Current Plant Biology (Dec 2020)
RNA-seq of aboveground sporophyte’s transcriptome of Huperzia serrata and transcriptional understanding of early steps associated with huperzine biosynthesis in forest
Abstract
Chinese toothed clubmoss (Huperzia serrata) is a primitive fern native in certain types of forests. It is severely endangered in China due to its difficult propagation and massive hunting for huperzine A to improve and prevent Alzheimer’s disease. In this study, we completed RNA-seq for young leaves (HSYL), old leaves (HSOL), and stems (HSS) of H. serrata (HS) plants collected from a forest in 2016. From these tissues, we generated 77,430,786 trimmed paired reads (paired 32,418,231,517 pb). Sequence assembly obtained 621,023 contigs and 755,420 transcripts, which were annotated to be 49,923 unigenes. Of all unigenes, 40,612 were expressed in the three tissues, while 9311 were differentially expressed. 1158, 1675, and 1326 unigenes are specifically expressed in HSYL, HSOL, and HSS, respectively. Sequence mining obtained two unigenes encoding lysine decarboxylase (LDC1 and 2) and three unigenes encoding copper amine oxidase (CAO1, 2, and 3), which were involved in two early steps of the huperzine pathway. Quantitative RT-PCR was carried out to validate these early pathway genes using samples collected in 2017. RPKM values and qRT-PCR analysis characterized that the transcriptional level of LDC1 was the highest in old leaves followed by young leaves and stems, while the transcriptional level of LDC2 was similar in three tissues. Of three CAO genes, qRT-PCR validated the expression of CAO1 and CAO2 but not CAO3. Metabolite analysis showed the formation and differentiation of huperzine A in the three tissues collected in 2019, demonstrating the expression of the biosynthetic pathway of huperzine. Furthermore, the gene expression and huperzine A formation are discussed to understand the biosynthesis of huperzine in the forest. Taken together, this study provides a valuable genome-wide transcriptome of the aboveground sporophyte tissues and shows a dynamically transcriptional and metabolic feature of the huperzine biosynthesis in the forest.
