Efficient gene knockout vector construction method and constructed engineering bacteria

A gene knockout and construction method technology, applied in the biological field, can solve the problems of cumbersome positive transformants, etc., and achieve the effect of shortening the research cycle and speeding up the research

Inactive Publication Date: 2017-02-01
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology

Mainly including Davidson's overlap PCR (Davidson et al., 2002), Catlett's split-marker deletion method (Catlett et al., 2002), PCR and restriction enzyme digestion and ligation( 2004), D. DelsGate (2007) Zahi Paz's OSCAR, Chuan Xu's improved OSCAR (ΔMrKu70). In all these methods, only how to optimize the construction of the fungal gene vector is considered, but for the knockout of the target gene, the positive transformant The screening work is the most important and tedious work

Method used

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  • Efficient gene knockout vector construction method and constructed engineering bacteria
  • Efficient gene knockout vector construction method and constructed engineering bacteria
  • Efficient gene knockout vector construction method and constructed engineering bacteria

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Embodiment 1

[0047] The construction of embodiment 1 recombinant vector

[0048] 1. The vector PGKO-Gateway (Methods in Molecular Biology, vol.344: Agrobacterium Protocols, 2 / e, volume 2) (20 years from the date of application can be obtained from the Institute of Microbiology, Chinese Academy of Sciences) was carried out through Xba Ⅰ and Hind Ⅲ Double enzyme digestion (see electrophoresis after enzyme digestion Figure 5 ), recover the large fragment of the vector after digestion. The reagents used for enzyme digestion are listed in Table 1.

[0049] Table 1

[0050] 10×RE Buffer 5μL Xba I 1μL HindⅢ 1μL PGKO-Gateway 10μL wxya 2 o

Make up 50μL

[0051] 2. The vector Psul-EGFP was digested by Xba Ⅰ and Hind Ⅲ (see the electrophoresis diagram after digestion). Image 6 ), recover the short fragments after enzyme digestion. The reagents used for enzyme digestion are listed in Table 2.

[0052] Table 2

[0053] 10×RE Buffer 5μ...

Embodiment 2

[0074] Example 2 Application and Verification of Recombinant Vectors in Gene Knockout

[0075] 2.1 Linearization of PGKO-HPT vector

[0076] Table 8

[0077] 10×cutSmart Buffer 30μL Pac I 10μL PKGO-HPT -μL (take 20-30μg)

[0078] wxya 2 o

Make up 300μL

[0079] The PGKO-HPT carrier prepared in Example 1 was digested overnight at 37°C. The reagents used for the digestion were shown in Table 8. The next morning, 4ul of PacI (New England Biolabs, Cat.no.R0547) and 4ul of Nt.BbvCI (New England Biolabs , Cat.no.R0632), and then placed in a 37°C water bath for 2 hours of enzyme digestion, and then 5ul was taken to run the gel to identify the enzyme digestion efficiency. All digested products were recovered with the SanPrep column DNA Gel Extraction Kit (Lot: 14023036Y) of Sangon Bioengineering (Shanghai) Co., Ltd., and the content was determined with a NanoDropND-1000 UV-Vis spectrophotometer after recovery. Determination. Aliqu...

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Abstract

The invention provides an efficient gene knockout vector construction method. According to the present invention, an efficient negative screening marker gene and a USER enzyme cloning technology are used to construct an enzyme cutting site in a vector so as to make the vector generate a sticky end after the enzyme cutting, the efficient negative screening marker gene is constructed in the vector, and the constructed vector is successfully used for knocking-out Verticillium dahliae V592 target gene, wherein the sticky end can be complementary to the upstream fragment and the downstream fragments of the target gene obtained after the enzyme cutting with the USER enzyme; and the experiment results prove that the research period of the Verticillium dahliae target gene is substantially shortened and the Verticillium dahliae function gene research is accelerated with the vector constructed by using the method of the present invention.

Description

technical field [0001] The present invention generally relates to the field of biotechnology, in particular to a high-efficiency gene knockout vector and its construction method and application. Background technique [0002] With the improvement of gene sequencing technology and the reduction of sequencing cost, more and more fungal genomes have been sequenced (http: / / www.broadinstitute.org / / scientific-community / data), and a large amount of genetic data has been obtained, but In the absence of strong experimental evidence to prove the function of related genes, there is an urgent need for the emergence of effective and rapid methods for detecting gene function. Two of the hottest gene editing technologies currently being studied: TALEN and CRISP-CAS9 have been used in the study of fungal genome functions, and there are few reports. Because fungi have a special gene repair mechanism: homologous recombination, this mechanism brings convenience to the study of fungal genome fu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/74C12R1/01
Inventor 郭惠珊王胜
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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