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A genetic transformation method mediated by Agrobacterium tumefaciens with Aspergillus flavus mycelium as receptor

A genetic transformation method, Agrobacterium-mediated technology, applied in the field of genetic transformation using Aspergillus flavus mycelium as a receptor, can solve the problems of cumbersome steps, genetic transformation of Aspergillus flavus strains, high cost, etc.

Active Publication Date: 2019-01-11
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] One of the purposes of the present invention is to provide a new Agrobacterium-mediated genetic transformation method using Aspergillus flavus hyphae as a receptor, to solve the problem that the genetic transformation of Aspergillus flavus usually requires the preparation of protoplasts, and the steps are cumbersome and expensive. , and at the same time, it can solve the problem of genetic transformation of non-spore-forming Aspergillus flavus strains

Method used

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  • A genetic transformation method mediated by Agrobacterium tumefaciens with Aspergillus flavus mycelium as receptor
  • A genetic transformation method mediated by Agrobacterium tumefaciens with Aspergillus flavus mycelium as receptor
  • A genetic transformation method mediated by Agrobacterium tumefaciens with Aspergillus flavus mycelium as receptor

Examples

Experimental program
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Effect test

Embodiment 1

[0052] Example 1, Construction of the pAg1-pt-mC binary vector carrying the fragment of interest

[0053] Use the binary vector pAg1-H3 with Hin d III and Sma The hygromycin resistance gene fragment was removed by I double enzyme digestion, and the pAg1 vector fragment was recovered; the pPTR I-mCherry plasmid (Nie X, Yu S, Qiu M et al., J. Agric. Food Chem ., 2016, 64(35):6772-6782) as a template, using the forward primer 5'-TCGGTACCAAGGCCCGGGCATCCGG

[0054] ATGTCGAAGGCTTG-3' (SEQ ID NO. 1); and reverse primer 5'-GGGAGTCACGAAGCTTGGGC

[0055] AATTGATTACGGGA-3' (SEQ ID NO.2) amplified 5227 bp target fragment ptrA- P gpdA -mcherry , the target fragment contains ptrA Gene and its promoter and terminator sequence (SEQ ID NO.3), derived from Aspergillus nidulans wxya promoter P gpdA Sequence (SEQ ID NO.4) and red fluorescent protein gene sequence derived from coralline animals mcherry (SEQ ID NO. 5).

[0056] Combine the pAg1 vector fragment recovered above w...

Embodiment 2

[0057] Embodiment 2, the preparation of the Agrobacterium bacterium liquid carrying target gene

[0058] Agrobacterium AGL-1 strain was taken out from -80 ℃, inoculated on LB solid medium containing rifampicin with a final concentration of 25 μg / mL, and activated at 28 ℃ until a single clone appeared; pick a single clone and inoculate it into 3 mL In LB liquid medium (containing rifampicin at a final concentration of 25 μg / mL), shake culture at 28 °C and 220 rpm for 24 h; take the above bacterial liquid and inoculate it into 100 mL of LB liquid medium (containing a final concentration of 25 μg / mL rifampicin), 28 ℃, 220 rpm shaking culture to OD 600 =0.5; ice-bathed bacterial solution for 30 min, centrifuged at 4200 rpm for 10 min at 4°C, collected the bacterial cells, removed the supernatant, and washed with pre-cooled 20 mMCaCl 2 Resuspend the washed cells, place on ice for 20 min; centrifuge at 4200 rpm, 4 °C for 10 min, collect the cells, remove the supernatant, and wash w...

Embodiment 3

[0061] Embodiment 3, the preparation of aspergillus flavus mycelia homogeneous liquid

[0062] Inoculate the spores or hyphae of Aspergillus flavus NRRL3357 strain on PDA solid medium, and culture in the dark at 37 °C for 2 days; the aerial hyphae on the surface of the eluted medium were inoculated in GMM liquid medium, and cultured at 37 °C for 12 hours with shaking at 180 rpm until the diameter of mycelial balls is 2 mm; remove the medium by filtering with sterile filter paper, collect 80 mg of mycelial balls in a 1.5 mL sterile centrifuge tube, add sterile small steel balls with a diameter of 2 mm, and homogenize with a homogenizer at the 12th gear Plastify mycelia for 20 min and resuspend in 5 mL of IM medium (containing a final concentration of 25 μg / mL rifampicin, a final concentration of 100 μg / mL kanamycin and a final concentration of 200 μM acetosyringone) Mix to obtain a homogeneous liquid of Aspergillus flavus mycelium.

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Abstract

The invention discloses a genetic transformation method mediated by Agrobacterium tumefaciens with Aspergillus flavus mycelium as a receptor, belonging to the field of microbiology. The method comprises the following steps: the divalent vector pAg1-H3 is digested to remove the hygromycin resistance gene fragment, and ligated with the target fragment containing the pyrithione resistance gene ptrA derived from Aspergillus oryzae to construct a pAg1-Pt binary vector; the binary vector pAg1-Pt plasmid is transformed into Agrobacterium tumefaciens to obtain Agrobacterium tumefaciens positive transformant and prepare bacterial liquid; Aspergillus flavus mycelium homogenate is prepared; Aspergillus flavus is transformed by co-culturing the prepared mycelium and mycelium homogenate; selective culture is performed, Aspergillus flavus transformants are screened and positive clones are identified. As the mycelium of Aspergillus flavus is used as the transformation acceptor, the transformation efficiency is high and stable without the restriction of the sporulation ability of the Aspergillus flavus strain, and the method is applicable to Aspergillus flavus strains with different genetic background and nutritional deficiencies.

Description

technical field [0001] The invention belongs to the field of microbiology, and in particular relates to an Agrobacterium-mediated genetic transformation method using Aspergillus flavus hyphae as a receptor. Background technique [0002] Aspergillus flavus ( Aspergillus flavus ) can parasitize in food, food and feed for growth and reproduction, and produce aflatoxin, among which aflatoxin B1 (Aflatoxin B1, AFB1) is the most harmful, with strong liver toxicity and carcinogenic effect. At present, 25% of the world's crops are polluted by mycotoxins every year, and the most serious one is aflatoxin pollution. It is common for peanuts and corn to be polluted by Aspergillus flavus in my country, and the contamination by Aspergillus flavus in livestock and poultry feed and aquatic feed is even more serious. Therefore, the prevention and control of Aspergillus flavus pollution is of great significance. At present, researchers have tried a variety of methods to prevent and control...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N15/65C12R1/67
CPCC12N15/65C12N15/80
Inventor 聂鑫怡张轶王银春李博文王秀娜汪世华
Owner FUJIAN AGRI & FORESTRY UNIV
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