Transcriptome analysis for molecular landscaping of genes controlling diterpene andrographolide biosynthesis in Andrographis paniculata (Burm . f.) Nees

Abstract

Kalmegh [Andrographis paniculata (Burm. f.) Nees.] is one of the essential medicinal plants due to an important terpenoid, i.e. andrographolide possesses immense therapeutic and pharmacological uses. The experiment was performed to elucidate the expression of candidate genes associated with andrographolide biosynthesis. Based on results obtained in chromatography for andrographolide content analysis of six genotypes, two contrast genotypes, i.e. IC-520361 (maximum andrographolide content-2.33%) and Anand Local (lowest andrographolide content-1.01%) were selected for the transcriptome analysis. A total of 1.04 Gb of raw data were produced using MiSeq Illumina platform, in which IC 520361 generated 645 million base pairs sequence along with 4,524,251 raw reads and Anand Local produced 419 million base pairs sequence along with 3,021,316 raw reads. The combined assembly of high quality reads generated for both the samples had 33,247,454 bp of total assembled bases and 38,292 of transcripts. The GC percent of assembled transcripts was 44.79%, an average read length was 800 bp and N₅₀ value was 1186 bp. Species-specific distribution using BLAST X (Nr), showed the highest Blast hits with Sesamum indicum. Out of 23,346 transcripts, 87% of transcripts annotated in UniProt KB (Universal Protein Resource KnowledgeBase) database and only 0.21% of transcripts were annotated in TAIR (The Arabidopsis Information Resources). Biological processes gene ontology classified based on Blast2GO showed, out of 6853 transcripts, 1370 of transcripts were represented by terpenoid biosynthetic pathway, which involved in secondary metabolite andrographolide biosynthesis. The heat map showed 1016 transcripts were differentially expressed between two kalmegh genotypes, in which nine important differentially expressed transcripts related to MEP (2C methyl-d-erythritol 4-phosphate) and MVA (Mevalonic acid) andrographolide biosynthesis pathways such as, geranyl diphosphate synthase small subunit, Isopentenyl-diphosphate delta-isomerase i-like, 4, 13-hydroxy-3-methylglutaryl-coenzyme a reductase etc. were upregulated in IC 520361 as compared to Anand Local, which were validated through RT-qPCR. The highest expression of gene 13-hydroxy-3-methylglutaryl-coenzyme a reductase (HMGR) was reported, which is responsible for accumulation of andrographolide in leaf. This comparative transcriptome analysis confirmed the expression level of genes were higher in accession IC 520361 as compare to Anand Local related to andrographolide biosynthesis pathways i.e. MEP and MVA. These up-regulated genes could be over-expressed to enhance the andrographolide content using genetic engineering of these metabolic pathways. It will also give an idea to the breeder for development of molecular markers for direct screening of the genotypes.

Keywords: Andrographolide; Differential gene expression; Kalmegh; Miseq platform; Transcriptome.

Fig. 1

Species distribution of the top BLAST hits of kalmegh transcripts in Nr database

Fig. 2

Biological processes gene ontology of kalmegh transcripts

Fig. 3

Cellular components gene ontology of kalmegh transcripts

Fig. 4

Molecular functions gene ontology of kalmegh transcripts

Fig. 5

Enzyme classification of kalmegh transcripts based on KEGG pathway study

Fig. 6

Top 21 metabolic pathways distinguished in kalmegh transcriptome

Fig. 7

KEGG pathway shows transcripts/enzymes involved in terpenoid backbone biosynthesis in kalmegh (Key enzymes with Enzyme Commission number are highlighted: (i) EC2.2.1.7.—1-deoxy-d-xylulose-5-phosphate synthase; (ii) EC1.1.267.—1-deoxy-d-xylulose-5-phosphate reductoisomerase; (iii) EC1.1.1.34.—hydroxymethylglutaryl-CoA reductase (NADPH); (iv) EC1.17.7.1—4-hydroxy-3-methylbut-2-enyl-diphosphate synthase; (v) EC1.17.1.2.—4-hydroxy-3-methylbut-2-enyl diphosphate reductase; (vi) EC4.1.1.33—di phosphomevalonate decarboxylase; (vii) EC5.3.3.2—isopentenyl-diphosphate Delta-isomerase)

Fig. 8

Putative MEP and MVA biosynthesis pathway of andrographolide in kalmegh (Red color highlighted genes/enzymes expressed in these pathways based on RT qPCR analysis). DXS 1-Deoxy-d-xylulose-5-phosphate synthase, DXR 1-Deoxy-d-xylulose-5-phosphate reductoisomerase, CMK 4-Diphosphocytidyl-2-C-methyl-d-erythritol kinase, MDS 2-C-Methyl-d-erythritol 2,4-cyclodiphosphate synthase, HMDS 4-Hydroxy-3-methylbut-2-enyl-diphosphate synthase, IDI Isopentenyl-diphosphate delta-isomerase, GGPS Geranylgeranyl diphosphate synthase, HMGR Hydroxymethylglutaryl-CoA reductase, MK mevalonate kinase, PMK Phosphomevalonate kinase

Fig. 9

The MA [M (log ratios) and A (mean average] plot showing differential expression and volcano plot reporting false discovery rate between two kalmegh genotypes

Fig. 10

Correlation expression profiles of transcripts based on qPCR between IC 520361 and Anand Local (Data are based on log2 fold change)