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Armillaria selection marker and transgenic strain construction method

A technology for screening markers and Armillaria armillaria, applied in the field of fungal molecular genetic engineering, can solve the problem of less research on the molecular mechanism of active substances of Armillaria armillaria

Inactive Publication Date: 2018-05-04
KUNMING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the research on Armillaria Gastrodia mainly focuses on the screening of Armillaria Gastrodiae, the optimization of culture conditions of Armillaria Gastrodiae, the influence of Armillaria Gastrodiae on the yield of Gastrodia elata, and the symbiotic relationship of Armillaria Gastrodiae. There are few studies on the molecular mechanism of gene expression, biosynthesis of active substances, and exchange of nutrients with Gastrodia elata.

Method used

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  • Armillaria selection marker and transgenic strain construction method
  • Armillaria selection marker and transgenic strain construction method
  • Armillaria selection marker and transgenic strain construction method

Examples

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Embodiment 1

[0026] Example 1: Armillaria antibiotic screening

[0027] The medium is PDA: 200g of potatoes, 20g of glucose, 20g of agar, and add water to make up to 1000mL. Add ampicillin Amp, kanamycin Km, spectinomycin Spe, and hygromycin Hyg to the PDA plate, the concentrations are: 0 µg / mL (Ck), 50 µg / mL, 100 µg / mL 150 µg / mL, 200 µg / mL, and 250 µg / mL were cultured for 5 days and then photographed to observe the growth of Armillaria armillaria as follows: figure 1 , the results showed that Armillaria can grow on all gradient concentrations of Amp, Km and Spe antibiotics, but not on the medium containing hygromycin.

Embodiment 2

[0028] Example 2: Cultivation on Solid Medium and Liquid Medium of Armillaria Armillaria

[0029] Inoculate the Armillaria strain on the slant medium PDA, and cultivate it at 26-27°C for about 10 days, after the mycelium is covered with the slant ( figure 2 A), transferred to the liquid medium, let it stand for 2 days, and cultured in a shaking table for 5 days, after the bacteria balls grow well ( figure 2 B) Store at 4°C for later use. The single colony hyphae can be seen from the solid medium, and after the hyphae are picked into the liquid medium, the bacterial balls are covered with the liquid medium.

Embodiment 3

[0030] Example 3: Expression vector pH2GW7-35S- EGFP Transform Agrobacterium

[0031] target gene sequence EGFP (such as SEQ ID NO: 1); use respectively xho I and Bam H I enzyme will purify pENTR * -2B and pMD18-T- EGFP Plasmid cutting, separation of cut vector and insert by agarose gel electrophoresis, recovery of pENTR * The vector fragment pENTR produced after -2B is cut * (3.8kb) and pMD18-T -EGFP DNA fragments produced by cleavage EGFP (0.717kb), and then use the ligase kit of TaKaRa to connect pENTR * and EGFP Generate intermediate vector pENTR * -T -EGFP ; conversion of high efficiency (10 8 ) Escherichia coli competent cells (DH5 α Tiangen Biochemical Technology), spread the transformed Escherichia coli on a plate added with kanamycin (Km, 50 μg / ml), culture overnight at 37°C, screen Km-resistant recombinant colonies, and recombined from Km-resistant Plasmids were extracted from the sub-colony with xho I and Bam H I double enzyme digestion de...

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Abstract

The invention discloses an Armillaria selection marker. Hygromycin is adopted as the Armillaria selection marker. Antibiotics capable of inhibiting Armillaria growth are screened out by experiments; the eukaryotic expression vector pH2GW7-35S-EGFP is selected for agrobacterium transformation, the hygromycin is taken as the selection marker, and an EGFP gene is transferred into Armillaria with an agrobacterium-mediated method; GFP fluorescence is seen under a fluorescence microscope, which proves expression of pH2GW7-35S-EGFP in Armillaria, and the Armillaria can serve as a host cell for transgenic operation; establishment of a transgenic method based on Armillaria lays foundation for further research of Armillaria gene expression, and a genetic engineering research method for deep researchof biosynthesis and regulation of active components in Armillaria, the molecular mechanism of exchange between Armillaria and gastrodia elata nutrients and the like is provided.

Description

technical field [0001] The invention relates to a screening marker of Armillaria armillaria and an Agrobacterium-mediated transgenic method of Armillaria armillaria, belonging to the field of fungal molecular genetic engineering. Background technique [0002] Armillaria ( Armillaria (Fr.: Fr.) Staude.), Also known as hazel mushroom, honey ring mushroom, and green oka mushroom, it is an important class of fungi with both medicinal and edible functions. It is symbiotic with Gastrodia elata and is the pillar for the survival of Gastrodia elata. The growth of Armillaria directly affects the inoculation status, yield and quality of Gastrodia elata. In addition to providing nutrients for the growth of Gastrodia elata, Armillaria mellea and its fermentation products have similar pharmacological effects to Gastrodia elata, including hypnosis and sedation, regulating blood circulation, enhancing immunity, scavenging free radicals, delaying aging, inhibiting tumors and other biologic...

Claims

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Application Information

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IPC IPC(8): C12N15/65C12N15/80C12N1/15C12R1/645
CPCC12N15/65C12N15/80
Inventor 李昆志张恒丽包燚谭彧文陈丽梅徐慧妮
Owner KUNMING UNIV OF SCI & TECH
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