Abstract
Virus-induced gene silencing (VIGS) is a potent way to study gene function in plants. In this study, a tobacco rattle virus (TRV)-mediated gene silencing system (TRV-VIGS) was established with the phytoene desaturase (HcPDS) as the marker gene in kenaf. Firstly, the seed soak agroinoculation method of the VIGS system was explored. HcPDS gene silencing led to photobleaching phenotype in kenaf leaves. According to qRT-PCR analysis, photobleached leaves had significantly lower gene expression levels compared with the control. Secondly, an optimizing approach with leaf infiltration was used to improve silencing efficiency. After the kenaf leaves were injected with Agrobacterium cultures with OD600 0.8, the plants were cultivated in chamber growth at 24 °C with approximately 60%-70% humidity (16 h day/8 h night), and the silencing efficiency could reach up to 90-100% at 2-3 weeks post-inoculation. To verify the reliability of this system, the HcPDS gene of 9 kenaf varieties (lines) from different genetic backgrounds was successfully silenced by the TRV-VIGS, implying the system can be widely used in kenaf. Furthermore, the knockdown of the anthocyanidin reductase gene (HcANR) by TRV-VIGS allowed for the significant decrease of the proanthocyanidins content. The HcANR silencing effects were analyzed by RNA-seq. The proanthocyanidins content reduced in silenced plants might be related to the dramatic transcriptional level changes of proanthocyanidins synthesis-related genes such as HcPAL, HcDFR, and HcF3'H. The establishment of TRV-VIGS will facilitate the functional study of target genes in kenaf.
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Data Availability
The original transcriptome data were stored in Genome Sequence Archive (GSA) with the accession number of CRA007065.
Abbreviations
- ANR:
-
Anthocyanidin reductase
- DEG:
-
Differentially Expressed Gene
- FPKM:
-
Fragments Per Kilobase Million
- PDS:
-
Phytoene desaturase
- TRV:
-
Tobacco rattle virus
- VIGS:
-
Virus-induced gene silencing
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Acknowledgements
We are grateful for the support of the National Natural Science Foundation of China (31972968) and the China Agriculture Research System of MOF and MARA (CARS-16). We are also grateful for the support of the platforms of Key Laboratory of Ministry of Agriculture and Rural Affairs for Biological Breeding of Fujian and Taiwan Crops, and Fujian International Science and Technology Cooperation Base for Genetics, Breeding and Multiple Utilization Development of Southern Economic Crops.
Funding
This work was supported by the National Natural Science Foundation of China (31972968), the Natural Science Foundation of Fujian, China (2023J01133679) and the China Agriculture Research System of MOF and MARA (CARS-16).
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Liwu Zhang designed the experiments. Xin Yang, Hu Li, Xingen Li and Lilan Zhang conducted the experiments. Jianmin Qi, Jiantang Xu, Aifen Tao, and Pingping Fang contributed to editing. Liwu Zhang and Sylvain Niyitanga contributed to editing. Xin Yang and Hu Li analyzed the data and wrote the manuscript.
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Yang, X., Li, H., Niyitanga, S. et al. Establishment of TRV-mediated Gene Silencing and Application for Elucidating Functions of Anthocyanidin Reductase Gene HcANR in Kenaf (Hibiscus cannabinus L.). Tropical Plant Biol. 16, 146–155 (2023). https://doi.org/10.1007/s12042-023-09341-1
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DOI: https://doi.org/10.1007/s12042-023-09341-1