Yeast-Agrobacterium shuttle vector, and construction method and application thereof

A technology of a shuttle vector and a construction method, applied in the field of plant viruses, can solve the problems of time-consuming and labor-intensive cloning efficiency, low success rate, and reducing the infection efficiency of plant virus infective clones.

Inactive Publication Date: 2018-03-23
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Time-consuming and labor-intensive, poor cloning efficiency and low success rate
In addition, since relatively large plant virus genomes often contain multiple enzyme cutting sites, the ability to select suitable restriction endonucleases for cloning has become the biggest obstacle restricting the development of this technology, and the use of in vitro assembly technology Need to use extremely expensive recombinase to complete gene cloning, which cannot meet the needs of high-throughput cloning
[0005] 2. The existence of some similar prokaryotic promoter sequences (TTGACA and TATAAT) in certain viral sequence

Method used

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  • Yeast-Agrobacterium shuttle vector, and construction method and application thereof
  • Yeast-Agrobacterium shuttle vector, and construction method and application thereof
  • Yeast-Agrobacterium shuttle vector, and construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0094] 1. The structure of the yeast-Agrobacterium shuttle vector pCB301-2μ-HDV vector

[0095] The yeast-Agrobacterium shuttle vector pCB301-2μ-HDV vector of this embodiment, such as figure 1 As shown, the shuttle vector contains elements and selectable markers that can be stably replicated and expressed in Agrobacterium and yeast, so it can be used for virus-infective cloning construction using the highly efficient homologous recombination system of yeast, and can also be used for Agrobacterium dual Metavectors contain the elements necessary for maintenance in Agrobacterium and transformation of plant cells, insertion into plants to complete plant infection.

[0096]comprising the following nucleic acid elements: a) a first nucleic acid element comprising a nucleotide sequence encoding a selectable marker functional in Agrobacterium sp.; b) a second nucleic acid element comprising a nucleotide sequence of a first origin of replication An element, the first origin of replica...

Embodiment 2

[0148] 1. Using the pCB301-2μ-HDV vector of Example 1 to construct Sonchus yellow net virus (Sonchus yellow net virus, SYNV) in yeast by one-step homologous recombination c DNA-infecting clones

[0149] Such as Figure 4 As shown in (a), the SYNV genome is a single-stranded negative-sense RNA with a length of about 13.7 kb. Invasive cloning of single-stranded negative-sense RNA needs to be transcribed in plants to produce an RNA sequence that is completely faithful to the viral genome, accurately cuts the start and end sites, and cannot contain any redundant nucleic acid sequences, and the relatively large genome is suitable for one-time It is a big challenge to construct an invasive clone that is completely faithful to the wild-type virus genome and to perform large-scale deletion and mutation of the virus genome. The ribozyme sequence of hepatitis delta virus (HDV) can be used to precisely cut transcripts in eukaryotic cells to produce full-length RNA that is completely fa...

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Abstract

The invention discloses a yeast-Agrobacterium shuttle vector, and a construction method and an application thereof, and belongs to the field of plant viruses. The shuttle vector simultaneously contains elements and a selective marker which can be stably replicated and expressed in Agrobacterium and yeast, so a virus infectious clone can be constructed by using the high-efficiency homologous recombination system of the yeast, and plant infection is completed by using the Agrobacterium binary vector containing the elements needed by the maintenance and transformation of plant cells and insertionof the plant in the Agrobacterium. The virus infection clone of a large genome or a complex genome can be constructed within two weeks the vector and the construction method. The vector can quickly and accurately construct the virus infection clone, so the yeast-Agrobacterium shuttle vector for rapid construction of the virus infection clone, and the application method thereof have very importantapplication values.

Description

technical field [0001] The invention relates to a shuttle vector, in particular to a yeast-Agrobacterium shuttle vector, and also relates to its construction method and application, belonging to the field of plant viruses. Background technique [0002] Since Ahlquist and colleagues discovered that brome mosaic virus (BMV) cDNA in vitro transcripts can infect plants, dozens of different plant RNA viruses have used bacterial RNA polymerase promoters (such as T7, T3 and SP6, etc.) were transcribed in vitro to construct invasive clones. However, this method requires mature skills, a good basis for molecular biology, and the high cost of experiments is not suitable for large-scale gene mutation and genetic manipulation, and its own limitations, such as this method is only applicable to some positive strands (Positive-strand) RNA virus is not suitable for negative-strand (negative-strand) RNA virus and some phloem-specific positive-strand RNA, and has not been widely used. Subse...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H6/82
CPCC12N15/8205C12N15/8261
Inventor 黄昌军李正和刘勇孙凯赵丹阳于海芹
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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