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Expression vector of fungal nucleus fluorescence labeling, reagent, preparation method and application

An expression vector and fluorescent labeling technology, applied in the field of fungal biology, can solve problems such as inability to accurately locate the nucleus, and achieve the effect of good application prospects and development space.

Active Publication Date: 2020-12-29
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Aiming at the above deficiencies or improvement needs of the prior art, the present invention provides an expression vector, reagent, preparation method and application of fluorescent markers for fungal cell nuclei, the purpose of which is to replace the existing Some fluorescent protein expression sequences modified by nuclear localization signals can more effectively localize the fusion protein to the nucleus, thereby transplanting the existing technology of transgenic permanent labeling of the nucleus to fungi to solve the technical problem that the nucleus cannot be accurately located

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  • Expression vector of fungal nucleus fluorescence labeling, reagent, preparation method and application
  • Expression vector of fungal nucleus fluorescence labeling, reagent, preparation method and application
  • Expression vector of fungal nucleus fluorescence labeling, reagent, preparation method and application

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preparation example Construction

[0066] The preparation method of the expression vector provided by the invention comprises the following steps:

[0067] Cloning the histone gene sequence containing the nuclear localization signal sequence, connecting the histone gene sequence containing the nuclear localization signal sequence to the expression region of the expression vector containing the fluorescent protein sequence, so that the histone gene sequence is connected to the fluorescent protein sequence upstream;

[0068] The histone gene sequence containing the nuclear localization signal sequence is H 2 B gene sequence, its nucleotide sequence is:

[0069] 4) SEQ ID NO.2;

[0070] 5) A nucleic acid sequence that has more than 80% homology with the sequence of SEQ ID NO.2 and is translated into the amino acid sequence shown in 1), 2), and 3);

[0071] 6) A nucleic acid sequence that can hybridize to the sequence in 4) under moderately stringent conditions.

[0072] In the present invention, a homology arm...

Embodiment 1

[0093] Embodiment 1: expression vector construction

[0094] designer histone H 2 The primer of B nucleotide sequence was sent to Wuhan Tianyi Huiyuan Biotechnology Co., Ltd. for synthesis, and the upstream primer sequence was H 2 B-F as shown in SEQ ID NO.3, downstream primer sequence H 2 B-R is shown in SEQ ID NO.4. PCR amplification was carried out with the DNA of Morchella spp. M04M26 as a template. Amplification reaction system (50μL): 100ng template DNA, 2μL each primer (10μM), 1μL dNTP Mix (10mM), 25μL 2×Phanta Max Buffer, 1μL Phanta Max Super-Fidelity DNAPolymerase (1U / μL), supplemented with ddH 2 0 to 50 μL. Reaction parameters: pre-denaturation at 95°C for 5min, denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 1min, cycle number 34, extension at 72°C for 10min. After the product was subjected to 1% agarose gel electrophoresis, the agarose gel containing the target band was cut out, and the target fragment was recovered with a San Pr...

Embodiment 2

[0099] Example 2: Obtaining morel hyphae with fluorescently labeled stable nuclei

[0100] The recombinant plasmid pHYG-H 2 B-mCherry-MI was transformed into Agrobacterium EHA105 (Vidi), and the transformation method was carried out according to the EHA105 Chemically Competent Cell product manual. Streak-culture the Agrobacterium containing the recombinant plasmid for 2 days, pick a single colony in 1 mL LB (20 μg / mL Rif+, 50 μg / mL Kan+) culture solution, culture at 200 r / min 28 ° C for 1 day, and then add it to 100 mL MM (50 μg / mL Kan+) liquid medium at 200r / min at 28°C for 1d. Collect the bacterial solution, centrifuge at 7000r / min for 5min, remove the supernatant, resuspend with an equal volume of IM culture solution, and dilute to OD with IM 600 =0.4, add acetosyringone (AS) to a concentration of 200 μmol / L, cultivate at 200 r / min at 28°C for 6 hours to OD 600 = 0.6-0.8.

[0101] After the hickory chick strain M04M26 mycelium was activated on the CYM solid medium, the ...

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Abstract

The invention discloses an expression vector of fungal nucleus fluorescence labeling, a reagent, a preparation method and application. An expression region of the vector comprises a recombinant fusionprotein expression sequence for expressing recombinant fusion protein of histone and fluorescent protein, and the recombinant fusion protein expression sequence comprises a histone expression sequence and a fluorescent protein expression sequence containing nuclear localization signals, wherein the histone expression sequence and the fluorescent protein expression sequence are located in a reading frame of the same promoter, and the histone expression sequence misses a termination codon and is connected to the upstream of the fluorescent protein expression sequence. The recombinant fusion protein expression sequence has the nuclear localization signals and is used for expressing the recombinant fusion protein. A recombinant which contains the expression vector and has the capability of infecting target fungal cells can be used as the labeling reagent for labeling cell nucleuses of the fungal cells, can be accurately positioned in the cell nucleuses, and emits fluorescence through excitation, so that the cell nucleuses of the target fungal cells are safely and permanently labeled.

Description

technical field [0001] The invention belongs to the field of fungal biotechnology, and more specifically relates to an expression carrier, a reagent, a preparation method and an application of a fungal nuclear fluorescent marker, especially a fluorescent marker expression vector, a reagent, and a preparation of a large fungus that forms a fruiting body. method and application. Background technique [0002] Nuclear labeling is of great significance for cell nucleus localization, cytology research, and the development of biological application technology. For example, marking the nucleus of morels can observe the behavior of the nucleus, especially in the research of mycelial mating nucleus migration and cross-breeding of edible fungi. [0003] However, most of the labeling methods for cell nuclei use organic fluorescent dyes, such as 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), although organic fluorescent dyes have high sensitivity , Strong specificity, ...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N15/65C12N15/64C12N1/21C12R1/645C12R1/01
CPCC12N15/80C12N15/65C12N15/64C07K14/37C07K14/43595C07K2319/09C07K2319/60
Inventor 康恒舒芳边银丙陈新
Owner HUAZHONG AGRI UNIV
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