Virus-induced turnip gene silencing system and application thereof

A technology of turnip and target gene, applied in the field of turnip gene silencing system, can solve the problems of in vitro culture and regeneration difficulty

Pending Publication Date: 2021-09-07
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Abstract
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  • Claims
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Problems solved by technology

[0004] Turnip (B.rapa var.rapa) is a biennial herb of the genus Brassica in the Brassicaceae family. It is a classic root vegetable that combines food, medicine and feed. It belongs to the same genus Brassica as the Chinese cabbage. The basic species of the genome, this type of vegetable has become the most difficult species in the Brassic

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  • Virus-induced turnip gene silencing system and application thereof
  • Virus-induced turnip gene silencing system and application thereof

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Embodiment 1

[0048] 1. Construction of VIGS Silencing Expression Vector of Turnip BrrPDS Gene

[0049] 1. Take the young leaves of Dongsheng small turnip, extract RNA, and reverse transcribe to obtain cDNA. The cDNA was used as a template, and the primer pair composed of BrrPDS-F and BrrPDS-R was used for PCR amplification to obtain PCR amplification products. After sequencing, the PCR amplification product is shown as sequence 3 in the sequence listing.

[0050] BrrPDS-F (Sequence 1 of the Sequence Listing): 5'-ATGGTTGTGTTTGGGAATGT-3';

[0051] BrrPDS-R (SEQ ID NO: 2 of the Sequence Listing): 5'-TCATGTTGATACAGTTATCTC-3'.

[0052] 2. Select the 40bp sequence and its reverse complementary sequence from the CDS of the BrrPDS gene as the interference sequence.

[0053] Selection principle: the 2nd to 4th bases are TGA, TAG or TAA; in order to facilitate the later screening and avoid recombination of the SnaBI restriction site, the 1-3 bases of the sequence should not be GTA, so 1 of the se...

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Abstract

The invention discloses a virus-induced turnip gene silencing system and application thereof. The invention provides a method for silencing a target gene in turnip. The method comprises the following steps of: (1) designing an interference sequence according to the target gene and a specific principle; (2) preparing an interference vector, wherein a starting vector of the interference vector is a pTY-S vector, and the interference vector has the interference sequence obtained in the step (1) and a reverse complementary sequence thereof; and (3) rubbing leaves of a turnip plant to make a wound, and then inoculating the interference vector. The virus-induced turnip gene silencing system has the beneficial effects that agrobacterium transfection is not needed, and complex steps such as gene gun bombardment are omitted; and the turnip virus-induced gene silencing system is established, simple rubbing inoculation is adopted, the duration is short, the operation is simple, the damage to plants is small, the silencing efficiency is greatly improved, and silencing phenotypes of a plurality of organs can be observed at the same time. The virus-induced turnip gene silencing system has important significance for variety improvement of brassica crops.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the technical field of plant genetic engineering, in particular to a virus-induced turnip gene silencing system and its application. Background technique [0002] Virus-induced gene silencing (VIGS), a post-transcriptional gene silencing (PTGS) phenomenon, is an RNA-mediated inherent and ubiquitous antiviral defense mechanism in plants . The mechanism is that after the recombinant plasmid formed by the virus vector and the target gene segment infects the host plant, the plant simultaneously targets the viral genome and the mRNA corresponding to the target gene segment, causing the silencing of the plant's endogenous gene. Changes in phenotype or physiological indicators to achieve the purpose of determining gene function. As a reverse genetics technology, compared with plant transgenic technologies such as gene knockout, transgene, and mutant screening, VIGS technology does not nee...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/53C12N9/02A01H5/00A01H6/20
CPCC12N15/8218C12N15/825C12N9/001C12Y103/99028C12Y103/99029C12Y103/9903C12Y103/99031
Inventor 王正李盼王利敏苏同兵
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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