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Copper ion permease gene promoter Ptcu1 in trichoderma reesei and application

A technology of copper ion permease and Trichoderma reesei, which is applied in the field of bioengineering, can solve the problems of difficult genetic transformation and difficulty in obtaining transformants, and achieve the effect of reducing production costs

Inactive Publication Date: 2015-04-29
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, we noticed that most of Trichoderma reesei exists in haploid form, and it is very difficult to genetically modify some genes that have a serious impact on growth, and it is difficult to obtain suitable transformants

Method used

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  • Copper ion permease gene promoter Ptcu1 in trichoderma reesei and application
  • Copper ion permease gene promoter Ptcu1 in trichoderma reesei and application
  • Copper ion permease gene promoter Ptcu1 in trichoderma reesei and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Isolation of tcu1 promoter gene in Trichoderma reesei in embodiment 1

[0033] 1. Inoculate 10 spores of Trichoderma reesei QM9414 in 20ml Trichoderma reesei MM culture medium 6 , cultivated at 30°C for 48 hours, collected mycelia, and extracted the genome.

[0034] 2. Trichoderma reesei MM medium, the components per liter are as follows:

[0035] Peptone, 10g;

[0036] Glucose, 20g;

[0037] (NH 4 ) 2 SO 4 , 5g;

[0038] K 2 HPO 4 , 15g with 1M NaOH to adjust the pH to 5.5, after constant volume value 1L.

[0039] Add to each bottle after sterilization

[0040] 200×MgSO 4 (120g / l) 5ml;

[0041] 200×CaCl 2 (120g / l) 5ml;

[0042] 1000×trace elements (FeSO 4 ·7H 2 O, 5g / l; MnSO 4 ·H 2 O, 1.6g / l; ZnSO 4 , 1.4g / l; CoCl 2 , 2g / l), 1ml.

[0043] 3. The genome was extracted using the fungal DNA mini Kit kit, which was purchased from Omega Company, and the steps are detailed in the product manual.

[0044] 4. Use the genome obtained in step 3 as a template...

Embodiment 2p

[0051] Embodiment 2 ptcu promoter starts endogenous copper ion permease gene (tcu1) transcriptional analysis

[0052] (1) Add 250ml Trichoderma reesei pre-cultivation medium in the glass Erlenmeyer flask of 1L, inoculate the spore 10 of Trichoderma reesei 6 Each, 30 ℃, 200 revs / min cultivated for 48 hours, collected mycelia with G1 sand core funnel and transferred to fresh 250ml Trichoderma reesei pre-cultivation medium and continued to cultivate for 12 hours.

[0053] (2) Collect mycelium in step (1) and transfer to CuSO containing 100, 200, 500, 1000, 5000, 10000 nM concentration with an inoculum size of 4 g / L 4 In the culture medium with a mass concentration of 1% glucose as the carbon source, samples were taken after culturing for 12 hours, RNA was extracted, and real-time quantitative PCR was performed to detect the transcription of tcu1. The results were as follows figure 1 As shown in A. The transcription of tcu1 decreased with the increase of copper ion concentration...

Embodiment 3

[0058] Example 3 Trichoderma reesei P tcu1 -GFP fluorescence signal detection analysis

[0059] (1) Construction of Trichoderma reesei P tcu1 ‐GFP expression strains. The pMD-simpleT-ptcu vector in Example 1 was digested with hindⅢ and NotI, and the terminator TrpC sequence containing hinddⅢ and NotI restriction sites at both ends was inserted into the above vector to form a pMD-simpleT-ptcu-trpC vector ;

[0060] (II) Insert the GFP sequence containing pmeI and speI restriction sites at both ends into the pMD-simpleT-ptcu-trpC vector to form the pMD-simpleT-ptcu-GFP-trpC vector;

[0061] (III) The pMD‐simpleT‐ptcu‐GFP‐trpC vector of step (II) and the pRLMex‐30 plasmid with the hygromycin resistance gene hph were transformed into Trichoderma reesei QM9414 by PEG-mediated protoplast transformation Trichoderma reesei P tcu1 ‐GFP strains;

[0062] (IV) Trichoderma reesei P tcu1 ‐GFP was cultured in MM medium containing 1% glucose as carbon source, and CuSO was added to the...

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Abstract

The invention relates to a copper ion permease gene promoter Ptcu1 in trichoderma reesei and an application. A nucleotide sequence of the copper ion permease gene promoter Ptcu1 in trichoderma reesei is shown in SEQ ID NO.1. The promoter Ptcu1 is strictly regulated by copper ion concentration, so that regulated transcription on some key genes can be carried out; meanwhile, by utilizing the high expression property of the Ptcu1, the Ptcu1 can be used as a good foreign protein expression system. Meanwhile, because of promoter overexpressive cellulase transcriptional regulation factors Xyr1, the dependency on induced carbon sources by the trichoderma reesei can be released, and the cellulase can be produced by utilizing commonly-used carbon sources such as glucose, other oligosaccharides and the like, so that the production cost of the cellulase is greatly reduced.

Description

technical field [0001] The invention relates to a copper ion permease gene promoter P in Trichoderma reesei tcu1 and applications, belonging to the technical field of bioengineering. Background technique [0002] On the earth, plants use solar photosynthesis to produce a large amount of cellulose and hemicellulose every year. The synthesis and degradation of cellulose and hemicellulose are important components of the earth's carbon cycle. Abundant cellulose and hemicellulose resources have important application potential in the production of renewable energy and basic compounds. Because of its high efficiency and diversity of cellulase production, Trichoderma reesei has been widely studied and used as an industrial strain. It mainly degrades external lignin by secreting cellulase and hemicellulase, which include exoglucosidase, endoglucosidase, β-glucosidase and xylanase Wait. The cellulase produced by fungi currently used is induced, and insoluble microcrystalline cellu...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/80C12N9/42C12R1/885
Inventor 刘巍峰吕新星郑芳林李春燕张伟欣
Owner SHANDONG UNIV