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Tobacco virus-resisting RNAi carrier

A technology of tobacco virus and CMV-RNA1, applied in the field of molecular biology to achieve the effect of preventing viral diseases

Inactive Publication Date: 2011-10-19
SHANDONG AGRICULTURAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Escherichia coli can express dsRNA, but because RNaseIII in Escherichia coli can cut dsRNA into 12-15bp fragments, these short dsRNAs cannot induce gene silencing [10]

Method used

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  • Tobacco virus-resisting RNAi carrier
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  • Tobacco virus-resisting RNAi carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Utilize RT-PCR to amplify virus single gene fragment

[0042] 1. Total RNA extraction

[0043]Tobacco plants infected with PVY, CMV, and TMV were collected from the field, and total RNA was extracted from infected tobacco leaves according to the method provided by the TransZol kit (Beijing TransGen Biotech Co., Ltd).

[0044] 2. RT-PCR reaction

[0045] The total plant RNA was used as a template, and PVY-R, CMV-R or TMV-R were used as primers for reverse transcription. The reaction system is as follows:

[0046]

[0047]

[0048] After mixing, incubate at 42°C for 50 minutes, and incubate at 70°C for 15 minutes to stop the reaction.

[0049] Using the obtained reverse transcription product as a template, single-fragment PCR amplification was performed.

[0050] The PVY fragment PCR amplification system is as follows:

[0051]

[0052] The CMV fragment PCR amplification system is as follows:

[0053]

[0054] TMV fragment PCR amplification...

Embodiment 2

[0059] Embodiment 2: Construction of the reverse fragment

[0060] 1. Perform 1% agarose gel electrophoresis on all PVY amplification products. Gel was cut and recovered using EasyPure Quick Gel Extraction Kit (TransGen Biotech, China). The fragment recovery product was connected to the pMD18-T simple vector, overnight at 4°C, and the connection system was as follows:

[0061]

[0062] 2. The ligation product was transformed into DH5α competent cells by heat shock method;

[0063] 3. Pick a single colony and shake it in liquid LB medium containing ampicillin at 37°C;

[0064] 4. Plasmid extraction by alkaline lysis;

[0065] 5. Use primers PVY-F and PVY-R to screen positive clones by PCR;

[0066] 6. Carry out double enzyme digestion on the identified positive recombinant plasmid. The enzyme digestion system is as follows:

[0067]

[0068] Electrophoresis, cutting the gel to recover the band around 3300bp;

[0069] 7. Take 5.0 μL of the CMV amplification product a...

Embodiment 3

[0086] Example 3: Amplification of forward fragments

[0087] 1. Using pMD18S-RPCT as a template, PCR amplifies the forward fragment F-PCT,

[0088] The system is as follows:

[0089]

[0090] The procedure is as follows:

[0091]

[0092] 2. Electrophoresis, gel cutting and recovery of 800bp fragments (F-PCT);

[0093] 3. Connect the recovered product of F-PCT to the pGEM-T easy (Promega, America) vector at 4°C overnight. The connection system is as follows:

[0094]

[0095] 2. The ligation product was transformed into DH5α competent cells by heat shock method;

[0096] 3. Pick a single colony and shake it in liquid LB medium containing ampicillin at 37°C;

[0097] 4. Plasmid extraction by alkaline lysis;

[0098] 5. PCR screens out the clones connected to F-PCT (system and program are the same as the amplification process of F-PCT fragments);

[0099] 6. BamHI / SpeI double enzyme digestion to identify the direction of the inserted clone. The enzyme digestion ...

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Abstract

The invention belongs to the field of biotechnology, and provides an RNAi vector pGEM-PCTRi capable of mediating the resistance to tobacco viral diseases in China. Analyzing the genome characteristics of main virus groups in China, which comprise Potato Virus Y (PVY), Cucumber Mosaic Virus (CMV) and Tobacco Mosaic Virus (TMV) and then selecting three segments of conserved effective sequences capable of inducing RNAi in PVY CP, CMV RNA1 and TMV CP; and integrating the three segments in a tabling manner and constructing an inverted repeat sequence so as to obtain an RNAi vector pGEM-PCTRi. Massproduction of dsRNA can be realized in escherichia coli by utilizing the vector, and the resistance of tobacco to PVY, CMV and TMV can be induced to generate after the tobacco treatment. The invention has the potential of being applicable to the prevention of viral diseases for tobacco farmland production, and endowed with the advantages of broad spectrum, high efficiency, no reference to transgenosis, and the like.

Description

technical field [0001] The invention belongs to the field of molecular biology. Specifically, the invention relates to an RNA interference (RNAi) vector and a construction method thereof, in particular to a construction method of an RNAi vector simultaneously resisting three viruses of PVY, CMV and TMV. Background technique [0002] Viral diseases are a large class of diseases that commonly occur in tobacco and cause serious harm. At present, 47 kinds of viruses infecting tobacco have been reported in the world, and 17 kinds are found in China, among which, potato virus Y (PVY) and cucumber mosaic virus (CMV) are widely distributed and seriously harmful. And tobacco mosaic virus (Tobacco mosaic virus, TMV) and so on. Field viral diseases often occur mixedly, often causing serious losses to tobacco production. In the year when the disease is prevalent, the loss of tobacco leaf production caused by viral diseases can reach 30% to 50%, and some areas even lose production. [1...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/113C12N1/21A01N61/00A01P1/00C12R1/19
Inventor 李向东贾金磊
Owner SHANDONG AGRICULTURAL UNIVERSITY
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