Overexpression of Tamarix hispida ThTrx5 Confers Salt Tolerance to Arabidopsis by Activating Stress Response Signals
Abstract
:1. Introduction
2. Results
2.1. Acquisition and Salt Resistance Analysis of ThTrx5-Overexpressing Transgenic Lines
2.2. Screening of Interacting Proteins
Pull Down
2.3. Transcriptome Analysis
2.3.1. Changes in ThTrx5 Gene Expression in Tamarix Hispida under NaCl Stress
2.3.2. Illumina Sequencing
2.3.3. Screening and Analysis of Differentially Expressed Genes
2.3.4. Analysis of GO (Gene Ontology) Enrichment of DEGs
2.3.5. Analysis of Transcription Factor (TF) Families Represented by DEGs Identified during Salt Stress Exposure
2.3.6. Analysis of DEGs Involved in Hormone Synthesis and Signal Transduction Pathways Triggered by Salt Stress Exposure
2.3.7. Analysis of DEGs Involved in Redox-Based Reaction Processes upon Salt Stress Exposure
2.3.8. qRT-PCR Analysis
3. Discussion
3.1. Overexpressing ThTRX5 Improves Salt Resistance of Arabidopsis Plants
3.2. Arabidopsis Proteins Interacting with ThTRX5 Participate with Stress Resistance
3.3. ThTrx5 Participates in the Redox Process, TF Expression, and Hormone Signal Transduction
3.4. ThTrx5 Regulates Biological and Metabolic Pathways via Protein Interactions
4. Materials and Methods
4.1. Materials
4.1.1. Plant Materials and Growth Conditions
4.1.2. Processing Method
4.2. Gene Cloning and Vector Construction
4.2.1. ThTrx5 Gene Cloning and Expression Vector Construction
4.2.2. Construction of Fusion Protein Expression Vector
4.2.3. qPT-PCR
4.2.4. Data Analysis
4.3. Preparation of Engineered Bacteria and Arabidopsis Transformation
4.4. Screening of Interacting Proteins
4.4.1. Induction of Protein Expression
4.4.2. Pull-Down
4.4.3. Mass Spectrometry
4.5. Transcriptome Analysis
4.5.1. RNA Extraction and Differential Expression Library Construction
4.5.2. De Novo Assembly and Functional Annotation
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
BLAST | the Basic Local Alignment Search Tool |
DEGs | differentially expressed genes |
GO | Gene ontology |
KEGG | Kyoto Encyclopedia of Genes and Genomes |
SwissProt | Swiss-prot sequence data bases |
ORFs | Open reading frames |
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Sample ID | WT-0h | WT-3h | OE-0h | OE-3h |
---|---|---|---|---|
Total Reads | 7,639,214(100.00%) | 7,499,090(100.00%) | 7,165,453(100.00%) | 7,318,039(100.00%) |
Total BasePairs | 366,468,011(100.00%) | 357,615,524(100.00%) | 346,511,389(100.00%) | 358,182,356(100.00%) |
Total Mapped Reads | 4,547,338(60.80%) | 5,339,751(73.16%) | 4,739,829(67.03%) | 5,396,632(73.83%) |
Perfect Match | 4,063,215(54.33%) | 4,756,366(65.17%) | 4,241,363(59.98%) | 4,835,471(66.15%) |
<=2bp Mismatch | 484,123(6.47%) | 583,385(7.99%) | 498,466(7.05%) | 561,161(7.68%) |
Unique Match | 4,291,451(57.38%) | 5,119,890(70.15%) | 4,490,200(63.50%) | 5,162,943(70.63%) |
Multi-position Match | 255,887(3.42%) | 219,861(3.01%) | 249,629(3.53%) | 233,689(3.20%) |
Total Unmapped Reads | 2,931,601(39.20%) | 1,958,525(26.84%) | 2,331,832(32.97%) | 1,913,212(26.17%) |
Clean Reads | 7,478,939 | 7,298,276 | 7,071,661 | 7,309,844 |
Hormone | Gene | Log2 (WT 0 h/Trx 0 h) | Log2 (WT 3 h/Trx 3 h) | Gene Annotation |
---|---|---|---|---|
ABA | AT1G52040.1 | −1.466825674 | −1.650566816 | myrosinase-binding protein 1 |
BR | AT4G34650.1 | 0.06962026 | −1.262992532 | squalene synthase 2 |
ETH | AT5G47220.1 | −0.468665009 | 1.963721496 | ethylene-responsive transcription factor 2 |
AT2G20880.1 | 0.519653186 | −1.327806826 | ethylene-responsive transcription factor ERF053 | |
AT1G69260.1 | −0.387237409 | −1.327806826 | ABI five binding protein | |
JA | AT3G55970.1 | −0.457626737 | −2.592176442 | jasmonate-regulated protein JRG21 |
AT3G50760.1 | 0.502974445 | −1.129387909 | Probable galacturonosyltransferase-like 2 | |
AT5G24780.1 | −2.57480625 | −2.023514997 | acid phosphatase VSP1 | |
AT5G24770.1 | −0.608451639 | −1.443297712 | acid phosphatase VSP2 | |
AT2G39030.1 | −3.23523431 | −2.243926852 | L-ornithine N5-acetyltransferase NATA1 | |
6-BA | AT1G21310.1 | −0.4968619 | 1.346863463 | response to cytokinin stimulus |
SA | AT3G20470.1 | 0.527147723 | 1.015449778 | glycine-rich protein 5 |
Primer Name | Forward Primer(5′→3′) | Reverse Primer(5′→3′) |
---|---|---|
α-Tubulin | 5′-GCACTGGCCTCCAAGGAT-3′ | 5′-TGGGTCGCTCAATGTCAAGG-3′ |
ThTrx5 | 5′-AATCTATGATTGCTCCTTCC-3′ | 5′-GCCAGCTCATTAACAACC-3′ |
AT4G25100.2 | 5′-GATGCTTTGGAGCCGCATATG-3′ | 5′-GAAGAACTCGTGGTTCCACG-3′ |
AT4G34400.1 | 5′-CAGCTCAGAGTTCATGGTGATC-3′ | 5′-GTGAGCTCCATTATAGGCAAAGG-3′ |
AT1G44970.1 | 5′-CGTGAGTGGATTCCCTAAACAATC-3′ | 5′-CACTCCAAACCCAGAGATTGG-3′ |
AT4G37990.1 | 5′-CGAAAGACAATTCCGGAGTTCTC-3′ | 5′-CAGTCACCACGCCCACGATC-3′ |
AT4G36190.1 | 5′-GGTTACTGCAACCTCGTAGGATTTC-3′ | 5′-GGTGATTCCATTGCAAGGTCC-3′ |
AT4G37410 | 5′-CCTCATCAAACCGCCGGTTC-3′ | 5′-AGCAATGTATTTGGAGGTTAGAAAACG-3′ |
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Luan, J.; Dong, J.; Song, X.; Jiang, J.; Li, H. Overexpression of Tamarix hispida ThTrx5 Confers Salt Tolerance to Arabidopsis by Activating Stress Response Signals. Int. J. Mol. Sci. 2020, 21, 1165. https://doi.org/10.3390/ijms21031165
Luan J, Dong J, Song X, Jiang J, Li H. Overexpression of Tamarix hispida ThTrx5 Confers Salt Tolerance to Arabidopsis by Activating Stress Response Signals. International Journal of Molecular Sciences. 2020; 21(3):1165. https://doi.org/10.3390/ijms21031165
Chicago/Turabian StyleLuan, Jiayu, Jingxiang Dong, Xin Song, Jing Jiang, and Huiyu Li. 2020. "Overexpression of Tamarix hispida ThTrx5 Confers Salt Tolerance to Arabidopsis by Activating Stress Response Signals" International Journal of Molecular Sciences 21, no. 3: 1165. https://doi.org/10.3390/ijms21031165