Analysis of lycopodiastrum casuarinoides transcriptome discovers lycodine-type alkaloid biosynthetic genes and genetic markers

OBJECTIVE o facilitate the basic acquaintance with the bioactive lycodine-type alkaloids biosynthetic pathways,we conducted the transcriptome analysis of L.casuarinoides by illumina sequencing.METHODS The plant of L.casuarinoides was collected and was subjected to RNA isolation,cDNA library construction,and illumina sequencing before bioinformatics analysis.After sequencing,the clean reads were obtained for de novo assembly by using Trinity software,and then further processed with TGICL sequencing clustering software to generate unigenes,The unigenes are aligned by Blast X alignment to six public protein database.In addition,all unigenes are functionally annotated by GO,KEGG and characterized putative genes involved in lycopodium alkaloids biosynthesis.RESULTS In total,124,524 high-quality unigenes were obtained with an average sequence length of 601 bp.Among the L.casuarinoides transcripts,61,304 showed significant similarity(E-value<1 e-5) to the known proteins in the public database.Among the total 124 524 unigenes,47,538 open reading frame(ORFs) were predicted.Based on the bioinformatics analysis,all possible enzymes involved in the Lycodine-type alkaloids biosynthetic pathway of L.casuarinoides were identified,including primary amine oxidase(PAO),and Malonly-CoA decarboxylase.In addition,a total of 64 putative cytochrome P450(CYP450) and 827 transcription factors were selected as the candidates of Lycodine-type alka.loids modifiers.Furthermore,a total of 13 352 simple sequence repeats(SSRs) were identified from the 124,524 unigenes,of which dinucleotide motifs AG/CT(50.1%),were the most abundant.CONCLU.SION This transcriptome analysis of L.casuarinoides,provides many valuable candidate genes involv.ing in the biosynthesis of novel secondary metabolites but also lays the foundation for genetic diversity analysis via SSRs markers in L.casuarinoides.