Potato Y virus attenuated strain, vector, preparation method and application thereof

A technology of attenuated strains and potatoes, which is applied in the field of carriers, preparations, and attenuated strains of potato Y virus to achieve the effects of delaying disease onset, reducing injury and reducing losses

Inactive Publication Date: 2020-03-03
SHANDONG AGRICULTURAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are also some PVY isolates whose HC-Pro protein contains two amino acid

Method used

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  • Potato Y virus attenuated strain, vector, preparation method and application thereof
  • Potato Y virus attenuated strain, vector, preparation method and application thereof
  • Potato Y virus attenuated strain, vector, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The construction of embodiment one potato virus Y infectious clone

[0043] Reverse transcription was performed with random primers using the RNA of potato virus Y as a template. According to the restriction enzyme map of the whole genome of the existing potato virus Y, it can be amplified in multiple parts, and assembled into a full-length cDNA clone of the potato virus Y after enzyme digestion.

[0044] PCR amplification was carried out using the obtained reverse transcription product as a template and corresponding primers. PCR products were subjected to 1% agarose gel electrophoresis. Multiple fragments were obtained after gel cutting and recovery, and multiple fragments were connected to the Agrobacterium-mediated expression vector pCAMBIA0390. The inventor named the invasive clone constructed by this strategy as pCamPVY N .

Embodiment 2

[0045] Embodiment two: the acquisition of attenuated toxic isolate

[0046]156 samples of potato virus disease were collected from Heilongjiang, and the results of ELISA showed that PVY was detected in 139 samples. 21 days after inoculation with common tobacco, 16 isolates including A12 only caused mild mosaic symptoms, but according to the results of amino acid sequence analysis, the HC-Pro of isolate A12 belonged to PVY N strain. By comparing isolate A12 with other PVY N HC-Pro amino acid sequence discovery of strains, generally PVY N The 182nd amino acid of strain HC-Pro is lysine (K), while the 182nd amino acid of isolate A12 is (R).

Embodiment 3

[0047] Example 3: Site-directed mutagenesis

[0048] Infectious Cloning of pCamPVY Using Potato Virus Y N As a template, PCR was used for mutation. The examples provided by the present invention are all according to conventional experimental conditions, wherein the primer sequences used are as follows:

[0049] Table 1 Site-directed mutagenesis primer sequences

[0050]

[0051] Among them, primers 1 and 2 were used to mutate the K at position 182 of HC-Pro into R. Bold letters represent mutated nucleotide positions. The nucleotide sequence of HC-Pro before mutation is shown in Seq ID No.3, and the amino acid sequence is shown in Seq ID No.4.

[0052] Infectious Cloning of pCamPVY Using Potato Virus Y N As a template, PCR was used for mutation, and the polymerase used was Phusion high-fidelity polymerase (Finnzymes).

[0053] The PCR reaction system is as follows:

[0054]

[0055] After the reaction was completed, 0.5 μL of DpnI (20 U / μL) was added to the PCR pro...

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PUM

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Abstract

The invention relates to the field of plant antiviral genetic engineering, and discloses a potato Y virus attenuated strain, a vector, a preparation method and applications thereof. In the attenuatedstrain of potato Y virus, the lysine at the 182nd site of the amino acid sequence of an HC-Pro gene is mutated to arginine. The nucleotide sequence of HC-Pro is shown as Seq ID No. 3, and the amino acid sequence of HC-Pro is shown as Seq ID No. 4. According to the invention, a potato Y virus attenuated strain mutant with obviously reduced pathogenicity is obtained by the method of site-directed mutation. The attenuated strain mutant can play an effective cross-protection role, significantly reduce the damage of plants infected with potato Y virus virulent strains, delay the onset of plants, and greatly reduce losses.

Description

technical field [0001] The invention belongs to the field of plant anti-virus genetic engineering, and in particular relates to an attenuated strain of potato virus Y, a carrier, a preparation method and an application thereof. Background technique [0002] Viral diseases are important diseases on crops, causing huge losses to agricultural production. Because there are many kinds of crop viral diseases, the transmission routes are complicated, there are no varieties with immunity or high resistance to viral diseases in production, and there are no effective medicaments for viral diseases on the market, so the prevention and treatment of viral diseases is very difficult. [0003] Potato virus Y (PVY) belongs to the genus Potyvirus and has a wide host range. It can infect a variety of Solanaceae crops including potato, tobacco, tomato and pepper, causing huge economic losses. [0004] At present, there are many studies on the division of PVY strains. According to its symptom...

Claims

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Application Information

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IPC IPC(8): C12N7/04C12N15/82C12N1/21A01H5/00A01H6/82C12R1/01
CPCC12N7/00C12N15/8205C12N15/8283C12N2770/34021
Inventor 李向东程林发刘茜万秀清田延平李现道郭思达耿超乔婵李若
Owner SHANDONG AGRICULTURAL UNIVERSITY
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