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Method for introducing agrobacterium tumefaciens-mediated exogenous gene into cordyceps militaris cells

An Agrobacterium-mediated, exogenous gene technology is applied in the field of Agrobacterium-mediated exogenous gene introduction into Cordyceps militaris cells, which can solve the problems of no literature report on the transgenic Cordyceps militaris, and achieve the effects of low cost and easy operation.

Inactive Publication Date: 2020-06-30
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Transgenic Cordyceps militaris mediated by Agrobacterium tumefaciens has not been reported in the literature

Method used

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  • Method for introducing agrobacterium tumefaciens-mediated exogenous gene into cordyceps militaris cells
  • Method for introducing agrobacterium tumefaciens-mediated exogenous gene into cordyceps militaris cells
  • Method for introducing agrobacterium tumefaciens-mediated exogenous gene into cordyceps militaris cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Construction of Agrobacterium tumefaciens AGL-1 containing plasmid PCAMBIA-PgpdA-Tcbh1-hph-PtrpC:

[0035] use -Uni Seamless Cloning and Assembly Kit combines hygromycin resistance gene hph gene (shown in SEQ ID NO.1) and enhanced green fluorescent protein gene egfp (nucleotide sequence shown in SEQ ID NO.2) with carrier PCAMBIA -PgpdA-Tcbh1-PtrpC was seamlessly cloned to obtain a solution of plasmid pCAMBIA-PgpdA-egfp-Tcbh1-hph-PtrpC, which was stored at -20°C.

[0036] Take 5 μL of the plasmid PCAMBIA-PgpdA-egfp-Tcbh1-hph-PtrpC solution and add it to 100 μL Agrobacterium tumefaciens AGL-1 competent, freeze in liquid nitrogen for 8 seconds, and bathe in water at 37°C for 5 minutes; add 800 μL LB liquid medium to the competent, 28°C, 180rpm, shaking culture for 1h; centrifuge at 3500rpm for 10min, discard the supernatant on the ultra-clean bench, and keep 100μL of bacterial liquid; resuspended bacterial liquid was spread on LB solid medium + 25mg L -1 Rif+50mg·L -1 ...

Embodiment 2

[0039] Table 1 Sources of materials

[0040]

[0041]

[0042] Table 2 Each medium component

[0043]

[0044] The composition of the PDA medium is: 200g / L potato, 20g / L glucose, 15-20g / L agar, distilled water as a solvent, and natural pH.

[0045] The composition of the LB liquid medium: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, solvent is distilled water, pH7.0.

[0046] The composition of the LB solid medium: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar 15-20g / L, solvent is distilled water, pH 7.0.

[0047] The composition of the IM medium: 0.145% KH 2 PO 4, 0.205% K 2 HPO 4 , 0.06% MgSO 4 ·7H 2 O, 0.03% NaCl, 0.01‰CaCl 2 , 0.001‰FeSO 4 , 0.05% NH 4 NO 3 , 5mL / L glycerin, 0.2% glucose, 5mL / L trace element stock solution, 40mL / L MES buffer solution (1mol / L, pH=5.5), solvent is distilled water; Described every 10ml trace element stock solution composition: ZnSO 4 ·7H 2 O 0.001g, CuSO 4 ·5H 2 O 0.001g, H 3 BO 4 0.001g, (NH 4 ) 2 SO 4...

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Abstract

The invention discloses a method for introducing an agrobacterium tumefaciens-mediated exogenous gene into cordyceps militaris cells. The method at least comprises the following steps of laminating asterile cellulose membrane on the surface of a co-culture solid medium, then mixing cordyceps militaris spore suspension with an equal volume of agrobacterium tumefaciens solution containing the exogenous gene, coating the sterile cellulose membrane with the mixed bacteria solution and carrying out light-tight co-culture at constant temperature of 24 DEG C for 48 h; transferring the mixed bacteriasolution together with the cellulose membrane into a selective medium; pouring a layer of selective medium on the surface of the tightly laminated cellulose membrane and carrying out light-tight co-culture at the constant temperature of 22-25 DEG C; and taking mycelia penetrating through the upper selective medium and transferring the mycelia into a subculture medium, carrying out light-tight co-culture at the constant temperature of 22-25 DEG C for 3-4 days and carrying out screening to obtain cordyceps militaris containing the exogenous genes. The genetic transformation efficiency of the exogenous genes of the cordyceps militaris is up to 100%, and the method is low in cost and easy to operate.

Description

[0001] (1) Technical field [0002] The invention relates to a method for Agrobacterium-mediated exogenous gene introduction into Cordyceps militaris cells. [0003] (2) Background technology [0004] With the vigorous development of life sciences, molecular biology and genetic engineering technology have also made great progress. The exogenous gene transformation method mediated by Agrobacterium tumefaciens has attracted widespread attention of researchers because it can introduce large fragments of DNA and the gene copy number is low, the transformation efficiency is high, and the expression effect is good. It has the advantages of low requirements for instruments and operating techniques, and is widely used in plant and fungal genetic engineering to study unknown gene functions and explore the mechanism of various gene regulatory elements. At present, Agrobacterium-mediated transformation of exogenous genes has been achieved in various fungi such as Rhizopus, Aspergillus, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N15/65C12N3/00C12R1/645
CPCC12N15/80C12N15/65C12N3/00
Inventor 杨胜利杨锡陈萍孔潇慧季小康
Owner ZHEJIANG UNIV OF TECH