Potato Y virus gene fragment for efficiently producing siRNA, attenuated vaccine, preparation method and application thereof

A technology of attenuated vaccines and virus genes, applied in the field of plant anti-virus genetic engineering, can solve the problems of limited safety of transgenics, inability to promote planting, severe symptoms, etc., and achieve the effect of delaying plant disease, stabilizing effect, and reducing damage

Active Publication Date: 2020-03-03
中国烟草总公司黑龙江省公司烟草科学研究所 +1
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

There is a synergistic effect between PYV and Potatovirus Y, once co-infected, it will cause severe symptoms and cause greater losses
Wu Yuanhua and Zhu Changxiang introduced potato virus Y replicase gene (NIB) and capsid protein gene (CP) into tobacco, and obtained PVY-resistant transgenic tobacco, but they could not be promoted because of the limited safety of transgene

Method used

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  • Potato Y virus gene fragment for efficiently producing siRNA, attenuated vaccine, preparation method and application thereof
  • Potato Y virus gene fragment for efficiently producing siRNA, attenuated vaccine, preparation method and application thereof
  • Potato Y virus gene fragment for efficiently producing siRNA, attenuated vaccine, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Construction of embodiment one multi-site TVBMV attenuated mutant

[0050] 1. Construction of an infectious clone of tobacco vein mosaic virus

[0051] Tobacco vein mosaic virus RNA was used as a template for reverse transcription with random primers. According to the existing restriction enzyme map of the whole genome of tobacco vein mosaic virus, it can be amplified in three parts, and assembled into a TVBMV full-length cDNA clone after enzyme digestion. First, the 35S promoter was fused to the upstream of the fragment from the 5' untranslated region of TVBMV to the Nru I restriction site of the HC-pro gene by Overlap-PCR, and the inventor named the fragment p35S-HC; PCR amplified HC- The fragment between the pro gene Nru I restriction site and the 6K2Xho I restriction site, the inventor named the fragment pHC-6K2; PCR amplification of the 6K2Xho I restriction site to the poly(A) tail Fragment, the inventor named the fragment p6K2-polyA (such as figure 1 shown).

...

Embodiment 2

[0069] Example 2 Amplification of Potato Virus Y Related Gene Fragments and Construction of Attenuated Vaccine

[0070] 1. Amplification of gene fragments related to potato virus Y

[0071] Using the cDNA genome of PVY as a template, each gene fragment was amplified by RT-PCR. The examples provided by the present invention are all according to conventional experimental conditions, wherein the primer sequences used are as follows:

[0072] Table 2 Sequences of primers for amplification of PVY gene fragments

[0073] Numbering sequence (5'-3') Seq ID Number 1 GCTCTAGAGCCACCACTTGCTTCTCAGACA Seq ID No.7 2 CTTAATTAACAAAGGGATGGTGGCAGGACTTC Seq ID No.8 3 GCTCTAGAGACCTACTTAGAGCCAGAGACTACGG Seq ID No.9 4 CTTAATTAAGTACATCACATTCGCACTACACACTTGG Seq ID No.10 5 GCTCTAGAA ATGGTATTCCTCAAGTCGCAGTGG Seq ID No.11 6 CTTAATTAAAAAGAAAGTTTCTGAGGCGGGGA Seq ID No.12

[0074] Primers 1 and 2 were used to amplify the PVY1 gene fragment,...

Embodiment 3

[0089] Embodiment three attenuated vaccine inoculation plant

[0090]The single attenuated vaccine vector constructed in Example 2 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), rifamycin (50 μg / mL), and tetracycline (50 μg / mL). Add 500 μL of bacterial liquid to 5 mL of LB medium containing 10 mmol / L 2-(N-morpholine)-ethanesulfonic acid (MES), 20 μmol / L acetosyringone (AS) and the above three antibiotics, at 28 °C Shake culture to logarithmic growth phase. Collect the bacteria by centrifugation and resuspend in 10mmol / L MgCl 2 , 10mmol / L MES, 150μmol / L AS, adjust the concentration so that its OD600 is about 0.5, and let stand at room temperature for 3 hours. Take a 5mL disposable syringe, remove the needle to absorb the Agrobacterium bacteria liquid, and infiltrate from the back of the leaves of ordinary tobacco (5-6 weeks old or 4-6 true leaves). Infiltrate ...

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Abstract

The invention relates to the field of plant antiviral genetic engineering, and discloses a potato Y virus gene fragment for efficiently producing siRNA, an attenuated vaccine, a preparation method andapplication thereof. The potato Y virus gene fragment that efficiently produces siRNA includes at least one of a PVY1 fragment, a PVY2 fragment and a PVY3 fragment. The nucleotide sequences of the PVY1 fragment, the PVY2 fragment and the PVY3 fragment are shown as Seq ID No. 13, Seq ID No. 14 and Seq ID No. 15, respectively. The gene fragment can produce siRNA efficiently after being inoculated with parasitic plants. The attenuated vaccine is based on the attenuated mutant of TVBMV. The attenuated mutant of TVBMV is embedded with an effective gene fragment that can induce cross protection against potato Y virus. The effective gene fragment comprises the potato Y virus gene fragment capable of producing siRNA. The attenuated vaccine against potato Y virus has stable effects, can play an effective cross-protection role, significantly reduces the damage of plants infected with potato Y virus virulent strains, delays the onset of plants, and greatly reduces losses.

Description

technical field [0001] The invention belongs to the field of plant anti-virus genetic engineering, and in particular relates to a gene fragment of potato virus Y efficiently producing siRNA, an attenuated vaccine, a preparation method and 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] Tobacco virus disease has always been an important factor restricting tobacco production in my country. At present, the main tobacco varieties in production are not ideal for the resistance to viral diseases, and the prevention and treatment of tobacco viral diseases ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C12N7/04C12N1/21C12N15/82A01H5/00A01H6/82C12R1/01
CPCC07K14/005C12N7/00C12N15/8283C12N15/8205C12N2770/34022C12N2770/34021
Inventor 田延平姜瀚林刘茜万秀清耿超李现道郭思达李向东李若乔婵
Owner 中国烟草总公司黑龙江省公司烟草科学研究所
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