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

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...

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 sequences often leads to the expression of proteins that are toxic to E. coli in the viral genome, such as viruses of the genus Potatovirus

Larger fragments make it more difficult for T-DNA to integrate into plants, reducing the infection efficiency of plant virus-infectious clones

Third, the reported cloning system is only reported to be suitable for positive-strand RNA viruses. Is it suitable for the infection line of negative-strand RNA viruses that need to be completely faithful to the viral genome, accurately cut the start and end sites, and cannot contain any redundant nucleic acid sequences? clone build unknown

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