Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (6): 1143-1153.doi: 10.3864/j.issn.0578-1752.2021.06.006

• PLANT PROTECTION • Previous Articles     Next Articles

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()   

  1. 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 E-mail:17854233569@163.com;wangfenglong@caas.cn;yangjinguang@caas.cn

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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

Fig. 1

Schematic diagram of TMV gene fragment"

Table 1

Primers used in this study"

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′
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′
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′
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′
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′
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′
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′
L4440-F 5′-CTTTATAGTCCTGTCGGGTTTCGC-3′ _ 重组表达载体测序
Sequencing of recombinant expression vector
L4440-R 5′-GGCGAGAAAGGAAGGGAAGAAAG-3′
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′
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′

Fig. 2

dsRNA electrophoresis of target genes 1: CP dsRNA; 2: MP dsRNA; 3: P126 dsRNA; 4: RdRP851-1238 dsRNA; 5: RdRP1461-1774 dsRNA; 6: RdRP1573-2330 dsRNA"

Fig. 3

Effects of different dsRNA treatments on the expression of wild-type TMV Analysis of variance (SPSS software) was conducted using Duncan’s multiple range test, different lowercase letters on the bars indicate significant difference (α=0.05)"

Fig. 4

Expression of CP protein of virus in infected leaves after different dsRNA treatments The expression of TMV CP protein after infiltration of PBS, TMV CP dsRNA, MP dsRNA, P126 dsRNA, RdRP851-1238 dsRNA, RdRP1461-1774 dsRNA, RdRP1573-2330 dsRNA"

Fig. 5

Symptoms of TMV-30B inoculation after different dsRNA treatments of N. benthamiana"

Fig. 6

Symptoms of Samsun NN leaves infiltrated with dsRNA after 3 d of TMV inoculation"

Fig. 7

Expression and identification of recombinant vector"

Fig. 8

Samsun NN after 3 d of TMV inoculation"

Fig. 9

Small RNA sequencing and personalized analysis map"

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