高效靶向降解烟草花叶病毒核酸的dsRNA筛选与大量制备

doi:10.3864/j.issn.0578-1752.2021.06.006

中国农业科学 ›› 2021, Vol. 54 ›› Issue (6): 1143-1153.doi: 10.3864/j.issn.0578-1752.2021.06.006

高效靶向降解烟草花叶病毒核酸的dsRNA筛选与大量制备

徐翔¹(),解屹¹,宋丽云¹,申莉莉¹,李莹¹,王勇²,刘明宏³,刘东阳²,王小彦³,赵存孝⁴,王凤龙¹(),杨金广¹()

¹中国农业科学院烟草研究所,山东青岛 266101
²四川省烟草公司凉山州公司,四川西昌 615000
³贵州省烟草公司遵义市公司,贵州遵义 563000
⁴甘肃省烟草公司庆阳市公司,甘肃庆阳 745099

收稿日期:2020-06-10 接受日期:2020-07-11 出版日期:2021-03-16 发布日期:2021-03-25
通讯作者: 王凤龙,杨金广
作者简介:徐翔,E-mail：17854233569@163.com。
基金资助:
烟草绿色防控重大专项(110201901041LS-04);四川省烟草公司科技项目(SCYC201804);四川省烟草公司科技项目(SCYC202008);甘肃省烟草公司科技项目(201862100020017);甘肃省烟草公司科技项目(201862100020016);贵州省烟草公司科技项目(201921)

Screening and Large-Scale Preparation of dsRNA for Highly Targeted Degradation of Tobacco Mosaic Virus (TMV) Nucleic Acids

Xiang XU¹(),Yi XIE¹,LiYun SONG¹,LiLi SHEN¹,Ying LI¹,Yong WANG²,MingHong LIU³,DongYang LIU²,XiaoYan WANG³,CunXiao ZHAO⁴,FengLong WANG¹(),JinGuang YANG¹()

¹Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, Shandong
²Liangshan Company, Sichuan Tobacco Company, Xichang 615000, Sichuan
³Zunyi City Company, Guizhou Tobacco Company, Zunyi 563000, Guizhou
⁴Qingyang Tobacco Company of Gansu Provincial Company, Qingyang 745099, Gansu

Received:2020-06-10 Accepted:2020-07-11 Online:2021-03-16 Published:2021-03-25
Contact: FengLong WANG,JinGuang YANG

摘要/Abstract

摘要：

【目的】筛选高效靶向降解烟草花叶病毒（tobacco mosaic virus,TMV）的dsRNA,实现其大量制备,并探究其作用机制。【方法】以TMV编码的CP、MP、RdRP功能基因为靶序列,体外转录合成相应的dsRNA,浸润本氏烟（Nicotiana benthamiana）,24 h后接种TMV,于接毒后2、3 d取样提取总RNA和蛋白质,以CP基因mRNA水平和蛋白水平为指标,结合TMV病毒生物学症状,综合评价各dsRNA对TMV的抑制效果。同时结合侵染性克隆TMV-30B在本氏烟烟株上的荧光表达现象和TMV在三生烟（Nicotiana tabacum var. Samsun NN）上的过敏性坏死反应（hypersensitive necrosis reaction）,通过比较TMV基因组上6个靶序列相对应的dsRNA,筛选出高效抑制TMV的dsRNA片段。为了获取大量的dsRNA,将dsRNA对应的基因片段插入到原核表达载体L4440的双T7启动子之间,转化至RNase III缺陷型大肠杆菌（Escherichia coli）HT115（DE3）中,并对原核表达制备的dsRNA喷施烟草后生成的siRNA进行深度测序,比较外源施用dsRNA后,对TMV侵染的small RNA表达特征和富集带的影响。【结果】筛选出高效影响TMV CP基因表达的dsRNA RdRP1461-1774,并构建了可诱导形成目的dsRNA的原核表达载体L4440-dsRdRP1461-1774,可在DE3中大量制备RdRP1461-1774的dsRNA,菌液中提取的dsRNA喷施于烟草上对TMV的防治效果显著。TMV-30B侵染本氏烟时荧光数量减少,并能够延长叶片萎蔫时间,在三生烟上施用时叶片枯斑数量明显减少。小RNA测序结果显示TMV侵染引起的RNAi过程中正义链和反义链以大致相等的频率产生siRNA,而外源性dsRNA的浸润会引起靶向区域siRNA的富集,siRNA反义链累积量骤增,对应的正义链累积量骤减,外源dsRNA的施用能够引起siRNA表达丰度的变化。【结论】通过比较dsRNA介导植物靶向抗TMV侵染的效果来筛选抗烟草花叶病毒的dsRNA序列,最终选定TMV RdRP基因上一段长313 bp的高效作用片段,该片段dsRNA能够高效与靶基因结合,降低染病植株烟草花叶病毒的表达量。同时构建了RdRP1461-1774基因的dsRNA原核表达系统,实现其低成本的高效量产,为后续dsRNA在植物病毒方面的防治应用打下了基础。

关键词: 烟草花叶病毒, RNA干扰, dsRNA, small RNA测序, 原核表达

Abstract:

【Objective】The objective of this study is to screen the dsRNAs with high efficiency and targeting degradation of tobacco mosaic virus (TMV), achieve its mass preparation, and to explore the mechanism. 【Method】Using CP, MP and RdRP genes encoded by TMV as target sequences, dsRNAs were synthesized in vitro and infiltrated into Nicotiana benthamiana. After 24 h, TMV was inoculated, total RNA and protein were extracted from samples 2 and 3 d after TMV inoculation, and the mRNA level and protein level of CP gene were used as indicators, combined with the biological symptoms of TMV, to comprehensively evaluate the inhibitory effect of each dsRNA on TMV. The fluorescence expression of TMV-30B on N. benthamianas and the hypersensitive necrosis reaction of TMV on Nicotiana tabacum var. Samsun NN were also combined. The dsRNA fragments that efficiently inhibit TMV were screened by comparing the dsRNAs corresponding to the six target sequences on the TMV genome. In order to obtain a large number of dsRNAs, the corresponding gene fragments of dsRNAs were inserted between the double T7 promoters of the prokaryotic expression vector L4440 and transformed into RNase III deficient Escherichia coli HT115 (DE3). And the siRNA generated after the prepared dsRNA spraying on tobacco was deeply sequenced to compare the effect of exogenous application of dsRNA on the small RNA expression characteristics and enrichment bands of TMV infestation. 【Result】RdRP1461-1774 dsRNAs with high effect on TMV CP gene expression were screened, and L4440-dsRdRP1461-1774 prokaryotic expression vector was constructed, which could induce the formation of target dsRNAs. The dsRNA extracted from bacteria solution could significantly inhibit TMV, when TMV-30B was applied to infect tobacco, the number of fluorescence decreased, the time of leaf wilting prolonged, and the number of necrosis spots decreased significantly. The results of small RNA sequencing showed that in the RNAi process induced by TMV infection, the sense and antisense chains produced siRNA with approximately equal frequency, while the infiltration of exogenous dsRNA resulted in the enrichment of siRNA in the target area, and the accumulation of antisense chain of siRNA increased sharply, the cumulative value of the corresponding sense chain decreased sharply. The application of exogenous dsRNA could change the abundance of siRNA expression. 【Conclusion】The targeted anti-TMV dsRNA sequences were screened by comparing the effects of dsRNA on the targeted anti-TMV infection in plants. Finally, a 313 bp long effective segment of RdRP gene was selected. This fragment of dsRNA can efficiently bind to the target gene and reduce the expression of TMV in infected plants. At the same time, a dsRNA prokaryotic expression system of RdRP1461-1774 gene was constructed to achieve low-cost and high-efficiency production, which provided the basis for the follow-up application of dsRNAs in the control of plant virus.

Key words: tobacco mosaic virus (TMV), RNA interference (RNAi), dsRNA, small RNA sequencing, prokaryotic expression

徐翔,解屹,宋丽云,申莉莉,李莹,王勇,刘明宏,刘东阳,王小彦,赵存孝,王凤龙,杨金广. 高效靶向降解烟草花叶病毒核酸的dsRNA筛选与大量制备[J]. 中国农业科学, 2021, 54(6): 1143-1153.

Xiang XU,Yi XIE,LiYun SONG,LiLi SHEN,Ying LI,Yong WANG,MingHong LIU,DongYang LIU,XiaoYan WANG,CunXiao ZHAO,FengLong WANG,JinGuang YANG. Screening and Large-Scale Preparation of dsRNA for Highly Targeted Degradation of Tobacco Mosaic Virus (TMV) Nucleic Acids[J]. Scientia Agricultura Sinica, 2021, 54(6): 1143-1153.

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图/表 10

图1

表1

本研究所用引物"

引物 Primer	引物序列 Primer sequence (5′-3′)	产物长度 Expected size (bp)	引物用途 Use of primer
TMVCP-F	5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGATGTCTTACAGTATCACTACTCC-3′	548	克隆带启动子的CP基因 CP gene with promoter fragment cloning
TMVCP-R	5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGAGTTGCAGGACCAGAGG-3′	548	克隆带启动子的CP基因 CP gene with promoter fragment cloning
TMVMP-F	5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGGGAAAGAGCCGACGAG-3′	401	克隆带启动子的MP基因 MP gene with promoter fragment cloning
TMVMP-R	5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGGCAAGCCTGATTGACATA-3′	401	克隆带启动子的MP基因 MP gene with promoter fragment cloning
TMVP126-F	5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGTCTTACCGTCGATGTTT-3′	709	克隆带启动子的P126基因 P126 gene with promoter fragment cloning
TMVP126-R	5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGGTTCTTGTTCGGCACT-3′	709	克隆带启动子的P126基因 P126 gene with promoter fragment cloning
TMVRdRP851-1238F	5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGCTTACTTCCCGGCCTCTA-3′	456	克隆带启动子的RdRP851-1238基因 RdRP851-1238 gene with promoter fragment cloning
TMVRdRP851-1238R	5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGGCTTTCGCCTGGTATGTT-3′	456
TMVRdRP1461-1774F	5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGATTTCGCTGGCGTTTG-3′	381	克隆带启动子的RdRP1461-1774基因 RdRP1461-1774 gene with promoter fragment cloning
TMVRdRP1461-1774R	5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGCTGCCGTCATTGGGTC-3′	381
TMVRdRP1573-2330F	5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGTGACCTTCCACGACAGA-3′	825	克隆带启动子的RdRP1573-2330基因 RdRP1573-2330 gene with promoter fragment cloning
TMVRdRP1573-2330R	5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGAGCGCCACATGATACTT-3′	825
L4440-dsRdRP1461-1774F	5′-GGAGACCGGCAGATCTATTTCGCTGGCGTTTGGG-3′	_	重组表达载体的构建 Construction of recombinant expression vector
L4440-dsRdRP1461-1774R	5′-TATAGGGCGAATTGGGTACCCTGCCGTCATTGGGTCAAC-3′	_	重组表达载体的构建 Construction of recombinant expression vector
L4440-F	5′-CTTTATAGTCCTGTCGGGTTTCGC-3′	_	重组表达载体测序 Sequencing of recombinant expression vector
L4440-R	5′-GGCGAGAAAGGAAGGGAAGAAAG-3′	_	重组表达载体测序 Sequencing of recombinant expression vector
Actin-F	5′-CAAGGAAATCACCGCTTTGG-3′	_	Actin基因转录水平的检测 Detection of Actin gene transcript level
Actin-R	5′-AAGGGATGCGAGGATGGA-3′	_	Actin基因转录水平的检测 Detection of Actin gene transcript level
TMV-F	5′-GAGTAGACGACGCAACGG-3′	_	TMV CP基因转录水平检测 Detection of TMV CP transcript level
TMV-R	5′-CCAGAGGTCCAAACCAAAC-3′	_	TMV CP基因转录水平检测 Detection of TMV CP transcript level

表1

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高效靶向降解烟草花叶病毒核酸的dsRNA筛选与大量制备

Screening and Large-Scale Preparation of dsRNA for Highly Targeted Degradation of Tobacco Mosaic Virus (TMV) Nucleic Acids

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