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Application of G418 resistance marker in agrobacterium tumefacien-mediated genetic transformation of trichoderma

An Agrobacterium-mediated, genetic transformation method, applied in the introduction of foreign genetic material, fungi, microorganism-based methods using vectors, etc., can solve the problems of cumbersome steps and insufficient selective markers, and achieve important biological engineering value. Effect

Inactive Publication Date: 2014-05-28
FUYANG NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] The first object of the present invention is to provide a kind of application method of G418 in the genetic transformation of Trichoderma mediated by Agrobacterium, the application method is to use the resistance of G418 as the selection marker of Trichoderma resistance, to solve the genetic transformation of Trichoderma The problem of insufficient selectable markers
[0005] The second object of the present invention is to provide a new Agrobacterium-mediated Trichoderma genetic transformation method to solve the problem that the protoplast transformation usually mediated by PEG has complicated steps

Method used

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  • Application of G418 resistance marker in agrobacterium tumefacien-mediated genetic transformation of trichoderma
  • Application of G418 resistance marker in agrobacterium tumefacien-mediated genetic transformation of trichoderma
  • Application of G418 resistance marker in agrobacterium tumefacien-mediated genetic transformation of trichoderma

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Experimental program
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Effect test

Embodiment 1

[0038] 1. Application of G418 antibiotic in Agrobacterium-mediated genetic transformation of Trichoderma

[0039] Since the G418 resistance selection gene is used in Trichoderma for the first time, a susceptibility test must be performed to determine whether Trichoderma is sensitive to G418 and the appropriate concentration of G418 in the selection medium. The results show that: with the increase of the concentration of G418 in the PDA medium, the inhibitory effect on the growth of wild Trichoderma is significantly enhanced, and on the PDA plate with a G418 concentration of 25 μg / mL, the wild-type Trichoderma dark green T23 strain was cultivated for 90 hours. Signs of growth were evident and growth was completely inhibited. It shows that the G418 concentration of 25 μg / mL can effectively inhibit the growth of wild-type Trichoderma dark green T23, so the resistance to G418 antibiotics can be used as a Trichoderma resistance selection marker. In order to effectively screen the ...

Embodiment 2

[0061] Except for the following technical features, other technical features are the same as in Embodiment 1.

[0062] (2) Inoculate the activated Agrobacterium liquid into the LB liquid medium containing folipin, and cultivate to OD at 29°C 600 The value is 0.3, after pre-cooling, centrifuge to collect the bacteria, discard the supernatant, and then use pre-cooling 20mmol / L CaCl 2 Resuspend the pellet.

[0063] (3) The mixture of p1300-neo carrier and Agrobacterium competent cells was incubated at 35°C for 4min; after adding it to LB liquid medium and incubating at 25°C for 4h, the bacterial solution was applied to the culture medium containing kanamycin and rifampicin Flatten the YEB option on the flatbed.

[0064] (4) Inoculate the recombinant Agrobacterium strain into LB liquid medium and culture it to OD with induction medium 660 up to 0.8.

[0065] (6) The cellophane in middle was transferred to the M-100 plate containing G418 and cephalosporin, cultured at 25°C for ...

Embodiment 2

[0067] Except for the following technical features, other technical features are the same as in Embodiment 1.

[0068] (2) Inoculate the activated Agrobacterium tumefaciens into the LB liquid medium containing folipin, and cultivate to OD at 29°C 600 The value is 0.5, after pre-cooling, centrifuge to collect the bacteria, discard the supernatant, and then use pre-cooling 25mmol / L CaCl 2 Resuspend the pellet.

[0069] (3) The mixture of p1300-neo carrier and Agrobacterium competent cells was incubated at 40°C for 5 minutes; added to LB liquid medium and cultured at 35°C for 5 hours, the bacterial liquid was spread on the YEB selection plate containing G418.

[0070] (4) Inoculate the recombinant Agrobacterium strain into LB liquid medium and culture it to OD with induction medium 660 up to 0.7.

[0071] (6) The cellophane in middle was transferred to the M-100 plate containing G418 and cephalosporin, cultivated at 30°C for 7 days, and the wild-type Trichoderma dark green tra...

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Abstract

The invention relates to application of a G418 resistance marker in agrobacterium tumefacien-mediated genetic transformation of trichoderma. The resistance of G418 is used as a selection marker of the resistance of trichoderma. An agrobacterium tumefacien-mediated genetic transformation method of trichoderma comprises the following steps: connecting a G418 resistant gene with a carrier p1300 subjected to enzyme digestion and dephosphorylation to form a carrier p1300-neo; activating agrobacterium strains and cultivating until the OD600 value is 0.3-0.6, and using 15-25 mmol / L of CaC12 to perform resuspension on bacterial cells to prepare agrobacterium competent cells; uniformly mixing the carrier p1300-neo with the agrobacterium competent cells, keeping the temperature of 35-40 DEG C for at least 3 min, using an LB (Luria-Bertani) culture medium for cultivation at the temperature of 25-35 DEG C, and then using a YEB culture medium containing kanamycin and rifampicin for cultivation and screening out recombinant agrobacterium tumefacien; uniformly mixing an activated recombinant agrobacterium tumefacien solution in a logarithmic phase, of which the OD660 value is 0.6-0.8 with a trichoderma viride conidial suspension and performing induction cultivation, and then using an M-100 culture medium containing G418 for cultivation at the temperature of 25-30 DEG C and screening out trichoderma resisting G418. The invention provides a novel resistance selection marker for trichoderma viride and brings considerable convenience to research of the gene function of trichoderma, and the cost is low.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to the application of a G418 resistance marker in the genetic transformation of Trichoderma mediated by Agrobacterium. Background technique [0002] Trichoderma widely exists in the soil environment and is an important group of soil microorganisms. It is also found on plant residues and animal manure, and also exists in large quantities in ecological environments such as plant roots, leaves, seeds and bulbs. Since the 1930s, this kind of beneficial microorganisms has been widely used in the biological control of plant diseases, the production of cellulase, and the function of bioremediation. Trichoderma can degrade cyanide, pesticides, formaldehyde, diesel, phenols and adsorb heavy metals. [0003] Agrobacterium-mediated genetic transformation provides an effective means for the genetic improvement and functional gene research of Trichoderma. In the genetic transform...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N1/15C12R1/885
Inventor 唐俊陈捷马越张雷
Owner FUYANG NORMAL UNIVERSITY
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