De novo Assembly of Leaf Transcriptome in the Medicinal Plant Andrographis paniculata

Abstract

Andrographis paniculata is an important medicinal plant containing various bioactive terpenoids and flavonoids. Despite its importance in herbal medicine, no ready-to-use transcript sequence information of this plant is made available in the public data base, this study mainly deals with the sequencing of RNA from A. paniculata leaf using Illumina HiSeq™ 2000 platform followed by the de novo transcriptome assembly. A total of 189.22 million high quality paired reads were generated and 1,70,724 transcripts were predicted in the primary assembly. Secondary assembly generated a transcriptome size of ~88 Mb with 83,800 clustered transcripts. Based on the similarity searches against plant non-redundant protein database, gene ontology, and eukaryotic orthologous groups, 49,363 transcripts were annotated constituting upto 58.91% of the identified unigenes. Annotation of transcripts-using kyoto encyclopedia of genes and genomes database-revealed 5606 transcripts plausibly involved in 140 pathways including biosynthesis of terpenoids and other secondary metabolites. Transcription factor analysis showed 6767 unique transcripts belonging to 97 different transcription factor families. A total number of 124 CYP450 transcripts belonging to seven divergent clans have been identified. Transcriptome revealed 146 different transcripts coding for enzymes involved in the biosynthesis of terpenoids of which 35 contained terpene synthase motifs. This study also revealed 32,341 simple sequence repeats (SSRs) in 23,168 transcripts. Assembled sequences of transcriptome of A. paniculata generated in this study are made available, for the first time, in the TSA database, which provides useful information for functional and comparative genomic analysis besides identification of key enzymes involved in the various pathways of secondary metabolism.

Keywords: Andrographis paniculata; cytochrome P450; de novo assembly; leaf transcriptome; simple sequence repeats; terpenoid biosynthesis.

Figure 1

Statistics of primary and secondary assembly of A. paniculata leaf transcriptome.

Figure 2

Size distribution of A. paniculata leaf transcripts (bp) obtained both in primary and secondary assembly.

Figure 3

Homologous species contributing for the functional annotation of clustered transcripts of A. paniculata leaf.

Figure 4

Classification of A. paniculata leaf transcripts annotated with Gene Ontology.

Figure 5

A. paniculata transcriptome encoded enzymes (highlighted) involved in terpenoid backbone biosynthesis.

Figure 6

A. paniculata transcriptome encoded enzymes (highlighted) pertaining to di-terpenoid biosynthetic pathway.

Figure 7

Distribution of transcripts annotated to both plant-specific and plant-non-specific transcription factor families.

Figure 8

PlantTFcat analysis of A. paniculata transcriptome showing distribution of transcription factors in top 10 families.

Figure 9

Phylogenetic tree of cytochrome P450 proteins expressed in the leaf of A. paniculata (tree was built with bootstrap testing using 1000 replications).

Figure 10

Conserved heme motif of deduced CYP450s expressed in the leaf of A. paniculata.

Figure 11

qRT-PCR of selected transcripts and their relative expression levels with reference to Actin (Apaniculata22671, len = 1798; control): CYP94B56 (Apaniculata22533, len = 6227), CYP96A96 (Apaniculata62700, len = 1891), CYP96A97 (Apaniculata270380, len = 675), CYP94B (Apaniculata474149, len = 415), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HMDR; Apaniculata23282, len = 1733), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (MED; Apaniculata24573, len = 1282).