[Transcriptome analysis of rhizome of Polygonatum cyrtonema and identification of candidate genes involved in biosynthetic pathway of steroidal saponin].

To enrich the transcriptome data in rhizome of Polygonatum cyrtonema seedlings, identify candidate functional genes involved in steroidal saponin biosynthesis and provide genetic resources for the research on anabolism pathway and regulatory mechanism of active components in P. cyrtonema, Illumina platform was applied to perform transcriptomic sequencing of rhizome of P. cyrtonema, followed by a series of bioinformatics analysis on RNA-seq data, including de novo assembly, annotation, classification and metabolic pathway analysis of the assembled unigene. Meanwhile, a deep analysis on the steroidal saponin biosynthesis in secondary metabolism pathway was performed. The results showed a total of 126 546 unigene were obtained by de novo transcriptome assembly, of which 47 226 were annotated. Of these, 16 499 unigene were mapped to 132 specific pathways, of which 2 768 were identified to be involved in 22 secondary metabolic pathways. One hundred and thirteen unigene were identified from the transcriptome database, which encoded 27 metabolic enzymes associated with steroidal saponin biosynthesis and shared similarity with 45 functional genes in Arabidopsis thaliana. In conclusion, a series of candidate functional genes, which might be involved in steroidal saponin biosynthesis, were selected from the transcriptome database of P. cyrtonema rhizome. Further investigation of these candidate genes will provide insight into their actual functions in the steroidal saponin biosynthetic pathway in P. cyrtonema. In addition, this study also provide abunant reference data for transcriptome characterization of P. cyrtonema and has important significance for functional genomics of P. cyrtonema.

[Selection and validation of internal reference genes for qPCR in Polygonatum cyrtonema tubers at different development stages and in response to abiotic stress].

In order to analyze the expression of genes involved in steroidal saponin biosynthesis pathway in Polygonatum cyrtonema tubers, it is very important to select internal reference genes that are stably expressed at different development stages and in response to abiotic stress. According to the previously established P. cyrtonema transcriptome database and reported internal reference genes in plant, this study systematically analyzed eight candidate internal reference genes including histone H2 A, glyceraldehyde-3-phosphate dehydrogenase, ACTIN, ß-tubulin, ubiquitin-conjugating enzyme-E2-10, elongation factor 1-alpha isoform, 18 S rRNA and α-tubulin 4 for expression stability in P. cyrtonema tubers at different development stages and in response to methyl jasmonate(MeJA) stress by using Real time fluorescence quantitative PCR(qPCR). Based on the statistical analysis of qPCR results by using GeNorm, NormFinder and BestKeeper softwares, the expression of ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform are the most stable in P. cyrtonema tubes at different development stages and in response to MeJA stress. The two internal reference genes were further validated by analyzing the expression of 4 genes involved in steroidal saponin biosynthesis pathways. In conclusion, ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform can be used as the most appropriate internal reference genes for qPCR analysis in P. cyrtonema. This study also provide a foundation for future investigate the molecular mechanism of steroidal saponin biosynthesis pathways in P. cyrtonema.

RNA-sequencing analysis of fungi-induced transcripts from the bamboo wireworm Melanotus cribricollis (Coleoptera: Elateridae) larvae.

Larvae of Melanotus cribricollis, feed on bamboo shoots and roots, causing serious damage to bamboo in Southern China. However, there is currently no effective control measure to limit the population of this underground pest. Previously, a new entomopathogenic fungal strain isolated from M. cribricollis larvae cadavers named Metarhizium pingshaense WP08 showed high pathogenic efficacy indoors, indicated that the fungus could be used as a bio-control measure. So far, the genetic backgrounds of both M. cribricollis and M. pingshaense WP08 were blank. Here, we analyzed the whole transcriptome of M. cribricollis larvae, infected with M. pingshaense WP08 or not, using high-throughput next generation sequencing technology. In addition, the transcriptome sequencing of M. pingshaense WP08 was also performed for data separation of those two non-model species. The reliability of the RNA-Seq data was also validated through qRT-PCR experiment. The de novo assembly, functional annotation, sequence comparison of four insect species, and analysis of DEGs, enriched pathways, GO terms and immune related candidate genes were operated. The results indicated that, multiple defense mechanisms of M. cribricollis larvae are initiated to protect against the more serious negative effects caused by fungal infection. To our knowledge, this was the first report of transcriptome analysis of Melanotus spp. infected with a fungus, and it could provide insights to further explore insect-fungi interaction mechanisms.