病毒介导的甘蓝基因沉默体系的建立

doi:10.7668/hbnxb.20195636

摘要/Abstract

摘要：

病毒诱导的基因沉默(VIGS)是一种转录后基因沉默技术,广泛应用于植物基因功能研究。然而,到目前为止,国内关于建立甘蓝VIGS基因沉默体系的报道较少。旨在甘蓝上建立以八氢番茄红素脱氢酶基因(Phytoene desaturase,PDS)作为有效视觉指示基因、病毒载体PCVA/PCVB介导的基因沉默体系。以甘蓝、大白菜和萝卜为植物材料,通过构建PCVA-PDS载体,对甘蓝PDS进行沉默。通过构建PCVA/PCVB-PDS-GFP转化农杆菌,并采用注射法侵染甘蓝和烟草叶片下表皮细胞、真空负压侵染甘蓝幼苗,结合实时荧光定量PCR技术,研究病毒介导的基因沉默体系在甘蓝中的适用性,并将该体系应用于另外2个具有代表性的十字花科作物—大白菜和萝卜。结果表明,载体PCVA/PCVB-PDS-GFP转化的农杆菌侵染甘蓝及烟草叶片细胞后,细胞膜可以观察到荧光出现;通过真空负压侵染甘蓝幼苗14 d后,新生叶片出现光漂白现象,并呈现范围逐渐扩大的趋势;实时荧光定量PCR分析显示,PDS同源基因在试验组中的相对表达量较对照组分别下降3.2,1.7倍;侵染大白菜和萝卜幼苗后,观察到大白菜和萝卜的叶脉及部分叶片出现白化现象,同时伴随一定程度的叶面卷曲。综上所述,通过对PDS基因沉默后甘蓝叶片出现光漂白现象,证明病毒介导的基因沉默体系在甘蓝植株中实现了有效的复制和传播,大白菜萝卜叶片出现白化也表明,VIGS系统同样可以应用于其他十字花科作物,进一步扩展了该沉默系统的应用范围。甘蓝VIGS沉默体系的建立,为十字花科作物相关基因功能研究提供了理论基础。

关键词: 甘蓝, 光漂白, 基因沉默, 病毒介导, 八氢番茄红素脱氢酶基因

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

中图分类号:

S635.1

王江, 刘永贵, 韩睿, 钟雄辉, 颉建明, 康俊根. 病毒介导的甘蓝基因沉默体系的建立[J]. 华北农学报, 2025, 40(3): 157-165. doi: 10.7668/hbnxb.20195636.

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.

http://www.hbnxb.net/CN/Y2025/V40/I3/157

图/表 8

表1 本研究中所用引物信息

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

图1 PDS同源基因Bo3g149210、Bo4g127210多序列碱基比对结果

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

图2 PCR扩增后WT、PCVA空载体、PCVA-PDS和甘蓝内参基因GAPDH条带情况

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

图3 PDS基因插入酶切位点及载体PCVA-PDS、PCVA-PDS-GFP的构建 A、B.大小为352 bp的2个PDS同源基因插入PCVA载体,利用引物PDS-silenced-qRT-7210-F/R与PDS-silenced-qRT-9210-F/R检测PDS基因;C. PDS基因片段插入PCVA载体,构建PCVA-PDS载体,酶切位点为Kpn Ⅰ与Xha Ⅰ,利用引物PCVA-detectio-F/R检测带有目的基因片段载体;D.含荧光蛋白GFP插入载体PCVA构建PCVA-PDS-GFP载体,使用引物为PCVA-detection-F/R。

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.

图4 载体PCVA-PDS-GFP在甘蓝、烟草细胞膜表达 A—C为该载体在甘蓝叶片细胞膜发荧光情况;D—F为该载体在烟草细胞膜发荧光情况。

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.

图5 病毒介导的甘蓝PDS基因沉默导致叶片的光漂白现象 A—I.基因沉默导致的甘蓝叶片叶脉及部分叶片出现白化表型;J—L.甘蓝植株生长过程中出现的白化现象。随时间推移甘蓝叶片光漂白由原先较小范围逐渐扩展至整个叶片。

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.

图6 甘蓝植株上病毒介导的沉默体系Bo4g127210、Bo3g149210相对表达量情况不同小写字母代表差异显著(P<0.05)。

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).

图7 甘蓝PDS基因沉默体系在大白菜和萝卜上的应用 A—H.萝卜植株叶片白化表型;I—P.大白菜植株叶片白化表型。图中萝卜叶片白化范围随叶脉向周围扩展;大白菜叶片白化面积较大并叶卷曲。

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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