Construction and application of infectious clone expression vector of watermelon mosaic virus

A watermelon mosaic virus and expression vector technology, applied in the field of plant genetic engineering, can solve the problems of reduced infectivity, instability, loss, etc., and achieve the effects of high-efficiency expression of foreign proteins, increased error rate, and reduced error rate

Inactive Publication Date: 2018-04-27
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are many technical difficulties in constructing plant virus-infectious clones, for example: 1) Due to the different genome structures of different plant viruses, when constructing virus-infectious clones, it is first necessary to reverse transcribe the viral RNA into cDNA, and then base the first-strand cDNA synthesizes full-length DNA. For viruses with longer genomes, it is difficult to synthesize full-length DNA due to the problems of RNA purity, reverse transcriptase and DNA polymerase amplification efficiency.
2) In the process of constructing virus-infectious clones, the introduction of non-viral nucleotide sequences at the 5' an

Method used

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  • Construction and application of infectious clone expression vector of watermelon mosaic virus
  • Construction and application of infectious clone expression vector of watermelon mosaic virus
  • Construction and application of infectious clone expression vector of watermelon mosaic virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1: the purification of watermelon mosaic virus virion virus RNA

[0054] Take 20g of zucchini diseased leaves infected by watermelon mosaic virus, grind them with liquid nitrogen, add 60mL of extraction buffer, mix well and put them on ice. Filter through magic filter cloth, transfer the supernatant to a centrifuge tube, centrifuge at 15000g, 4°C for 20min. After the supernatant was filtered through a 0.22 μM membrane, it was ultracentrifuged at 40,000 g for 1.5 h at 4°C. Discard the supernatant, add 1.5mL 0.02M phosphate buffer (pH7.0) to dissolve the precipitate.

[0055] Take 300 μL of virus particles and add an equal amount of lysate (40 mM Tris-HCl, pH=9.0; 2 mM EDTA; 2% SDS), and vortex to mix. Add 100U RNase inhibitor and an equal volume of water-saturated phenol, vortex and mix well, and incubate at 60°C for 1min. Let stand at room temperature for 5 minutes, centrifuge at 13000g for 5 minutes at 4°C. Transfer the supernatant to a new 1.5mL centrif...

Embodiment 2

[0056] Embodiment 2: Construction of watermelon mosaic virus infectious clone

[0057] Using the viral RNA obtained in Example 1 as a template, reverse transcription was performed with random primers and Oligo dT. The reverse transcription reaction procedure is as follows:

[0058]

[0059] Incubate at 65°C for 5 minutes, quickly place on ice to cool for 2 minutes, and spin off.

[0060]

[0061] 25°C, 5min: 50°C, 45min: 85°C, 5min to terminate the reaction.

[0062] Using the obtained reverse transcription product as a template, use Phusion high-fidelity polymerase for PCR amplification. The PCR reaction system is as follows:

[0063]

[0064] Reaction procedure:

[0065]

[0066] The PCR product was subjected to 1% agarose gel electrophoresis, and the gel was cut and recovered for later use to obtain the whole genome PCR product of WMV.

[0067] The 35S promoter gene amplified by PCR and the pCambia0390 plasmid were ligated by Xma I and Pst I after double dig...

Embodiment 3

[0068] Example 3: Watermelon Mosaic Virus Infectious Cloning and Expression of Foreign Protein Vector Construction

[0069] Using Phusion high-fidelity polymerase PCR amplification to obtain GFP, the sequence is shown in SEQ ID No.10, and the reaction system is as follows:

[0070]

[0071] Reaction procedure:

[0072]

[0073]

[0074] The PCR product was subjected to 1% agarose gel electrophoresis, and after gel cutting and recovery, it was connected to the vector pCaWMV by homologous recombination, and the invasive expression vector was named pCaWMV-GFP ( figure 1 ).

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Abstract

The invention discloses an infectious clone expression vector of watermelon mosaic virus, wherein the infectious clone expression vector is prepared by cloning whole genome of the watermelon mosaic virus, in a homologous recombination manner, to a binary vector, pCambia 0390, which contains the 35S promoter of cauliflower mosaic virus. The nucleotide sequence of the infectious clone expression vector of the watermelon mosaic virus is represented as the SEQ ID No.9. In the invention, for the first time, the infectious clone of the watermelon mosaic virus, which can be inoculated in a manner ofagrobacteria infiltration and stably and high-effectively infect muskmelons, Cucurbita pepo and the like host plants, can be obtained; the infectious clone also can high-effectively express a heterologous protein.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to the construction and application of a watermelon mosaic virus invasive clone expression vector. Background technique [0002] An invasive clone refers to an invasive cDNA or an invasive RNA that can be produced by in vitro transcription (Boyer et al., 1994). Infectious clones of plant viruses can be divided into two types: one is invasive RNA, which constructs a vector containing the cDNA sequence of the full-length viral genome. After linearization, it is transcribed in vitro by the corresponding RNA polymerase into a Infection active RNA; the other is the infectious cDNA, the virus genome cDNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter, which can be directly used for mechanical friction inoculation, gene gun inoculation or agricultural Bacillus co-infiltration inoculation. [0003] Watermelon mosaic virus (WMV) is one of the main m...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N1/21C12R1/01
CPCC12N15/8205
Inventor 田延平冀树娴李向东
Owner SHANDONG AGRICULTURAL UNIVERSITY
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