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Trichoderma viride alkali protease as well as eukaryotic expression method and application thereof

A technology of Trichoderma viride and an expression method, applied in the directions of application, botanical equipment and method, biochemical equipment and method, etc., can solve problems such as unreported alkaline protease, and achieve the effect of inhibiting the growth of Aspergillus flavus

Inactive Publication Date: 2011-08-31
CROP RES INST GUANGDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, the research on alkaline protease in Trichoderma viridans has not been reported

Method used

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  • Trichoderma viride alkali protease as well as eukaryotic expression method and application thereof
  • Trichoderma viride alkali protease as well as eukaryotic expression method and application thereof
  • Trichoderma viride alkali protease as well as eukaryotic expression method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] (1) Isolation and identification of Trichoderma viride strains

[0048] ①The isolated soil samples were taken from the soil of peanut planting field in Shantou City, Guangdong Province. Weigh 10 g of soil sample, put it into a triangular flask with glass beads containing 100 ml of sterile water, and shake for 10 min to fully mix the soil and water. Under aseptic operation conditions, use a pipette to draw 1ml from the triangular flask, add it to another test tube containing 9ml of sterile water, mix well, and so on to make 10 -2 , 10 -3 , 10 -4 Soil solutions of different dilutions.

[0049] ②Coating culture and streak separation: take 10 -2 and 10 -3The soil dilution of two concentrations was used as the object of coating plate culture, and it was coated on PDA (potato dextrose agar) solid medium (potato 200g, glucose 20g, agar 15g, pH6.8, distilled water was settled to 1000ml) on , 3 replicates for each sample, and after culturing at 28°C for 3 days, the microor...

Embodiment 2

[0093] Get Trichoderma viride alkaline protease

[0094] (1) Synthesize PCR primers

[0095]AM: 5′-CAATCTGGTGCTCCTTGGGGT-3′

[0096] BM: 5′-CGTATGTAGATCCACCAATTGTTCC-3′

[0097] Q1: 5′-GTCATCGTCAATCTGGGTCACAAGC-3′

[0098] Q2: 5′-GTATGTAGATCCACCAATTGTTCCG-3′

[0099] M1: 5′-ATGGCCATCATCCGTCGTCTT-3′

[0100] M2: 5′-TTAAGCACTGTGTCCGTTGAAG-3′

[0101] The above primers were synthesized by Sigma Company.

[0102] (2) Obtain the cDNA of Trichoderma viride alkaline protease

[0103] 1, operate according to the instructions, extract the total RNA of Trichoderma viride GZ101 with TRIZOL reagent (purchased from Invitrogen), carry out the amplification of the first strand of cDNA with RT-PCR kit (purchased from TaKaRa company, operate according to the instructions), The primer was OLIGO 18T to obtain the first strand of cDNA. Then take the first strand of cDNA as a template, carry out PCR amplification, and the PCR system of amplification is as follows:

[0104] Reagent Volume ...

Embodiment 3

[0177] In vitro activity of TvALP1 protein against Aspergillus flavus

[0178] After the concentration of purified TvALP1 protease was determined by Coomassie brilliant blue, the activity of inhibiting the growth of Aspergillus flavus in vitro was detected with Aspergillus flavus GIM3.17 (purchased from the Institute of Microbiology, Chinese Academy of Sciences) as the indicator bacteria. The specific steps are: add 100 μl of PDA liquid medium (200 g of potato, 20 g of glucose, pH 6.8, distilled water to 1000 ml) and 20 μl of 1×10 5 A suspension of Aspergillus flavus GIM3.17 spores, and then 100 μl of different concentrations of protease solutions prepared by double dilution were added to the wells, so that the final concentration of protease was 300 μg / ml to 1 μg / ml (10 concentrations) ; Treat with 100 μl of PBS (0.2mol / L, pH7.0) as a negative control; treat with 100 μl of 5 μg / ml of amphotericin B as a positive control, a total of 12 treatments, each with 3 replicates. The ...

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Abstract

The invention discloses trichoderma viride alkali protease as well as an eukaryotic expression method and application thereof. The coding sequence of the trichoderma viride alkali protease is obtained through an RACE (Rapid Amplification of cDNA Ends) method, the length of an amino acid sequence is 410 amino acids, and the length of the coding sequence is 1233 nucleotides. The coding nucleotide sequence of the trichoderma viride alkali protease is constructed in an eukaryotic expression vector to perform eukaryotic expression, and trichoderma viride alkali protease capable of inhibiting growth of aflatoxin can be obtained. The trichoderma viride alkali protease has the function of inhibiting the growth of the aflatoxin.

Description

technical field [0001] The invention belongs to the technical field of anti-Aspergillus flavus growth, and in particular relates to a Trichoderma viride alkaline protease and a eukaryotic expression method and application thereof. Background technique [0002] Trichoderma viride (Trichoderma viride) belongs to the subphylum Deuteromycetes, the order Cleospora, and the family Cleospora. Trichoderma viride has attracted much attention due to its abundant resources, long lasting effect, unique mode of action, safety to non-target organisms and no environmental pollution. In research at home and abroad, the use of Trichoderma to control crop damping, blight, botrytis, sheath blight, etc. has achieved good results. [0003] Alkaline Protease (Alkaline Protease) belongs to serine extraspore high alkaline protease. It can hydrolyze protein molecules and has a strong ability to decompose proteins. It is an important cell wall cleavage enzyme. In the interaction between Trichoderma ...

Claims

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Application Information

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IPC IPC(8): C12N9/58C12N15/55C12N15/79A01N63/04A01P3/00
Inventor 刘付香梁炫强李玲陈小平刘海燕张二华温世杰
Owner CROP RES INST GUANGDONG ACAD OF AGRI SCI
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