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

Abstract

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

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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https://www.chinaagrisci.com/EN/Y2021/V54/I6/1143

Figures/Tables 10

Fig. 1

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′	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

Table 1

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

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