Abstract

   Baphicacanthus cusia (Nees) Bremek is an herb widely used for the
   clinical treatment of colds, fever, and influenza in Traditional
   Chinese Medicine. The roots, stems and leaves can be used as natural
   medicine, in which indigo and indirubin are two main active
   ingredients. In this study, quantification of indigo, indirubin,
   indican and adenosine among various tissues of B. cusia was conducted
   using HPLC-DAD. Leaves have significantly higher contents than stems
   and roots (380.66, 315.15, 20,978.26, 4323.15 μg/g in leaves, 306.36,
   71.71, 3,056.78, 139.45 μg/g in stems, and 9.31, 7.82, 170.45, 197.48
   μg/g in roots, respectively). De novo transcriptome sequencing of B.
   cusia was performed for the first time. The sequencing yielded
   137,216,248, 122,837,394 and 140,240,688 clean reads from leaves, stems
   and roots respectively, which were assembled into 51,381 unique
   sequences. A total of 33,317 unigenes could be annotated using the
   databases of Nr, Swiss-Prot, KEGG and KOG. These analyses provided a
   detailed view of the enzymes involved in indican backbone biosynthesis,
   such as cytochrome P450, UDP-glycosyltransferase, glucosidase and
   tryptophan synthase. Analysis results showed that tryptophan synthase
   was the candidate gene involved in the tissue-specific biosynthesis of
   indican. We also detected sixteen types of simple sequence repeats in
   RNA-Seq data for use in future molecular mark assisted breeding
   studies. The results will be helpful in further analysis of B. cusia
   functional genomics, especially in increasing biosynthesis of indican
   through biotechnological approaches and metabolic regulation.

Introduction

   Baphicacanthus cusia (Nees) Bremek (B. cusia) also named Strobilanthes
   cusia is a perennial medicinal herb distributed broadly in Fujian,
   Zhejiang, Guangdong, Hainan, Guangxi, Hong Kong, Taiwan and other
   southern areas of China. It is also distributed across Bangladesh,
   India, and Myanmar, from the Himalayas to Indo-China [[34]1]. It has
   been cultivated in Fujian Province for more than 1000 years since the
   Song Dynasty. Indigo Naturalis is the leaf and stem extract of B.
   cusia, whose commercial name is “Qingdai” [[35]2], and Baphicacanthus
   cusiae Rhizome et Radix is derived from its root [[36]3], with the
   commercial name of “Nanbanlangen”. Previous studies have shown that
   Qingdai as an active ingredient was used to treat dental ulcers
   [[37]4], ulcerative colitis [[38]5–[39]7], and psoriasis
   [[40]8–[41]10], and that Nanbanlangen has amany pharmacological
   applications, including antibacterial [[42]11], antivirus [[43]12], and
   anti-inflammatory effects [[44]13, [45]14].

   Chemical research has indicated that Qingdai and Nanbanlangen both
   contain many active ingredients, such as indigo [[46]15] and indirubin
   [[47]16]. Indican has been known to be a precursor of indigo and
   indirubin [[48]17–[49]19], the content of which was different in the
   leaf and root of B. cusia [[50]15, [51]20]. To better understand the
   biosynthesis of indigo, gene expression possibly involved in indigo
   biosynthesis have been analysed by RNA-Seq in Indigofera tinctoria and
   Polygonum tinctorium [[52]21], species closely related to B. cusia.
   However, these organisms belong to different genera and species
   classifications and differ in morphological traits.

   The Qingdai produced in Fujian Province of China, named Jianqingdai, is
   a well-known genuine regional drug in China because of its good
   quality, but its effective constituent biosynthesis mechanism in vivo
   is still unclear. Until now, only nine proteins have been identified in
   the B. cusia leaf [[53]22, [54]23], and 98 nucleotide sequences are
   available for Baphicacanthus cusia, but none of the nucleotide
   sequences of B. cusia can be found in NCBI GenBank, it is hard to
   clarify its molecular mechanisms due to the lack of information of
   genomic sequences, especially sequences of indican biosynthesis and
   metabolism genes. Therefore, our primary work is to obtain and
   characterize the transcriptome of B. cusia.

   RNA-Seq next-generation sequencing (NGS) has been widely used in the
   analysis of transcriptomes in crops or vegetables, such as rice
   [[55]24], barley [[56]25], Sorghum bicolor [[57]26], Withania somnifera
   [[58]27], Brassica rapa [[59]28] and Chinese cherry [[60]29]. It has
   also been used to illustrate active components of biosynthetic and
   metabolic pathways in traditional Chinese medicine or identify
   differentially expressed genes (DEGs) between distinct sample groups
   such as Isatis indigotica [[61]30–[62]34], Salvia miltiorrhiza [[63]35,
   [64]36], Taxillus chinensis [[65]37], Lonicera japonica [[66]38], Toona
   sinensis Roem [[67]39], Pseudostellariae radix [[68]40], Eucommia
   ulmoides Oliver [[69]41], Cistanche deserticola [[70]42], Lycium
   chinense Mill [[71]43] and Gentiana macrophylla [[72]44].

   In this study, we measured the amounts of indigo, indirubin, adenosine
   and indican in the leaves, stems and roots of B. cusia. To understand
   the biosynthesis and metabolism pathways of indican in root, stem and
   leaf tissues of B. cusia, we generated multi-tissue transcriptomic data
   of B. cusia using RNA-Seq and annotated the multi-tissue transcriptome
   using publicly available databases and tools; we then ascertained genes
   that may be related to indican biosynthesis and metabolism pathways.
   Here, we report simple sequence repeats (SSR) in leaves, stems and
   roots from B. cusia and a number of differentially expressed genes
   (DEGs), particularly, cytochrome P450 (CYP450), UDP-glycosyltransferase
   (UGT), glucosidase and tryptophan synthase, that may be related to
   differential expression in indican biosynthesis and metabolism among
   leaves, stems and roots. Based on our results, the functional
   characterization of the B. cusia genes mentioned above should be
   further investigated.

Results

Quantitative analysis with HPLC

   The calibration curves of indigo, indirubin, adenosine and indican were
   formulated with five different concentration references. The regression