Establishment of Virus-Mediated Gene Silencing System in Cabbage

doi:10.7668/hbnxb.20195636

Abstract

Abstract:

Virus-induced gene silencing(VIGS)is a post-transcriptional gene silencing technique widely used in plant gene function research.However,there are few reports on the establishment of a VIGS system for cabbage in China.The aim of this study was to establish a PCVA/PCVB-mediated gene silencing system using phytoene desaturase(PDS) as an effective visual indicator gene in cabbage.Cabbage,Chinese cabbage,and radish were used as plant materials.The PDS gene was silenced by constructing the PCVA-PDS vector.The PCVA/PCVB-PDS-GFP vector was then generated and transformed into Agrobacterium tumefaciens,which was used to infect cabbage and tobacco epidermal cells via injection and to perform vacuum infiltration in cabbage seedlings.Real-time Quantitative PCR was used to study the applicability of the VIGS system in cabbage,and the system was also applied to another representative Brassicaceae crops-Chinese cabbage and radish.The results showed that green fluorescence could be observed on the cell membranes of cabbage and tobacco leaf cells after infection with Agrobacterium tumefaciens transformed with PCVA/PCVB-PDS-GFP.After 14 d of vacuum infiltration in cabbage seedlings,photobleaching appeared on the new leaves,with the affected area gradually expanding.Real-time Quantitative PCR analysis indicated that the relative expression of PDS homologous genes in the experimental groups decreased 3.2-fold and 1.7-fold compared to the control group,respectively.After infecting Chinese cabbage and radish seedlings,whitening of the leaf veins and some leaves was observed,along with some leaf curling.In conclusion,the photobleaching observed in cabbage leaves after PDS gene silencing demonstrates that the VIGS system effectively replicates and spreads within cabbage plants.The whitening of leaves in Chinese cabbage and radish also indicates that the VIGS system can be applied to other Brassicaceae crops,thus expanding the application scope of this silencing system.The establishment of the cabbage VIGS system provides a theoretical foundation for functional gene studies in Brassicaceae crops.

Key words: Cabbage, Photobleaching, Gene silencing, Virus-mediation, PDS

CLC Number:

S635.1

WANG Jiang, LIU Yonggui, HAN Rui, ZHONG Xionghui, XIE Jianming, KANG Jungen. Establishment of Virus-Mediated Gene Silencing System in Cabbage[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3): 157-165. doi: 10.7668/hbnxb.20195636.

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Figures/Tables 8

Tab.1 Primers used in this study

引物名称 Primer name	碱基序列(5'—3') Sequence(5'—3')
PDS-F	AACAACGAGATGCTGACATGG
PDS-R	GCGATTTCGTCAGGGAAGAG
PDS352KpnIFORPCVA-F	GCCTAAGCGGCTAGCGGTACCGCCACTCCAGTCGATATCCTG
PDS352XbaIFORPCVA-R	TAGGCTAGCGAGCTCAGATCTTTTGGTCAGCAGCGATTTCG
PCVA-detection-F	TGGGACCACGCATTAAATGA
PCVA-detection-R	TTTCGCAACGGACAGATCCT
PDS-silenced-qRT-7210-F	GTCCTTTGCAGGTAGTGTGTG
PDS-silenced-qRT-7210-R	CATCCTTCCATGCAGCTATC
PDS-silenced-qRT-9210-F	GGCAACTCAGTGGAAGGAAC
PDS-silenced-qRT-9210-R	CCTCATCAGTCACTCGATCAG
PCVA-PDS-GFP-F	TCGCTGCTGACCAAAAGATCTATGGTGAGCAAGGGCGAGGA
PCVA-PDS-GFP-R	TAGGCTAGCGAGCTCAGATCTCTTGTACAGCTCGTCCATGC

Fig.1 Results of multiple sequence alignment of PDS homologs Bo3g149210 and Bo4g127210

Fig.2 PCR amplification results showing bands for WT,PCVA empty vector,PCVA-PDS and the internal reference gene GAPDH in cabbage

Fig.3 PDS gene insertion into the restriction site and construction of the vectors PCVA-PDS and PCVA-PDS-GFP A,B.Two PDS homolog gene with a size of 352 bp was inserted into PCVA vector,and PDS gene was detected using primers PDS-silenced-7210-F/R and PDS-silenced-9210-F/R;C. PDS gene fragment was inserted into PCVA vector to construct PCVA-PDS vector,the restriction sites were Kpn Ⅰ and Xha Ⅰ,and the primers PCVA-detection-F/R were used to detect the vector with target gene fragment;D.PCVA-PDS-GFP vector containing fluorescent protein GFP insert vector PCVA was constructed using primers PCVA-detection-F/R.

Fig.4 Expression of vector PCVA-PDS-GFP in cell membranes of cabbage and tobacco A—C show the fluorescence of the carrier in the cell membrane of cabbage leaves;D—F show the fluorescence of the carrier in tobacco cell membrane.

Fig.5 Leaf photobleaching caused by virus-mediated PDS gene silencing in cabbage A—I.The white phenotype of cabbage leaves veins and some leaves caused by gene silencing;J—L.The albino phenomenon that occurred during the growth of cabbage plants.The photobleaching of cabbage leaves gradually expanded from a small area to the whole leaf with time.

Fig 6 Relative expression levels of virus-mediated silencing system Bo4g127210 and Bo3g149210 in cabbage plants Different lowercase letters represent significant difference(P <0.05).

Fig.7 Application of the cabbage PDS gene silencing system in Chinese cabbage and radish A—H.The leaf albino phenotype of radish plants;I—P.The leaf albino phenotype of Chinese cabbage.In the figure,the whitening range of radish leaves expands with the veins;the leaves of Chinese cabbage have large albino area and curly leaves.

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