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Potato leaf roll virus infectious clone and construction method thereof

A leaf-roll virus and a construction method are applied in the field of plant genetic engineering and can solve the problems of reduced infectivity, unstable cDNA transfer, and difficulty in obtaining infective clones.

Pending Publication Date: 2019-05-24
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the construction of full-length infectious cDNA clones is highly technical, and there are many technical difficulties in constructing plant virus infectious clones. Reverse transcribe viral RNA into cDNA, and then synthesize full-length DNA based on the first-strand cDNA. For viruses with longer genomes, it is difficult to synthesize full-length due to the problems of RNA purity, reverse transcriptase and DNA polymerase amplification efficiency. DNA
2) In the process of constructing virus-infectious clones, the introduction of non-viral nucleotide sequences at the 5' and 3' ends of the viral genome will also have a great impact on the infectivity of transcripts, reducing infectivity or even lost
4) The synthetic cDNA is very unstable when transferred into the host bacteria, especially RNA viruses with large genomes, which makes it difficult to obtain infectious clones
Based on the above reasons, only a small number of plant viruses have obtained infectious clones, and so far there has been no report on the construction of potato leafroll virus infectious clones

Method used

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  • Potato leaf roll virus infectious clone and construction method thereof
  • Potato leaf roll virus infectious clone and construction method thereof
  • Potato leaf roll virus infectious clone and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: the extraction of potato leafroll virus RNA

[0046] Total plant RNA was extracted using the TRIzol method, and the extraction method was referred to the description of the plant RNA extraction kit.

[0047] Take 0.1g of potato diseased leaves infected by potato leafroll virus, add it to a pre-cooled mortar, grind it into powder with liquid nitrogen, transfer it to a sterilized 1.5ml centrifuge tube, add 1mL TRIzol and shake vigorously for 30s. After incubating at room temperature for 5 min, 0.2 ml of chloroform was added, shaken vigorously for 15 s, and then incubated at room temperature for 3 min. After centrifugation at 10,000 g for 15 min at 4°C, take 0.5 ml of the upper aqueous phase into a new centrifuge tube, add 0.5 ml of isopropanol to mix by inversion, and incubate at room temperature for 10 min. Centrifuge at 10000g for 10min at 4°C to remove the supernatant, add 1ml of 75% ethanol and shake vigorously. After centrifugation at 7500g for 5 minu...

Embodiment 2

[0048] Example 2: Construction of Potato Leafroll Virus Infectious Clones

[0049] Using the viral RNA obtained in Example 1 as a template, reverse transcription was performed with random primers and primer 4. The reverse transcription reaction procedure is as follows:

[0050]

[0051]

[0052] Incubate at 65°C for 5 minutes, quickly place on ice to cool for 2 minutes, and spin off.

[0053]

[0054] 25°C, 5min; 50°C, 45min; 85°C, 5min to terminate the reaction. Using the obtained reverse transcription product (cDNA) as a template, use Phusion high-fidelity polymerase for PCR amplification. The PCR reaction system is as follows:

[0055]

[0056] Reaction procedure:

[0057]

[0058] The PCR product was subjected to 1% agarose gel electrophoresis, the gel was cut and recovered for future use, and the PCR product of the PLRV 1-3161bp fragment was obtained. Using primers 5 and 6 to amplify the pCB301 vector sequence, the annealing temperature was 60°C, and th...

Embodiment 3

[0059] Example 3: Determination of the infectivity effect of pCB-PLRV

[0060] The pCB-PLRV plasmid was transformed into Agrobacterium GV3101. After colony PCR verification, pick a single spot and inoculate it in liquid LB medium containing kanamycin (50 μg / mL) and rifamycin (50 μg / mL). Take 500 μL of the bacterial solution and add it to 5 mL of 10 mmol / L 2-(N-morpholine)-ethanesulfonic acid (MES) (50 μL) and 20 μmol / L acetosyringone (AS) (1 μL) and the same antibiotic as in the previous step In LB medium, shake culture at 28°C until logarithmic growth phase. The cells were collected by centrifugation and resuspended in Agrobacterium supersuspension solution (10mmol / L MgCl 2 (100mL), 10mmol / L MES (1mL), 0.15μmol / L AS (150μL)), adjust the concentration to make OD 600 Between 0.5-0.6, stand at room temperature for 3 hours. Take a 1mL disposable syringe, remove the needle to absorb the Agrobacterium liquid, and infiltrate Nicotiana benthamiana and potatoes. Infiltrate 2 leav...

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Abstract

The invention discloses a potato leaf roll virus infectious clone and a construction method thereof. The potato leaf roll virus infectious clone is obtained by cloning a whole genome of a potato leafroll virus into a pCB301 binary vector containing a cauliflower mosaic virus double 35S promoter in a homologous recombination mode. The potato leaf roll virus infectious clone which can be inoculatedby an agrobacterium infiltration method and stably and efficiently infects host plants such as nicotiana benthamiana and potatoes is prepared for the first time, and the potato leaf roll virus infectious clone has important significance for preventing and treating potato leaf roll virus diseases.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a potato leafroll virus invasive clone and a construction method thereof. Background technique [0002] Potato leafroll virus (PLRV) is a representative species of the genus Polerovirus. After infecting potatoes, it causes symptoms such as rolling up and yellowing of young leaves along the veins, purple bases of leaves, and reticular necrosis of tubers. , is one of the most serious viruses affecting potato production in my country. [0003] PLRV is mainly confined to the phloem of the host and is transmitted in a persistent manner by aphids. PLRV cannot be transmitted mechanically by friction, but can be infected by Agrobacterium-mediated inoculation. Therefore, obtaining an infectious clone of potato leafroll virus has broad application prospects. [0004] Infectious cloning refers to the construction of the full-length genome sequence of the virus on a speci...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N1/21C12R1/01
Inventor 田延平王健李向东耿超
Owner SHANDONG AGRICULTURAL UNIVERSITY
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