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作物学报 ›› 2026, Vol. 52 ›› Issue (10): 2851-2863.doi: 10.3724/SP.J.1006.2026.65011

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝型油菜TRV诱导基因沉默体系的建立及应用

刘瑞帆(), 刘书洁, 侯豆豆, 易斌, 戴成, 马朝芝()   

  1. 华中农业大学作物遗传改良全国重点实验室, 湖北武汉 430070
  • 收稿日期:2026-02-24 接受日期:2026-07-15 出版日期:2026-10-12 网络出版日期:2026-07-20
  • 通讯作者: 马朝芝, E-mail: yuanbeauty@mail.hzau.edu.cn
  • 作者简介:刘瑞帆, E-mail: liuruifan2024@163.com
  • 基金资助:
    国家自然科学基金项目(32472138)

Establishment and application of TRV-induced gene silencing system in Brassica napus

Liu Rui-Fan(), Liu Shu-Jie, Hou Dou-Dou, Yi Bin, Dai Cheng, Ma Chao-Zhi()   

  1. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, Hubei, China
  • Received:2026-02-24 Accepted:2026-07-15 Published:2026-10-12 Published online:2026-07-20
  • Contact: Ma Chao-Zhi, E-mail: yuanbeauty@mail.hzau.edu.cn
  • Supported by:
    National Natural Science Foundation of China(32472138)

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摘要：

病毒诱导的基因沉默(VIGS)作为一种转录后基因沉默技术, 在植物基因功能研究中得到广泛应用。为探究烟草脆裂病毒(TRV)诱导基因沉默技术是否能够精确靶向甘蓝型油菜基因并显著下调其表达、从而有效提升基因功能研究的效率, 本研究选取BnaJMJ13、BnaSNAP33等4个基因为靶点, 扩增其cDNA中300~500 bp的核心区段, 并将其连接至TRV2载体, 通过真空渗透法处理萌发2 d的甘蓝型油菜种子, 分别对植株的叶片和花蕾组织进行定量分析。结果显示, TRV诱导的基因沉默能显著降低目标基因在甘蓝型油菜叶片和花蕾中的表达水平。花期表型鉴定表明, BnaJMJ13表达下调的植株开花时间显著提前, 表明其是油菜花期的负调控因子。半体外授粉试验表明, BnaSNAP33通过影响花粉附着和水合进而可能影响甘蓝型油菜结实。为了验证TRV诱导基因沉默的有效性, 创建了BnaSNAP33的缺失突变体, 通过半体外授粉试验证明BnaSNAP33通过降低花粉附着和水合从而影响结实, 说明了该技术在甘蓝型油菜中的有效性。综上, 本研究所建立的甘蓝型油菜TRV-VIGS体系具有操作简便、效率高及周期短等优势, 将促进甘蓝型油菜功能基因组学研究的发展。

关键词: 甘蓝型油菜, 烟草脆裂病毒, 基因沉默, BnaJMJ13, BnaSNAP33

Abstract:

Virus-induced gene silencing (VIGS), a post-transcriptional gene silencing technique, is widely used for the functional characterization of plant genes. To determine whether TRV-mediated VIGS can specifically target genes in Brassica napus and significantly downregulate their expression, thereby improving the efficiency of gene function studies, four genes, including BnaJMJ13 and BnaSNAP33, were selected as target genes in this study. A 300-500 bp core cDNA fragment of each gene was amplified and cloned into the TRV2 vector. Two-day-old germinated seeds of B. napus were then subjected to vacuum infiltration, and leaves and flower buds were collected for RT-qPCR analysis. The results showed that TRV-VIGS significantly reduced the transcript levels of the target genes in both tissues. Phenotypic analysis at the flowering stage showed that plants with downregulated BnaJMJ13 expression flowered significantly earlier, suggesting that BnaJMJ13 functions as a negative regulator of flowering time in B. napus. Semi-in vivo pollination assays revealed that BnaSNAP33 may affect seed setting in B. napus by modulating pollen adhesion and hydration. To further verify the efficiency of TRV-induced gene silencing, loss-of-function mutants of BnaSNAP33 were generated in this study. Semi-in vivo pollination assays further confirmed that BnaSNAP33 regulates seed setting by affecting pollen adhesion and hydration. These results demonstrate the applicability of this silencing system in B. napus. The TRV-VIGS system established in this study is easy to operate, highly efficient, and time-saving, and will facilitate functional genomics research in B. napus.

Key words: Brassica napus, Tobacco rattle virus, gene silence, BnaJMJ13, BnaSNAP33

表1

TRV诱导沉默的基因列表"

基因名称Gene name 基因号Gene ID
BnaJMJ13 BnaA02G0300900WE
BnaA09G0198400WE
BnaC02G0396300WE
BnaC07G0250500WE
BnaC09G0229300WE
BnaSNAP33 BnaA03G0417000WE
BnaC02G0506400WE
BnaC07G0373400WE
BnaVAMP722 BnaA03G0174900WE
BnaA04G0202300WE
BnaA05G0111300WE
BnaC03G0124200WE
BnaC04G0139500WE
BnaC04G0509100WE
BnaCDF3 BnaA06G0170600WE
BnaC03G0533800WE

表2

本研究所用引物"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')
TRV2-BnaJMJ13-F CTGTGAGTAAGGTTACCGAATTCTGTCCTGTCTACAGACCAACAA
TRV2-BnaJMJ13-R GTGAGCTCGGTACCGGATCCCACCTTTTCAAATTTTAGAGCTGCAG
TRV2-BnaSNAP33-F CTGTGAGTAAGGTTACCGAATTCGACTTCTAACTCCAGATACCAGTA
TRV2-BnaSNAP33-R GTGAGCTCGGTACCGGATCCGGGTTTAGTCCCAGTTTCTCCCT
TRV2-BnaCDF3-F CTGTGAGTAAGGTTACCGAATTCATCACCAGAGAAGGTAACTACAGA
TRV2-BnaCDF3-R GTGAGCTCGGTACCGGATCCCCCGGATCGAGCCTTGC
TRV2-BnaVamp722-F CTGTGAGTAAGGTTACCGAATTCGTGCCTCCAGAAGCTTCCG
TRV2-BnaVamp722-R GTGAGCTCGGTACCGGATCCTGTGTTCCTTGTGTCCTGAAATCTTGC
QPCR-BnaJMJ13-F TGTACAAGGCTGTACAGAAACCTG
QPCR-BnaJMJ13-R TCAATTCTTCATGAGGAAGCAATGG
QPCR-BnaSNAP33-F CCTGTCATTACTAGAGATCACTCACC
QPCR-BnaSNAP33-R CTCCACTCTCTGATACGCATCA
QPCR-BnaCDF3-F GATGGAAAGTAGAGATCCAGCTATTAAGC
QPCR-BnaCDF3-R GGAGTGGCTTGCTCTGTAGTT
QPCR-BnaVAMP722-F ATCCTCGTGGAGTTCACCG
QPCR-BnaVAMP722-R CAACTGCAACAACACAATAAGTGAA
Bnactin7-F CTATCCTCCGTCTCGATCTCGC
Bnactin7-R CTTAGCCGTCTCCAGCTCTTGC
CR-SNAP33-DT1-BsF ATATATGGTCTCGATTGGTCTGTTCAGGAGCTTTGAGTT
CR-SNAP33-DT1-F0 TGGTCTGTTCAGGAGCTTTGAGTTTTAGAGCTAGAAATAGC
CR-SNAP33-DT2-R0 AACCTGTACAAGGTTGTCTCAACAATCTCTTAGTCGACTCTAC
CR-SNAP33-DT2-BsR ATTATTGGTCTCGAAACCTGTACAAGGTTGTCTCAACAA

表3

sgRNA序列"

sgRNA名称
sgRNA name		 序列
Sequence (5'-3')
sgRNA1 CAGTCTGTTCAGGAGCTTGAGG
sgRNA2 GTACAAGGTTGTCTCAAGGTGG

图1

TRV诱导的基因沉默体系在甘蓝型油菜(Brassica napus)中应用的技术流程图 A: 选取目标基因300-500 bp核心序列构建TRV2载体, 与pTRV2空载、pTRV1分别转化农杆菌GV3101, 接种于含利福平(Rif)、庆大霉素(GMS)、卡那霉素(Kana)的LB液体培养基中培养, 离心收集后重悬, 将pTRV1与pTRV2菌悬液按1∶1 (v/v)混合。B: 30-50粒甘蓝型油菜种子经75%乙醇消毒1-2 min、无菌水冲洗3次, 用5 mmol L-1 GA3处理1-2 h促萌发, 再冲洗3次, 置于湿润滤纸培养皿中暗培养48 h诱导萌发。C: 萌发种子浸入新鲜农杆菌侵染液, 15 psi真空渗透10 min, 平铺于1/2 MS固体培养基暗培养24 h, 冲洗后播种于温室土壤, 16 h光照/8 h黑暗培养至成熟。"

图2

甘蓝型油菜与拟南芥中JMJ13的氨基酸序列比对 拟南芥AtJMJ13与其在甘蓝型油菜中的同源基因的多序列碱基比对。使用ClustalW算法对序列进行比对, 并使用ESPript 3.0进行可视化。在所比对的序列中, 相同的残基用黑色背景突出显示, 相似的残基用灰色阴影表示, 为了优化比对而引入的缺口用短划线表示。"

图3

甘蓝型油菜与拟南芥中SNAP33、VAMP722及CDF3的氨基酸序列比对 拟南芥AtSNAP33、AtVAMP722及AtCDF3与其在甘蓝型油菜中的同源基因的多序列碱基比对。使用ClustalW算法对序列进行比对, 并使用ESPript 3.0进行可视化。在所比对的序列中, 相同的残基用黑色背景突出显示, 相似的残基用灰色阴影表示, 为了优化比对而引入的缺口用短划线表示。"

图4

拟南芥与甘蓝型油菜JMJ13、SNAP33、VAMP722及CDF3的保守结构域分析 A-D: JMJ13、SNAP33、VAMP722及CDF3所包含的保守结构域示意图。横轴代表蛋白质序列长度, 不同颜色的方框代表不同的保守结构域。"

图5

TRV诱导的基因沉默后苗期叶片中靶基因表达量的检测 A-D: 通过RT-qPCR测定TRV接种后4个靶基因在叶片组织中的相对表达水平。Ev: 空载体。以Actin作为内参基因进行数据标准化。数据表示为3次独立生物学重复的平均值±标准差。ns: 差异不显著; *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001。"

图6

TRV诱导的基因沉默后花蕾中靶基因表达量的检测 A-B: 通过RT-qPCR测定TRV接种后BnaJMJ13和BnaSNAP33在花蕾中的相对表达水平。Ev: 空载体。以Actin作为内参基因进行数据标准化。数据表示为3次独立生物学重复的平均值±标准差。ns: 差异不显著; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001。"

图7

TRV诱导的BnaJMJ13基因沉默株系花期提前 Ev: 转化空载体的植株; ****: P < 0.0001。"

图8

TRV诱导BnaSNAP33基因沉默株系的花粉附着和萌发率 A-B: 对照组(Ev)以及VIGS-bnasnap33株系花粉同时授于对照组(Ev)的柱头后花粉的附着和萌发的结果。上图为明场视野, 下图为荧光视野。每个株系至少观察6个柱头。Ev: 空载体。标尺为200 μm。C: 自花授粉和异花授粉后附着在柱头上的花粉粒数量统计。D: 自花授粉和异花授粉后柱头上花粉粒萌发数量统计。所有组合中, 母本均为对照组(Ev, 转化空载体的植株), 父本分别为Ev及VIGS-bnasnap33株系。**: P < 0.01; ***: P < 0.001。"

图9

bnasnap33突变体编辑情况 A: BnaSNA33基因结构和靶点位置。B: T1代BnaSNA33基因靶点突变情况。蓝色代表变异, 红色代表靶点序列。Ref: 参考序列; i: 插入突变; d: 缺失突变。"

图10

bnasnap33突变体花粉附着和萌发率 A-C: 野生型(WT)以及bnasnap33突变体花粉同时授于野生型(WT)的柱头后花粉的附着和萌发情况。上图为明场视野, 下图为荧光视野。每个株系至少观察10个柱头。标尺为200 μm。D: 自花授粉和异花授粉后附着在柱头上的花粉粒数量统计。E: 自花授粉和异花授粉后柱头上花粉粒萌发数量统计。所有组合中, 母本均为野生型, 父本包括野生型以及bnasnap33突变体。****: P < 0.0001。"

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