51,157unigenes were obtained with mean unigene size being517nt, including many disease/insect-resistance related genes, such as lipoxygenase (LOX) gene, phenylalanine ammonialyase (PAL) gene, serine protease inhibitor (serpin) gene, cysteine protease inhibitor (cystatin) gene and chitinase gene. We matched unigene sequences against Nr protein databases obtaining32,445unigenes from which9,920sequences had a COG classification and were divided into25COG categories. Further, we obtained the Gene Ontology (GO) functional annotation with Nr annotation. Based on the sequence homology,13,317sequences were categorized into44functional groups. We assigned14,462sequences to119KEGG pathways,36.76%of these unigenes involving in metabolic pathways and the biosynthesis of secondary metabolites. In total,32,047unigenes were predicted by using Blastx based on sequencing results and we use blast results information to extract CDS from Unigene sequences and translate them into peptide sequencs, providing a lot of fundamental data for further researching related genes functions.We sequenced5digital gene expression libraries and generated between3.5and5.9million raw tags for each of the5samples, obtained52,040reference genes after removal of redundancy and found that less than30%of the differentially expressed genes are orphan sequences. We found that oxidoreductase activity is the most significantly enriched GO-term of molecular functions in5digital gene expression libraries. The mRNA expression changes at different growth phase of Larix gmelinii inducing by different elicitors in general. The number of differentially expressed genes in the tree needles induced by jasmonic acid and methyl jasmonate is similar, with the majority are down-regulated. The number of differentially expressed genes,whether up-regulated or down-regulated, induced by elicitors in the120-day needles of Larix gmelinii are more than in the60-day needles.The number of differentially expressed genes of metabolism-related is twice as much as secondary metabolism-related in Larix gmelinii induced by jasmonic acid and methyl jasmonate. The differentially expressed genes of Larix gmelinii in one growth phase induced by different elicitors and in different growth phases induced by the same elicitor were analyzed putting emphasis on the analysis of differentially expressed about the disease/insect-resistance related key enzyme genes in the pathways of phenylalanine, tyrosine and tryptophan biosynthesis, phenylpropanoid biosynthesis, glucosinolate biosynthesis and alpha-Linolenic acid metabolism.Among the samples of Larix gmelinii sprout after60days, the expression of aspartate transaminase gene, phenylalanine ammonialyase gene, trans-cinnamate4-monooxygenase gene, coniferyl-aldehyde dehydrogenase gene, transferase activity gene, glucosyl transferase gene and lipoxygenase gene which treated by jasmonic acid or methyl jasmonate was significantly higher than the control. The expression of3-dehydroquinate dehydratase gene, shikimate dehydrogenase gene and coumarate3-hydroxylase gene was significantly different, and the result showed that the gene expression was down-regulated among jasmonic acid treatment samples, whereas up-regulated among methyl jasmonate samples.Among the samples of120-day needles, treated by jasmonic acid or methyl jasmonate, the expression of3-dehydroquinate dehydratase gene, shikimate dehydrogenase gene, aspartate transaminase gene, coumarate3-hydroxylase gene, coniferyl-aldehyde dehydrogenase gene, transferase activity gene, glucosyl transferase gene and lipoxygenase gene was significantly higher than the control. The expression of phenylalanine ammonialyase gene and trans-cinnamate4-monooxygenase gene was significantly different, showing that the gene expression was up-regulated among jasmonic acid treatment samples, whereas down-regulated among methyl jasmonate samples.The analysis of differentially expressed genes was conducted by the same elicitor treatment to the needles of different growth phases. The result showed that the120-day needles induced by jasmonic acid were focused on energy synthesis and metabolism more than the60-day needles that, induced by methyl jasmonate, however, were more focused on plant chemical defense. Whether the samples were induced by jasmonic acid or methyl jasmonate, the expression of phenylalanine ammonialyase gene and lipoxygenase gene for the120-day needles were significantly higher than that of60-day needles.The analysis was conducted for the differentially expressed resistance related genes, such as serine protease inhibitor gene, cysteine protease inhibitor gene, pathogenesis-related protein gene and chitinase gene, indicating that serine protease inhibitor gene, treated by jasmonic acid or methyl jasmonate, was significantly changed than the control, but cysteine protease inhibitor gene and sulfotransferase gene were not significantly changed.The data of transcriptome and digital gene expression libraries obtained in this study provide important fundamental resources for further researching on growth, development, physiological metabolism and structures and functions of resistance related genes of Larix gmelinii. The analysis of differentially expressed disease/insect-resistance related genes was conducted using digital gene expression libraries sequencing for Larix gmelinii induced by jasmonic acid or methyl jasmonate. The result showed that the disease/insect-resistance related genes was significantly changed for the60-day needles than the control, and methyl jasmonate was better than jasmonic acid in induction of disease/insect-resistance related genes for the120-day needles. The results provide important theoretical foundations for selection of elicitors related to plant resistance to insects in industry practices and further research on mechanism of elicitors.