Background
Results and Discussion
Use | Primer | Sequence |
---|---|---|
Anchored Primer | ||
DD10 | 5'-TTTTTTTTTTTG-3' | |
cDNA | DD11 | 5'-TTTTTTTTTTTC-3' |
DD12 | 5'-TTTTTTTTTTTA-3' | |
Arbitrary Primer | ||
B-01 | 5'-GTTTCGCTCC-3' | |
B-02 | 5'-TGATCCCTGG-3' | |
B-03 | 5'-CATCCCCCTG-3' | |
B-04 | 5'-GGACTGGAGT-3' | |
B-05 | 5'-TGCGCCCTTC-3' | |
B-06 | 5'-TGCTCTGCCC-3' | |
B-07 | 5'-GGTGACGCAG-3' | |
B-08 | 5'-GTCCACACGG-3' | |
B-09 | 5'-TGGGGGACTC-3' | |
DDRT-PCR | B-10 | 5'-CTGCTGGGAC-3' |
B-11 | 5'-GTAGACCCGT-3' | |
B-12 | 5'-CCTTGACGCA-3' | |
B-13 | 5'-TTCCCCCGCT-3' | |
B-14 | 5'-TCCGCTCTGG-3' | |
B-15 | 5'-GGAGGGTGTT-3' | |
B-16 | 5'-TTTGCCCGGA-3' | |
B-17 | 5'-AGGGAACGAG-3' | |
B-18 | 5'-CCACAGCAGT-3' | |
B-19 | 5'-ACCCCCGAAG-3' | |
B-20 | 5'-GGACCCTTAC-3' |
Analysis of DEGs identified at two days post infiltration
DEG | Length bps | Identity | Up/Down regulation |
---|---|---|---|
SAA
| (287) | S. lycopersicum WRKY transcription factor IId-1 splice | Upregulated |
SAB
| (231) | Putative Rieske iron-sulfur protein [A. thaliana]Length = 539, Rieske iron-sulfur protein Tic55 [P. sativum]Length = 553 | Upregulated |
SAC
| (386) | N. tabacum mitochondrial DNA, complete genome Length = 430597 NADH dehydrogenase subunit 1 NADH dehydrogenase subunit 2, 846 bp at 5' side: | Down regulated |
SAD
| (262) | Quinonprotein alcohol dehydrogenase like M. truncatula | Upregulated |
SA1
| (442) | Trigger factor (chaperone in protein export) | Upregulated |
SA2
| (688) | A. thaliana calmodulin-binding receptor-like kinase | Upregulated |
SA3
| (772) | Polyribonucleotide nucleotidyltransferase | Upregulated |
SA4
| (283) | Chromosomal replication initiator protein DnaA | Down regulated |
Genes | Identity | Relative quantification against Unit mass | Relative quantification Normalized to a reference gene | |||
---|---|---|---|---|---|---|
Control | Healthy | Livak method | ΔCT Method | Pfaffi Method | ||
SAA
| S. lycopersicum WRKY transcription factor IId-1 splice | 2.32 | 1.32 | 0.737C/0.381H | 0.942C/0.838H | 0.737C/0.381H |
SAB
| Putative Rieske iron-sulfur protein [A. thaliana]Length = 539, Rieske iron-sulfur protein Tic55 [P. sativum]Length = 553 | 2.751 | 0.566 | 0.870C/0.162H | 0.972C/0.728H | 0.870C/0.162H |
SAC
| N. tabacum mitochondrial DNA, complete genome Length = 430597 NADH dehydrogenase subunit 1 NADH dehydrogenase subunit 2, 846 bp at 5' side | 0.010 | 1.905 | 0.003C/0.547H | -0.672C/1.355H | 0.003C/0.547H |
SAD
| Quinonprotein alcohol dehydrogenase like [M. truncatula] | 5.205 | 0.829 | 1.647C/0.238H | 1.167C/0.707H | 1.647C/0.238H |
Gene | Identity | Relative quantification against Unit mass | Relative quantification normalized to a reference gene | |||
---|---|---|---|---|---|---|
Control | Healthy | Livak method | ΔCT Method | Pfaffi Method | ||
SA1
| Trigger factor (chaperone in protein export) | 2.88 | 17.75 | 1.443C/7.727H | 1.08C/1.74H | 1.443C/7.727H |
SA2
| A. thaliana calmodulin-binding receptor-like kinase | 3.759 | 3.759 | 8.564 | - | 8.564 |
SA3
| Polyribonucleotide nucleotidyltransferase | 1.32 | 1.70 | 0.664C/0.742H | 0.790C/.837H | 0.664C/0.742H |
SA4
| Chromosomal replication initiator protein DnaA | 0.659 | 0.882 | 0.329C/0.253H | 0.674C/0.706H | 0.329C/0.253H |
Analysis of DEGs identified at four days post infiltration
DEG | Length of amplified fragment | Identity | Accession No. | Identity |
---|---|---|---|---|
Genes differentially expressed at two days after inoculation | ||||
SAA
| 287 | S. lycopersicum WRKY transcription factor IId-1 splice | AY157059 | (76%) |
SAB
| 231 | Putative Rieske iron-sulfur protein [A. thaliana]Length = 539, Rieske iron-sulfur protein Tic55 [P. sativum] Length = 553 | NM128041 AJ000520 | (68%)/(73%) |
SAC
| 386 | N. tabacum mitochondrial DNA, complete genome Length = 430597: NADH dehydrogenase subunit 1 and NADH dehydrogenase subunit 2, 846 bp at 5' side | BA000042 | (93%) |
SAD
| 262 | Quinonprotein alcohol dehydrogenase like [M. truncatula] | ABE84009 ABE86610 | (47%) |
Genes differentially expressed at four days after inoculation
| ||||
SA1
| 442 | Trigger factor (Chaperone protein) | AE017283 | (99%) |
SA2
| 688 | A. thaliana calmodulin-binding receptor-like kinase 2 (CRCK2) | NM116255 | (72%) |
SA3
| 772 | Polyribonucleotide nucleotidyltransferase | CP000323 | (92%) |
SA4
| 263 | Chromosomal replication initiator protein DnaA | CP000653 | (90%) |
Analysis of DEGs using KOBAS
Sequence identifier | KO term | KO definition | Rank | E-value | Score | Identity (%) | Blast hit |
---|---|---|---|---|---|---|---|
SA3 | K00962 | Polyribonucleotide nucleotidyltransferase | 1 | 1e-111 | 404.0 | 96.64 | Pcr. Pcryo 0080 |
Query gene | Pathway | Count and ratio | p-value | q-value | Web site |
---|---|---|---|---|---|
SA3 | Pyrimidine metabolism |
1/100% 44/1.53%
| 0.0153417015342 | 0.023709902371 | |
SA3 | Purine metabolism |
1/100% 68/2.37%
| 0.023709902371 | 0.023709902371 |
Conclusions
Methods
Cloning of βC1 of Chilli leaf curl betasatellite in pJIT163
Transfer of expression cassette to binary vector and transformation of Agrobacterium tumefaciens
Agroinfiltration of plants
Isolation of messenger RNA and cDNA synthesis
Differential display analysis
Cloning and sequencing of differentially expressed genes (DEGs)
Analysis of DEGs using NCBI, KOBAS and RT-PCR
Name of genes | Sequences of primers | MERS |
---|---|---|
Primer sequences two days post inoculation of βC1 of ChLCB for Q-RTPCR analysis | ||
SAA
| SAA F: GAGACCCGGGATGTCCTGGCAAGAAAGCAT |
(30 MERS)
|
SAAQPCR:AATTACAAAAGAGCCCCTAAATCCCTAAGC |
(30 MERS)
| |
SAA F2: GGAGAGGGCAACCGATGA |
(18 MERS)
| |
SAA QPCR2: CCCCTAAATCCCTAAGC |
(17 MERS)
| |
SAA F3: GGGACGATCGCCGGCGCCGG |
(20 MERS)
| |
SAA QPCR3: TCACTACCCACCGTATC |
(17 MERS)
| |
SAB
| SAB F: AATCCCCGGGATGTATGCTCCGAATCCCGC |
(30 MERS)
|
SABQPCR:CATAGTGATGTCGAAAGCAAAAGTAGGGCC |
(30 MERS)
| |
SAB F2: GTATGCTCCGAATCCCG |
(17 MERS)
| |
SAB QPCR2: CAAAAGTAGGGCCTTCC |
(17 MERS)
| |
SAB F3: CCAGCTAAGGGAGGAATC |
(18 MERS)
| |
SAB QPCR3: GGCCTTCCACTGTCTTCCTG |
(20 MERS)
| |
SAC
| SAC F: TCCCCCCGGGATGTTTCAGGTTCACATGAA |
(30 MERS)
|
SACQPCR:TAGGCTATAGGTGGGGGACAATGTAGACTG |
(30 MERS)
| |
SAC F2: CACAACACGACTCCCTAC |
(18 MERS)
| |
SAC QPCR2: GAAGTTGGGCCCACCTG |
(17 MERS)
| |
SAC F3: CTCCACGAGTCTTCATCCCC |
(20 MERS)
| |
SAC QPCR3: CCGAGATCGAGAGCTTTC |
(18 MERS)
| |
SAD
| SA D F; GGTGCCCGGGATGGCAGATCAGTGGAGTTG |
(30 MERS)
|
SADQPCR: GATTAGGTTCCCGTAGATAGATGCATAACC |
(30 MERS)
| |
SAD F2: AAGTTCTAATTCGGAGGG |
(18 MERS)
| |
SAD QPCR2: TAGATAGATGCATAA |
(17 MERS)
| |
SAD F3: GTTAGCTTACTTAAACAG |
(20 MERS)
| |
SAD QPCR3: TAGATGCATAACC |
(17 MERS)
|
Name of genes | Sequences of primers | MERS |
---|---|---|
Primer sequences four days post inoculation of βC1 of ChLCB for Q-RTPCR analysis | ||
SA1
| SA1 F: GTCACCCGGGATGTGACGCCGACGGTCAAT |
(30 MERS)
|
SA1 QPCR: GGGCCGCACCATGGTCCTGCTGACTTACCG |
(30 MERS)
| |
SA1 F2: GACGGTCAATCCATGTAT |
(18 MERS)
| |
SA1 QPCR2: GGTGTCAGGAGACCCCTTCCA |
(17 MERS)
| |
SA1 F3: GGTAGAGCCCCAGTCTTCCA |
(20 MERS)
| |
SA1 QPCR3: GCACCCGCCCAACTCCACGG |
(17 MERS)
| |
SA2
| SA2 F: AATACCCGGGATGATAAACATTTGGGGG |
(30 MERS)
|
SA2 QPCR: CCAATGTCTAGTCTTGATGCAAAATCAA |
(30 MERS)
| |
SA2 F2: CTAGTAAAGTTTTATGGATTCTTGGA |
(17 MERS)
| |
SA2 QPCR2: ATGGATAATAGGGTGATCAGT |
(17 MERS)
| |
SA2 F3: CACTTGGACTGTGGTCCTG |
(18 MERS)
| |
SA2 QPCR3: GTCAGCCACCTTAGCTCG |
(20 MERS)
| |
SA3
| SA3 F: CGCGCCCGGGATGCATCTAGATTGTCCACA |
(30 MERS)
|
SA3 QPCR: TCAATCAGACGCGAGGTTAAGGTTTCAGAC |
(30 MERS)
| |
SA3 F2: GAAGGCTATGTAAACGAG |
(18 MERS)
| |
SA3 QPCR2: GCTCTTCAAGGGTCGGGTTCAG |
(17 MERS)
| |
SA3 F3: GACTTGGTCGTCGCTGGTA |
(20 MERS)
| |
SA3 QPCR3: GCTTGATCGCGTACAGG |
(18 MERS)
| |
SA4
| SA4 F: GCGACCCGGGAtGCATCTAGATTTGGGGGA |
(30 MERS)
|
SA4 QPCR: AGAAACAGAAGATCTCTGGCTCAGTTTAGG |
(30 MERS)
| |
SA4 F2: TTCATGATTGTTGGCGCAC |
(18 MERS)
| |
SA4 QPCR2: CTGATCTTCCTGTGGA |
(17 MERS)
| |
SA4 F3: CGGCATGACCCTGTGTAA |
(20 MERS)
| |
SA4 QPCR: GGGGGACTCGCGCCAGG |
(17 MERS)
|