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Genetic transformation method for PEG-medicated fusarium oxysporum sesame special type protoplast

A technology of Fusarium oxysporum and protoplast, which is applied in the field of genetic transformation of Fusarium oxysporum sesame specialized protoplast, can solve problems such as lack of genetic transformation system, achieve high transformation efficiency and improve the effect of fluorescence intensity

Active Publication Date: 2014-06-18
HENAN ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] In recent years, with the completion of the genome sequencing of Fusarium oxysporum and the attempt to study the functional genomics of Fusarium wilt, the lack of a suitable genetic transformation system has become an important limiting factor for further in-depth research.

Method used

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  • Genetic transformation method for PEG-medicated fusarium oxysporum sesame special type protoplast
  • Genetic transformation method for PEG-medicated fusarium oxysporum sesame special type protoplast
  • Genetic transformation method for PEG-medicated fusarium oxysporum sesame special type protoplast

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Experimental program
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Effect test

Embodiment 1

[0053] Embodiment 1, the collection of Fusarium oxysporum sesame specialization mycelia

[0054] Pick the mycelium of Fusarium oxysporum sesame specialization preserved on the PDA plate and inoculate it in 100mL PDB liquid medium. After shaking and culturing at 26°C and 130rpm for 3 days, filter the medium with double-layer sterile fine-pore gauze, and centrifuge at 4000rpm at room temperature. 6min to collect spores. Transfer the spore mass to 100mLYEPD liquid medium, culture at 26°C and 130rpm for 15h, filter the medium with double-layer sterile fine-pored gauze, and collect mycelium. The form of mycelium is as follows: figure 1 shown.

Embodiment 2

[0055] Embodiment 2, the preparation of Fusarium oxysporum sesame specialized strain protoplast

[0056] 1) Take 0.3g of collected fresh mycelium and put it into a 50mL centrifuge tube, add 5mL of protoplast lysis buffer, shake and culture at 30°C and 100rpm for 3h, observe the protoplast under a microscope, the morphology is as follows: figure 2 shown.

[0057] 2) Filter the enzymatic hydrolysis mixture into a 50mL centrifuge tube with three layers of lens paper, wash as much protoplasts as possible with 1.2M KCl, collect by centrifugation at 3600rpm at room temperature for 6min, and discard the supernatant.

[0058] 3) Add 10 mL of STC buffer to gently resuspend the protoplast pellet, centrifuge at 3600 rpm for 6 min at room temperature, and discard the supernatant. Repeat this step 1 time.

[0059] 4) Add 1mL STC buffer to resuspend the protoplasts, then transfer to a 2mL centrifuge tube, centrifuge at 3600rpm at room temperature for 6min, then add an appropriate amount ...

Embodiment 3

[0060] Embodiment 3, transformation of Fusarium oxysporum sesame specialized strain protoplast

[0061] 1) Add 6 μg of pBHt2-eGFP plasmid to a 50 mL centrifuge tube containing 200 μL of protoplast suspension, mix gently, and let stand at room temperature for 20 minutes. Then slowly add 1mL of 40% PTC buffer to the centrifuge tube, mix gently, let stand at room temperature for 20min, then add 5mL of TB3 medium, shake at room temperature for 10h at 100rpm.

[0062] 2) Centrifuge at 5000rpm for 5min, discard the supernatant, resuspend with 1mL STC buffer, then add 10mL Bottom Agar medium (containing 200μg / mL hygromycin and 50μg / mL cephalosporin) cooled to 50°C, After mixing evenly, place the plate on the plate, and after incubating at room temperature for 12 hours, cover the Bottom Agar plate with 10 mL of Top Agar medium (containing 300 μg / mL hygromycin and 50 μg / mL cephalosporin) cooled to 50 °C, and incubate at 26 °C until Transformants grow out as image 3 shown. A total o...

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Abstract

The invention discloses a genetic transformation method for PEG-medicated fusarium oxysporum sesame special type protoplast, and belongs to the technical field of biology. The PEG-medicated protoplast transformation method is utilized, and through the preparation and transformation of fusarium oxysporum sesame special type protoplast and the screening of transformant, foreign DNA sections containing screening labels are conversed into the fusarium oxysporum sesame special type genome. The transformation of PEG-medicated fusarium oxysporum sesame special type protoplast is achieved, a whole genetic transformation and screening method is established, a great amount of transformated bacterium strains is obtained, wherein the bacterium strains can intensely express hygromycin resistant protein and green fluorescent protein, and the character can be stably inherited; and the transformation efficiency can reach 7667 transformants per mg of DNA. Moreover, the carrier used in the transformation comprises enhanced green fluorescent protein genes and strong promoter gpd in fungus, and thus the strength of fluorescent given off by the transformated bacterium strains is effectively strengthened.

Description

technical field [0001] The invention relates to a method for genetic transformation of Fusarium oxysporum sesame specialized protoplasts mediated by PEG, and belongs to the field of biotechnology. Background technique [0002] Sesame Fusarium wilt is an important soil-borne fungal disease caused by Fusarium oxysporum f.sp.sesami (Zaprometoff) Castellani, which occurs to varying degrees in sesame planting regions around the world. In our country, the harm of sesame fusarium wilt to sesame production is relatively common. The incidence rate in general popular years is about 5-15%, and it can reach more than 30% in large-scale occurrences, which seriously affects the yield and quality of sesame. The study of the pathogenic characteristics of the chemotype is very important. Due to the late start of research on sesame diseases and the weak research foundation, there are few reports on the pathogenic mechanism of Fusarium wilt of sesame. [0003] In recent years, with the compl...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N1/15C12R1/77
Inventor 苗红梅段迎辉常淑娴张海洋
Owner HENAN ACAD OF AGRI SCI
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