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Overexpression and VIGS system for functional gene validation in oriental melon (Cucumis melo var. makuwa Makino)

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Abstract

As one of the effective and powerful methods for gene function analysis in plants, transient overexpression and virus-induced gene silencing (VIGS) system were developed for oriental melon (Cucumis melo var. makuwa Makino). Here, the full-length of CmLOX10, the conserved domains of CmLOX10 and CmPDS were isolated from oriental melon. The vectors for overexpression of CmLOX10, and the VIGS vector of CmLOX10 and CmPDS were constructed and transformed into agrobacterium, respectively. Especially, the sprout absorption method was performed on oriental melon, and CmPDS was selected as a reporter gene in VIGS. The expression level of CmLOX10 significantly increased in the oriental melon with the CmLOX10 overexpression construct, and lipoxygenases (LOXs) contributed to the leaf cell death in the oriental melon. The expression levels of CmLOX10 and CmPDS were significantly decreased in plants with the VIGS construct, and there was albino phenotype in CmPDS silencing leaves. In this study, CmLOX10 gene was silenced using Tobacco rattle virus based VIGS system firstly. These results proved that the transient expression technology provides an excellent tool to explore gene function in oriental melon.

Key message

Transient over-expression and virus-induced gene silencing (VIGS) system could be performed successfully in oriental melon by leaf agro-infiltration and sprout absorption methods, respectively.

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Abbreviations

ALSV:

Apple latent spherical virus

AS:

Acetosyringone

BPMV:

Bean pod mottle virus

BSMV:

Barley stripe mosaic virus

CMV:

Cucumber mosaic virus

DAB:

Diaminobenzidine

GFP:

Green fluorescent protein

LB:

Luria-Bertani

LOXs:

Lipoxygenases

MDA:

Malondialdehyde

MES:

4-Morpholineethanesulfonic acid

PDS:

Phytoene desaturase

PEG:

Polyethylene glycol

PVX:

Potato virus X

qRT-PCR:

Quantitative real-time PCR

ROS:

Reactive oxygen species

TBA:

Thiobarbituric acid

TCA:

Trichloroacetic acid

TRV:

Tobacco rattle virus

VIGS:

Virus-induced gene silencing

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Acknowledgements

We are grateful to Dr Hao Jinghong (Beijing University of Agriculture, China) for kindly offering pTRV1 and pTRV2 vectors. This study has been supported by China Agriculture Research System (Grant No.: CARS-25), Shenyang Science and Technology Project (Grant No.: 17-143-3-00) and Liaoning Innovative Research Team in University (Grant No.: LZ2015025).

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Authors and Affiliations

  1. College of Horticulture and Shenyang Agricultural University/Key Laboratory of Protected Horticulture, Ministry of Education/Northern National & Local Joint Engineering Research Center of Horticultural Facilities Design and Application Technology, Shenyang, 110866, Liaoning, China

    Jing Jing Liao, Cheng Hui Wang, Qiao Juan Xing, Yue Peng Li, Xian Feng Liu & Hong Yan Qi

  2. Institute of Medicinal Plant and Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, 100193, China

    Jing Jing Liao

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  1. Jing Jing Liao
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Contributions

JJL and HYQ conceived and designed the experiments. JJL, CGW, QJX, YPL and XFL performed the experiments. JJL analyzed the data and wrote the manuscript, and HQ improved the manuscript.

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Correspondence to Hong Yan Qi.

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Communicated by K.X. Tang.

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Liao, J.J., Wang, C.H., Xing, Q.J. et al. Overexpression and VIGS system for functional gene validation in oriental melon (Cucumis melo var. makuwa Makino). Plant Cell Tiss Organ Cult 137, 275–284 (2019). https://doi.org/10.1007/s11240-019-01568-9

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