Pseudostellaria heterophylla (Miq.) Pax is a mild tonic herb widely cultivated in the Southern part of China. The tuberous roots of P. heterophylla accumulate high levels of secondary metabolism products of medicinal value such as saponins, flavonoids, and isoquinoline alkaloids. Despite numerous studies on the pharmacological importance and purification of these compounds in P. heterophylla, their biosynthesis is not well understood. In the present study, we used Illumina HiSeq 4000 sequencing platform to sequence the RNA from flowers, leaves, stem, root cortex and xylem tissues of P. heterophylla. We obtained 616,413,316 clean reads that we assembled into 127, 334 unique sequences with an N50 length of 951 bp. Among these unigenes, 53,184 unigenes (41.76%) were annotated in a public database and 39, 795 unigenes were assigned to 356 KEGG pathways; 23,714 unigenes (8.82%) had high homology with the genes from Beta vulgaris. We discovered 32, 095 DEGs in different tissues and performed GO and KEGG enrichment analysis. The most enriched KEGG pathway of secondary metabolism showed up-regulated expression in tuberous roots as compared with the ground parts of P. heterophylla. Moreover, we identified 72 candidate genes involved in triterpenoids saponins biosynthesis in P. heterophylla. The expression profiles of 11 candidate unigenes were analyzed by quantitative real-time PCR (RT-qPCR). Our study established a global transcriptome database of P. heterophylla for gene identification and regulation. We also identified the candidate unigenes involved in triterpenoids saponins biosynthesis. Our results provide an invaluable resource for the secondary metabolites and physiological processes in different tissues of P. heterophylla.

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To investigate the molecular mechanism of quality formation of Pseudostellaria heterophylla, the carotenoid cleavage dioxygenases (CCDs) genes were cloned from the transcriptome database of P. heterophylla, and analyzed them with bioinformatics analysis and expression analysis. The sequence length of four new gene were 1 617, 1 461, 1 746, 1 875 bp, and subsequently, named as PhCCD1,PhNCED2,PhNCED3 and PhCCD4 according to its genetic relationship with Arabidopsis thaliana. The sequence analysis showed that four new gene were all containing REP65 domains and binding sites of ferrous ion, such as histidine, glutamates and aspartates. Analysis phylogeny showed that PhNCED2 and PhNCED3 were the cluster of NCEDs, PhCCD1 and PhCCD4 were the cluster of CCDs. In addition, PhCCD1 and AtCDD1 of Arabidopsis thaliana, PhCCD4 and AtCCD4 of A. thaliana,PhNCED2, PhNCED3 and AtNCED3 of A. thaliana have high similarities. Analysis of real-time fluorescence quantitative showed that PhNCED2 and PhNCED3 were expressed mainly in underground part, the expression quantity of PhNCED2 reached the highest in fibrous root, PhNCED3 keeps higher in phloem and xylem, it may be the key enzymes of ABA biosynthesis genes. Moreover,PhCCD1 and PhCCD4 were expressed mainly in aerial part,the expression quantity of PhCCD1 reached the highest in leaf,PhCCD4 keeps higher in stem and leaf.It may be involved in the biosynthesis of carotenoids for P. heterophylla. The study obtained CDDs gene of P. heterophylla for the first time,this would lay the foundation of developing the response mechanism of P. heterophylla about external stress further,and then exploring the biological approach of quality formation in P. heterophylla.

Abscisic acid 8'-hydroxylase was one of key enzymes genes in the metabolism of abscisic acid (ABA). Seven menbers of abscisic acid 8'-hydroxylase were identified from Pseudostellaria heterophylla transcriptome sequencing results by using sequence homology. The expression profiles of these genes were analyzed by transcriptome data. The coding sequence of ABA8ox1 was cloned and analyzed by informational technology. The full-length cDNA of ABA8ox1 was 1 401 bp,with 480 encoded amino acids. The predicated isoelectric point (pI) and relative molecular mass (MW) were 8.55 and 53 kDa,respectively. Transmembrane structure analysis showed that there were 21 amino acids in-side and 445 amino acids out-side. High level of transcripts can detect in bark of root and fibrous root. Multi-alignment and phylogenetic analysis both show that ABA8ox1 had a high similarity with the CYP707As from other plants,especially with AtCYP707A1 and AtCYP707A3 in Arabidopsis thaliana. These results lay a foundation for molecular mechanism of tuberous root expanding and response to adversity stress.