Trichoderma engineering strain capable of efficiently expressing beta-1, 4-glucanase coding gene and application thereof

A technology for engineering strains and glucanase, which is applied in the field of bioengineering to achieve the effects of enhanced conidia ability, stable expression of glucanase activity, and improved control effect

Active Publication Date: 2013-04-24
BIOTECH CENT OF SHANDONG ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, more than 20 kinds of coding genes from Trichoderma itself and exogenous bioactive molecules have been successfully transferred into Trichoderma, which significantly improved the antagonistic ability of Trichoderma. Less reported strains

Method used

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  • Trichoderma engineering strain capable of efficiently expressing beta-1, 4-glucanase coding gene and application thereof
  • Trichoderma engineering strain capable of efficiently expressing beta-1, 4-glucanase coding gene and application thereof
  • Trichoderma engineering strain capable of efficiently expressing beta-1, 4-glucanase coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1: Trichoderma expression vector pSilent / glu14 build

[0047] The expression vector pSilent / obtained in this embodiment glu14 , not only for the transformation of Trichoderma fungi, but also for the transformation of other filamentous fungi.

[0048]β-1,4-glucanase gene glu14 The size is about 1.5kb, and it has been registered in Genbank with the serial number HM130670. The genomic DNA of Bacillus megaterium Ap25 was extracted by the CTAB / NaCl method. Using the genomic DNA as a template, primers were designed according to the β-1,4-glucanase gene published on Genbank and PCR amplification was performed. After repeated experiments, Finally, the suitable for amplification of Ap25 genomic DNA was determined. glu14 The primer sequence is glu14 / F:5'-GCGC AAGCTT ATGAAACGGTCAAATCTCTAT-3' and glu14 / R:5'-GCGC AGATCT CTAs

[0049] ATTTGGTTCTGTTCC-3' for PCR amplification (the underline represents the added Hin dⅢ and Bgl Ⅱ restriction site), th...

Embodiment 2

[0051] Embodiment 2: Protoplast preparation and regeneration

[0052] In this example, the young aerial hyphae of Trichoderma was used as the inoculum, which can eliminate the interference of the subsequent experiments by using the ungerminated spores as protoplasts during the cultivation process after the inoculation of conidia. At the same time, the regeneration-promoting reagent JC (0.3 mol / L inositol, 5 g / L chitin powder) was added to the regeneration medium, which was beneficial to the production of cell walls by protoplasts and increased the regeneration rate.

[0053] Pick the young aerial hyphae of Trichoderma viride LTR-2 from the plate to inoculate PDA liquid medium, culture at 28°C for 13 hours with shaking, filter with 3 layers of sterile lens-cleaning paper, wash with sterile water twice, and the osmotic pressure is stable Flush 2 times. Suspend an appropriate amount of mycelium in the lysate (Lysing Enzymes / Helicase / Cellulase: 5mg / mL each, dissolved in osmotic p...

Embodiment 3

[0060] Embodiment 3: REMI method transforms Trichoderma viride LTR-2

[0061] Compared with other transformation methods, the transformation of filamentous fungi using the REMI method has high transformation efficiency and an increased probability of single-copy insertion. It can quickly isolate the tagged target gene through plasmid rescue and PCR techniques.

[0062] According to the recombinant expression vector pSilent / glu14 enzyme cutting site on the Sac Ⅰ Carry out enzyme digestion, and the digested products are gel-purified for later use. Take 30 μg of linearized plasmid, restriction endonuclease Sac Ⅰ 4 μL, STC 150 μL, mix well, ice bath; add protoplasts (10 7 pcs / mL) 150μL, mix well, ice bath 20min; slowly add PTC (pre-cooling) 1.5mL, mix well, ice bath 5min, room temperature 20min; add STC (precooling) 5mL, mix well, 4℃, 7000r / min, centrifuge for 10 min; add 2 mL of liquid regeneration medium to the precipitate, and incubate for 12 hours at 28°C, 50r / min; ...

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Abstract

The invention discloses a trichoderma engineering strain capable of efficiently expressing a beta-1, 4-glucanase coding gene glu14 of bacillus megaterium Ap25 source. The engineering strain L-10 is obtained by integrating fungus expression vector pSilent / glu14 containing beta-1, 4-glucanase coding gene glu14 in trichoderma viride LTR-2 chromosome with a biological control function of plant disease through a REMI (Restriction Enzyme Mediated Integration) transformation method. Beta-1, 4-glucanase coding gene glu14 can be constitutively and efficiently expressed by the engineering strain L-10, and compared with an original strain LTR-2, the growth rate and the ability of producing conidium are obviously improved. In the industrial production of the engineering strain L-10, a wettable powder is prepared by adopting liquid fermentation and a novel technology of chlamydospore induced and produced by an inducer, and adopting vector such as medical stone, so that the storage life reaches 2 years. Compared with the original strain LTR-2, a greenhouse experiment shows that the prevention effect of the engineering strain L-10 to wheat sharp eyespot and borrytis cinerea is remarkably improved.

Description

technical field [0001] The invention belongs to the field of biological engineering. In particular, it relates to a Trichoderma biocontrol recombinant engineering strain highly expressing the glucanase coding gene of Bacillus megaterium Ap25 and its application. Background technique [0002] Trichoderma ( Trichoderma ) belongs to the subphylum Deuteromycota, Hyphospora, Hyphospora, Myxospora, mycelia have septa, can produce conidia, cells are mononuclear structure, and the behavior is Hypocrea ( Hypocrea ). Trichoderma is a biocontrol fungus widely distributed in nature. It has antagonistic effects on at least 20 kinds of pathogenic fungi and a variety of pathogenic bacteria in 18 genera. At present, more than 100 biological agents containing Trichoderma components are used in more than 60 countries on five continents in the world product. [0003] Trichoderma can produce a variety of cell wall degrading enzymes, among which dextranase has become a research hotspot and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/80C12N3/00A01N63/04A01N25/14A01P3/00C12R1/645
Inventor 李纪顺陈凯杨合同扈进冬李红梅魏艳丽张广志周红姿赵晓燕王贻莲郭凯
Owner BIOTECH CENT OF SHANDONG ACAD OF SCI
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