An endo-β-1,3-glucanase coding gene, enzyme, preparation method and application thereof

A technology of glucanase and coding genes, which is applied in botany equipment and methods, biochemical equipment and methods, applications, etc., and can solve problems such as rampant pests and diseases, irrational use, and pesticide residues

Active Publication Date: 2020-06-30
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the early 1940s, a large number of organic synthetic pesticides were used. Although they played an important role in ensuring the stability and high yield of agriculture and livestock, due to the shortcomings of chemical pesticides and their long-term unreasonable use, some serious problems gradually emerged, such as Drug resistance, pesticide residues, and pests and diseases are rampant again, that is, the 3R problem (Resistance, Residue, Resurgence)

Method used

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  • An endo-β-1,3-glucanase coding gene, enzyme, preparation method and application thereof
  • An endo-β-1,3-glucanase coding gene, enzyme, preparation method and application thereof
  • An endo-β-1,3-glucanase coding gene, enzyme, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Endo-β-1,3-glucanase full-length gene cloning

[0047] Genomic DNA of Microbacterium fibroblasts was extracted according to the operation steps of Genomic DNA Purification Kit (Thermo, LOT 00105781). After performing multiple sequence alignment analysis on the endo-β-1,3-glucanase gene sequence in The National Center for Biotechnology Information (NCBI) database, a degenerate primer glu-F: 5'-GATATACATATGGCRCCSGGSGACMTCSTSTGGTCSGACGAG-3' was designed; glu-R: 5'-GTATAACTCGAGGARSGTCCACTGCTGGKYGGTCGWGCCGTKGCA-3', using the extracted genomic DNA of Microbacteria fibroticus as a template, amplified the gene sequence encoding the mature protein of endo-β-1,3-glucanase (excluding the signal peptide gene). The PCR reaction conditions were as follows: 94°C for 2min, 1 cycle; 98°C for 30s, 68°C for 30s, 0.5°C for each cycle, 72°C for 2min, 30 cycles; 72°C for 5min, 1 cycle. PCR products were analyzed by agarose gel electrophoresis (see figure 1 ), the target fragment...

Embodiment 2

[0048] Example 2 Endo-β-1,3-Glucanase Gene Sequence Analysis

[0049] The results of the sequencing were analyzed using the Basic Local Alignment Search Tool (BLAST) in the GenBank database, and the Vector NTI Suite 8.0 software was used for multiple sequence alignment and sequence information analysis.

[0050] The coding region of the obtained endo-β-1,3-glucanase gene (named gluE) is 1179 bp long, and its nucleotide sequence is shown in SEQ ID NO 1. gluE encodes 412 amino acids and a stop codon, its amino acid sequence is shown in SEQ ID NO 2, the theoretical protein molecular weight is 44kDa, and the predicted isoelectric point is 6.33. The amino acid sequence of gluE has the highest identity (99%) with the β-1,3-glucanase (Accession No. activity research. The domain characteristics of gluE are more similar to members of the glycoside hydrolase 16 family, thus indicating that gluE is a new member of the GH16 family.

Embodiment 3

[0051] Embodiment 3 Recombinant expression and purification of gluE gene in Escherichia coli

[0052] In order to facilitate the recombinant expression of the gene, NdeI and XhoI restriction sites were respectively introduced into the designed upstream and downstream primers. The PCR amplified product and the expression vector pET28a were digested with NdeI and XhoI, respectively. After the digested products were separated by electrophoresis and recovered from the gel, the PCR product that had undergone double digestion and the pET28a vector that had also undergone double digestion were digested with T 4 DNA ligase connection (ligation system: 5mL (T 4 DNA Ligase 0.5mL, 10′T 4 DNALigase Buffer 0.5mL, pET28a 2.5mL, PCR product 1.5mL), connection conditions: room temperature connection. ). 5 mL of the ligation product was transformed into E. coli TOP10 competent cells, spread on solid Luria-Bertani medium containing 100 μg / mL ampicillin, and cultured at 37°C for 12-16 hours. ...

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Abstract

The invention discloses an endo-β-1,3-glucanase coding gene derived from Cellulosimerbium cellsulans and its enzyme, preparation method and application. The gene of the endo-β-1,3-glucanase is cloned into an Escherichia coli expression vector to obtain a recombinant E. coli strain capable of expressing the enzyme heterologously, and the endo-β-1,3-glucanase prepared by heterologous expression of the strain is obtained. 3‑glucanase, which can directly and efficiently degrade solid Curdlan (codran polysaccharide). The invention also provides a biocontrol agent for preventing and controlling animal and plant diseases—endo-β-1,3-glucanase, which belongs to the field of biological control and is expected to become a new type of biological pesticide. The endo-β-1,3-glucanase provided by the invention can be widely used in the fields of agriculture, food, feed additive, medicine, glucooligosaccharide preparation and the like.

Description

technical field [0001] The invention relates to a gene sequence of an endo-beta-1,3-glucanase and its preparation method and application. The invention provides the recombinant plasmid and recombinant genetic engineering strain of the endo-β-1,3-glucanase and its application in polysaccharide degradation and animal and plant disease prevention and control. The endo-β-1,3-glucanase provided by the invention can be widely used in the fields of agriculture, food, feed addition, medicine, oligosaccharide preparation and the like. Background technique [0002] β-1,3-Glucooligosaccharides have great application potential in nutrition and health care, disease diagnosis and prevention, animal husbandry, plant growth regulation and induction of plant resistance, etc. The development and utilization of functional oligosaccharides have also been used in food , medicine, feed and agriculture and other fields. Compared with other β-glucans, such as laminarin, zymosan, etc., the product...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N9/24C12P19/14C12P19/02A01N47/44A01P1/00A01P3/00
CPCA01N47/44C12N9/244C12P19/02C12P19/14C12Y302/01006
Inventor 尹恒李悝悝王文霞谭海东张鑫
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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