ABSTRACT
Heat shock protein 20 (HSP20) serves as a chaperone and plays roles in numerous biological processes, but the codon usage bias (CUB) of its genes has remained unexplored. This study identified 140 HSP20 genes from four cruciferous species, Arabidopsis thaliana, Brassica napus, Brassica rapa, and Camelina sativa, that were identified from the Ensembl plants database, and we subsequently investigated their CUB. As a result, the base composition analysis revealed that the overall GC content of HSP20 genes was below 50%. The overall GC content significantly correlated with the constituents at three codon positions, implying that both mutation pressure and natural selection might contribute to the CUB. The relatively high ENc values suggested that the CUB of the HSP20 genes in four cruciferous species was relatively weak. Subsequently, ENc exhibited a negative correlation with gene expression levels. Analyses, including ENc-plot analysis, neutral analysis, and PR2 bias, revealed that natural selection mainly shaped the CUB patterns of HSP20 genes in these species. In addition, a total of 12 optimal codons (ΔRSCU > 0.08 and RSCU > 1) were identified across the four species. A neighbor-joining phylogenetic analysis based on coding sequences (CDS) showed that the 140 HSP20 genes were strictly and distinctly clustered into 12 subfamilies. Principal component analysis and cluster analysis based on relative synonymous codon usage (RSCU) values supported the fact that the CUB pattern was consistent with the genetic relationship at the gene level and (or) species levels. These results will not only enrich the HSP20 gene resource but also advance our understanding of the CUB of HSP20 genes, which may underlie the theoretical basis for exploration of their genetic and evolutionary pattern.
ABSTRACT
Fritillaria unibracteata is an endangered medicinal plant whose bulb has been used as a Chinese herb to suppress cough, asthma and excessive phlegm for centuries. Steroidal alkaloids, which are synthesized via the steroid synthesis pathways, are their significant bioactive constituents. However, few studies on genes involved in steroidal alkaloid biosynthesis in F. unibracteata have been reported, mainly due to the lack of the F. unibracteata genome. In this paper, comparative transcriptomic and metabolomic analyses of four different tissues of F. unibracteata (leaves, flowers, stems, and bulbs) were performed. Imperialine, peiminine, and peimisine were among the significant bioactive compounds that were considerably abundant in bulb tissue, according to the metabolomic findings. Then, 83.60 Gb transcriptome sequencing of four different tissues was performed, of which one gene encoding phosphomevalonate kinase was directly functionally characterized to verify the accuracy of sequences obtained from the transcriptome. A total of 9217 differentially expressed unigenes (DEGs) were identified in four different tissues of F. unibracteata. GO and KEGG enrichments revealed that phenylpropanoid biosynthesis, MVA-mediated terpenoid backbone biosynthesis, and steroid biosynthesis were enriched in bulb tissue, whereas enrichment of MEP-mediated terpenoid backbone biosynthesis, photosynthesis, photosynthesis-antenna protein and carotenoid biosynthesis was observed in aerial tissues. Moreover, clustering analysis indicated that the downstream steroid biosynthesis pathway was more important in steroidal alkaloid biosynthesis compared to the upstream terpenoid backbone biosynthesis pathway. Hence, MVA-mediated biosynthesis of steroidal alkaloids was proposed, in which 15 bulb-clustered DEGs were positively correlated with a high accumulation of bioactive steroid alkaloids, further validating our proposal. In addition, 36 CYP450s showing a positive correlation with bioactive steroidal alkaloids provided candidate enzymes to catalyze the subsequent steps of steroidal alkaloid biosynthesis. In addition, the transcription factors and ABC transporters clustered in bulb tissue might be responsible for the regulation and transportation of steroidal alkaloid biosynthesis. Protein-protein interaction analysis implied a highly complex steroid alkaloid biosynthesis network in which delta (24)-sterol reductase might be one of the central catalysts. Based on the integrated transcriptome and metabolome, this current study is a first step in understanding the tissue-specific biosynthesis of steroidal alkaloids in F. unibracteata. Furthermore, key genes and regulators identified herein could facilitate metabolic engineering to improve steroidal alkaloids in F. unibracteata.
