Chitin Biosynthesis Inhibition of Meloidogyne incognita by RNAi-Mediated Gene Silencing Increases Resistance to Transgenic Tobacco Plants
Abstract
:1. Introduction
2. Results
2.1. In Silico Mining and Cloning of Chitin Biosynthesis Pathway Genes
2.2. Plant Transformation Vector Construction and Tobacco Transformation
2.3. Chitin Genes Expression Analysis by qRT-PCR
2.4. Efficiency of Regeneration and GUS Staining of Transgenic Plants
2.5. RKN Inoculation of Transgenic Lines and Suppression Analysis
2.6. Downregulation of Chitin Biosynthetic Genes of the Female Nematode in RNAi Transgenic Roots
2.7. Effect of RNAi-Mediated Gene Silencing on Egg Number and Female Morphology in Transgenic Lines
3. Discussion
4. Materials and Methods
4.1. Isolation and Cloning of Chitin Synthesis Genes
4.2. RNAi Vector Construction
4.3. Stage-Wise Nematode Sample Preparation
4.4. RNA Isolation from Nematode
4.5. cDNA Synthesis
4.6. Quantitative Real-Time-PCR (qRT-PCR) Analysis
4.7. Plant Transformation and PCR Analysis
4.8. RKN Inoculation and Suppression Analysis
4.9. Statistical Analyses
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
RKN | Root knot nematode |
J2 | Juvenile 2 |
RNAi | RNA interference |
qRT-PCR | Quantitative real-time polymerase chain reaction |
UBQ | Ubiquitin |
GPI | Glucose-6-phosphate isomerase |
dsRNA | Double-stranded RNA |
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Name | Sequence (5′ to 3′) |
---|---|
CS-F1-F | ATGGTTAAAGGCCCCTCAACTG |
CS-F1-R | TTATAAAAAAACCTGTGACCACC |
CS-F2-F | ATGGTTAAAGGCCCCTCAACTG |
CS-F2-R | CTCATAAAGTTCTTGAAAAAGACC |
CS-F3-F | AAATATGGCATGAGAAAGCTCAATC |
CS-F3-R | ATTCGAAGAGGGCTTTCCTCAG |
GPI-F1-F | ATGACTTCAACAATTACTGGTCTA |
GPI-F1-R | TCAATCCTTGTAATTTTTAATTAAATTA |
GPI-F2-F | ATGCCTGATGTTAATGCTGTTC |
GPI-F2-R | TCAATCTTTGTAATTTTTAATTAAATT |
GPI-F3-F | ATGCCTGATGTTAATGCTGTTC |
GPI-F3-R | CGTATGGTGAAGACC TCCAC |
TH1-F1-F | ATGCTTTATTATGTTGTTTCTTTGC |
TH1-F1-R | TTAAAATACATTATTTAAATAAATTCTTT |
TH1-F2-F | ATGCTTTATTATGTTGTTTCATTGC |
TH1-F2-R | TCAATTCATATGCACCATTGGAG |
TH1-F3-F | TAATTGAAGGCTTCCGTACCAG |
TH1-F3-R | TTAAAATACATTATTTAAATAAATTCTTT |
Name | Sequence (5′ to 3′) |
---|---|
Phos-gusf1 | ATGTTACGTCCTGTAGAAACCCC |
Phos-gusr1 | TCATTGTTTGCCTCCCTGCTGC |
RNAi UBQ1 F9 | cggaattcAGGTGCCAAATCTTTGATTGGAGTTG |
RNAi UBQ1 R12 | aagcttCTTTTGTGTTTCGTCTTCTCTCACG |
RNAi UBQ1 R13 | ggtaccctcgagaagcttCTTTTGTGTTTCGTCTTCTC |
RNAi UBQ1 R14 | cggaattcggtaccctcgagaagcttCTTTTGTG |
MCSF8 | CCGAGCTCGCCCAAGCTTACGCGTGGATCCCTGCAG |
MCSR8 | CTGCAGGGATCCACGCGTAAGCTTGGGCGAGCTCGG |
Name | Sequence (5′ to 3′) |
---|---|
pK7-F | TTTGCGGACTCTAGCATGGCCGCG |
Int-R1 | CTTGAAAGTCAAATTGTCGAATTTG |
Int-R2 | GATCGGTGTGATACAAAACCTAATC |
UBQ-F | CCATCTTAGACTTAGCTAAGTTT |
Name | Sequence (5′ to 3′) |
---|---|
Mi-actin F | TTATTCTTTCACCGCAACCG |
Mi-actin R | TTGACCGTCAGGCAATTCAT |
CS-F | CACTTGTGCCTTTCACTGTTTC |
CS-R | TGATGGTAGACTTGCGGTAATG |
GPI-F | TGGCCAATGGACTGGTTATAC |
GPI-R | TTGAGTGCTTCAGTGACCATTA |
TH1-F | CAGAAGGGTAAAGGACGATGTT |
TH1-R | AACGACCACCAGGAATGATAAA |
CS-RNAi-F | CGTATTTGGAGACCAAGCAAAG |
CS-RNAi-R | ACACTGGATGGATACACGTAAA |
GPI-RNAi-F | TACTCCAAATACATTGGGCTCTT |
GPI-RNAi-R | GCTAATTGTTTGCCTAATTCAACAC |
TH1-RNAi-F | CCCTGGACATGAACTACAAGAA |
TH1-RNAi-R | CCCAACGCCGAAGTTGATA |
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Mani, V.; Reddy, C.S.; Lee, S.-K.; Park, S.; Ko, H.-R.; Kim, D.-G.; Hahn, B.-S. Chitin Biosynthesis Inhibition of Meloidogyne incognita by RNAi-Mediated Gene Silencing Increases Resistance to Transgenic Tobacco Plants. Int. J. Mol. Sci. 2020, 21, 6626. https://doi.org/10.3390/ijms21186626
Mani V, Reddy CS, Lee S-K, Park S, Ko H-R, Kim D-G, Hahn B-S. Chitin Biosynthesis Inhibition of Meloidogyne incognita by RNAi-Mediated Gene Silencing Increases Resistance to Transgenic Tobacco Plants. International Journal of Molecular Sciences. 2020; 21(18):6626. https://doi.org/10.3390/ijms21186626
Chicago/Turabian StyleMani, Vimalraj, Chinreddy Subramanyam Reddy, Seon-Kyeong Lee, Soyoung Park, Hyoung-Rai Ko, Dong-Gwan Kim, and Bum-Soo Hahn. 2020. "Chitin Biosynthesis Inhibition of Meloidogyne incognita by RNAi-Mediated Gene Silencing Increases Resistance to Transgenic Tobacco Plants" International Journal of Molecular Sciences 21, no. 18: 6626. https://doi.org/10.3390/ijms21186626