Identification of Powdery Mildew Responsive Genes in Hevea brasiliensis through mRNA Differential Display
Abstract
:1. Introduction
2. Results
2.1. Comparison of Powdery Mildew Resistance between RRIC52 and Reyan 7-33-97
2.2. Identification and Isolation of Differentially Expressed Transcripts
2.3. Sequence Analysis Revealed the Significances of Differentially Expressed RRIC52 Transcripts
2.4. Characterization of the Differentially Expressed ESTs
ESTs | dbEST_Id | Accession No | Homology Found with | Homologous Species | Identity % | Description | E-Value |
---|---|---|---|---|---|---|---|
ESTs in cell wall and membrane pathways | |||||||
HBOH2 | 79640384 | JZ822682 | XM_002522813.1 | Ricinus communis | 85.47 | pectinesterase-2 precursor, putative | 2 × 10−87 |
HBOH3 | 79721589 | JZ893198 | U48687.1 | Castanea sativa | 78.07 | endochitinase | 3 × 10−164 |
HBOH5 | 79721590 | JZ893199 | AB367524.1 | Arabidopsis thaliana | 66 | CERK1 mRNA for chitin elicitor receptor kinase 1 | 1.00 × 10−100 |
HBOH7 | 79640387 | JZ822685 | XM_002530816.1 | Ricinus communis | 84.47 | glucan endo-1,3-beta-glucosidase precursor, putative | 3 × 10−149 |
HBOH15 | 79721594 | JZ8931203 | XM_006375808.1 | Populus trichocarpa | 80.33 | germin-like protein 1 | 6 × 10−72 |
HBOH66 | 79761733 | JZ897003 | XM_006376356.1 | Populus trichocarpa | 81.84 | class IV chitinase family protein | 2 × 10−132 |
HBOH67 | 79761734 | JZ897004 | XM_002532766.1 | Ricinus communis | 80.94 | chitinase 1 precursor, putative | 3 × 10−118 |
HBOH68 | 79761735 | JZ897005 | AY973617.1 | Manihot esculenta | 88.24 | germin-like protein GLP partial CdS | 2 × 10−163 |
ESTs involved in transcription factor and regulatory proteins | |||||||
HBOH4 | 79640385 | JZ822683 | XM_002526622.1 | Ricinus communis | 86.35 | ferritin, plant, putative | 7 × 10−171 |
HBOH13 | 79721592 | JZ8931201 | XM_002303816.2 | Populus trichocarpa | 78.24 | WRKY transcription factor 40 family protein | 3 × 10−85 |
HBOH14 | 79721593 | JZ8931202 | XM_012596852.1 | Gossypium raimondii | 58 | WRKY transcription factor 18-like | 7 × 10−15 |
HBOH31 | 79761701 | JZ896971 | XM_002534015.1 | Ricinus communis | 87.56 | eukaryotic initiation factor iso-4F subunit p82-34, putative | 3 × 10−62 |
HBOH33 | 79761703 | JZ896973 | XM_002517578.1 | Ricinus communis | 78.79 | DNA binding protein, putative | 2 × 10−140 |
HBOH36 | 79761705 | JZ896975 | XM_002527643.1 | Ricinus communis | 84.82 | transcription factor btf3, putative | 0 |
HBOH55 | 79761724 | JZ896994 | XM_002524212.1 | Ricinus communis | 82.11 | rop guanine nucleotide exchange factor, putative | 0 |
HBOH60 | 79761727 | JZ896997 | XM_006368449.1 | Populus trichocarpa | 85.21 | WRKY transcription factor 40 family protein | 4 × 10−140 |
ESTs involved in transporter | |||||||
HBOH8 | 79640388 | JZ822686 | XM_002521455.1 | Ricinus communis | 85.71 | dimethyladenosine transferase, putative | 1 × 10−53 |
HBOH28 | 79761698 | JZ896968 | XM_002527741.1 | Ricinus communis | 88.11 | clathrin assembly protein, putative | 2 × 10−144 |
HBOH38 | 79761707 | JZ896977 | XM_002529119.1 | Ricinus communis | 82.69 | DNA-directed RNA polymerase II, putative | 6 × 10−154 |
HBOH39 | 79761708 | JZ896977 | XM_002529119.1 | Ricinus communis | 83.15 | DNA-directed RNA polymerase II, putative | 3 × 10−158 |
HBOH44 | 79761713 | JZ896983 | XM_002269429.2 | Vitis vinifera | 66 | clathrin light chain 2 | 5 × 10−73 |
HBOH46 | 79761715 | JZ896985 | XM_002531006.1 | Ricinus communis | 76.31 | transferase, transferring glycosyl groups, putative | 4 × 10−90 |
HBOH52 | 79761721 | JZ896991 | HM461981.1 | Vernicia fordii | 84.48 | glycerol-3-phosphate acyltransferase 9 (GPAT9) gene | 0.00003 |
HBOH54 | 79761723 | JZ896993 | HQ285842.1 | Hevea brasiliensis | 98.95 | chloroplast, complete genome | 0 |
HBOH75 | 79761741 | JZ897011 | XM_002524326.1 | Ricinus communis | 72.5 | flavonol 4'-sulfotransferase, putative | 4 × 10−51 |
HBOH81 | 79761747 | JZ897017 | XM_007017019.1 | Theobroma cacao | 72.69 | non-specific lipid-transfer protein, putative | 2 × 10−26 |
ESTs involved in signal transduction | |||||||
HBOH1 | 79640383 | JZ822681 | XM_002516001.1 | Ricinus communis | 68.49 | serine-threonine protein kinase, plant-type, putative | 4 × 10−63 |
HBOH6 | 79640386 | JZ822684 | XM_002324361.2 | Populus trichocarpa | 87.66 | H+-ATPase family protein mRNA, complete cds | 2 × 10−130 |
HBOH9 | 79721591 | JZ893120 | XM_003605902.1 | Medicago truncatula | 40 | hypothetical protein mRNA, complete cds | 0.0001 |
HBOH10 | 79640389 | JZ822687 | XM_007019829.1 | Theobroma cacao | 86.79 | cysteine-rich RLK (RECEPTOR-like protein kinase) 8 | 0.00002 |
HBOH22 | 79761693 | JZ896963 | XM_002514747.1 | Ricinus communis | 87.7 | 4-nitrophenylphosphatase, putative | 0 |
HBOH23 | 79761694 | JZ896964 | XM_007019349.1 | Theobroma cacao | 66 | cysteine-rich RLK (RECEPTOR-like protein kinase) 8 | 5 × 10−43 |
HBOH26 | 79761697 | JZ896967 | XM_002509773.1 | Ricinus communis | 83.05 | ATP binding protein, putative | 3 × 10−170 |
HBOH41 | 79761710 | JZ896980 | XM_002308527.2 | Populus trichocarpa | 71.47 | kinase family protein | 9 × 10−35 |
HBOH42 | 79761711 | JZ896981 | AM478326.2 | Vitis vinifera | 84.55 | contig VV78X154662.12, whole genome shotgun sequence | 5 × 10−24 |
HBOH45 | 79761714 | JZ896984 | XM_007026459.1 | Theobroma cacao | 95.35 | signal transducer and transcription activator isoform 1 | 1 × 10−28 |
HBOH53 | 79761722 | JZ896992 | XM_002524179.1 | Ricinus communis | 75.7 | kinase, putative | 3 × 10−10 |
HBOH56 | 79761725 | JZ896995 | XM_002524179.1 | Ricinus communis | 75.26 | kinase, putative | 6 × 10−47 |
HBOH73 | 79761739 | JZ897009 | XM_002531397.1 | Ricinus communis | 83.52 | serine-threonine protein kinase, plant-type, putative | 1 × 10−74 |
HBOH74 | 79761740 | JZ897010 | XM_002523439.1 | Ricinus communis | 82.36 | serine-threonine protein kinase, plant-type, putative | 5 × 10−139 |
HBOH78 | 79761744 | JZ897014 | XM_002530595.1 | Ricinus communis | 69.87 | B-Raf proto-oncogene serine/threonine-protein kinase, putative | 9 × 10−27 |
HBOH80 | 79761746 | JZ897016 | XM_002513521.1 | Ricinus communis | 88.85 | kinase, putative | 3 × 10−107 |
ESTs involved in phytoalexin biosynthesis | |||||||
HBOH48 | 79761717 | JZ896987 | DQ371802.1 | Populus alba | 83.36 | chalcone synthase (CHS) gene, CHS-M2 allele | 5 × 10−159 |
HBOH61 | 79761728 | JZ896998 | XM_002300417.2 | Populus trichocarpa | 78.07 | senescence-related gene 1 family protein | 5 × 10−180 |
HBOH62 | 79761729 | JZ896999 | XM_002517028.1 | Ricinus communis | 85.4 | acireductone dioxygenase, putative | 6 × 10−35 |
HBOH63 | 79761730 | JZ897000 | XM_002519376.1 | Ricinus communis | 84.65 | UDP-glucosyltransferase, putative | 0 |
HBOH70 | 79761737 | JZ897007 | XM_002533589.1 | Ricinus communis | 83.4 | leucoanthocyanidin dioxygenase, putative | 0 |
HBOH72 | 79761738 | JZ897008 | XM_002524028.1 | Ricinus communis | 76.42 | short chain alcohol dehydrogenase, putative | 4 × 10−98 |
HBOH76 | 79761742 | JZ897012 | AY207387.1 | Hevea brasiliensis | 98.12 | 1-aminocyclopropane-1-carboxylate oxidase | 5 × 10−98 |
HBOH77 | 79761743 | JZ897013 | XM_012216610.1 | Jatropha curcas | 99 | cytochrome P450 82C4-like (LOC105633910), mRNA | 1 × 10−61 |
HBOH79 | 79761745 | JZ897015 | XM_002311738.2 | Populus trichocarpa | 84.97 | galactinol synthase family protein | 0 |
HBOH83 | 79761749 | JZ897019 | XM_002531145.1 | Ricinus communis | 81.68 | geranyl geranyl pyrophosphate synthase, putative | 2 × 10−146 |
HBOH85 | 79761751 | JZ897021 | XM_002531048.1 | Ricinus communis | 100 | cytochrome P450, putative, mRNA | 1 × 10−81 |
ESTs involved in other metabolism | |||||||
HBOH11 | 79640390 | JZ822688 | XM_002512371.1 | Ricinus communis | 77.2 | histone h2a, putative | 3 × 10−59 |
HBOH12 | 79640391 | JZ822689 | XM_002297827.2 | Populus trichocarpa | 84.94 | Humj1 family protein | 2 × 10−124 |
HBOH18 | 79761690 | JZ896960 | HM363448.1 | Hevea brasiliensis | 94.58 | 60S ribosomal protein L27B (RPL27B) | 3 × 10−143 |
HBOH19 | 79761691 | JZ896961 | XM_007008726.1 | Theobroma cacao | 85.13 | ubiquitin-specific protease 24 isoform 4 | 1 × 10−53 |
HBOH29 | 79761699 | JZ896969 | XM_002519479.1 | Ricinus communis | 71.98 | ribonucleoprotein, chloroplast, putative | 8 × 10−28 |
HBOH30 | 79761700 | JZ896970 | KC533451.1 | Jatropha curcas | 70.7 | SCAR marker SCAR27-740-NT genomic sequence | 3 × 10−16 |
HBOH32 | 79761702 | JZ896972 | XM_003540551.2 | Glycine max | 76.5 | RNA-binding protein 24-like | 3 × 10−59 |
HBOH40 | 79761709 | JZ896979 | XM_002520681.1 | Ricinus communis | 85.64 | photosystem I reaction center subunit II, chloroplast precursor, putative | 0 |
HBOH47 | 79761716 | JZ896986 | AY247789.1 | Hevea brasiliensis | 72.73 | HEV2.1 gene | 1 × 10−31 |
HBOH49 | 79761718 | JZ896988 | XM_002512371.1 | Ricinus communis | 80.46 | histone h2a, putative | 7 × 10−68 |
HBOH57 | 79761726 | JZ896996 | M60274.1HEVRBSS | Hevea brasiliensis | 100 | ribulose-1,5-bisphosphate carboxylase small subunit (RbsS) | 7 × 10−89 |
HBOH64 | 79761731 | JZ897001 | HQ285842.1 | Hevea brasiliensis | 100 | chloroplast, complete genome | 3 × 10−171 |
HBOH84 | 79761750 | JZ897020 | HG975448.1 | Solanum pennellii | 82.22 | chromosome ch09, complete genome | 4 × 10−28 |
Unclear ESTs | |||||||
HBOH17 | 79761689 | JZ896959 | XM_002321005.2 | Populus trichocarpa | 73.09 | hypothetical protein | 2 × 10−24 |
HBOH21 | 79761692 | JZ896962 | XM_002529035.1 | Ricinus communis | 84.38 | conserved hypothetical protein | 3 × 10−107 |
HBOH24 | 79761695 | JZ896965 | XM_002514837.1 | Ricinus communis | 73.53 | conserved hypothetical protein | 6 × 10−90 |
HBOH25 | 79761696 | JZ896966 | CT028466.1 | Poplar | 78.79 | cDNA sequences | 7 × 10−24 |
HBOH35 | 79761704 | JZ896974 | FN552731.1 | Hevea brasiliensis | 90 | microsatellite DNA, clone mHbCIRA2697 | 2 × 10−51 |
HBOH37 | 79761706 | JZ896976 | XM_002519498.1 | Ricinus communis | 82.3 | conserved hypothetical protein | 0 |
HBOH43 | 79761712 | JZ896982 | HQ285842.1 | Hevea brasiliensis | 99.72 | chloroplast, complete genome | 0 |
HBOH50 | 79761719 | JZ896989 | XM_002517090.1 | Ricinus communis | 86.52 | protein FRIGIDA, putative | 3 × 10−168 |
HBOH51 | 79761720 | JZ896990 | EF146205.1 | Populus trichocarpa | 69.32 | clone WS0116_D21 unknown mRNA | 2 × 10−12 |
HBOH65 | 79761732 | JZ897002 | AY439300.1 | Hevea brasiliensis | 83.91 | clone hmct18 microsatellite sequence | 2 × 10−15 |
HBOH69 | 79761736 | JZ897006 | XM_002519826.1 | Ricinus communis | 87.7 | conserved hypothetical protein | 2 × 10−106 |
HBOH82 | 79761748 | JZ897018 | AC213413.1 | Populus trichocarpa | 79.66 | clone POP014-E15, complete sequence | 7 × 10−19 |
2.5. Analysis of Selected EST Gene Expression Profiles in RRIC52 and Reyan 7-33-97 after Powdery Mildew Infection Using qRT-PCR
Clone_ID | Primer Pair Used in DDRT-PCR | Size (bp) | Similar Genes Found in the Database | Functional Relevance | Plant Defense Mechanism Strategies |
---|---|---|---|---|---|
HBOH3 | T11A/B0308 | 759 | endochitinase (Castanea sativa) | Chitinases are enzymes that catalyze the hydrolysis of the beta-1,4-N-acetyl-d-glucosamine linkages in chitin polymers | catalyzes the hydrolysis of chitin polymers on fungal cell wall |
HBOH4 | T11G/B0314 | 685 | ferritin, plant, putative, mRNA (Ricinus communis) | iron ion binding | regulation of cellular iron under oxidative stress |
HBOH5 | T11A/B0314 | 895 | CERK1 mRNA for chitin elicitor receptor kinase 1 (Arabidopsis thaliana) | involved in receptor and signaling pathways, regulating innate immune responses and inflammation; involved in plant resistance to pathogen infection and herbivore attack | catalyzes the hydrolysis of chitin polymers on fungal cell wall |
HBOH7 | T11C/B0304 | 494 | Glucan endo-1,3-β-glucosidase precursor, putative (Ricinus communis) | involved in carbohydrate binding | hydrolyses β-1,3 gucans on fungal cell wall |
HBOH9 | T11G/B0313 | 362 | hypothetical protein mRNA (Medicago truncatula) | a single-pass membrane protein, sphingolipids play important roles in regulating cellular responses, involved in signaling pathways | signal transduction |
HBOH13 | T11C/B0322 | 525 | WRKY transcription factor 40 family protein (Populus trichocarpa) | involved in the regulation of various physiological programs that are unique to plants, including pathogen defense, senescence and trichome development | regulation of immune effector process |
HBOH14 | T11G/B0308 | 251 | WRKY transcription factor 18-like (Gossypium raimondii) | involved in the regulation of various physiological programs that are unique to plants, including pathogen defense, senescence and trichome development | regulation of immune effector process |
HBOH15 | T11A/AP34 | 305 | germin-like protein 1 (Populus trichocarpa) | involved in plant cell wall reinforcement and papillae formation | protected epidermal cells from attack by fungus |
3. Discussion
3.1. Cell Wall Pathway and PR Protein
3.2. Transcription Factor
3.3. Transporter
3.4. Signal Transduction
3.5. Phytoalexin Biosynthesis
3.6. ESTs Involved in Other Metabolic Pathways
4. Materials and Methods
4.1. Rubber Trees and O. heveae
4.2. Inoculation of Rubber Trees with O. heveae HO-73
4.3. RNA Extraction and cDNA Synthesis
4.4. Differential Display Analysis
4.5. Isolation of Differentially Displayed cDNA Bands from Agarose Gel
4.6. Reverse Northern Dot Blot, Cloning, and Sequence Analysis
4.7. Expression Validation by Real-Time Quantitative RT-PCR
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
© 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Li, X.; Bi, Z.; Di, R.; Liang, P.; He, Q.; Liu, W.; Miao, W.; Zheng, F. Identification of Powdery Mildew Responsive Genes in Hevea brasiliensis through mRNA Differential Display. Int. J. Mol. Sci. 2016, 17, 181. https://doi.org/10.3390/ijms17020181
Li X, Bi Z, Di R, Liang P, He Q, Liu W, Miao W, Zheng F. Identification of Powdery Mildew Responsive Genes in Hevea brasiliensis through mRNA Differential Display. International Journal of Molecular Sciences. 2016; 17(2):181. https://doi.org/10.3390/ijms17020181
Chicago/Turabian StyleLi, **ang, Zhenghong Bi, Rong Di, Peng Liang, Qiguang He, Wenbo Liu, Weiguo Miao, and Fucong Zheng. 2016. "Identification of Powdery Mildew Responsive Genes in Hevea brasiliensis through mRNA Differential Display" International Journal of Molecular Sciences 17, no. 2: 181. https://doi.org/10.3390/ijms17020181