International Journal of Plant Genomics

International Journal of Plant Genomics / 2014 / Article

Research Article | Open Access

Volume 2014 |Article ID 651912 |

Ravi Ranjan Kumar, Shailesh Yadav, Shourabh Joshi, Prithviraj P. Bhandare, Vinod Kumar Patil, Pramod B. Kulkarni, Swati Sonkawade, G. R. Naik, "Identification and Validation of Expressed Sequence Tags from Pigeonpea (Cajanus cajan L.) Root", International Journal of Plant Genomics, vol. 2014, Article ID 651912, 7 pages, 2014.

Identification and Validation of Expressed Sequence Tags from Pigeonpea (Cajanus cajan L.) Root

Academic Editor: Søren K. Rasmussen
Received14 Nov 2013
Accepted15 Apr 2014
Published06 May 2014


Pigeonpea (Cajanus cajan (L) Millsp.) is an important food legume crop of rain fed agriculture in the arid and semiarid tropics of the world. It has deep and extensive root system which serves a number of important physiological and metabolic functions in plant development and growth. In order to identify genes associated with pigeonpea root, ESTs were generated from the root tissues of pigeonpea (GRG-295 genotype) by normalized cDNA library. A total of 105 high quality ESTs were generated by sequencing of 250 random clones which resulted in 72 unigenes comprising 25 contigs and 47 singlets. The ESTs were assigned to 9 functional categories on the basis of their putative function. In order to validate the possible expression of transcripts, four genes, namely, S-adenosylmethionine synthetase, phosphoglycerate kinase, serine carboxypeptidase, and methionine aminopeptidase, were further analyzed by reverse transcriptase PCR. The possible role of the identified transcripts and their functions associated with root will also be a valuable resource for the functional genomics study in legume crop.

1. Introduction

Pigeonpea (Cajanus cajan L.) Millsp. () is a major grain legume of the arid and semiarid regions of the world [1]. Though considered a minor crop, pigeonpea is of considerable importance in areas of South Asia (mainly on the Indian subcontinent), Africa, the Caribbean, and Latin America, where it is a prominent source of protein in the human diet, as well as wood for fuel and light duty structural applications such as thatch for roofing [2]. Pigeonpea has now moved from an “orphan legume crop” to one of the promising pluses where genomics-assisted breeding approaches for a sustainable crop improvement are routine by Pigeonpea Genome Initiative, an effort of various researchers [3]. The first pigeonpea EST dataset provides a transcriptomic resource for gene discovery and development of functional markers associated with biotic stress resistance [4]. Root is the major part of water and nutrition uptake in pigeonpea which has a deep and extensive root system that provides access to water stored deep in the soil profile when that in the surface layer is depleted; this source of water is particularly important for long duration crops. In order to identify the associated genes in pigeonpea root tissues, a normalized cDNA library was constructed from pigeonpea root and expression analysis of the identified genes was carried out by reverse transcriptase PCR (RT-PCR) technique.

2. Materials and Methods

The pigeonpea genotype, namely, GRG-295 was selected to construct cDNA library and identification of expressed sequence tags (ESTs). The seeds of GRG-295 pigeonpea genotype were grown in petri dish for 15 days and irrigated with water. At the end of the 15th day, total RNA was isolated from root tissues by using the Trizol reagent (Invitrogen, Carlsbad, CA, USA), and mRNA was further isolated by using the PolyATract mRNA Isolation System (Promega, Madison, WI, USA). The quantification of RNA was verified by absorption ratio of OD260/280 and by formaldehyde gel electrophoresis. The first and second strands of cDNA were synthesized using Clontech SMARTer PCR cDNA Synthesis Kit. The cDNAs were purified by the MinElute PCR purification kit (Qiagen, Valencia, CA, USA) and ligated into a pGEM-T easy vector (Promega, Madison, WI, USA). Ligated plasmid DNAs were used for transformation into competent E. coli DH5α strain. Positive clones were selected on an ampicillin/IPTG/X-Gal LB plate. Plasmid DNA from positive clones were isolated by using REAL 96 plasmid isolation kit (Qiagen, Netherlands), and purified DNA was used for single-pass Sanger sequencing by using M13F/R universal sequencing primers on ABI sequencing machine 3500XL Genetic analyzer. All the ESTs were processed using VecScreen ( to remove vector and cloning oligo sequences and various contaminants to trim a high quality region. Based on the qualified sequences, the predicted amino acid sequences were used to search for similar peptide sequences to search for similar protein sequences in public database NCBI ( using the BLASTx search algorithm [5] by using default parameters of the program. The similarity scores between the cDNA clones and known sequences were represented by BLASTx probability values. Further the ESTs were classified into different functional categories based on the knowledge of biochemistry, plant physiology, and molecular biology (, GO (, and COG ( tools and by searching related abstracts in PubMed.

2.1. RT-PCR Analysis

Total root RNAs isolated from pigeonpea root tissues were used for reverse transcription polymerase chain reaction (RT-PCR) analysis. Genomic DNA contamination was removed by DNase I. First-strand cDNA was synthesized from each 2 μg of total RNA sample using Clontech SMARTer PCR cDNA Synthesis Kit according to the manufacturer’s protocol. The cDNAs were purified using a commercial column (Qiagen). To determine the expression of candidate genes, PCR was performed with 2 μL of the first-strand cDNA template and gene-specific primer pairs. Gene-specific RT-PCR primers were designed with Primer 3.0 according to the EST sequences and were synthesized commercially. General PCR was conducted with annealing as required for the specific primer pairs (Table 1). RT-PCR experiments were repeated three times, and the PCR products were detected on 1.5% agarose gel.

Accession numberPutative functionOptimum (°C)Primer sequence (5′-3′)

JK973671 S-Adenosylmethionine synthetase61F-AGAGGAAAT CGGT GCTGGTG

JK973674Phosphoglycerate kinase59F-TCCCGATCCCGATACCCTAC

JK973715Serine carboxypeptidase60F-ACATGAAGCTCAGTGGAGGAG

JK973726Methionine aminopeptidase60F-GGCAT TGAAAGTTGGGCAGG

3. Results and Discussion

Plant root systems serve a number of important functions, including anchoring the plant, absorbing water and nutrients, producing amino acids and hormones, and secreting organic acids, enzymes, and alkaloids [6]. The physiological significance of roots is belied by their relative structural simplicity as compared to other plant organs; major metabolic pathways such as photosynthesis lacking in root tissues have a stereotypical morphology that is conserved across taxa and throughout the life cycle of individuals. This combination of physiological relevance and structural simplicity has made roots obvious targets for functional genomics analyses [7]. As a major grain legume of semiarid tropics and a deep and extensive root system of pigeonpea represents an excellent source of identification of ESTs associated with their root tissues. So, the present work was focused on the study of genes associated with pigeonpea root tissues.

In the present investigation, the cDNA library was constructed in order to identify ESTs associated with pigeonpea roots and their functional analysis was carried out. The total RNA from the pigeonpea root tissue was isolated and the first and second cDNA strand was synthesized. The presence of the gene in plasmid construct of colonies was confirmed by colony PCR. The colony PCR showed that the size of these inserts ranged from 200 to 800 bp. Out of 400 bacterial clones, the plasmid construct of 250 positive recombinant clones was sequenced in single passed sequencing reaction from 3′ end using M13 forward/reverse primer and the sequence data was subjected to BLAST analysis. The leading sequences, tailing of the sequence, and poor quality sequences were excluded firstly. Finally, 105 high quality ESTs were retained which were clustered into 72 unigenes comprising 25 contigs and 47 singlets (Table 2) and were compared with NCBI nonredundant protein database using BLASTx algorithm and default parameters. In BLASTx analysis, it was shown that most of the sequences were having a significant homology with known proteins. Sequences that had no significant homology with protein database were compared to nucleotide BLAST using default parameters. The ESTs were deposited to NCBI dbEST with the accession number of JK973637 to JK973741 (Table 3).

Total clone sequenced250
ESTs taken for analysis105
Number of unigenes72
Number of contigs25
Number of singlets47
Average length of unigenes442 bp
Average length of ESTs437 bp
% GC content of unigenes50.3
% GC content of ESTs51.2

Sl. numberGen bank accession numberLength (in bp)Homologous protein -value

1JK973637560Vesicle-associated membrane protein 727-like [Glycine max]
2JK973638231 Mitochondrial inner membrane protein OXA1-like [Vitis vinifera]
3JK973639630Uncharacterized protein [Arabidopsis thaliana]
4JK973640621Hypothetical protein [Sorghum bicolor]
5JK973641570GTP-binding protein hflx [Medicago truncatula]
6JK973642412Unknown [Glycine max]
7JK973643264Secretory carrier-associated membrane protein 3-like [Vitis vinifera]
8JK973644438Histone 2 [Populus trichocarpa]
9JK973645200RNA recognition motif-containing protein [Arabidopsis thaliana]
10JK973646870Beta-glucosidase [Arabidopsis thaliana]
11JK973647555Epsin N-terminal homology (ENTH) domain-containing protein [Medicago truncatula]
12JK973648210Gamma-gliadin precursor [Ricinus communis]
13JK973649210AP2/ERF and B3 domain-containing transcription factor [Arabidopsis thaliana]
14JK973650474 Glycine dehydrogenase (decarborcylating mitochondrial like) [Glycine max]
15JK973651432Signal recognition particle subunit [Arabidopsis thaliana]
16JK973652227TBP-associated factor 6B [Arabidopsis thaliana]
17JK973653290TBP-associated factor 6B [Arabidopsis thaliana]
18JK973654303Transcription factor [Medicago truncatula]
19JK973655432Uncharacterized protein [Glycine max]
20JK973656237Nuclear cap-binding protein subunit [Arabidopsis thaliana]
21JK973657501Glucan endo-1,3-beta-glucosidase-like protein 3-like [Glycine max]
22JK973658499DNA-binding protein RAV1 [Zea mays]
23JK973659560Two-component response regulator ARR9 [Vitis vinifera]
24JK973660210Ribonucleoside-diphosphate reductase small chain like [Glycine max]
25JK973661748Hypothetical protein [Oryza sativa]
26JK973662742Cyclin-dependent protein kinase complex component [Aspergillus kawachii]
27JK973663406Elongation factor 2 [Nicotiana tobacum]
28JK973664841Expressed protein [Oryza sativa]
29JK973665385Hypothetical protein [Vitis vinifera]
30JK973666544ADP-ribosylation factor-like 8d [Nicotiana tobacum]
31JK973667510Jmjc domain-containing protein 4-like [Brachypodium distachyon]
32JK973668630Nosignificant match
33JK973669804Gag-pro[Pisum sativum]
34JK973670320Hypothetical protein MTR [Medicago truncatula]
35JK973671509Putative S-adenosylmethionine synthetase [Capsicum annum]
36JK973672371ABC transporter F family member 1-like [Brachypodium distachyon]
37JK973673513Predicted protein [Hordeum vulgare]
38JK973674465Phosphoglycerate kinase, cytosolic [Triticum aestivum]
39JK973675465Phosphoglycerate kinase, cytosolic [Triticum aestivum]
40JK973676457Unknown [Picea sitchensis]
41JK973677785GTP-binding signal recognition particle SRP54 [Medicago truncatula]
42JK973678241Signal recognition particle 54 kDA subunit precursor [Pisum sativum]
43JK973679475Protein SET-like [Brachypodium distachyon]
44JK973680350EIN3-binding F-box protein [Brachypodium distachyon]
45JK973681389Predicted protein [Hordeum vulgare]
46JK97368227240S ribosomal protein S24-2-like [Brachypodium distachyon]
47JK973683210Putative calcium exchanger [Triticum dicoccoides]
48JK973684478Thioredoxin [Medicago truncatula]
49JK973685210Nosignificant match
50JK973686553Root nodule extension [Pisum sativa]
51JK973687600Signal recognition particle protein [Oryza sativa]
52JK973688544ADP-ribosylation factor-like 8d [Nicotiana tobacum]
53JK973689395MLO-like protein [Medicago truncatula]
54JK973690250Phosphatidylcholine transfer protein [Ricinus communis]
55JK973691215Phosphatidylcholine transfer protein [Ricinus communis]
56JK973692411Unknown [Glycine max]
57JK973693246Phosphatidylcholine transfer protein [Ricinus communis]
58JK973694246Phosphatidylcholine transfer protein [Ricinus communis]
59JK973695420Uncharacterized protein [Zea mays]
60JK973696350Uncharacterized protein [Zea mays]
61JK973697244Jmjc domain-containing protein 4-like [Brachypodium distachyon]
62JK973698415Os01g0678900 [Oryza sativa]
63JK973699282 D-3-Phosphoglycerate dehydrogenase [Ricinus communis]
64JK97370055860S ribosomal protein [Vitis vinifera]
65JK973701490Nosignificant match
66JK973702556Root nodule extension [Pisum sativum]
67JK973703313Coiled-coil domain-containing protein [Ricinus communis]
68JK973704311Coiled-coil domain-containing protein [Ricinus communis]
69JK973705320Hypothetical protein MTR_4g076190 [Medicago truncatula]
70JK973706509Putative S-adenosylmethionine synthetase [Capsicum annuum]
71JK973707460Transmembrane emp24 domain-containing protein 10-like [Brachypodium distachyon]
72JK97370848260S ribosomal protein [Zea mays]
73JK973709395MLO5-like protein [Medicago truncatula]
74JK973710480Type 2 metallothionein [Prosopis   juliflora]
75JK973711556Root nodule extension [Pisum sativa]
76JK973712174Hypothetical protein [Oryza sativa]
77JK973713278Nosignificant match
78JK973714439Nosignificant match
79JK973715439Serine carboxypeptidase-like 19-like [Glycine max]
80JK973716551Serine carboxypeptidase-like 19-like [Glycine max]
81JK973717347Nosignificant match
82JK973718661Gamma-glutamyl hydrolase [Vitis vinifera]
83JK973719499Type 2 metallothionein [Prosopis juliflora]
84JK973720463Nodulin mtn21/eama-like transporter protein [Arabidopsis thaliana]
85JK973721200RNA recognition motif-containing protein [Arabidopsis thaliana]
86JK973722280 Beta-glucosidase 44-like [Glycine max]
87JK973723424V-type proton ATpase 21 kDA proteolipid subunit [Medicago truncatula]
88JK973724210AP2/ERF and B3 domain-containing transcription factor [Arabidopsis thaliana]
89JK973725540Polyribonucleotide nucleotidyltransferase [Vitis vinifera]
90JK973726649Methionine aminopeptidase 2B-like [Brachypodium distachyon]
91JK973727585Hypothetical protein [Vitisvinifera]
92JK973728487Unknown [Lotus japonica]
93JK97372955860S ribosomal protein [Vitisvinifera]
94JK973730314 D-3-Phosphoglycerate dehydrogenase [Ricinus communis]
95JK973731350EIN3-binding F-box protein 1-like [Brachypodium distachyon]
96JK973732499DNA-binding protein RAV1 [Zea mays]
97JK973733239NOT2/NOT3/NOT5 family protein [Oryza sativa]
98JK97373427240S ribosomal protein S24-2-like [Brachypodium distachyon]
99JK973735559Signal recognition particle subunit [Arabidopsis thaliana]
100JK973736793Vesicle-associated membrane protein 727-like [Glycine max]
101JK973737649Methionine aminopeptidase 2B [Brachypodium distachyon]
102JK973738210Gamma-gliadin precursor [Ricinus communis]
103JK973739553Unknown protein product [Glycine max]
104JK973740600Glycine dehydrogenase [Glycine max]
105JK973741572Glucan endo-1,3-beta-glucosidase-like protein 3-like [Glycine max]

The ESTs were categorized into 8 diverse functional group consisting of 10% transporter genes, 6% signal transduction genes, 20% cell growth and transcriptional regulator genes, and 26% metabolism genes. In other genes, 8% genes were uncharacterized, 8% hypothetical genes, 10% genes with no significant match, and 14% genes was involved in other functions. There were 8% of the ESTs found that did not show any match to known proteins in BLASTx program and that suggest novel nature of those genes (Figure 1).

In order to validate the ESTs generated from cDNA library, the expression of the four genes which were involved in different metabolic pathways was analysed by RT-PCR. RT-PCR results showed that the expression levels of four candidate genes, namely; S-adenosylmethionine synthetase, phosphoglycerate kinase, serine carboxypeptidase, and methionine aminopeptidase, were clearly expressed in pigeonpea roots (Figure 2). It was concluded that overall, there was a good agreement between the cDNA library data and the RT-PCR results.

S-Adenosylmethionine synthetase (SAMS) comprises of two cDNAs in Pinus contorta among which SAMS1 is expressed in roots and exhibits a specific expression pattern in the meristem at the onset of adventitious root development [8]. SAMS also catalyses the nucleophilic substitution reaction from between methionine and ATP into S-adenosylmethionine which have central role in several biological process in plants, namely, methyl group donor in trimethylation of lignin, DNA, and alkaloids as well as donor of aminopropyl moieties in ethylene and polyamine synthesis [8, 9].

Phosphoglycerate kinase superfamily has a diverse function in numerous metabolic processes like generation of precursor metabolites and energy, carbohydrate metabolism, phosphorous metabolism, glycolysis, kinase activity, ATP binding activity, and so forth. The presence of phosphoglycerate kinase transcripts in the cDNA library supports its diverse function in pigeonpea root.

Serine carboxypeptidase differentially expressed in root and other tissues is responsible for the synthesis of sinapoylcholine and sinapoylmalate in Arabidopsis which encodes 51 proteins annotated as serine carboxypeptidase-like enzymes and emerged as a new group of acyltransferase enzymes that are able to modify plant natural products [1012]. Methionine aminopeptidase, a ubiquitous enzyme, differentially expressed in root tissues is one of the central enzymes in protein synthesis that catalyzes N-terminal methionine from proteins [13].

A few important ESTs, namely, type 2 metallothionein, TBP-associated factor, ABC transporter, and phosphatidylcholine transfer protein, were also abundantly found in the cDNA library. Reddy et al. [14] found abundant metallothionein genes in normalized library from rice leaves and suggested that they might perform essential functions of plant growth beside metal detoxification. TATA-binding protein and TBP-associated factors are transcriptional factors which are predominantly involved in RNA polymerase II mediated transcription process [15]. ABC transporters play an important role in organ growth, plant nutrition, plant development, response to abiotic stress, and the interaction of the plant with its environment [16]. Phosphatidylcholine is usually the most abundant phospholipids in animals and plants, often amounting to almost 50% of the total, and as such it is obviously the key building block of membrane bilayers making up a very high proportion of the outer leaflet of the plasma membrane [17]. Similarly numerous ESTs like glycine dehydrogenase, signal proteins, root nodule extensions, membrane proteins, and β-glucosidase were observed in the cDNA library constructed from pigeonpea root tissues, which is thought to play possible roles in plant metabolism, growth, and development.

Apart from the known ESTs, 6 EST transcripts (JK973668, JK973685, JK973701, JK973714, JK973715, and JK973718) were observed as not having any significant match in NCBI database. Along with that uncharacterized and hypothetical proteins were also observed in cDNA library which were thought to impart in the cardinal role in root tissues of pigeonpea.

4. Conclusion

Our present investigation contains a precise repertoire of transcripts associated with the various metabolic functions in pigeonpea root. These ESTs appear to be involved in multiple metabolism pathways in the plant’s physiological and biochemical processes. In addition to known genes, some ESTs were unknown and uncharacterized, whose functional roles remain unclear and require further investigation in future. The root transcriptome characterized in this study markedly provides a unique resource for investigating the functional specificities of the root system. These EST tags may be useful for functional gene annotation, analysis of splice site variants and intron/exon determination, and evaluation of gene homologies or KEGG pathway confirmation.


ESTs:Expressed sequence tags
RT PCR:Reverse transcription polymerase chain reaction
SAMS:S-Adenosyl methionine synthetase.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.


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