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Cloning and analysis of endo-beta-1,4-glucanase(Aus cel5A) gene

An endoglucanase and gene technology, applied in genetic engineering, plant genetic improvement, enzymes, etc., can solve problems that have not been reported before, and achieve the effect of large-scale industrial production and application potential and economic value

Inactive Publication Date: 2011-07-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fungi are an important source of industrial cellulase, mainly Trichoderma reesei, Trichoderma viride, Trichodermakoningii, Aspergillus ntger, Penicillium decumbens), Chaetomiumthermophile and Thermoascus aurantiacus, etc., but related to the cloning and expression of the β-endoglucanase gene (Aus cel5A) derived from Aspergillususamii GH5 No other studies have reported

Method used

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  • Cloning and analysis of endo-beta-1,4-glucanase(Aus cel5A) gene
  • Cloning and analysis of endo-beta-1,4-glucanase(Aus cel5A) gene
  • Cloning and analysis of endo-beta-1,4-glucanase(Aus cel5A) gene

Examples

Experimental program
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Effect test

Embodiment 1A

[0030]Cloning of Example 1 Aus cel5A 3' end mRNA sequence

[0031] The first strand of cDNA was synthesized by reverse transcription with Oligo dT-Adaptor Primer as primers; the first round of PCR was performed with M13 Primer M4 and Cel5A-3F1 as primers, and the reaction conditions were: 94°C for 2min, 30 cycles (94°C 30s, 52°C for 30s, 72°C for 90s), 72°C for 10min; use M13 Primer M4 and Cel5A-3F2 as primers for the second round of PCR, the reaction conditions are: 94°C for 2min, 30 cycles (94°C for 30s, 52°C 30s, 72°C 90s), 72°C 10min. The two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, the target band was recovered and ligated with pUCm-T (pUCm-T-cel5A3′), transformed into JM109, and then sent to Shanghai Sangon for sequencing.

Embodiment 2A

[0032] Cloning of embodiment 2Aus cel5A 5' end mRNA sequence

[0033] The first round of PCR amplification was performed using the Outer Primer and Cel5A-5R1 of the 5′-Full RACE Kit as primers. The reaction conditions were: 94°C for 3 minutes, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 60s), 10 min at 72°C; the second round of PCR was performed using Inner Primer and Cel5A-5R2 as primers, and the reaction conditions were: 3 min at 94°C, 30 cycles (30s at 94°C, 30s at 55°C, 60s at 72°C), and 10 min at 72°C. The two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, the target band was recovered and ligated with pUCm-T (pUCm-T-cel5A5′), transformed into JM109, and then sent to Shanghai Sangon for sequencing.

Embodiment 3A

[0034] Cloning of Example 3 Aus cel5A 5' end promoter sequence

[0035] Using the processed A.usamii genomic DNA as a template, the first round of PCR used T-PrimerF and Cel5A-5R1 as primers, and the reaction conditions were: 94°C for 4min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 60s ), 72°C for 10min; the second round of PCR uses T-PrimerF and Cel5A-5R2 as primers, and the reaction conditions are: 94°C, 4min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 60s), 72°C for 10min . The two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, the target band was recovered and ligated with pUCm-T (pUCm-T-cel5AP), transformed into JM109, and sent to Shanghai Sangon for sequencing after enzyme digestion and identification.

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Abstract

The invention provides a method for cloning and analyzing complete micro ribonucleic acid (mRNA) and deoxyribonucleic acid (DNA) sequences of a novel endo-beta-1,4-glucanase gene derived from an aspergillus usamii E001 strain. Nucleotide sequences of the complete mRNA and DNA sequences are shown as SEQ ID NO. 1 and SEQ ID NO. 2 respectively. Bioinformatic analysis shows that the endo-beta-1,4-glucanase belongs to the group V of glycoside hydrolase and is called Aus Cel5A, wherein the amino acid sequence of the glucanase is shown as SEQ ID No. 3; and a corresponding gene is called Aus cel5A. A theoretical basis is laid for heterogeneous expression and industrial production of the gene; greater industrial production and application potential and economic value are achieved; and a theoretical basis is laid for research of other endo-beta-1,4-glucanases.

Description

technical field [0001] The present invention relates to the cloning and analysis of the complete mRNA and DNA sequence of a β-1,4-endoglucanase gene (Aus cel5A) of a glycoside hydrolase family 5 derived from Aspergillus usamii (Aspergillus usamii) E001 strain, It belongs to the technical field of bioengineering. Background technique [0002] Cellulase refers to the general term of a group of enzymes that hydrolyze cellulose β-1,4 glucosidic bonds to generate cellobiose and glucose. A complete cellulase system is mainly composed of endoglucanase (endo-1, 4-β-glucanase, EC 3.2.1.4), exoglucanase (exo-1, 4-β-glucanase, EC 3.2.1.91) and β-glucosidase (β-glucosidase, EC 3.2.1.21). Endoglucanase first randomly hydrolyzes the β-1,4 glucosidic bonds inside the cellulose to generate a large number of free wood ends, and then hydrolyzes the cellulose under the synergistic action of exoglucanase and β-glucosidase into glucose. Because cellulase can degrade naturally occurring cellu...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/42C12N15/10
Inventor 邬敏辰唐存多韦敬土赵顺阁郭静
Owner JIANGNAN UNIV
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