Tobacco mosaic virus gene fragment for efficiently producing siRNA, attenuated vaccine, preparation method and application thereof

A technology of tobacco mosaic virus and gene fragments, which is applied in the field of plant anti-virus genetic engineering, can solve the problems of inconvenient promotion and use of inoculation and multiplication, limitations of genetically modified tobacco, and inability to promote planting, so as to facilitate large-scale application and reduce Injury, delayed effect of plant disease

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
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is a synergistic effect between TMV and cucumber mosaic virus, once combined infection will cause severe symptoms and cause greater losses
[0006] Tian Yingchuan and Fang Rongxiang reported that they obtained transgenic TMV-resistant engineering plants. Tian Bo and Chen Zhangliang also introduced the TMV-CP gene and satellite RNA into tobacco varieties NC89 and C-28 respectively to create transgenic tobacco resistant to TMV. Restrict but cannot promote planting
Yang Gong and Shao Biying screened attenuated TMV strains N14 and TMV-152 and developed them into attenuated vaccines, but they were not popularized due to the inconvenience of vaccination and multiplication

Method used

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  • Tobacco mosaic virus gene fragment for efficiently producing siRNA, attenuated vaccine, preparation method and application thereof
  • Tobacco mosaic virus gene fragment for efficiently producing siRNA, attenuated vaccine, preparation method and application thereof
  • Tobacco mosaic 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 Tobacco Mosaic Virus Related Gene Fragments and Construction of Attenuated Vaccine

[0070] 1. Amplification of gene fragments related to tobacco mosaic virus

[0071] Using the cDNA genome of TMV 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 TMV gene fragment amplification primer sequence

[0073]

[0074] Primers 1 and 2 were used to amplify the TMV1 gene fragment, primers 3 and 4 were used to amplify the TMV 2 gene fragment, and primers 5 and 6 were used to amplify the TMV 3 gene fragment. The nucleotide sequence of the amplified TMV1 gene fragment is shown in Seq ID No.13, the nucleotide sequence of the TMV 2 gene fragment is shown in Seq ID No.14, and the nucleotide sequence of the TMV 3 gene fragment is shown in Seq ID Shown in No.15.

[0075] The ...

Embodiment 3

[0088] Embodiment three attenuated vaccine inoculation plant

[0089]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 tobacco mosaic virus gene fragment for efficiently producing siRNA, an attenuated vaccine, a preparation method and application thereof. The tobacco mosaic virus gene fragment that efficiently produces siRNA includes at least one of a TMV1 fragment, a TMV2 fragment and a TMV3 fragment. The nucleotide sequences of the TMV1 fragment, the TMV2 fragment and the TMV3 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 beinginoculated 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 tobacco mosaic virus. The effective gene fragment comprises the tobacco mosaic virus gene fragment capable of producing siRNA. The attenuated vaccine against tobacco mosaic virus hasstable effects, can play an effective cross-protection role, significantly reduces the damage of plants infected with tobacco mosaic 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 specifically relates to a tobacco mosaic virus gene fragment for efficiently producing siRNA, an attenuated vaccine, 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] 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C12N1/21C12N15/82A01H5/00A01H6/82A01G22/45A01G7/06
CPCC07K14/005C12N15/8283A01G22/45A01G7/06C12N2770/00022
Inventor 耿超姜瀚林刘茜郭兆奎田延平刘锦万秀清刘文涛李向东李若乔婵
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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