Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI) in Potyviruses Resistance
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cloning CmSPI Gene cDNA and Full Length Genomic Fragment of Cucumis metuliferus
Primer Name | Oligonucleotides |
---|---|
5′ and 3′ RACE reaction | |
HMK2007-14 | 5′-AAT TCC AAC ACA AAT CAT CAT CTT-3′ |
HMK2007-15 | 5′-TAA CAA ACA ACC AAA CTC GAT CAC-3′ |
Full length genome walking | |
HMK2010-35 | 5′-CTA ATA CgA CTC ACT ATA ggg CTC gAg Cgg CCg CCC ggg CAg gT-3′ |
HMK2010-36 | 5′-ACC TgC CC-3′ |
HMK2010-37 | 5′-ggA TCC TAA TAC gAC TCA CTA TAg ggC-3′ |
HMK2010-38 | 5′-ACT CAC TAT Agg gCT CgA gCg ggC-3′ |
HMK2011-118 | 5′-TAT TCC AAC AAG TTC CGG CCA CTG-3′ |
HMK2011-119 | 5′-GAA AGT TGT TGA AAT TCC GAA GGT TG-3′ |
HMK2011-127 | 5′-gct gga act gga acc act aaa ga-3′ |
HMK2011-128 | 5′-cca taa atg gaa aca tac cag gag-3′ |
CmSPI RNAi construction | |
HMK2007-93 | 5′-ggA TCC CCA Tgg CCC ggg CgA ATT CCA AgC TT-3′ |
HMK2007-94 | 5′-TgT ACA CTC gAg TAg Agg ggA TCC AgA TCT-3′ |
Full length genomic CmSPI construction | |
HMK2012-6 | 5′-CAA ATA GGA GAA GAT GTT CTC G-3′ |
HMK2012-23 | 5′-CAG GTG ACA CAT GCG TAT AAC-3′ |
Genomic PCR and probe synthesis | |
HMK2011-102 | 5′-gaa ctt tct gga tct act tta ttt g-3′ |
HMK2011-103 | 5′-aca caa act tca tct aac ctt aaa c-3′ |
RT-PCR and probe synthesis | |
HMK2013-60 | 5′-acg cGA ATT CGT CGA CCT CGA GAA CCT TCG GAA TTT CAA CAA C-3′ |
HMK2013-61 | 5′-acg cGG ATC CGA GCT CAA GCT TAT GGC TGA TAT TTG TCC TCC T-3′ |
2.2. Sequence Analysis and Construction of Phylogenetic Tree
2.3. Construction of Binary Vector and Bacterial Strain
2.4. Plant Material and Transformation
2.5. Inoculation of Plant Viruses
2.6. Gene Expression Detection
2.6.1. Genomic PCR
2.6.2. Southern Hybridization
2.6.3. RT-PCR (Reverse Transcription PCR)
2.6.4. Northern Hybridization
2.6.5. Western Hybridization
2.6.6. Detection of Small Interfering RNA
3. Results
3.1. Cloning and Functional Characterization of C. metuliferus CmSPI Gene
3.2. Generation of CmSPI RNAi Transgenic Plant
3.3. Alteration of Virus Resistance in CmSPI RNAi Transgenic C. metuliferus
3.4. Resistance to Virus in Transgenic N. benthamiana
Transgenic Lines | Symptomatic Plants/Total of Inoculated Plants |
---|---|
T1F-3 | 28/28 |
T1F-5 | 25/25 |
T1F-14 | 21/22 |
T1F-21 | 18/30 |
T1F-51 | 17/30 |
T1F-52 | 27/30 |
T1F-53 | 29/29 |
T1F-54 | 30/30 |
T1F-55 | 27/28 |
Non-transformed control | 27/27 |
Transgenic Lines | Symptomatic Plants/Total of Inoculated Plants |
---|---|
T2F-21-2 | 33/44 |
T2F-51-10 | 2/44 |
Non-transformed control | 15/15 |
4. Discussion
4.1. CmSPI Is Involved in Plant Physiological Responses and Plant Defense
4.2. Silencing of CmSPI Alters PRSV Resistance in C. metuliferus
4.3. Expression of CmSPI in N. benthamiana Plants Can Supply Resistance for PVY
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Lin, C.-W.; Su, M.-H.; Lin, Y.-T.; Chung, C.-H.; Ku, H.-M. Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI) in Potyviruses Resistance. Viruses 2015, 7, 3816-3834. https://doi.org/10.3390/v7072799
Lin C-W, Su M-H, Lin Y-T, Chung C-H, Ku H-M. Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI) in Potyviruses Resistance. Viruses. 2015; 7(7):3816-3834. https://doi.org/10.3390/v7072799
Chicago/Turabian StyleLin, Chia-Wei, Mei-Hsiu Su, Yu-Tsung Lin, Chien-Hung Chung, and Hsin-Mei Ku. 2015. "Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI) in Potyviruses Resistance" Viruses 7, no. 7: 3816-3834. https://doi.org/10.3390/v7072799