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.

Mining lycodine-type alkaloid biosynthetic genes and genetic markers from transcriptome of Lycopodiastrum casuarinoides

Objective:Lycopodiastrum casuarinoides,a fern of the Lycopodiaceae family,is a traditional Chinese medicine,which has similar efficacy to that of Huperzia serrata in treating rheumatoid arthritis(RA).However,they are different in the contents and compositions of lycopodium alkaloids.In this study,the biosynthesis related genes of lycopodium alkaloids and genetic markers are discovered in L.casuarinoides transcriptome.Methods:The plant of L.casuarinoides was collected and was subjected to the RNA isolation,c DNA library construction,high throughput RNA sequencing and bioinformatics analysis.Results:Totally 124,524 high-quality unigenes were assembled from RNA sequencing reads,with an average sequence length of 601 bp.Among the L.casuarinoides transcripts,61,304 shared the significant similarity(E-value<10-5)with existing protein sequences in the public databases.From 124,524 unigenes,47,538 open reading frames(ORFs)were predicted.Based on the bioinformatics analysis,all possible enzyme genes involved in the lycodine-type alkaloids biosynthetic pathway of L.casuarinoides were identified,including lysine decarboxylase(LDC),primary amine oxidase(PAO),malonyl-Co A decarboxylase,etc.Sixty-four putative cytochrome p450(CYP)and 827 putative transcription factors were selected from the transcriptome unigenes as the candidates of lycodine-type alkaloids biosynthesis modifiers.Furthermore,13,352 simple sequence repeats(SSRs)were identified from 124,524 unigenes,of which dinucleotide motifs AG/CT were the most abundant(50.1%).Meanwhile,we confirmed the amplification effectiveness of25 PCR primer pairs for randomly selected SSRs.Conclusion:We obtained the comprehensive transcriptomic information from the high throughput RNA sequencing and bioinformatics analysis,which provided a valuable resource of transcript sequences of L.casuarinoides in public databases.