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Preparation and application of small interfering RNA for preventing tobacco mosaic virus diseases

A technology of tobacco mosaic virus and small interference, applied in the direction of DNA preparation, chemicals for biological control, DNA/RNA fragments, etc., can solve problems such as difficult control, high inactivation temperature, and threat to high-quality tobacco leaf production

Inactive Publication Date: 2009-11-04
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The economic loss caused by TMV in the world exceeds 100 million U.S. dollars every year; in my country, it occurs in both the north and south tobacco areas, especially in the south tobacco area. The incidence of field strains is generally 5% to 20%, and individual fields can reach 30% to 100%, the loss of early disease can reach 50% to 70%, or even loss of harvest; in addition, the quality grade of infected tobacco leaves will also decline, and the quality will deteriorate, posing a great threat to the production of high-quality tobacco leaves
At the same time, the inactivation temperature of TMV in vitro is relatively high. The virus in the juice loses its pathogenicity after being treated at 75°C for 40 days, and the dry diseased leaves do not lose their pathogenicity after 30 minutes at 120°C. It is quite difficult to control

Method used

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  • Preparation and application of small interfering RNA for preventing tobacco mosaic virus diseases
  • Preparation and application of small interfering RNA for preventing tobacco mosaic virus diseases
  • Preparation and application of small interfering RNA for preventing tobacco mosaic virus diseases

Examples

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preparation example Construction

[0067] (1) Preparation of CaCl2 Competent Cells

[0068] The recipient bacteria cultured overnight were transferred to 50ml LB at a ratio of 2%, shaken at 37°C for 1.5h to logarithmic growth phase, centrifuged at 4000rpm for 2min, supernatant was discarded, and the bacteria were suspended in 10ml of ice-cold 100mM CaCl2. Cool in ice for 30min, centrifuge at 4000rpm for 2min at 4°C, suspend the bacteria in 2ml ice-cooled 100mM CaCl 2 Store in an ice bath for later use.

[0069] (2)CaCl 2 Transformation of Competent Cells

[0070] Transformation of plasmid DNA and its ligation products: Take 20ng of plasmid DNA or an appropriate amount of ligation products, add 100μl competent cells, mix gently, place in ice bath for 30min, heat shock at 42°C for 90s, add 1ml LB culture medium, and incubate at 37°C After incubation for 45 min, 200 μl was spread on LB plates containing appropriate antibiotics, and cultured upside down at 37°C overnight.

[0071] (3) Propagation of Escherichia...

Embodiment 1

[0088] Design and synthesis of 19 oligonucleotide (nt) sequences corresponding to the TMV coat protein coding region:

[0089] According to the mRNA corresponding to the TMV coat protein, design and synthesize the siRNA expressing the small interfering RNA, and the design and synthesis are designed according to the following principles: (1) design from the 3' end of the ORF of the target protein after 300 bp, avoiding the regulatory recognition region; (2) ) Select the 19nt Oligo starting with AA, and make the G+C content 30-50%; (3) Loop base is TTCAAGAGA; (4) 4-6 TT regions of RNaseIII binding termination transcription site are added at the end of Oligo , the tail avoids G. According to the requirements of the pBI121 vector, add corresponding restriction endonuclease recognition sequences at both ends of Oligo, such as adding BamH I recognition sequence at the 3' end and Sst I recognition sequence at the 5' end. A HindIII recognition sequence was added downstream of BamH I ...

Embodiment 2

[0093] Development of biological agents acting on the TMV replicase gene:

[0094] (1) According to the 1519-1537 and 2129-2147 fragments of the coding region corresponding to the TMV 126kDa protein (underlined part, the same below), design and synthesize the siRNA1519 sequence expressing small interfering RNA:

[0095] F: 5'-GATCCAAGCTTCG ACTTATCAGAGTGGCAGGC TTCAAGAGA GCCTGCCACTCTGATAAGT TTTTTGAGCT-3'

[0096] R: 5'-CAAAAA ACTTATCAGAGTGGCAGGC TCTCTTGAA GCCTGCCACTCTGATAAGT CGAAGCTTG-3'

[0097] siRNA2129:

[0098] F: 5'-GCTGGATCCAAGCTTCG TTCGTAAGCAGATGAGCTC TTCAAGAGA GAGCTCATCTGCTTACGAA TTTTTGAGCTCCTG-3'

[0099] R: 5'-CAGGAGCTCAAAAA TTCGTAAGCAGATGAGCT CTCTCTTGAA GAGCTCATCTGCTTACGAA CGAAGCTTGGATCCAGC-3'

[0100] (2) The pBI121 plasmid was digested with BamH I and Sst I, and the annealed Oligo DNA was ligated with the digested product with T4 DNA ligase to construct a recombinant expression vector pBI121 / siRNA for siRNA.

[0101] (3) Transformation into compet...

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Abstract

The invention relates to preparation and application of small interfering RNA for preventing tobacco mosaic virus diseases, wherein the small interfering RNA can effectively interfere in the infection of the tobacco mosaic virus and prevent various plant virus diseases caused by the tobacco mosaic virus. The preparation method comprise the following steps: selecting a replicative enzyme gene, a coat protein gene and a movement protein gene of tobacco mosaic virus as target sites; and designing the small interfering RNA according to the following method: after 300 bp of an open reading frame 3' end, avoiding an adjust and control recognition zone; selecting 19nt oligonucleotide taking an AA as a beginning and containing 30 to 50 percent of G+C; using TTCAAGAGA as a cyclo-base; adding 4 to 6 TT zones of an RNase III combined termination transcription site to an Oligo tail end; avoiding G at the tail part; adding a BamH I recognition sequence at an Oligo 3' end and an Sst I recognition sequence at a 5' end; adding an Hind III recognition sequence to the downstream of the BamH I; and biologically synthesizing the small interfering RNA which is homologous with a TMV genome.

Description

1. Technical field: [0001] The present invention relates to a novel biological preparation for preventing tobacco mosaic virus (Tobacco Masaic Virus-TMV) infection, in particular to a kind of small interfering RNA (small interfering RNA-siRNA) for preventing tobacco mosaic virus disease The preparation belongs to the technical field of biopharmaceuticals. 2. Background technology: [0002] The RNA interference phenomenon refers to that when a double-stranded RNA (dsRNA) homologous to a certain sequence of the endogenous mRNA coding region is introduced into the cell, the mRNA is specifically degraded, resulting in the silencing of the gene expression. It belongs to the category of posttranscriptional gene silencing (PTGS) mechanism, and is considered to be a very advanced defense method developed by plants during the evolution process to resist the invasion of foreign genes (viruses, transposons, etc.). At present, RNAi phenomena have been found in Drosophila, Trypanosomes,...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10C12N15/70C12N15/74A01N63/02A01P1/00C12R1/01C12R1/19C12N15/113
Inventor 安德荣赵明敏张文斌
Owner NORTHWEST A & F UNIV
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