Optimizing virus-induced gene silencing efficiency with Cymbidium mosaic virus in Phalaenopsis flower.
Virus-induced gene silencing (VIGS) is a good way to study floral gene functions of orchids, especially those with a long life cycle. To explore the applicability and improve viral silencing efficiency for application of Cymbidium mosaic virus (CymMV)-induced gene silencing, we examined several variables, including the optimal length of the DNA fragment, the effect of developmental maturation status of inflorescence, and suitable inoculation sites. A CymMV-based VIGS system can be used with orchids to silence genes including PeUFGT3, PeMADS5 and PeMADS6 and induce prominent phenotypes with silencing efficiency up to 95.8% reduction. The DNA fragment size used for silencing can be as small as 78-85 bp and still reach 61.5-95.8% reduction. The effect of cDNA location as a target in VIGS varies among genes because of non-target gene influence when using the 5' terminus of the coding region of both PeMADS5 and PeMADS6. Use of VIGS to knock down a B-class MADS-box gene (PeMADS6) in orchids with different maturation status of inflorescence allowed for observing discernable knockdown phenotypes in flowers. Furthermore, silencing effects with Agro-infiltration did not differ with both leaf and inflorescence injections, but injection in the leaf saved time and produced less damage to plants. We propose an optimized approach for VIGS using CymMV as a silencing vector for floral functional genomics in Phalaenopsis orchid with Agro-infiltration: (1) DNA fragment length about 80 bp, (2) a more mature status of inflorescence and (3) leaf injection.
Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan.
Ming-Hsien
MH
Hsieh
Hsiang-Chia
HC
Lu
Zhao-Jun
ZJ
Pan
Hsin-Hung
HH
Yeh
Shyh-Shyan
SS
Wang
Wen-Huei
WH
Chen
Hong-Hwa
HH
Chen
D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D035264:Flowers / Q000235:genetics* / Q000254:growth & development / Q000648:ultrastructure; D020868:Gene Silencing; D017343:Genes, Plant; D005822:Genetic Vectors / Q000235:genetics; D026161:MADS Domain Proteins / Q000235:genetics; D008855:Microscopy, Electron, Scanning; D009029:Mosaic Viruses; D029595:Orchidaceae / Q000033:anatomy & histology / Q000235:genetics* / Q000254:growth & development; D010641:Phenotype; D019441:Plant Epidermis / Q000648:ultrastructure; D018515:Plant Leaves / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D015203:Reproducibility of Results; D013997:Time Factors
D018076:DNA, Complementary; D018744:DNA, Plant; D026161:MADS Domain Proteins; D010940:Plant Proteins