不同接种方式对烟草根、叶差异表达基因沉默效应的影响

doi:10.16178/j.issn.0528-9017.20240176

摘要/Abstract

摘要：

不同基因在烟草不同组织中的表达存在差异,TRV病毒诱导的基因沉默(virus induced gene silencing,VIGS)技术在烟草中所用接种方式是否适宜有待明确。采用叶片注射法与灌根接种法分别对烤烟云烟87中根、叶差异表达基因NtBBL、NtPPO7进行沉默,明确不同接种方式的沉默效率以及系统分析产生的效应。结果表明,NtBBL基因在根部的相对表达量显著高于叶部,NtPPO7基因在叶部的相对表达量显著高于根部,符合试验需要;对于沉默根、叶部基因,叶片注射法适用于叶部沉默,灌根接种法适用于根部沉默;叶片注射法对叶部高表达基因沉默效率更高,灌根接种法对根部高表达基因沉默效率更高;不同接种方式沉默高表达部位基因后,烟草中BBL活性和PPO活性均有一定程度的影响,叶片注射法接种能更多降低叶部PPO活性,灌根接种法能更多降低根部BBL活性;灌根接种法对降低烟草叶片烟碱含量更有效。针对烟草中不同组织中的高表达基因需选择适宜的接种方式,不同接种方式对不同基因产生的沉默效应不同,本研究为VIGS技术在烟草中的应用提供支撑。

关键词: VIGS, 烟草, 接种方式, 基因表达量, 酶活性

Abstract:

The expression of different genes in different tissues of tobacco is different, and whether the inoculation method using TRV induced gene silencing (VIGS) technology in tobacco is suitable remains to be determined. Leaf injection method and root inoculation method were used to silence NtBBL and NtPPO7 differentially expressed genes in root and leaf of Yunyan 87, respectively, to determine the silencing efficiency of different inoculation methods and the effects of systematic analysis. The results showed that the relative expression level of NtBBL gene in the root was significantly higher than that in the leaf, and the relative expression level of NtPPO7 gene in the leaf was significantly higher than that in the root, which met the needs of the experiment. For silencing genes in root and leaf, leaf injection was suitable for silencing in leaf, and root inoculation was suitable for silencing in root. Leaf injection method has a higher silencing efficiency for high expression genes in leaves, while root inoculation method has a higher silencing efficiency for high expression genes in roots. BBL and PPO activity in tobacco were affected to a certain extent by different inoculation methods after silencing genes at high expression sites. The PPO activity in leaf was decreased by leaf injection method, while BBL activity in root was decreased by root inoculation method. Root inoculation method was more effective in reducing the nicotine content of tobacco leaves. It is necessary to select appropriate inoculation methods for the highly expressed genes in different tissues of tobacco, and different inoculation methods have different silencing effects on different genes. This study provides support for the application of VIGS technology in tobacco.

Key words: VIGS, tobacco, inoculation method, gene expression, enzyme activity

中图分类号:

S572

李伟, 杨再军, 许燕彪, 谢可, 徐秉聪, 吴剑平, 叶性荣, 周建军, 祖琼瑶, 郑聪. 不同接种方式对烟草根、叶差异表达基因沉默效应的影响[J]. 浙江农业科学, 2025, 66(5): 1151-1157.

LI Wei, YANG Zaijun, XU Yanbiao, XIE Ke, XU Bingcong, WU Jianping, YE Xingrong, ZHOU Jianjun, ZU Qiongyao, ZHENG Cong. Silencing effect of different inoculation methods on differentially expressed genes in root and leaf of tobacco[J]. Journal of Zhejiang Agricultural Sciences, 2025, 66(5): 1151-1157.

图/表 8

表1 试验处理

Table 1 Test treatment

处理	侵染液
CK	不接种侵染液
CK1	叶片注射接种含pTRV2的侵染液(空载体对照)
CK2	灌根接种含pTRV2的侵染液(空载体对照)
A1	叶片注射接种含pTRV2-NtBBL侵染液
A2	灌根接种含pTRV2-NtBBL侵染液
B1	叶片注射接种含pTRV2-NtPPO7侵染液
B2	灌根接种含pTRV2-NtPPO7侵染液

表2 RT-qPCR所用引物序列

Table 2 Primers for RT-qPCR

引物名称	引物序列(5'- 3')
NtBBL -F NtBBL -R	TTGCCAAGGTAAGTGACGTTC CCGTCCATCAGCATCAATAAG
NtPPO7 -F NtPPO7 -R	GCACGTTCAGTTCCTCCAGTTA TCGCTGATCGATGTTCAGTTC
26S rRNA-F 26S rRNA-R	GAAGAAGGTCCCAAGGGTTC TCTCCCTTTAACACCAACGG

图1 NtBBL与 NtPPO7在根、叶中的相对表达量图中同组柱上无相同小写字母表示同一基因在不同部位表达差异显著(P<0.05)。图2~3同。

Fig.1 Relative expression levels of NtBBL and NtPPO7 in roots and leaves

图2 不同接种处理的根、叶中NtBBL基因相对表达量

Fig.2 Relative gene expression of NtBBL in root and leaf treated by different inoculation methods

图3 不同接种处理的根、叶中NtPPO7基因相对表达量

Fig.3 Relative gene expression of NtPPO7 in root and leaf treated by different inoculation methods

图4 不同接种处理的烟草根中BBL活性同组柱上无相同小写字母表示不同处理间差异显著(P<0.05),图5~6同。

Fig.4 BBL activity in tobacco root treated with different inoculation methods

图5 不同接种处理的烟草叶中PPO活性

Fig.5 PPO activity in tobacco leaf treated with different inoculation methods

图6 不同接种方式沉默NtBBL基因后的烟草叶片烟碱含量

Fig.6 Nicotine content in tobacco leaf after NtBBL gene silencing by different inoculation methods

参考文献 33

[1]	饶红宇, 黄敏仁, 王明庥. 转基因植物中外源基因的沉默[J]. 南京林业大学学报, 2000, 24(3): 56-60.
[2]	宋震, 李中安, 周常勇. 病毒诱导的基因沉默(VIGS)研究进展[J]. 园艺学报, 2014, 41(9): 1885-1894.
[3]	郝梦媛, 杭琦, 师恭曜. VIGS基因沉默技术在作物基因功能研究中的应用与展望[J]. 中国农业科技导报, 2022, 24(1): 1-13.
[4]	SENTHIL-KUMAR M, MYSORE K S. Virus-induced gene silencing can persist for more than 2 years and also be transmitted to progeny seedlings in Nicotiana benthamiana and tomato[J]. Plant Biotechnology Journal, 2011, 9(7): 797-806.
[5]	李琳琳, 王超群, 李春霞, 等. 木薯MeHsfB3a基因的克隆及其抗细菌性枯萎病功能鉴定[J]. 热带作物学报, 2023, 44(1): 9-16.
[6]	KIM K H, LIM S, KANG Y J, et al. Optimization of a virus-induced gene silencing system with soybean yellow common mosaic virus for gene function studies in soybeans[J]. The Plant Pathology Journal, 2016, 32(2): 112-122.
[7]	李月, 吾尕力汗·阿不都维力, 周垚均, 等. 陆地棉小GTP结合蛋白基因GhRop4的克隆及其表达分析[J]. 棉花学报, 2020, 32(1): 21-29.
[8]	SHI G Y, HAO M Y, TIAN B M, et al. A methodological advance of tobacco rattle virus-induced gene silencing for functional genomics in plants[J]. Frontiers in Plant Science, 2021, 12: 671091.
[9]	傅达奇. 植物中病毒诱导基因沉默技术操作指导[M]. 北京: 中国农业大学出版社, 2016: 4-6.
[10]	TAVARES-ESASHIKA M L, CAMPOS R N S, BLAWID R, et al. Characterization of an infectious clone of pepper ringspot virus and its use as a viral vector[J]. Archives of Virology, 2020, 165(2): 367-375.
[11]	CHENG C X, GAO J P, MA N. Investigation of petal senescence by TRV-mediated virus-induced gene silencing in rose[J]. Methods in Molecular Biology, 2018, 1744: 49-63.
[12]	MENG L H, WANG R H, ZHU B Z, et al. Efficient virus-induced gene silencing in Solanum rostratum[J]. PLos One, 2016, 11(6): e0156228.
[13]	ZHANG J X, WANG F R, ZHANG C Y, et al. A novel VIGS method by agroinoculation of cotton seeds and application for elucidating functions of GhBI-1 in salt-stress response[J]. Plant Cell Reports, 2018, 37(8): 1091-1100.
[14]	RATCLIFF F, MARTIN-HERNANDEZ A M, BAULCOMBE D C. Technical advance: tobacco rattle virus as a vector for analysis of gene function by silencing[J]. The Plant Journal, 2001, 25(2): 237-245.
[15]	FU D Q, ZHU B Z, ZHU H L, et al. Enhancement of virus-induced gene silencing in tomato by low temperature and low humidity[J]. Molecules and Cells, 2006, 21(1) : 153-160.
[16]	RYU C M, ANAND A, KANG L, et al. Agrodrench: a novel and effective agroinoculation method for virus-induced gene silencing in roots and diverse Solanaceous species[J]. The Plant Journal, 2004, 40(2): 322-331.
[17]	DING X S, SCHNEIDER W L, CHALUVADI S R, et al. Characterization of a Brome mosaic virus strain and its use as a vector for gene silencing in monocotyledonous hosts[J]. Molecular Plant-Microbe Interactions, 2006, 19(11): 1229-1239.
[18]	TAKIZAWA M, HORI K, INAI K, et al. A virus-induced gene silencing approach for the suppression of nicotine content in Nicotiana benthamiana[J]. Plant Biotechnology, 2007, 24(3): 295-300.
[19]	刘天波, 蔡海林, 曾维爱, 等. 抗马铃薯Y病毒病的VIGS生防剂发酵条件及施用方法优化[J]. 中国农业科技导报, 2018, 20(11): 29-35.
[20]	李文辰, 刘鑫, 康越, 等. TRV病毒诱导大豆基因沉默体系优化及应用[J]. 生物技术通报, 2023, 39(7): 143-150.
[21]	LEWIS R S, LOPEZ H O, BOWEN S W, et al. Transgenic and mutation-based suppression of a berberine bridge enzyme-like (BBL) gene family reduces alkaloid content in field-grown tobacco[J]. PLoS One, 2015, 10(2): e0117273.
[22]	李玉娥, 尹启生, 宋纪真, 等. 烟草酶促棕色化反应及调控技术研究进展[J]. 中国烟草科学, 2008, 29(6): 71-77.
[23]	BURCH-SMITH T M, ANDERSON J C, MARTIN G B, et al. Applications and advantages of virus-induced gene silencing for gene function studies in plants[J]. The Plant Journal, 2004, 39(5): 734-746.
[24]	THOMAS C L, JONES L, BAULCOMBE D C, et al. Size constraints for targeting post-transcriptional gene silencing and for RNA-directed methylation in Nicotiana benthamiana using a potato virus X vector[J]. The Plant Journal, 2001, 25(4): 417-425.
[25]	LIU E W, PAGE J E. Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus[J]. Plant Methods, 2008, 4: 5.
[26]	TUTTLE J R, IDRIS A M, BROWN J K, et al. Geminivirus-mediated gene silencing from Cotton leaf crumple virus is enhanced by low temperature in cotton[J]. Plant Physiology, 2008, 148(1): 41-50.
[27]	苏龙. 烟草BBL基因对烟草生长发育和烟碱合成的影响[D]. 贵阳: 贵州大学, 2022.
[28]	李琼, 王雪云, 宋卫武, 等. 普通烟草PPO基因家族的鉴定与表达分析[J]. 烟草科技, 2018, 51(8): 9-13.
[29]	郭玉鸽, 张路阳, 党伟, 等. VIGS诱导GS同工酶基因沉默对烤烟氮代谢的影响[J]. 中国烟草学报, 2023, 29(1): 79-87.
[30]	王心宇, 吕坤, 蔡彩平, 等. TRV病毒介导的基因沉默体系在棉花中的建立及应用[J]. 作物学报, 2014, 40(8): 1356-1363.
[31]	姚怡帆, 代卓毅, 江智敏, 等. RNA干扰对烟草NtPPO8基因沉默的效应分析[J]. 作物杂志, 2022(3): 80-86.
[32]	王传义, 吕国新, 朱启法, 等. 烤烟烘烤特性与烟草多酚氧化酶活性相关性研究[J]. 湖北农业科学, 2016, 55(6): 1495-1499, 1503.
[33]	KAJIKAWA M, SHOJI T, KATO A, et al. Vacuole-localized berberine bridge enzyme-like proteins are required for a late step of nicotine biosynthesis in tobacco[J]. Plant Physiology, 2011, 155(4): 2010-2022.