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RNAi nano preparation and preparation method and application thereof in prevention and treatment of TMV

A nano preparation, chitosan nano technology, applied in genetic engineering technology and its application field, can solve the problems of lack, complicated preparation process of pharmaceuticals, poor dsRNA stability and delivery efficiency, etc., to reduce the expression amount and promote the practice and development. , The effect of not being easily degraded

Active Publication Date: 2020-08-11
TOBACCO RES INST CHIN AGRI SCI ACAD +2
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In order to solve the problems of the current lack of efficient RNAi nano-preparation against TMV, the preparation process of the medicament is complicated, and the stability and delivery efficiency of dsRNA are poor, the present invention provides a RNAi nano-preparation and its preparation method and its application in the prevention and treatment of TMV. Screening out a highly effective anti-TMV replication and proliferation fragment, providing a convenient preparation method for this RNAi nano-preparation and applying it to the process of TMV prevention and treatment

Method used

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  • RNAi nano preparation and preparation method and application thereof in prevention and treatment of TMV
  • RNAi nano preparation and preparation method and application thereof in prevention and treatment of TMV
  • RNAi nano preparation and preparation method and application thereof in prevention and treatment of TMV

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Embodiment 1

[0039] Example 1: This example provides a method for screening high-efficiency fragments and its application. After selecting six groups of candidate gene fragments and synthesizing dsRNA in vitro, their antiviral ability was evaluated by biological methods, and on this basis, dsRNA that efficiently degrades TMV-targeted nucleic acids was screened.

[0040] The six groups of candidate gene fragments of TMV are CP, MP, P126, RdRP2, RdRP3, and RdRP4, and their gene sequences are as shown in the sequence list: SEQ ID NO.4, 5, 6, 7, 1, and 8.

[0041] The dsRNA screening method for efficient prevention and treatment of TMV specifically comprises the following steps:

[0042] S1: extraction of total RNA from tobacco leaves infected with TMV. Reverse transcription was performed using the extracted total RNA as a template to obtain cDNA of TMV;

[0043] S2: Using cDNA as a template, design specific amplification primers for the six sequences of CP, MP, P126, RdRP2, RdRP3, and RdRP4, ...

Embodiment 2

[0118] Embodiment 2: the application of the high-efficiency dsRNA further screened in embodiment 1 in the prevention of TMV virus, the specific application method is as follows:

[0119] S1: Planting and transplanting of tobacco in the laboratory: the steps refer to S5-1 of Example 1, and the cultivated tobacco plant grows to an appropriate size.

[0120] S2: Injection of dsRNA and inoculation of TMV-30b: In this experiment, dsRNA was injected first, followed by inoculation of TMV-30b virus 24 hours later. Select benthic tobacco leaves of uniform size in advance and mark them. Dissolve 200 μg of dsRNA of MP and RdRP3 in 0.7ml H 2 O was injected into the leaves, and only 0.7ml H was injected into the leaves of the control group. 2 O. After 24 hours of inoculation, weigh a small amount of TMV-30b poison source leaves, grind them into juice in a mortar, add 40 times the volume of PBS buffer solution (pH 6.8) and mix well, and sprinkle a layer of 100-mesh quartz sand, use a co...

Embodiment 3

[0123] Embodiment 3: the preparation method of the nano-sized RNAi preparation for preventing and treating TMV, comprises the following steps:

[0124] (S1) The combination of chitosan nanomaterials and dsRNA, the specific operation steps are as follows:

[0125] S1-1: dissolving chitosan in glacial acetic acid to prepare chitosan solution A with a final concentration of 2 μg / μl;

[0126] S1-2: Slowly add TMV dsRNA solution with a concentration of 1 μg / μl into chitosan solution A, the volume ratio of chitosan and dsRNA is 10: (1-6). If the mass ratio is too low, many dsRNAs will not be combined with chitosan, which will eventually affect the effect of TMV virus prevention and treatment. If the ratio is too high, many chitosan nanomaterials will not be attached to dsRNA, resulting in unnecessary waste.

[0127] The dsRNA of RdRP3 gene sets 6 mixing ratios respectively, and chitosan glacial acetic acid solution: dsRNA volume ratio is respectively 10:1, 10:2, 10:3, 10:4, 10:5, 1...

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Abstract

The invention belongs to the field of gene engineering technology and application thereof. The invention aims to solve the problems that an anti-TMV efficient RNAi nano preparation is lacked at present, the preparation process of medicament is complicated, the stability and the delivery efficiency of dsRNA are poor, and the like. The invention provides an RNAi nano preparation as well as a preparation method and application thereof in prevention and treatment of TMV. The RNAi nano preparation is prepared from dsRNA and a chitosan nano material, wherein the dsRNA is a screened RdRP3 gene with the length of 313bp and high TMV resistance, the nucleotide sequence of the RdRP3 gene is shown as SEQ ID NO.1, and the volume ratio of chitosan to 1mu g / mu l of the dsRNA is 10: (1-6). According to the RNAi nano preparation disclosed by the invention, the stability and durability of dsRNA can be higher, and meanwhile, the RNAi nano preparation has the advantages of good biocompatibility, good biodegradability, no phytotoxicity to crops, environmental friendliness and the like, and has a good application prospect in the field of TMV virus prevention and treatment.

Description

technical field [0001] The invention belongs to the field of genetic engineering technology and its application, in particular to an RNAi nano-preparation and its preparation method and its application in the prevention and treatment of TMV, in particular to an efficient targeted gene screening, a simple preparation method of RNAi nano-preparation and its New applications in TMV prevention and treatment. Background technique [0002] Chitosan (chitosan) is obtained by deacetylation of chitin (chitin), which exists widely in nature, and has been widely studied and applied because of its good safety, biocompatibility, and microbial degradability. In the fields of plant protection, biological therapy, genetic engineering, food and medicine. [0003] Tobacco mosaic virus (TMV) is widely distributed in various smoking areas in my country. It is the most widely distributed and most common viral disease on tobacco, and it is extremely harmful to tobacco. After the plants are infe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N57/16A01N25/10A01P1/00C12N15/113C12N15/10
CPCA01N57/16A01N25/10C12N15/113C12N2310/14A01P1/00A01N63/60A01N25/04A01N25/22A01N25/28A01N25/30C12N15/1131C12N2310/141C12N2320/32
Inventor 杨金广王凤龙徐翔李莹宋丽云申莉莉雷强冯长春李斌王勇刘东阳江连强
Owner TOBACCO RES INST CHIN AGRI SCI ACAD
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