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

Authors: Ravi Ranjan Kumar; Shailesh Yadav; Shourabh Joshi; Prithviraj P. Bhandare; Vinod Kumar Patil; Pramod B. Kulkarni; Swati Sonkawade; G. R. Naik
Journal: International Journal of Plant Genomics
 (2014)

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## Abstract

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.


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## Body

## 1. Introduction

Pigeonpea (Cajanus cajan L.) Millsp. (2
n
=
22) 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 competentE. 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 (http://www.ncbi.nlm.nih.gov/VecScreen/VecScreen.html) 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 (http://www.ncbi.nlm.nih.gov) 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 E values. Further the ESTs were classified into different functional categories based on the knowledge of biochemistry, plant physiology, and molecular biology (http://www.MetaCyc.org/), GO (http://www.ebi.ac.uk/GOA/), and COG (http://www.ncbi.nlm.nih.gov/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 2μg 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.Table 1
Primer sequences of subset of ESTs for RT-PCR analysis.
Accession number
Putative function
OptimumT
m (°C)
Primer sequence (5′-3′)
JK973671
S-Adenosylmethionine  synthetase
61
F-AGAGGAAAT CGGT GCTGGTG
R-GCAGCAATTTGGTCGTTGGT
JK973674
Phosphoglycerate kinase
59
F-TCCCGATCCCGATACCCTAC
R-CAGCACGCTTTTCAGCAGTT
JK973715
Serine carboxypeptidase
60
F-ACATGAAGCTCAGTGGAGGAG
R-AGCCATGGCCTCCAATCTTC
JK973726
Methionine  aminopeptidase
60
F-GGCAT TGAAAGTTGGGCAGG
R-GATTGCAGCACCGACATCAC


## 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 2μg 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.Table 1
Primer sequences of subset of ESTs for RT-PCR analysis.
Accession number
Putative function
OptimumT
m (°C)
Primer sequence (5′-3′)
JK973671
S-Adenosylmethionine  synthetase
61
F-AGAGGAAAT CGGT GCTGGTG
R-GCAGCAATTTGGTCGTTGGT
JK973674
Phosphoglycerate kinase
59
F-TCCCGATCCCGATACCCTAC
R-CAGCACGCTTTTCAGCAGTT
JK973715
Serine carboxypeptidase
60
F-ACATGAAGCTCAGTGGAGGAG
R-AGCCATGGCCTCCAATCTTC
JK973726
Methionine  aminopeptidase
60
F-GGCAT TGAAAGTTGGGCAGG
R-GATTGCAGCACCGACATCAC


## 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 (Table2) 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).Table 2
Summary of ESTs library.
Total clone sequenced
250
ESTs taken for analysis
105
Number of unigenes
72
Number of contigs
25
Number of singlets
47
Average length of unigenes
442 bp
Average length of ESTs
437 bp
% GC content of unigenes
50.3
% GC content of ESTs
51.2Table 3
Homology search of the ESTs generated from pigeonpea root along with theirE value using BLASTx programme.
Sl. number
Gen bank accession number
Length (in bp)
Homologous protein
E-value
1
JK973637
560
Vesicle-associated membrane protein 727-like [Glycine max]
1
e
-
111
2
JK973638
231
Mitochondrial inner membrane protein OXA1-like [Vitis vinifera]
5
e
-
41
3
JK973639
630
Uncharacterized protein [Arabidopsis thaliana]
9
e
-
159
4
JK973640
621
Hypothetical protein [Sorghum bicolor]
9
e
-
139
5
JK973641
570
GTP-binding protein hflx [Medicago truncatula]
6
e
-
13
6
JK973642
412
Unknown [Glycine max]
1.1
7
JK973643
264
Secretory carrier-associated membrane protein 3-like [Vitis vinifera]
2
e
-
55
8
JK973644
438
Histone 2 [Populus trichocarpa]
5
e
-
63
9
JK973645
200
RNA recognition motif-containing protein [Arabidopsis thaliana]
4
e
-
10
10
JK973646
870
Beta-glucosidase [Arabidopsis thaliana]
0.0
11
JK973647
555
Epsin N-terminal homology (ENTH) domain-containing protein [Medicago truncatula]
4
e
-
36
12
JK973648
210
Gamma-gliadin precursor [Ricinus communis]
7
e
-
10
13
JK973649
210
AP2/ERF and B3 domain-containing transcription factor [Arabidopsis thaliana]
4
e
-
40
14
JK973650
474
Glycine dehydrogenase (decarborcylating mitochondrial like) [Glycine max]
4
e
-
102
15
JK973651
432
Signal recognition particle subunit [Arabidopsis thaliana]
9
e
-
80
16
JK973652
227
TBP-associated factor 6B [Arabidopsis thaliana]
0.051
17
JK973653
290
TBP-associated factor 6B [Arabidopsis thaliana]
8
e
-
48
18
JK973654
303
Transcription factor [Medicago truncatula]
6
e
-
29
19
JK973655
432
Uncharacterized protein [Glycine max]
8
e
-
11
20
JK973656
237
Nuclear cap-binding protein subunit [Arabidopsis thaliana]
8
e
-
48
21
JK973657
501
Glucan endo-1,3-beta-glucosidase-like protein 3-like [Glycine max]
2
e
-
04
22
JK973658
499
DNA-binding protein RAV1 [Zea mays]
4
e
-
25
23
JK973659
560
Two-component response regulator ARR9 [Vitis vinifera]
1
e
-
45
24
JK973660
210
Ribonucleoside-diphosphate reductase small chain like [Glycine max]
2
e
-
34
25
JK973661
748
Hypothetical protein [Oryza sativa]
26
JK973662
742
Cyclin-dependent protein kinase complex component [Aspergillus kawachii]
0.12
27
JK973663
406
Elongation factor 2 [Nicotiana tobacum]
5
e
-
39
28
JK973664
841
Expressed protein [Oryza sativa]
7.3
29
JK973665
385
Hypothetical protein [Vitis vinifera]
2.1
30
JK973666
544
ADP-ribosylation factor-like 8d [Nicotiana tobacum]
2
e
-
105
31
JK973667
510
Jmjc domain-containing protein 4-like [Brachypodium distachyon]
6
e
-
77
32
JK973668
630
Nosignificant match
33
JK973669
804
Gag-pro[Pisum sativum]
9
e
-
18
34
JK973670
320
Hypothetical protein MTR [Medicago truncatula]
2
e
-
13
35
JK973671
509
Putative S-adenosylmethionine synthetase [Capsicum annum]
6
e
-
114
36
JK973672
371
ABC transporter F family member 1-like [Brachypodium distachyon]
4
e
-
60
37
JK973673
513
Predicted protein [Hordeum vulgare]
2
e
-
89
38
JK973674
465
Phosphoglycerate kinase, cytosolic [Triticum aestivum]
2
e
-
97
39
JK973675
465
Phosphoglycerate kinase, cytosolic [Triticum aestivum]
2
e
-
97
40
JK973676
457
Unknown [Picea sitchensis]
0.97
41
JK973677
785
GTP-binding signal recognition particle SRP54 [Medicago truncatula]
7
e
-
51
42
JK973678
241
Signal recognition particle 54 kDA subunit precursor [Pisum sativum]
0.044
43
JK973679
475
Protein SET-like [Brachypodium distachyon]
7
e
-
33
44
JK973680
350
EIN3-binding F-box protein [Brachypodium distachyon]
8
e
-
65
45
JK973681
389
Predicted protein [Hordeum vulgare]
4
e
-
27
46
JK973682
272
40S ribosomal protein S24-2-like [Brachypodium distachyon]
2
e
-
57
47
JK973683
210
Putative calcium exchanger [Triticum dicoccoides]
4
e
-
27
48
JK973684
478
Thioredoxin [Medicago truncatula]
2
e
-
75
49
JK973685
210
Nosignificant match
50
JK973686
553
Root nodule extension [Pisum sativa]
5
e
-
04
51
JK973687
600
Signal recognition particle protein [Oryza sativa]
5
e
-
08
52
JK973688
544
ADP-ribosylation factor-like 8d [Nicotiana tobacum]
2
e
-
105
53
JK973689
395
MLO-like protein [Medicago truncatula]
2
e
-
24
54
JK973690
250
Phosphatidylcholine transfer protein [Ricinus communis]
3
e
-
09
55
JK973691
215
Phosphatidylcholine transfer protein [Ricinus communis]
3
e
-
09
56
JK973692
411
Unknown [Glycine max]
1.1
57
JK973693
246
Phosphatidylcholine transfer protein [Ricinus communis]
5
e
-
09
58
JK973694
246
Phosphatidylcholine transfer protein [Ricinus communis]
5
e
-
09
59
JK973695
420
Uncharacterized protein [Zea mays]
9
e
-
83
60
JK973696
350
Uncharacterized protein [Zea mays]
5
e
-
51
61
JK973697
244
Jmjc domain-containing protein 4-like [Brachypodium distachyon]
5
e
-
27
62
JK973698
415
Os01g0678900 [Oryza sativa]
8
e
-
11
63
JK973699
282
D-3-Phosphoglycerate dehydrogenase [Ricinus communis]
3
e
-
51
64
JK973700
558
60S ribosomal protein [Vitis vinifera]
8
e
-
65
65
JK973701
490
Nosignificant match
66
JK973702
556
Root nodule extension [Pisum sativum]
3
e
-
07
67
JK973703
313
Coiled-coil domain-containing protein [Ricinus communis]
2
e
-
65
68
JK973704
311
Coiled-coil domain-containing protein [Ricinus communis]
1
e
-
64
69
JK973705
320
Hypothetical protein MTR_4g076190 [Medicago truncatula]
2
e
-
13
70
JK973706
509
Putative S-adenosylmethionine synthetase [Capsicum annuum]
9
e
-
119
71
JK973707
460
Transmembrane emp24 domain-containing protein 10-like [Brachypodium distachyon]
2
e
-
45
72
JK973708
482
60S ribosomal protein [Zea mays]
3
e
-
52
73
JK973709
395
MLO5-like protein [Medicago truncatula]
2
e
-
24
74
JK973710
480
Type 2 metallothionein [Prosopis  juliflora]
4
e
-
28
75
JK973711
556
Root nodule extension [Pisum sativa]
3
e
-
07
76
JK973712
174
Hypothetical protein [Oryza sativa]
0.86
77
JK973713
278
Nosignificant match
78
JK973714
439
Nosignificant match
79
JK973715
439
Serine carboxypeptidase-like 19-like [Glycine max]
2
e
-
31
80
JK973716
551
Serine carboxypeptidase-like 19-like [Glycine max]
9
e
-
39
81
JK973717
347
Nosignificant match
82
JK973718
661
Gamma-glutamyl hydrolase [Vitis vinifera]
1
e
-
54
83
JK973719
499
Type 2 metallothionein [Prosopis juliflora]
2
e
-
30
84
JK973720
463
Nodulin mtn21/eama-like transporter protein [Arabidopsis thaliana]
2
e
-
44
85
JK973721
200
RNA recognition motif-containing protein [Arabidopsis thaliana]
4
e
-
10
86
JK973722
280
Beta-glucosidase 44-like [Glycine max]
3
e
-
52
87
JK973723
424
V-type proton ATpase 21 kDA proteolipid subunit [Medicago truncatula]
2
e
-
29
88
JK973724
210
AP2/ERF and B3 domain-containing transcription factor [Arabidopsis thaliana]
4
e
-
40
89
JK973725
540
Polyribonucleotide nucleotidyltransferase [Vitis vinifera]
3
e
-
07
90
JK973726
649
Methionine aminopeptidase 2B-like [Brachypodium distachyon]
3
e
-
40
91
JK973727
585
Hypothetical protein [Vitisvinifera]
6.9
92
JK973728
487
Unknown [Lotus japonica]
1
93
JK973729
558
60S ribosomal protein [Vitisvinifera]
8
e
-
65
94
JK973730
314
D-3-Phosphoglycerate dehydrogenase [Ricinus communis]
1
e
-
55
95
JK973731
350
EIN3-binding F-box protein 1-like [Brachypodium distachyon]
8
e
-
65
96
JK973732
499
DNA-binding protein RAV1 [Zea mays]
4
e
-
25
97
JK973733
239
NOT2/NOT3/NOT5 family protein [Oryza sativa]
3
e
-
62
98
JK973734
272
40S ribosomal protein S24-2-like [Brachypodium distachyon]
2
e
-
57
99
JK973735
559
Signal recognition particle subunit [Arabidopsis thaliana]
9
e
-
80
100
JK973736
793
Vesicle-associated membrane protein 727-like [Glycine max]
1
e
-
111
101
JK973737
649
Methionine aminopeptidase 2B [Brachypodium distachyon]
1
e
-
24
102
JK973738
210
Gamma-gliadin precursor [Ricinus communis]
7
e
-
10
103
JK973739
553
Unknown protein product [Glycine max]
1
e
-
04
104
JK973740
600
Glycine dehydrogenase [Glycine max]
4
e
-
102
105
JK973741
572
Glucan endo-1,3-beta-glucosidase-like protein 3-like [Glycine max]
2
e
-
04The 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 (Figure1).Figure 1
Functional classification of ESTs of pigeonpea generated by single pass sequencing.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, andmethionine 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.Figure 2
Validation of cDNA library results by RT-PCR (M-100 bp ladder, 1-S-adenosylmethionine synthetase; 2-phosphoglycerate kinase; 3-serine carboxypeptidase; and 4-methionine aminopeptidase).S-Adenosylmethionine synthetase (SAMS) comprises of two cDNAs inPinus 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 ofphosphoglycerate 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 inArabidopsis which encodes 51 proteins annotated asserine carboxypeptidase-like enzymes and emerged as a new group of acyltransferase enzymes that are able to modify plant natural products [10–12].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 likeglycine 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.



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*Source: 651912-2014-05-06.xml*