Identification of Exogenous Nitric Oxide-Responsive miRNAs from Alfalfa (Medicago sativa L.) under Drought Stress by High-Throughput Sequencing
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Materials and Growth Conditions
2.2. Small RNA Library Construction and Sequencing
2.3. Bioinformatic Identification of Conserved and Novel miRNAs
2.4. Differential Expression Analysis of Known and Novel miRNAs
2.5. Prediction of miRNA Targets, Gene Ontology (GO), and KEGG Pathway Analysis
2.6. Validation of miRNA and Target Gene Expression with qRT-PCR
3. Results
3.1. Deep Sequencing Results of Alfalfa Small RNA (sRNA) Libraries
3.2. Conserved miRNAs from Alfalfa
3.3. Check of Unknown miRNAs in Medicago sativa L. “Sandeli”
3.4. The Response of miRNA to Drought Stress and Exogenous Nitric Oxide under Drought Stress
3.5. Prediction and Functional Classification of Target Genes of Drought Stress Response Type and Exogenous Nitric Oxide Reactive miRNA under Drought Stress
3.6. Validation of the Expression of miRNAs and Their Targets
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Category | CK | PEG | PEG + SNP | |||
---|---|---|---|---|---|---|
Count | Percentage | Count | Percentage | Count | Percentage | |
Raw reads | 26,849,326 | 100.00% | 24,252,135 | 100.00% | 20,514,941 | 100.00% |
Clean reads | 23,528,741 | 87.63% | 18,046,004 | 74.41% | 15,076,692 | 73.49% |
Mapped to genome | 10,366,134 | 38.61% | 7,651,367 | 31.55% | 6,487,853 | 31.63% |
miRNAs | 1,267,908 | 4.72% | 1,180,088 | 4.87% | 871,935 | 4.25% |
rRNA | 2,931,163 | 10.92% | 3,527,200 | 14.54% | 1,974,535 | 9.62% |
snoRNA | 3746 | 0.01% | 7001 | 0.03% | 10,497 | 0.05% |
tRNA | 234,634 | 0.87% | 333,859 | 1.38% | 417,378 | 2.03% |
Without annotation | 20,273,991 | 75.51% | 14,113,193 | 58.19% | 12,602,624 | 61.43% |
Name | Sequence | Score_Total | Expressional Reads | G + C Contents | Hairpin Energy | Length |
---|---|---|---|---|---|---|
c104339.graph_c0_3174 | UUUCAUUCCAUAUGUCAUCUAG | 17,723.5 | 14,392 | 31.82% | −53.6 | 22 |
c111080.graph_c1_10087 | GGCGGAUGUAGCCAAGUGGA | 1,721,964.4 | 730,326 | 60.00% | −53.2 | 20 |
c114875.graph_c0_14536 | UUCAUUUCUAAAAUAGGCAUUG | 9543.7 | 7780 | 27.27% | −59.2 | 22 |
c116131.graph_c0_16318 | CGGGAUCGGAGAUUAGAGAAU | 146,625.1 | 106,408 | 47.62% | −73.0 | 21 |
c86020.graph_c0_42677 | UACGUCUCUGUCUUUCGGGUUG | 16,890.1 | 13,750 | 50.00% | −74.6 | 22 |
c99918.graph_c0_48282 | UUAAUCAAGGAAAUCACAGUC | 13,503.9 | 13,046 | 33.33% | −55.0 | 21 |
c102504.graph_c0_1594 | AUGGUUCUUGUUCAGUAGAGU | 30,812.8 | 6815 | 38.10% | −78.4 | 21 |
c91116.graph_c0_44142 | UUGUGGAACAUAGAAGCACGUG | 9771.1 | 2317 | 45.45% | −63.6 | 22 |
c121624.graph_c0_25405 | UUGAUUCUCAUCACAACUUGG | 14,278.7 | 7926 | 38.10% | −66.2 | 21 |
c111105.graph_c0_10123 | UUUGGCAUUCUGUCCACCUCC | 8328.7 | 7532 | 52.38% | −55.6 | 21 |
miRNAs | Sequences | Target Gene or Protein | Description of Function | Reference |
---|---|---|---|---|
mtr-miR156a | tgacagaagagagagagcaca | SQUAMOSA promoter-binding-like protein (SPL) genes. | Anthocyanin biosynthesis; vegetative phase transition. | [30,36,37] |
mtr-miR399a | tgccaaaggagatttgcccag | Phosphate transporter; high affinity inorganic phosphate transporter. | Pi uptake. | [38,39] |
mtr-miR399c | tgccaaaggagatttg | |||
mtr-miR399q | ccctgtgccaaaggagagctgctctt | |||
mtr-miR5213-5p | tacgtgtgtcttcacctctgaa | Disease resistance protein. | Response to salt/alkali stress. | [40,41] |
mtr-miR5752a | cattgtttggtttagtacaaa | Starch synthase; amino acid binding; metal ion binding. | Starch, galactolipid, amylopectin biosynthetic; response to hypoxia; regulation of ethylene-activated; metabolic process; metal ion transport. | [42] |
mtr-miR7696a-5p | tcaagttctcataattcaaaa | Chitin binding; protein kinase. | Innate immune response; protein phosphorylation; cell wall macromolecule catabolic. | [43] |
mtr-miR398a-5p | ggagtgacactgagaacacaag | Cu/Zn-superoxide dismutase copper chaperone. | Defense against reactive oxygen toxicity. | [44] |
mtr-miR5232 | tacatgtcgctctcacctgaa | Glycoside hydrolase; type IIB calcium ATPase protein kinase. | Participate in metabolism. | [41] |
mtr-miR5559-5p | tacttggtgaattgttggatc | inorganic diphosphatase magnesium ion binding. | Response to cadmium ion; phosphate-containing compound metabolic. | [29] |
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Zhao, Y.; Ma, W.; Wei, X.; Long, Y.; Zhao, Y.; Su, M.; Luo, Q. Identification of Exogenous Nitric Oxide-Responsive miRNAs from Alfalfa (Medicago sativa L.) under Drought Stress by High-Throughput Sequencing. Genes 2020, 11, 30. https://doi.org/10.3390/genes11010030
Zhao Y, Ma W, Wei X, Long Y, Zhao Y, Su M, Luo Q. Identification of Exogenous Nitric Oxide-Responsive miRNAs from Alfalfa (Medicago sativa L.) under Drought Stress by High-Throughput Sequencing. Genes. 2020; 11(1):30. https://doi.org/10.3390/genes11010030
Chicago/Turabian StyleZhao, Yaodong, Wenjing Ma, Xiaohong Wei, Yu Long, Ying Zhao, Meifei Su, and Qiaojuan Luo. 2020. "Identification of Exogenous Nitric Oxide-Responsive miRNAs from Alfalfa (Medicago sativa L.) under Drought Stress by High-Throughput Sequencing" Genes 11, no. 1: 30. https://doi.org/10.3390/genes11010030
APA StyleZhao, Y., Ma, W., Wei, X., Long, Y., Zhao, Y., Su, M., & Luo, Q. (2020). Identification of Exogenous Nitric Oxide-Responsive miRNAs from Alfalfa (Medicago sativa L.) under Drought Stress by High-Throughput Sequencing. Genes, 11(1), 30. https://doi.org/10.3390/genes11010030