TRV介导的上海青和芥菜VIGS体系的构建

doi:10.13304/j.nykjdb.2025.0124

中国农业科技导报 ›› 2025, Vol. 27 ›› Issue (8): 239-249.DOI: 10.13304/j.nykjdb.2025.0124

• 方法与技术创新 • 上一篇

TRV介导的上海青和芥菜VIGS体系的构建

马燕勤¹^,²(), 周玉洁¹^,², 龙海成¹^,², 李菊¹^,², 王海娥¹^,², 常伟³, 李志¹^,², 钟建¹^,², 苗明军¹^,², 杨亮¹^,²()

^1.四川省农业科学院园艺研究所，蔬菜种质与品种创新四川省重点实验室，农业农村部西南地区园艺作物生物学与种质创制重点实验室，成都 610066
^2.四川省蔬菜工程技术研究中心，四川彭州 611934
^3.四川省食用菌研究所，成都 610066

收稿日期:2025-02-26 接受日期:2025-06-23 出版日期:2025-08-15 发布日期:2025-08-26
通讯作者: 杨亮
作者简介:马燕勤 E-mail：dora0514@sina.cn；
基金资助:
四川省农业科学院科研启动费项目(NKYRCZX2025009);四川省农业科学院“1 + 9”揭榜挂帅科技攻关项目（1+9KJGG001）;国家现代农业产业技术体系四川特色蔬菜创新团队项目(SCCXTD-2024-22);四川省科技计划项目(2021YFYZ0022);四川省科技计划项目(2023NSFSC0162);四川省财政自主创新专项(2022ZZCX050)

Construction of TRV-mediated VIGS System in Brassica rapa subsp. chinensis and Brassica juncea

Yanqin MA¹^,²(), Yujie ZHOU¹^,², Haicheng LONG¹^,², Ju LI¹^,², Haie WANG¹^,², Wei CHANG³, Zhi LI¹^,², Jian ZHONG¹^,², Mingjun MIAO¹^,², Liang YANG¹^,²()

^1.Vegetable Germplasm Innovation and Variety Improvement Key Laboratory of Sichuan Province，Key Laboratory of Horticultural Crops Biology and Germplasm Enhancement in Southwest Regions of Ministry of Agriculture and Rural Affairs，Horticulture Research Institute，Sichuan Academy of Agricultural Sciences，Chengdu 610066，China
^2.Sichuan Province Engineering Technology Research Center of Vegetables，Sichuan Pengzhou 611934，China
^3.Sichuan Institute of Edible Fungi，Chengdu 610066，China

Received:2025-02-26 Accepted:2025-06-23 Online:2025-08-15 Published:2025-08-26
Contact: Liang YANG

摘要/Abstract

摘要：

为构建烟草脆裂病毒（tobacco rattle virus，TRV）介导的十字花科植物的病毒诱导的基因沉默（virus-induced gene silencing，VIGS）体系，以十字花科蔬菜上海青和芥菜为研究对象，以内源八氢番茄红素脱氢酶（phytoene desaturase，PDS）基因为标记基因，构建pTRV2-BrPDS、pTRV2-BjuPDS-g和pTRV2-BjuPDS-c载体，并进一步对农杆菌的侵染水平进行优化，构建上海青和芥菜的高效VIGS系统。结果显示，pTRV2-BrPDS、pTRV2-BjuPDS-g和pTRV2-BjuPDS-c载体构建成功。当农杆菌OD₆₀₀=1.0时，上海青植株白化表型最明显，当农杆菌OD₆₀₀= 0.8时，芥菜的沉默效果最佳。通过qRT-PCR证实，白化表型是由TRV重组病毒侵染植株后，内源性PDS基因发生沉默而引起的。以上研究结果为利用VIGS技术研究十字花科植物的基因功能提供了理论依据。

关键词: 上海青, 芥菜, VIGS, PDS基因, 白化表型

Abstract:

To establish an efficient virus-induced gene silencing （VIGS） system in important Brassicaceae vegetables， Brassica rapa subsp. chinensis and Brassica juncea were chosen as the material and the endogenous phytoene desaturase （PDS） gene was selected as the marker gene. The pTRV2-BrPDS， pTRV2-BjuPDS-g and pTRV2-BjuPDS-c vectors were constructed， and the infection efficiency of Agrobacterium was optimized to construst VIGS system with high efficient. The results showed that the pTRV2-BrPDS， pTRV2-BjuPDS-g and pTRV2-BjuPDS-c vectors were successfully constructed. The albinism phenotype of B. rapa subsp. chinensis plants was the most obvious with OD₆₀₀=1.0 of Agrobacterium， while the silencing efficiency of B. juncea was optimal with OD₆₀₀=0.8 of Agrobacterium. The qRT-PCR analysis confirmed that the observed albinism phenotype was resulted from the silencing of the endogenous PDS gene in plant infected with the TRV-based recombinant virus. Above results provided theoretical basis for studing the gene functions of B. rapa subsp. chinensis and B. juncea using VIGS technology.

Key words: Brassica rapa subsp. Chinensis, Brassica juncea, VIGS, PDS gene, albinism phenotype

中图分类号:

马燕勤, 周玉洁, 龙海成, 李菊, 王海娥, 常伟, 李志, 钟建, 苗明军, 杨亮. TRV介导的上海青和芥菜VIGS体系的构建[J]. 中国农业科技导报, 2025, 27(8): 239-249.

Yanqin MA, Yujie ZHOU, Haicheng LONG, Ju LI, Haie WANG, Wei CHANG, Zhi LI, Jian ZHONG, Mingjun MIAO, Liang YANG. Construction of TRV-mediated VIGS System in Brassica rapa subsp. chinensis and Brassica juncea[J]. Journal of Agricultural Science and Technology, 2025, 27(8): 239-249.

图/表 11

图1 基于TRV的VIGS载体结构注：Replicase—依赖于RNA的RNA聚合酶；16 KD—16 kD富半胱氨酸蛋白；MP—移动蛋白；CP—外壳蛋白；LB 和RB—T-DNA左、右边界；Rz—自剪切核酸酶；MCS—多克隆位点。

Fig. 1 Structure of TRV-based VIGS vectorNote： Replicase—RNA-dependent RNA polymerase； 16 KD—16 kD cysteine-rich protein； MP—Move protein； CP—Coat protein； LB and RB—left and right border of T-DNA； Rz—Ribozyme； MCS—Multiple clone site.

表1 载体构建引物

Table 1 Primers for vector construction

引物名称 Primer name	序列 Sequence （5’-3’）	备注 Reference
BrPDS-F	ATGGTTGTGTTTGGGAATGT	扩增BrPDS开放阅读框序列
BrPDS-R	TCATGTTGATACAGTTGTCTC	Amplify BrPDS open reading frame
BjuPDS-g-F	ATGGTTGTGTTTGGGAATGT	扩增BjuPDS-g 开放阅读框序列
BjuPDS-g-R	TCATGTTGATACAGTTGTCTC	Amplify BjuPDS-g open reading frame
BjuPDS-c-F	ATGGTTGTGTTTGGGAATGT	扩增BjuPDS-c 开放阅读框序列
BjuPDS-c-R	TCATGTTGATACAGTTGTCTC	Amplify BjuPDS-g open reading frame
BrPDS-F1	CTGTGAGTAAGGTTACCGAATTCACCTGATCGCGTGACTGATG	构建pTRV2-BrPDS沉默表达载体 Construct TRV2-BrPDS vector
BrPDS-R1	GTGAGCTCGGTACCGGATCCAGCGTCTCCTTGGATAGTGG	构建pTRV2-BrPDS沉默表达载体 Construct TRV2-BrPDS vector
BjuPDS-g-F1	CTGTGAGTAAGGTTACCGAATTCACCTGATCGCGTGACTGATG	构建pTRV2-BjuPDS-g沉默表达载体 Construct TRV2-BjuPDS-g vector
BjuPDS-g-R1	GTGAGCTCGGTACCGGATCCAGCGTCTCCTTGGATAGTGG	构建pTRV2-BjuPDS-g沉默表达载体 Construct TRV2-BjuPDS-g vector
BjuPDS-c-F1	CTGTGAGTAAGGTTACCGAATTCACCTGATCGCGTGACTGATG	构建pTRV2- BjuPDS-c沉默表达载体 Construct TRV2-BjuPDS-c vector
BjuPDS-c-R1	GTGAGCTCGGTACCGGATCCAGCGTCTCCTTGGATAGTGG	构建pTRV2- BjuPDS-c沉默表达载体 Construct TRV2-BjuPDS-c vector
YL192-F	CTTGAAGAAGAAGACTTTCGAAGTCTC	鉴定TRV1，约900 bp Identify TRV1， 900 bp
YL192-R	GTAAAATCATTGATAACAACACAGACAAAC	鉴定TRV1，约900 bp Identify TRV1， 900 bp
YL156-F	GGTCAAGGTACGTAGTAGAG	鉴定TRV2，约390 bp Identify TRV1， 390 bp
YL156-R	CGAGAATGTCAATCTCGTAGG	鉴定TRV2，约390 bp Identify TRV1， 390 bp
BrPDS-F2	CCTGATCGCGTGACTGATG	内源BrPDS表达检测 Detect expression of BrPDS
BrPDS-R2	TGTTCAACAATCGGCATGCA	内源BrPDS表达检测 Detect expression of BrPDS
BrActin-F	GTCTCCATCTCCTGCTCATAGT	上海青内参基因
BrActin-R	GCTGACCGTATGAGCAAAGA	actin gene of B. rapa subsp. chinensis
BjuPDS-g-F2	CTGATCGCGTGACTGATGAG	内源BjuPDS-g表达检测
BjuPDS-g-R2	CCATGTTTCTCCTGAAGAAACC	Detect expression of BjuPDS-g
BjuPDS-c-F2	TATAGCCATGTCAAAGGCGC	内源BjuPDS-c表达检测
BjuPDS-c-R2	GCTCAATCTTCCTTATCCTTG	Detect expression of BjuPDS-c
BjuActin-R	GCTGACCGTATGAGCAAAGA	芥菜内参基因
BjuActin-R	GTTGGAAAGTGCTGAGGGAT	actin gene of B. juncea

图2 不同PDS蛋白氨基酸序列比对

Fig. 2 Amino acid sequence alignment of different PDS proteins

图3 VIGS载体的构建A：内源PDS基因扩增片段，-表示阴性对照，1~3分别为BrPDS、BjuPDS-g和BjuPDS-c基因片段；B：重组载体转化大肠杆菌DH5α菌落PCR检测，1~3为pTRV2-BrPDS DH5α菌落PCR 检测，4~6为pTRV2-BjuPDS-g DH5α菌落PCR 检测，7~9为pTRV2-BjuPDS-c DH5α菌落PCR检测；C：重组载体转化根癌农杆菌GV3101菌株菌落PCR检测，1~2为pTRV2-BrPDS GV3101菌落PCR 检测，3~4为pTRV2-BjuPDS-g大GV3101菌落PCR 检测，5~6为pTRV2-BjuPDS-c GV3101菌落PCR 检测。M—DL2000 DNA Marker。

Fig. 3 Construction of VIGS vectorsA： Amplification fragments of Endogenous PDS gene， - indicates negative control； 1~3 are BrPDS， BjuPDS-g and BjuPDS-c gene fragments， respectively； B： PCR detection of recombinant vector-transformed Escherichia coli DH5α colonies， 1~3 are pTRV2-BrPDS DH5αcolonies PCR detection， respectively； 4~6 are pTRV2-BjuPDS-g DH5αcolonies PCR detection， respectively； 7~9 are pTRV2-BjuPDS-c DH5αcolonies PCR detection， respectively； C： PCR detection of recombinant vector-transformed Agrobacterium tumefaciens GV3101 strain colonies， 1~2 are pTRV2-BrPDS GV3101 colonies PCR detection， respectively； 3~4 are pTRV2-BjuPDS-g GV3101 colonies PCR detection， respectively； 5~6 are pTRV2-BjuPDS-c GV3101 colonies PCR detection. M—DL2000 DNA Marker

图4 不同农杆菌水平下植株的白化率注：不同小写字母表示不同处理间在P<0.05水平差异显著。

Fig. 4 Albinism rate of plant under different Agrobacterium levelsNote： Different lowercase letters indicate significant differences between different treatments at P<0.05 level.

图5 上海青BrPDS沉默植株的白化表型A：OD600=0.3；B：OD600=0.5；C：OD600=0.8；D：OD600=1.0。标尺=1 cm

Fig. 5 Albinism phenotype of Chinese cabbage plant with silenced BrPDSA： OD600=0.3； B： OD600=0.5； C： OD600=0.8； D： OD600=1.0. Scale bar=1 cm

图6 芥菜PDS基因下调导致植株叶片白化现象A：抱子芥OD600=0.3；B：抱子芥OD600=0.5；C：抱子芥OD600=0.8；D：抱子芥OD600=1.0； E：结球芥OD600=0.3；F：结球芥OD600=0.5；G：结球芥OD600=0.8；H：结球芥OD600=1.0。标尺=1 cm

Fig. 6 Leaf albinismof B. juncea plant with downregulation of PDSA： B. juncea var. gemmifera with OD600=0.3； B： B. juncea var. gemmifera with OD600=0.5； C： B. juncea var. gemmifera with OD600=0.8； D： B. juncea var. gemmifera with OD600=1.0；E： B. juncea var. capitate with OD600=0.3； F： B. juncea var. capitate with OD600=0.5； G： B. juncea var. capitate with OD600=0.8； H： B. juncea var. capitate with OD600=1.0. Scale bar =1 cm

图7 芥菜内源PDS基因下调导致植株花序白化现象A：抱子芥；B：结球芥。标尺=1 mm

Fig. 7 Inflorescence albinism of B. juncea plant with downregulation of PDSA： B. juncea var. gemmifera； B： B.juncea var. capitata. Scale bar =1 mm

图8 芥菜内源PDS基因下调导致植株花冠白化现象A：抱子芥；B：结球芥。标尺=1 mm

Fig. 8 Corolla albinism of B. juncea plant with downregulation of PDSA： B. juncea var. gemmifera； B： B.juncea var. capitata. Scale bar =1 mm.

图9 叶片中TRV RNA的表达分析A：上海青；B：抱子芥；C：结球芥。TRV1为叶片中TRV1的表达量检测，TRV2为叶片中TRV2的表达量检测

Fig. 9 Analysis of TRV RNA expression in leafA： B. rapa subsp. Chinensis； B： B. juncea var. gemmifera； C： B. juncea var. capitata. TRV1 is the detection of TRV1 expression in leaves， TRV2 is the detection of TRV1 expression in leaves

图10 内源PDS基因的相对表达量A~B：上海青；C~D：抱子芥；E~F：结球芥

Fig. 10 Relative expression of endogenous PDS geneA~B： B. rapa subsp. Chinensis； C~D： B. juncea var. gemmifera； E~F： B. juncea var. capitata

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TRV介导的上海青和芥菜VIGS体系的构建

Construction of TRV-mediated VIGS System in Brassica rapa subsp. chinensis and Brassica juncea

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