Application of TuMV-CP gene fragment-mediated RNAi carrier in cultivation of anti-TuMV transgenic plant

A technology of transgenic plants and fragments, applied in the field of RNAi vectors, can solve the problems of reduced quality of rapeseed, loss of rapeseed yield and the like

Inactive Publication Date: 2014-02-19
BEIJING AGRO BIOTECH RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The incidence of diseased fields is generally 20% to 30%, and the incidence rate of severe cases is more than 50%, resulting in a major loss of rapeseed yield, and the occurrence of viral diseases also leads to a decrease in the quality of rapeseed

Method used

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  • Application of TuMV-CP gene fragment-mediated RNAi carrier in cultivation of anti-TuMV transgenic plant
  • Application of TuMV-CP gene fragment-mediated RNAi carrier in cultivation of anti-TuMV transgenic plant
  • Application of TuMV-CP gene fragment-mediated RNAi carrier in cultivation of anti-TuMV transgenic plant

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, have the discovery of the fragment that suppresses turnip mosaic virus function

[0037] Sequence comparison of 122 TuMV strains registered in GenBank revealed that there is a conserved region in the CP gene of turnip mosaic virus (the CP gene is shown in sequence 2 in the sequence listing, encoding the coat protein shown in sequence 1 in the sequence listing) , a fragment of 377bp was finally determined in this conserved region (named CP377 fragment, as shown in sequence 3 of the sequence listing, which is the 446-822 nucleotides from the 5' end of the CP gene shown in sequence 2 of the sequence listing ), the RNA encoded by this fragment is expected to inhibit the turnip mosaic virus. The RNA transcribed by the CP377 fragment is a single-stranded RNA shown in sequence 4 of the sequence listing.

Embodiment 2

[0038] Embodiment 2, the construction of RNAi expression vector

[0039] 1. Construction of plant expression vector pBBBasta

[0040] The construction method of the plant expression vector pBBBasta (referred to as pBBBasta vector) vector is as follows:

[0041] 1. Digest plasmid pDHB321.1 with restriction endonuclease SacI, and recover a fragment of about 2100 bp (the sequence between SacI digestion is shown in sequence 6 of the sequence table, including "LB-bar expression box-RB"). It is also possible to directly artificially synthesize the double-stranded DNA molecule shown in sequence 6 of the sequence table with SacI restriction sites at both ends, and then digest it with restriction endonuclease SacI to recover a fragment of about 2100bp (containing "LB-bar Expression box-RB", the sequence between the SacI restriction sites is shown in sequence 6 of the sequence table).

[0042] 2. Digest the plasmid pBBR1MCS-2 with restriction endonuclease SspI, and recover a fragment ...

Embodiment 3

[0066] Embodiment 3, the acquisition and identification of transgenic plants

[0067] 1. Obtaining of transgenic plants

[0068] 1. Introduce recombinant plasmid pBBBTu-CP377 into Agrobacterium GV3101:pMP90 to obtain recombinant Agrobacterium.

[0069]2. Introduce recombinant Agrobacterium into Colombian ecotype Arabidopsis thaliana by inflorescence dipping method (Clough, S.J., and Bent, A.F. (1998). Floral dip: asimplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743.) mustard, get T 0 generation seeds.

[0070] 3. Harvest T 0 Seeds obtained by selfing of generation plants, which grow T after sowing 1 For the first generation seedlings, spray Basta herbicide with a concentration of 1-2‰ (volume ratio) after the seedlings grow two cotyledons, and the plants that can survive and continue to grow are T 1 Positive plants. A total of 64 T 1 Positive plants.

[0071] 4. Take T 1 Cut the leaves of the positive plants for mol...

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Abstract

The invention discloses application of a TuMV-CP gene fragment-mediated RNAi carrier in cultivation of an anti-TuMV transgenic plant. The sequence 4 in a protection sequence table provided in the invention shows RNA fragments and its coding sequence. The RNA molecule can interfere with replication of TuMV RNA, thus inhibiting the TuMV. The invention also protects a transgenic plant cultivation method, which includes the following step of: expressing the RNA molecule in a starting plant so as to obtain a transgenic plant with higher TuMV resistance than that of the starting plant. The invention is of great value for cultivation of anti-TuMV plants (especially cruciferous plants).

Description

technical field [0001] The invention relates to the application of an RNAi carrier mediated by TuMV-CP gene fragments in cultivating anti-TuMV transgenic plants. Background technique [0002] Turnip mosaic virus (TuMV) belongs to the family Potyviridae and the genus Potyvirus. Virus particles are curved and linear, about 720nm long and 15-20nm wide, consisting of 95% coat protein and 5% RNA. The viral nucleic acid is a single-stranded positive-sense RNA consisting of approximately 10,000 nucleotides. The virus has a wide range of hosts. Under artificial inoculation conditions, it can infect 43 families, 156 genera, more than 318 dicotyledonous plants and some monocotyledonous plants. [0003] Most viruses in the genus Potatovirus Y have a narrow host range, but TuMV has a wide host range, infecting many important economic crops, especially many cruciferous vegetables and oil-bearing feed crops including cabbage, cabbage, rape, etc. Second only to cucumber mosaic virus (CM...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/63C12N15/82C12N15/84A01H5/00A01N43/90A01P1/00
Inventor 姚磊曾钢叶艳英曹鸣庆马荣才
Owner BEIJING AGRO BIOTECH RES CENT
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