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A kind of β-1,6-glucanase and its coding gene and application

A glucanase and gene technology, applied in the field of agricultural microorganisms and plant protection, can solve the problem of low activity

Active Publication Date: 2019-06-14
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, β-1,6-glucanase derived from microorganisms mainly comes from Trichoderma harzianum and Actinomycetes, but its application is affected by its low activity and related properties

Method used

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  • A kind of β-1,6-glucanase and its coding gene and application
  • A kind of β-1,6-glucanase and its coding gene and application
  • A kind of β-1,6-glucanase and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Purification and gene cloning of embodiment 1β-1,6-glucanase

[0068] 1. Separation and purification of 1β-1,6-glucanase

[0069] Inoculate the strain Corαllococcus sp.EGB (CCTCC NO: M2012528) into VY / 4 liquid medium (VY / 4 medium: 0.25% yeast cells, 0.1% CaCl 2 , pH 7.0), cultured on a shaker at 30°C for 2-3 days, collected the fermentation supernatant by centrifugation, concentrated the supernatant by 40%-80% ammonium sulfate gradient precipitation, passed through DEAE weak anion exchange column, hydrophobic column, sephardex G200 molecular sieve Combined with dextran substrate adsorption and desorption and other methods, the target protein is purified. By zymographic analysis (such as figure 1 ), determine that the band with a molecular weight of about 97KD on the SDS-PAGE protein electrophoresis is the target band.

[0070] Yeast glucan, β-1,3-glucan, laminarin, agarin, glucan BIWP2 (doi:10.1016 / j.carbpol.2012.12.036), oat glucan, cellulose, wood Glycans were use...

Embodiment 2

[0089] Example 2. Heterologous expression of β-1,6-glucanase gene gluM

[0090] 2.1 Construction of expression vector gluM-pET-29a(+)

[0091] Digest the recombinant plasmid extracted in 1.3.3 and pET-29a(+) (Merck-Novαgen, Cat NO.69871) with NdeI and XhoI

[0092] Enzyme cutting system:

[0093]

[0094] In a 37°C water bath, the enzyme digestion reaction was carried out overnight. The digested products were recovered by 0.75% agarose gel electrophoresis. The pET-29a(+) digested by the recovered fragment was enzyme-ligated to obtain the pET-29a(+) recombinant plasmid containing the β-1,6-glucanase gene.

[0095] The enzyme-linked pET-29a(+) recombinant plasmid containing β-1,6-glucanase gene was transformed into the expression host strain E.coli BL21(DE3) (NBE, Cat NO.C2527H) to obtain the recombinant microorganism E. coli BL21(DE3), spread LB plates containing 50mg / L kanamycin, pick a single colony to extract the plasmid and verify the gene sequence is correct by sequ...

Embodiment 3

[0099] Example 3. Functional verification of β-1,6-glucanase GluM

[0100] Use spore-forming medium to culture Magnaporthe grisea, and collect Magnaporthe grisea spores, which are derived from induced expression of recombinant β-1,6-glucanase GluM-BL21 (full-length gene of β-1,6-glucanase , gene sequence position: 79bp-3222bp, SEQ ID NO.1; amino acid sequence position: 27AA-1073AA, SEQ ID NO.2; remove signal peptide) and β-1,6-glucan derived from supernatant crude enzyme solution The enzyme GluM-S was co-cultured with blast spores on a hydrophobic membrane, and the germination of blast spore tubes and the formation of attached spores were observed at intervals. Simultaneously with bacterial strain supernatant crude enzyme liquid, supernatant enzyme liquid ultrafiltration liquid (molecular weight cut-off 10KD) and heat-inactivated enzyme liquid as contrast treatment, the result is as follows Figure 5 . The results showed that after 4 hours of treatment, compared with the nor...

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Abstract

The invention discloses a novel beta-1,6-glucanase, an encoding gene thereof, and applications of the encoding gene. The invention provides the beta-1,6-glucanase gene belonging to the glucoside hydrolase system, wherein the nucleotide sequence of the beta-1,6-glucanase gene is shown in SEQ ID NO.1, and the protein (amino acid) sequence of the encoded outer membrane glucoside hydrolase is shown in SEQ ID NO.2. The beta-1,6-glucanase can effectively prevent the infection of plant pathogenic fungi to plants. An engineered strain established by the gene realizes the prokaryotic expression of the beta-1,6-glucanase gene, and verifies the function of the glucoside hydrolase of the beta-1,6-glucanase gene, the result shows that GluM can effectively inhibit the germination of rice blast spores, and decompose the rice blast spores. After the beta-1,6-glucanase gene is transplanted into the dicotyledonous model plant arabidopsis thaliana and the monocotyledonous model plant rice, the result shows that the obtained transgenic plants have good anti-infection effects for grey mould and rice blast.

Description

technical field [0001] The invention belongs to the field of agricultural microorganisms and plant protection, and discloses a new β-1,6-glucanase, its coding gene and application. Background technique [0002] Plant diseases are one of the restrictive factors for high-quality and high-quality crops. According to statistics, the global crop yield loss due to plant diseases accounts for about 16%, and the annual direct economic loss reaches hundreds of millions of dollars. Among plant diseases, 70%-80% of the diseases are caused by the infection of plant pathogenic fungi. Plant fungal diseases not only directly cause the decline of crop yield and quality, but also some pathogenic fungi can secrete a variety of toxins and metabolites harmful to humans and animals during the process of infecting crops, posing a great threat to the safety of agricultural products. [0003] The control of plant fungal diseases is difficult, and effective control methods are urgently needed in pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N9/46C12N1/21A01H5/00A01H6/46A01N63/02A01P3/00C12R1/19
Inventor 崔中利李周坤张碧滢叶现丰黄彦
Owner NANJING AGRICULTURAL UNIVERSITY
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