Enhanced Synthesis of Antioxidant Enzymes, Defense Proteins and Leghemoglobin in Rhizobium-Free Cowpea Roots after Challenging with Meloydogine incognita
Abstract
:1. Introduction
2. Experimental Section
2.1. Nematode Inoculums
2.2. Plant Material and Nematode Inoculation
2.3. Extraction of Proteins from the Cowpea Roots
2.4. Two-Dimensional Polyacrylamide Gel Electrophoresis (2D-SDS-PAGE)
2.5. In-Gel Digestion
2.6. Mass Spectrometry Analysis
2.7. Gene Expression Analysis
3. Results
3.1. Proteomic Analysis of RKN-Inoculated and Non-Inoculated Cowpea Roots
Target Gene (GenBank Accession Number) | Oligonucleotide Sequences 2 | Position 3 | Amplicon Size (bp) | Annealing Temperature (°C)/Cycle | Reference |
---|---|---|---|---|---|
Asparaginyl endopeptidase (D89971) | 5'-AACGGCTATTGGAACTAC-3' (f) | 217–234 | 876 | 52.5/35 | This work |
5'-GAGATCAGCATCCCTTTG-3' (r) | 1074–1092 | ||||
ACC synthase (Z12135) | 5'-CAAATGGGTCTTGCTGAGAAT-3' (f) | 70–90 | 858 | 58.9/20 | This work |
5'-TCTCAGCCTCTCCCTGTT-3' (r) | 910–927 4 | ||||
ARG 10 (AB012110) | 5'-CGAACACCATCGCCAAAG-3' (f) | 104–121 | 498 | 56.4/35 | This work |
5'-AGGGAAAGAAGCAAGCGA-3' (r) | 584–601 | ||||
Chalcone-flavanone isomerase (AB073787) | 5'-GAGAGGGGTTGACGATT-3' (f) | 112–129 | 477 | 54.2/35 | This work |
5'-GCCAATCATCGTCTCCAA-3' (r) | 571–588 | ||||
Cysteinyl endopeptidase (U49445) | 5'-TACGAGAGATGGAGGAGT-3' (f) | 119–136 | 765 | 51.2/25 | This work |
5'-TCCGACAATTGCTACACC-3' (r) | 866–883 | ||||
Leghemoglobin (U33205) | 5'-ATGGTTGCTTTCTCTGACAAG-3' (f) | 46–66 | 375 | 64.7/35 | This work |
5'-TTCATCACTCCATTTGTCTCC-3' (r) | 400–420 | ||||
CuZn-superoxide dismutase (AJ278668) | 5'-AAAGCGGTGGCGGTGCTGAAA-3' (f) | 195–215 | 393 | 61.0/35 | This work |
5'-GCTCAGTTCATGGCCGCCTTT-3' (r) | 567–587 | ||||
nodC (AE006469) | 5'-TGATYGAYATGGARTAYTGGCT-3' (f) | 545–566 | 640 | 55.6/35 | Sarita et al. 2005 [21] |
5'-CGYGACARCCARTCGCTRTTG-3' (r) | 1164–1184 | ||||
Chitinase I (X88800) | 5'-AGGATGATATGGAGCGTAGC-3' (f) | 14–33 | 972 | 58.0/28 | This work |
5'-GACAGGGTGAGATGTAGATC-3' (r) | 966–985 | ||||
Chitinase IIIa (X88802) | 5'-CTATCAACAACTGCAACGTG-3' (f) | 152–171 | 576 | 55.0/28 | This work |
5'-ATTTGGAAGAACCCTTGATG-3' (r) | 708–727 | ||||
Chitinase IIIb (X88801) | 5'-ACGTCAACATAGCTTTCCTC-3' (f) | 175–194 | 563 | 55.0/28 | This work |
5'-CTTCCCAGCAGGTACTGTAC-3' (r) | 718–737 | ||||
Chitinase IV (X88803) | 5'-GCTCAGAACTGTGGTTGTGC-3' (f) | 8–27 | 743 | 57.0/28 | This work |
5'-TAGCAAGTAAGATTATCACC-3' (r) | 731–750 | ||||
Actin (AF143208) | 5'-GCGTGATCTCACTGATGC-3' (f) | 669–686 | 530 | 59.0/35 | Costa et al., 2004 [23] |
5'-TCGCAATCCACATCTGTTGG-3' (r) | 1179–1198 |
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Spot *No. | Accession No. (NCBI) | Protein Identification | Organism | pI/MW (kDa) | Score | Sequence Covered (%) | Sequences | |
---|---|---|---|---|---|---|---|---|
Experimental | Theoretical | |||||||
Functional category: Disease/defense | ||||||||
2↑ | gi/297493 | ACC synthase | Vigna radiata | 4.89/39.678 | 5.40/43.768 | 79 | 7.98 | YFDGWK |
VHIVYSLSK | ||||||||
VGTIYSYNDSVVTTAR | ||||||||
3↑ | gi/86197901 | ACC oxidase | Vigna radiate | 5.05/38.056 | 5.86/40.236 | 111 | 12.30 | GAAMEMIK |
EMVANK | ||||||||
VSNYPPCPTPDLIK | ||||||||
DDQWIDVPPMR | ||||||||
11↑ | gi/42795352 | Ascorbate peroxidase | Vigna unguiculata | 6.62/28238 | 6.67/31.746 | 108 | 11.84 | NCAPLMLR |
EIVALSGGHTLGR | ||||||||
SGFDGPWTEDPLK | ||||||||
15↑ | gi/13274148 | CuZn-superoxide dismutase | Populus tremula | 6.76/23.114 | 5.87/29.203 | 142 | 14.74 | AVAVLK |
LTHGAPEDEIR | ||||||||
GGHELSSTTGNAGGR | ||||||||
16↑ | gi/123539 | 17.5 kDa HSP | Glycine max | 5.87/22.268 | 6.12/17.412 | 177 | 18.18 | DFHVPTSSVSAENSAFVSTR |
VLQISGER | ||||||||
17↑ | gi/130829 | PR-1 | Phaseolus vulgaris | 4.44/16.126 | 4.83/16.528 | 168 | 18.58 | ALPDSFK |
ISFVEDGETK | ||||||||
LSDGPNGGSLIK | ||||||||
18↑ | gi/4850337 | PR-3 (Chitinase) | Vigna unguiculata | 4.55/15.971 | 4.75/16.265 | 122 | 14.28 | ISFLEDGETK |
LSDGSNGGSVVK | ||||||||
19↑ | gi/ 130835 | PR-2 (β-1,3-glucanase) | Phaseolus vulgaris | 5.11/16.352 | 4.85/16.402 | 86 | 10.96 | ISIDSK |
GDAPPNEDELK | ||||||||
Functional category: Secondary metabolism | ||||||||
6↑ | gi/27530706 | Chalcone-flavone isomerase | Lotus japonicus | 6.41/35666 | 5.94 /36.987 | 97 | 11.11 | SYFLGGAGER |
STGTYGEAEAAAIGK | ||||||||
Functional category: Metabolism | ||||||||
21↑ | gi/ 20138591 | Leghemoglobin | Vigna unguiculata | 6.00/14.722 | 5.68/15.341 | 225 | 31.03 | ADIPK |
NLFSFLANGVDATNPK | ||||||||
ASGGVVADAALGAVHSQK | ||||||||
EAVGDK | ||||||||
22↑ | gi/ 26245403 | Nucleoside diphosphate Kinase | Glycine max | 6.89/15.964 | 6.30/16.254 | 183 | 21.08 | PDGVQSGLIGEIISR |
IIGATNPAQSEPGTIR | ||||||||
Functional category: Protein destination and storage | ||||||||
1↓ | gi/ 4589396 | Asparaginyl endopeptidase | Vigna mungo | 4.38/49.316 | 5.14/52.982 | 77 | 6.83 | FPIIFVVANLITLVSGGR |
NSLVPPSK | ||||||||
APLGSSR | ||||||||
4↑ | gi/ 1223922 | Cysteinyl endopeptidase | Vigna radiata | 5.10/35.289 | 5.44/37.332 | 64 | 6.35 | LLWVVLSLSLVLGVANSFDFHEK |
Functional category: Unknown/Predicted/Uncharacterized | ||||||||
7↑ | gi/297849580 | Predicted protein | Arabidopsis lyrata | 5.03/25.370 | 6.22/33.633 | 119 | 12.58 | EVETLPEEAFEEEEDK |
EILENHGGEER | ||||||||
IMDEAVNASR | ||||||||
9↑ | gi/ 297824991 | Predicted protein | Arabidopsis lyrata | 5.26/25.967 | 7.97/39.819 | 58 | 5.54 | QVVDETEPK |
VYGSIEEHYYR | ||||||||
10↓ | gi/2970051 | ARG 10 | Vigna radiata | 5.33/30.326 | 5.62/25.480 | 133 | 14.34 | DEIFCLFEGALDNLGSLR |
VVCHLSGSFAFIVFDK | ||||||||
12↑ | gi/416640 | Auxin induced protein | Vigna radiata | 4.81/19.354 | 4.65/21.345 | 104 | 11.34 | EGLGLEITELR |
GYSDLAFALEK |
3.2. RT-PCR Analyses of Cowpea Root Gene Expression
4. Discussion
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Oliveira, J.T.A.; Araujo-Filho, J.H.; Grangeiro, T.B.; Gondim, D.M.F.; Segalin, J.; Pinto, P.M.; Carlini, C.R.R.S.; Silva, F.D.A.; Lobo, M.D.P.; Costa, J.H.; et al. Enhanced Synthesis of Antioxidant Enzymes, Defense Proteins and Leghemoglobin in Rhizobium-Free Cowpea Roots after Challenging with Meloydogine incognita . Proteomes 2014, 2, 527-549. https://doi.org/10.3390/proteomes2040527
Oliveira JTA, Araujo-Filho JH, Grangeiro TB, Gondim DMF, Segalin J, Pinto PM, Carlini CRRS, Silva FDA, Lobo MDP, Costa JH, et al. Enhanced Synthesis of Antioxidant Enzymes, Defense Proteins and Leghemoglobin in Rhizobium-Free Cowpea Roots after Challenging with Meloydogine incognita . Proteomes. 2014; 2(4):527-549. https://doi.org/10.3390/proteomes2040527
Chicago/Turabian StyleOliveira, Jose T. A., Jose H. Araujo-Filho, Thalles B. Grangeiro, Darcy M. F. Gondim, Jeferson Segalin, Paulo M. Pinto, Celia R. R. S. Carlini, Fredy D. A. Silva, Marina D. P. Lobo, Jose H. Costa, and et al. 2014. "Enhanced Synthesis of Antioxidant Enzymes, Defense Proteins and Leghemoglobin in Rhizobium-Free Cowpea Roots after Challenging with Meloydogine incognita " Proteomes 2, no. 4: 527-549. https://doi.org/10.3390/proteomes2040527