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Modified cellulase gene and expression vector and application thereof

A cellulase and cellulose technology, applied in the field of genetic engineering, can solve the problems of low utilization rate, low enzyme activity, and increased cost, and achieve the effects of reducing production cost, improving nutritional value, and improving palatability.

Active Publication Date: 2018-12-07
HENAN AGRICULTURAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Crude fiber is the main component of crop straw, and the common problem in the process of micro-storage of straw used as animal feed is that although the palatability of straw has been improved by biological fermentation, the digestibility has not improved significantly, which is mainly due to the bacteria and bacteria. Due to the poor synergy of enzymes, most microorganisms have low enzyme activity in the strongly acidic environment of straw micro-storage
In addition, although the direct addition of cellulase in the process of straw treatment improves the utilization rate of straw, it greatly increases the cost and is difficult to popularize and apply in production.
[0005] In summary, when the cellulose in straw is used as feed, the utilization rate is low, and the utilization rate of straw cannot be increased at a low cost, and the enzyme activity is low in an acidic environment, and further improvement is urgently needed

Method used

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  • Modified cellulase gene and expression vector and application thereof
  • Modified cellulase gene and expression vector and application thereof
  • Modified cellulase gene and expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] In this example, Aspergillus niger and Trichoderma konii were used as strains preserved in the laboratory. Escherichia coli DH5α and cloning plasmid pMD19-T were purchased from TAKARA Company. PCR amplification primers were designed. The complete gene sequences of Aspergillus niger and Trichoderma konei were obtained from NCBI, and primers were designed downstream of the signal peptide sequence in the coding region of the CBHI gene, and an XbaⅠ restriction site was added to the forward primer. A PstI restriction site was added to the reverse primer, and protective bases GC and CCG were added to the 5' end of the reverse primer respectively (these primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.).

[0038] Trichoderma konshii primers:

[0039] Forward primer K1 is shown in SEQ NO.5: 5'-GC T CTA GA A TGT ATC GGA AGT TGG CCG TC Xba Ⅰ

[0040] The reverse primer K2 is shown in SEQ NO.6: 5'-CCG CTC GAG TTA CAG GCA CTG AGA GTA GTA Pst Ⅰ

[0041] Asp...

Embodiment 2

[0044] Example 2 PCR Amplification of Cellulase Gene

[0045] Trichoderma konshii and Aspergillus niger were inoculated in PDA medium respectively, cultured on a shaker at 30°C at 120rpm for 48 hours, the bacteria were filtered with sterile filter paper and dried, and the RNA extraction kit was used to extract Trichoderma konii and Aspergillus niger of total RNA.

[0046] Among them, the PDA medium is prepared by weighing MgSO 4 0.15g, soluble starch 3g, glucose 10g, KH 2 PO 4 1g, 1g of yeast extract powder, 2.5g of tryptone, dissolved in 400mL of distilled water, stirred thoroughly to dissolve, and distilled to 500mL with distilled water. 121°C, 1.05×10 5 Pa high-pressure steam sterilization for 20min, set aside.

[0047] To establish the cDNA library of the cellulase gene, treat the total RNA with DNase I, react at 37°C for 20-30min, and then inactivate it in a PCR instrument at 80°C for 10min; configure the following system in the microtube according to Table 1.

[...

Embodiment 3

[0060] Example 3 Ligation transformation of cellulase gene

[0061] For the connection of the cellulase gene to the pMD19-vector, configure the connection system of the cellulase gene PCR recovery product according to Table 4, and connect overnight at 4°C.

[0062] Table 4 PCR product and pMD19-T vector ligation reaction system (μL)

[0063]

[0064] Place well-grown E. coli plates in a refrigerator at 4°C, and develop color for several hours. Such as figure 2 As shown, the Escherichia coli without the recombinant plasmid has β-galactosidase activity, the center of the colony is light blue, and the periphery is dark blue; while the Escherichia coli containing the recombinant plasmid has no β-galactosidase activity, the colony Milky white for initial screening.

[0065] For gene preparation, perform PCR amplification on the cellulase gene that has been sequenced, and use an agarose purification and recovery kit to recover the PCR product to obtain the cellulase gene.

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Abstract

The invention discloses a modified cellulase gene and an expression vector and application thereof. A protein has an amino acid composition sequence shown in the formula of SEQ. 1 or is a protein derived from the protein shown in the formula of SEQ. 1 through replacement and / or detection and / or addition of one or more amino acid residues and having cellulase activity. Recombinant plant lactobacillus improves the palatability of the fermented straws, improves the nutritional value of the straws, is free of cellulase in the straw treatment process and greatly reduces a production cost.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant cellulase gene and its expression vector and application. Background technique [0002] Crude fiber is the main component of crop straw, including macromolecular polysaccharide cellulose, monosaccharide heteropolymer hemicellulose and aromatic polymer lignin. Cellulose is arranged and combined in a certain way in space to form a fine vascular bundle structure, and the intricate vascular bundles constitute a network-like cell wall. Ordinary crop stalks contain crude fibers with a mass fraction of 70%-85%. In addition, crop stalks also contain a small amount of other substances, such as 5% ash (mainly composed of silica, densely distributed on the surface of the straw), 2- 8% crude protein, 1-2% lipid, 10% water, etc. [0003] Cellulose is a natural polymer compound, which aggregates a large number of monosaccharides and constitutes the main nutritional ...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/74C12N1/21C12R1/25
CPCC12N9/2437C12N15/74
Inventor 常娟韩浩然王平尹清强孙贵宾刘超齐党晓伟朱群李庆华张志敏
Owner HENAN AGRICULTURAL UNIVERSITY
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