CN1699576A - An endo-β-1,3 glucanase gene and its cloning method - Google Patents

Abstract

The invention relates to an endo beta-1 glucanase gene and process for cloning, which belongs to the field of biological genetic engineering. the invention provides an endo-beta-1,3-glucanase glu originated from Neurospora crassa AS 3.1604, the nucleic acid sequence of the gene glu and the amino acid sequence of the corresponding protein, bacillus coli expression carrier pET28a-glu containing gene glu and the highly effective expression in bacillus coli, the invention can be applied to the construction of genetic engineering bacterium for the industrial production of the endo-beta-1,3-glucanase, and the increase of level and quality for endo-beta-1,3-glucanase by means of biofermentation.

Description

An endo-β-1,3 glucanase gene and its cloning method

technical field

An endo-beta-1,3 glucanase gene and its cloning method belong to the field of microbial genetic engineering.

Background technique

β-1,3-glucanase is divided into endo-β-1,3-glucanase (EC3.2.1.39) and exo-β-1,3-glucanase (EC3.2.1.58 ), widely distributed in bacteria, fungi and higher plants. Endo-β-1,3-glucanase specifically hydrolyzes high-molecular-weight glucan polymerized by β-1,3-glucosidic bonds by internal random cutting, and the product is oligosaccharide or glucose.

The physiological function of β-1,3-glucanase varies depending on the source: in plants, although studies have shown that it plays a role in cell differentiation, it is mainly used as a defense system for pathogenic fungi; in bacteria Among them, it plays a nutritional role, because most bacteria do not contain β-1, 3-glucan; in fungi, β-1, 3-glucanase has many different functions. First, studies have shown that it plays a role in It has a certain physiological role in the formation and disintegration of fungal morphology during development and differentiation; secondly, as a lytic enzyme, β-1,3-glucanase interacts with β-1,3-glucan when carbon sources and energy sources are exhausted transfer is related.

The β-1,3-glucosidic bond exists in the non-starch polysaccharide of hemicellulose in the cell wall of gramineous plants (corn, rice, sorghum, etc.), and is generally on the branch chain, which is one of the important reasons for the increase of the solution viscosity. The addition of endo-β-1,3-glucanase can disintegrate the network structure formed by the branched chain of β-1,3-glucosidic bonds, increase the water solubility of the polysaccharide, and then be effectively utilized. For example, in the beverage industry, dextran is one of the factors that cause turbidity of beer and fruit juice, especially in the process of wort filtration, the presence of dextran seriously hinders the filtration and greatly slows down the filtration speed. The addition of highly active and stable dextranase undoubtedly greatly facilitates filtration and clarification. In addition, in the feed industry, on the one hand, the viscosity of chyme increases after β-glucan dissolves, which is not conducive to the digestion and absorption of other nutrients; on the other hand, undigested and absorbed glucan has a strong The high water absorption greatly increases the amount of defecation of animals, which is extremely unfriendly to the environment. The addition of endo-β-1,3-glucanase can not only reduce the viscosity of the feed, increase the feed intake of livestock, poultry and fish, but also reduce the volume of their defecation, thereby reducing the pollution to the environment. It can be seen that the development and research of endo-β-1,3-glucanase is of great significance.

Scholars at home and abroad have done little research on the heterologous expression of endoβ-1,3-glucanase. Vladimir V.Zverlov etc. cloned from Thermotoga neapolitana and successfully expressed a thermophilic β-1,3-glucanase (LminA) in Escherichia coli. Streptomyces

(Streptomyces sioyaensis) was successfully cloned and expressed in Escherichia coli endo-β-1,3-glucanase. Lan Haiyan et al. cloned the tobacco-derived β-1,3-glucanase cDNA and successfully expressed it in Escherichia coli.

Contents of the invention

The purpose of the present invention is to find a new β-1,3-glucanase and its coding gene, then clone the enzyme gene by means of gene amplification, and finally express and prepare the β-1 in Escherichia coli , 3-glucanase.

The invention provides a β-1,3-glucanase gene sequence and its corresponding amino acid sequence derived from Neurospora crassa. The expression vector pET28a-glu containing the β-1,3-glucanase gene of Neurospora crassa expressed in Escherichia coli and its gene recombinant strain EC-Glu are provided. The achievements of the invention can be used for the construction and large-scale preparation of genetically engineered bacteria for the industrial production of β-1,3-glucanase.

Technical scheme of the present invention: use BLAST software to search the microbial genome sequence with the amino acid sequence of endo-β-1,3-glucanase, and the open reading frame B23B10.170 coding product in the genome sequence of Neuromonas crassa may be an endo-β-1,3-glucanase. beta-1,3-glucanase. This sequence was extracted from the genome sequence, and the GenScan software was used to analyze that the gene contained two introns, and the SignalP V2.0 program was used to analyze the coding product of this gene. It was identified as a secreted protein and the cleavage site of the signal peptide was determined. On the basis of the above analysis, primers P1 and P2 were designed, using the chromosomal DNA of Neuromonas crassa AS 3.1604 as a template, PCR amplified 2432bp nucleotide fragment gluA without coding signal peptide sequence, PCR amplification conditions were 95 5 min at 94 °C, 50 s at 54 °C, 2 min at 72 °C for 30 s, 35 cycles; 10 min at 72 °C; the amplified target fragment gluA was digested with EcoRI and ligated with pUC18 digested with the same enzyme to obtain the recombinant plasmid pUC-gluA. Nucleotide sequence determination confirmed the acquisition of the endo-β-1,3 glucanase gene gluA, in which the 61bp after the 267th base is the first intron, and the 34bp after the 448th base is the second Intron.

Exon splicing was performed by PCR method to remove two introns in the target gene. At first, primers P3, P4, P5 and P6 were designed, wherein P3 and P4, and 20 and 23 bases at the 5' ends of the two pairs of primers of P5 and P6 were complementary to facilitate splicing. Using pUC-gluA as template, P1 and P3, P4 and P2 as primers for PCR, PCR products P1P3 and P4P2 were amplified respectively. The PCR amplification conditions for obtaining the product P1P3 are 95°C for 5min; 94°C for 30s, 54°C for 50s, 72°C for 30s, 35 cycles; 72°C for 10min; the PCR amplification conditions for obtaining the PCR product P4P2 are 95°C for 5min; 94°C for 30s , 54°C for 50s, 72°C for 2min, 35 cycles; 72°C for 10min. Then, using the mixture of P1P3 and P4P2 as a template, PCR splicing was performed by primers P1 and P2 to remove the first intron intr1. Use the obtained product after removing the first intron as a template, then use P1, P2, P5 and P6 combination, repeat the above process and perform PCR amplification under the same conditions, first obtain products P1P5 and P6P2, and then use primers P1 and P2 to perform PCR amplification. PCR splicing, the second intron intr2 was removed, the intron-removed glu was cloned into pUC18, and the recombinant plasmid pUC-glu was obtained and sequenced to confirm that the intron had been correctly removed. The nucleotide sequence of the gene glu is as follows

atg tctccat tgctggacgt ctccgcttca gtctccttga actggaccag cgctcaaggt 60

gtttccgacc gaccgaccag gttttggtac tccagtatcg accacagcac tccccttgtg 120

cgcggcttcg ctcccgacct tgacggagat gtcaactatg ccgtcttcaa ggcagtgaaa 180

cccggcgatg gggcgagtat ccaaacagcg atcaactcgg ggaccaacgg tgctaagaga 240

cacggtctat ggtttgcttc ccagccacga gttgtgtaca taccgccggg aacatacgag 300

atctctgaga ccatcttcat gaacactgac acagttctga tgggcgacgc aacagatgta 360

aggacgatgg cctctcccat gcaagcggaa ccacccatca tcaaagcatc ttcgaacttc 420

tccgggaatc aaacgttgat ctctggccaa gaccccgcaa ctggtatttc cggtgagcta 480

tcgttcgccg tctccttgaa gaacttaatc ctcgacacca ccaatatccc aggagaccaa 540

gccttcacag ctctctggtg gggtgttgct caaggagctc agttgcagaa tgtgaagatc 600

cgtatggcgc ctgccatcga tggtgaggga cacagtggta ttcgcctcgg ccgtggctcg 660

actctcggag tttcggatgt tcgtatcgaa tacgggcaaa acggtatctg gtataacggc 720

catcagcaag cagttttcaa gagcatctac ttcttcaaaa atgctgtggg aatgttcatt 780

gacggtggcg ccacgatcag catcgtcaac ccgacctttg acggctgtgg cttgggcgtc 840

taccacgtcg cgggcaaccc ttggattggt ctaatcgatg ccatctctat caactccggt 900

acgacgctga agacgacaga ctggccaaac tacctggtcg agaaccttcg tgtcatcagc 960

ggaaaaaccg agaacgcggt cgaagggccc ggcgactttg ttctggcaac caagccaaac 1020

gtagcccagc tctcgtacgc caacactgtt ggccatgatc ccatctatgg ccccattgaa 1080

gcagcgcagt tgaaccgtcc atcatcgctg gcgcctggac ctgatgggcg ttatgcatac 1140

cttccagcac cgaactatgc cgagctcagc gtccaagact ttctcaacgt caaagatcct 1200

cttcagaacg gaggctgcct cgtctttggc gacaacaccc gagacgagtc ctccaccctc 1260

aacgccatcc ttcgtctggc cgctcgtcag aacaagatcg cttactttcc cttcggcaag 1320

taccgcgtcg actcgactct tttcgttccc tccggctccc gtattgtcgg cgaagcgtgg 1380

gccacaatca cgggatatgg ccccttcttc acagacagcg ctcatcccca accgatcatc 1440

aaggtcggca accccggcga tattggcacc gcgcacatcc aggatatgcg cttcaccgta 1500

tcggacgtgc ttcccggagc catcatcctg cagttcaacc tcgccggtgc gcagccgggg 1560

gacgtggcaa tctggaactc gcttgtcaca gtcggaggaa cgcggggtgc gaaggcgtta 1620

acggacaagt gcgtcaatcc ggagacggac gaggcgtgca aggctgcttt cttgggtatc 1680

catctcgctt cgacgtcgtc cgtgtatctc gagaacgtat ggaactgggt agccgaccac 1740

atcgccgaag aaccgatatc gccgggcggg agcaacatcg ccggaaaggg cggagtgctc 1800

gtcgaagcga ccaaggggac ctggctgcac gcgctgggat cggagcactg gtggctgtac 1860

cagcttaatc tgcgaaaggc gtcgaatgtg ttagtgacga tgttgcaaag cgagaccaac 1920

tatgaccagg gagacaacgc ggtgcaggtg gtgccgcatc cgtggacgcc ggacgtggag 1980

ggatggggcg atccggactt tggctggtgc gcggggcagg cgaacgagaa gaggtgtcga 2040

atggggttcg cgaactacat caatggcgga agcaacattc ggacctacgc cagtgcctcg 2100

tgggcgttct tcagcgggcc gggataccaa gggtgcgcgg ggcagtatca gtgccaacgg 2160

tatatgcatt gggtggagga gacaccggcc aatttgcagg cgtttgggtt gtgctcgaag 2220

gatacgtggg cgacgttgag gttggagaat ggaaccgaga ttgtcacgaa cgaggggttc 2280

accggtcgt ggtctgggtc gggaggcgat gtcggcaggt acactccgga ggcatcg tga 2340

Wherein, atg is a start codon, and tga is a stop codon.

According to the above nucleotide sequence, the amino acid sequence of β-1,3-glucanase is as follows:

Met Ser Pro Leu Leu Asp Val Ser Ala Ser Val Ser Leu Asn Trp

1 5 10 15

Thr Ser Ala Gln Gly Val Ser Asp Arg Pro Thr Arg Phe Trp Tyr

20 25 30

Ser Ser Ile Asp His Ser Thr Pro Leu Val Arg Gly Phe Ala Pro

35 40 45

Asp Leu Asp Gly Asp Val Asn Tyr Ala Val Phe Lys Ala Val Lys

50 55 60

Pro Gly Asp Gly Ala Ser Ile Gln Thr Ala Ile Asn Ser Gly Thr

65 70 75

Asn Gly Ala Lys Arg His Gly Leu Trp Phe Ala Ser Gln Pro Arg

80 85 90

Val Val Tyr Ile Pro Pro Gly Thr Tyr Glu Ile Ser Glu Thr Ile

95 100 105

Phe Met Asn Thr Asp Thr Val Leu Met Gly Asp Ala Thr Asp Val

110 115 120

Arg Thr Met Ala Ser Pro Met Gln Ala Glu Pro Pro Ile Ile Lys

125 130 135

Ala Ser Ser Asn Phe Ser Gly Asn Gln Thr Leu Ile Ser Gly Gln

140 145 150

Asp Pro Ala Thr Gly Ile Ser Gly Glu Leu Ser Phe Ala Val Ser

155 160 165

Leu Lys Asn Leu Ile Leu Asp Thr Thr Asn Ile Pro Gly Asp Gln

170 175 180

Ala Phe Thr Ala Leu Trp Trp Gly Val Ala Gln Gly Ala Gln Leu

185 190 195

Gln Asn Val Lys Ile Arg Met Ala Pro Ala Ile Asp Gly Glu Gly

200 205 210

His Ser Gly Ile Arg Leu Gly Arg Gly Ser Thr Leu Gly Val Ser

215 220 225

Asp Val Arg Ile Glu Tyr Gly Gln Asn Gly Ile Trp Tyr Asn Gly

230 235 240

His Gln Gln Ala Val Phe Lys Ser Ile Tyr Phe Phe Lys Asn Ala

245 250 255

Val Gly Met Phe Ile Asp Gly Gly Ala Thr Ile Ser Ile Val Asn

260 265 270

Pro Thr Phe Asp Gly Cys Gly Leu Gly Val Tyr His Val Ala Gly

275 280 285

Asn Pro Trp Ile Gly Leu Ile Asp Ala Ile Ser Ile Asn Ser Gly

290 295 300

Thr Thr Leu Lys Thr Thr Asp Trp Pro Asn Tyr Leu Val Glu Asn

305 310 315

Leu Arg Val Ile Ser Gly Lys Thr Glu Asn Ala Val Glu Gly Pro

320 325 330

Gly Asp Phe Val Leu Ala Thr Lys Pro Asn Val Ala Gln Leu Ser

335 340 345

Tyr Ala Asn Thr Val Gly His Asp Pro Ile Tyr Gly Pro Ile Glu

350 355 360

Ala Ala Gln Leu Asn Arg Pro Ser Ser Leu Ala Pro Gly Pro Asp

365 370 375

Gly Arg Tyr Ala Tyr Leu Pro Ala Pro Asn Tyr Ala Glu Leu Ser

380 385 390

Val Gln Asp Phe Leu Asn Val Lys Asp Pro Leu Gln Asn Gly Gly

395 400 405

Cys Leu Val Phe Gly Asp Asn Thr Arg Asp Glu Ser Ser Thr Leu

410 415 420

Asn Ala Ile Leu Arg Leu Ala Ala Arg Gln Asn Lys Ile Ala Tyr

425 430 435

Phe Pro Phe Gly Lys Tyr Arg Val Asp Ser Thr Leu Phe Val Pro

440 445 450

Ser Gly Ser Arg Ile Val Gly Glu Ala Trp Ala Thr Ile Thr Gly

455 460 465

Tyr Gly Pro Phe Phe Thr Asp Ser Ala His Pro Gln Pro Ile Ile

470 475 480

Lys Val Gly Asn Pro Gly Asp Ile Gly Thr Ala His Ile Gln Asp

485 490 495

Met Arg Phe Thr Val Ser Asp Val Leu Pro Gly Ala Ile Ile Leu

500 505 510

Gln Phe Asn Leu Ala Gly Ala Gln Pro Gly Asp Val Ala Ile Trp

515 520 525

Asn Ser Leu Val Thr Val Gly Gly Thr Arg Gly Ala Lys Ala Leu

530 535 540

Thr Asp Lys Cys Val Asn Pro Glu Thr Asp Glu Ala Cys Lys Ala

545 550 555

Ala Phe Leu Gly Ile His Leu Ala Ser Thr Ser Ser Val Tyr Leu

560 565 570

Glu Asn Val Trp Asn Trp Val Ala Asp His Ile Ala Glu Glu Pro

575 580 585

Ile Ser Pro Gly Gly Ser Asn Ile Ala Gly Lys Gly Gly Val Leu

590 595 600

Val Glu Ala Thr Lys Gly Thr Trp Leu His Ala Leu Gly Ser Glu

605 610 615

His Trp Trp Leu Tyr Gln Leu Asn Leu Arg Lys Ala Ser Asn Val

620 625 630

Leu Val Thr Met Leu Gln Ser Glu Thr Asn Tyr Asp Gln Gly Asp

635 640 645

Asn Ala Val Gln Val Val Pro His Pro Trp Thr Pro Asp Val Glu

650 655 660

Gly Trp Gly Asp Pro Asp Phe Gly Trp Cys Ala Gly Gln Ala Asn

665 670 675

Glu Lys Arg Cys Arg Met Gly Phe Ala Asn Tyr Ile Asn Gly Gly

680 685 690

Ser Asn Ile Arg Thr Tyr Ala Ser Ala Ser Trp Ala Phe Phe Ser

695 700 705

Gly Pro Gly Tyr Gln Gly Cys Ala Gly Gln Tyr Gln Cys Gln Arg

710 715 720

Tyr Met His Trp Val Glu Glu Thr Pro Ala Asn Leu Gln Ala Phe

725 730 735

Gly Leu Cys Ser Lys Asp Thr Trp Ala Thr Leu Arg Leu Glu Asn

740 745 750

Gly Thr Glu Ile Val Thr Asn Glu Gly Phe Thr Gly Ser Trp Ser

755 760 765

Gly Ser Gly Gly Asp Val Gly Arg Tyr Thr Pro Glu Ala Ser

770 775 779

This β-mannanase consists of 779 amino acid residues.

The recombinant plasmid pUC-glu was digested with EcoRI, and the gene glu fragment was obtained by gel recovery. The gene glu was cloned into the EcoRI site of the plasmid pET28a to obtain the recombinant plasmid pET28a-glu in which the gene glu was inserted forward. The recombinant plasmid pET28a-glu was transformed into Escherichia coli DE3 (RILplus), and the gene recombinant strain EC-Glu was obtained.

Beneficial effects of the present invention: the present invention provides a kind of endo-beta-1,3 glucanase gene glu derived from Neurospora crassa (Neurospora crassa) AS 3.1604, provides the nucleotide sequence of gene glu and corresponding The amino acid sequence of the protein provides the Escherichia coli expression vector pET28a-glu containing the gene glu and its high expression in Escherichia coli. The invention can be used for the construction of genetically engineered bacteria for the industrial production of endo-β-1,3 glucanase, and improves the level and quality of endo-β-1,3-glucanase produced by microbial fermentation.

Description of drawings

Figure 1 The process of splicing to remove introns. P1-P6 are primers, and P1P3, P4P2, P1P5 and P6P2 are corresponding PCR amplification products.

Fig. 2 Physical map of recombinant expression plasmid pET28a-glu.

Figure 3 SDS-PAGE analysis of recombinant bacteria expressing β-1,3-glucanase. 1. Protein molecular weight standard; 2. Empty vector control; 3. EC-Glu.

Detailed ways

Example 1 Cloning and transformation of endo-β-1,3 glucanase gene glu

P1 accggaattcatgtctccattgctggacgt

P2 cgtgaattcacgatgcctccggagtgt

P3 cccggcggtatgtacacaactcgtggctgggaagcaaacc

P4 gttgtgtacataccgccgggaacat

P5 gatgctttgatgatgggtggttccgcttgcatgggagg

P6 ccacccatcatcaaagcatctcg

Use BLAST software to search the microbial genome sequence with the amino acid sequence of the endo-β-1,3-glucanase. The open reading frame B23B10.170 coding product in the genome sequence of Neuromonas crassa may be the endo-β-1,3- Glucanase. This sequence was extracted from the genome sequence, and the GenScan software was used to analyze that the gene contained two introns, and the coded product of this gene was analyzed by the program Signa1PV2.0. It was identified as a secreted protein and the cleavage site of the signal peptide was determined. On the basis of the above analysis, primers P1 and P2 were designed, and the chromosomal DNA of Neuromonas crassa AS3.1604 was used as a template to amplify a 2432bp nucleotide fragment gluA without coding signal peptide sequence by PCR. The PCR amplification conditions were as follows: 95°C for 5 min; 94°C for 30 s, 54°C for 50 s, 72°C for 2 min for 30 s, 35 cycles; 72°C for 10 min; the amplified target fragment gluA was digested with EcoRI and ligated with pUC18 digested with the same enzyme to obtain the recombinant plasmid pUC-gluA. Nucleotide sequence determination confirmed the endo-β-1,3 glucanase gene gluA, in which the 61bp after the 267th base is the first intron, and the 34bp after the 448th base is the second introns. Exon splicing was performed by PCR method to remove two introns in the target gene (Figure 1). At first, primers P3, P4, P5 and P6 were designed, wherein P3 and P4, and 20 and 23 bases at the 5' ends of the two pairs of primers of P5 and P6 were complementary, so as to facilitate splicing. Using pUC-gluA as template, P1 and P3, P4 and P2 as primers for PCR, PCR products P1P3 and P4P2 were amplified respectively. The PCR amplification conditions for obtaining the product P1P3 are 95°C for 5min; 94°C for 30s, 54°C for 50s, 72°C for 30s, 35 cycles; 72°C for 10min; the PCR amplification conditions for obtaining the PCR product P4P2 are 95°C for 5min; 94°C for 30s , 54°C for 50s, 72°C for 2min, 35 cycles; 72°C for 10min. Use the obtained product after removing the first intron as a template, then use appropriate combinations of P1, P2, P5 and P6, repeat the above process and perform PCR amplification under the same conditions, first obtain products P1P5 and P6P2, and then use primers P1, P2 Perform PCR splicing, remove the second intron, clone the intron-removed glu into pUC18, obtain the recombinant plasmid pUC-glu and perform sequence determination to confirm that the intron has been correctly removed.

Example 2 Expression of endo-β-1,3-glucanase gene glu

The recombinant plasmid pUC-glu was digested with EcoRI, and the gene glu fragment was obtained by gel recovery. The gene glu was cloned into the EcoRI site of the plasmid pET28a to obtain the recombinant plasmid pET28a-glu with the gene glu inserted forward (Figure 2). The recombinant plasmid pET28a-glu was transformed into Escherichia coli DE3 (RILplus), and the gene recombinant strain EC-Glu was obtained. The recombinant strain EC-Glu was inoculated in 35mL LB medium, cultured at 37°C with shaking at 200r/min until the OD value was about 0.6, and induced by adding a final concentration of 0.5mmol/L isopropyl-β-D-galactoside for 4h. The cells were collected by centrifugation, and the cells were disrupted by adding lysozyme at a final concentration of 12.5 μg/mL to obtain a crude enzyme solution of endo-β-1,3 glucanase. The expression product was detected by SDS-PAGE (Fig. 3), and the recombinant strain EC-Glu expressed an obvious protein band at 80kD. Recombinant beta-1,3 glucanase exhibited the best ability to liquefy barley glucan at 45-55°C.

Claims (3)

1. An endo-β-1,3-glucanase gene glu derived from Neurospora crassa AS 3.1604, the nucleotide sequence of which is: atg tctccat tgctggacgt ctccgcttca gtctccttga actggaccag cgctcaaggt 60 gtttccgacc gaccgaccag gttttggtac tccagtatcg accacagcac tccccttgtg 120 cgcggcttcg ctcccgacct tgacggagat gtcaactatg ccgtcttcaa ggcagtgaaa 180 cccggcgatg gggcgagtat ccaaacagcg atcaactcgg ggaccaacgg tgctaagaga 240 cacggtctat ggtttgcttc ccagccacga gttgtgtaca taccgccggg aacatacgag 300 atctctgaga ccatcttcat gaacactgac acagttctga tgggcgacgc aacagatgta 360 aggacgatgg cctctcccat gcaagcggaa ccacccatca tcaaagcatc ttcgaacttc 420 tccgggaatc aaacgttgat ctctggccaa gaccccgcaa ctggtatttc cggtgagcta 480 tcgttcgccg tctccttgaa gaacttaatc ctcgacacca ccaatatccc aggagaccaa 540 gccttcacag ctctctggtg gggtgttgct caaggagctc agttgcagaa tgtgaagatc 600 cgtatggcgc ctgccatcga tggtgaggga cacagtggta ttcgcctcgg ccgtggctcg 660 actctcggag tttcggatgt tcgtatcgaa tacgggcaaa acggtatctg gtataacggc 720 catcagcaag cagttttcaa gagcatctac ttcttcaaaa atgctgtggg aatgttcatt 780 gacggtggcg ccacgatcag catcgtcaac ccgacctttg acggctgtgg cttgggcgtc 840 taccacgtcg cgggcaaccc ttggattggt ctaatcgatg ccatctctat caactccggt 900 acgacgctga agacgacaga ctggccaaac tacctggtcg agaaccttcg tgtcatcagc 960 ggaaaaaccg agaacgcggt cgaagggccc ggcgactttg ttctggcaac caagccaaac 1020 gtagcccagc tctcgtacgc caacactgtt ggccatgatc ccatctatgg ccccattgaa 1080 gcagcgcagt tgaaccgtcc atcatcgctg gcgcctggac ctgatgggcg ttatgcatac 1140 cttccagcac cgaactatgc cgagctcagc gtccaagact ttctcaacgt caaagatcct 1200 cttcagaacg gaggctgcct cgtctttggc gacaacaccc gagacgagtc ctccaccctc 1260 aacgccatcc ttcgtctggc cgctcgtcag aacaagatcg cttactttcc cttcggcaag 1320 taccgcgtcg actcgactct tttcgttccc tccggctccc gtattgtcgg cgaagcgtgg 1380 gccacaatca cgggatatgg ccccttcttc acagacagcg ctcatcccca accgatcatc 1440 aaggtcggca accccggcga tattggcacc gcgcacatcc aggatatgcg cttcaccgta 1500 tcggacgtgc ttcccggagc catcatcctg cagttcaacc tcgccggtgc gcagccgggg 1560 gacgtggcaa tctggaactc gcttgtcaca gtcggaggaa cgcggggtgc gaaggcgtta 1620 acggacaagt gcgtcaatcc ggagacggac gaggcgtgca aggctgcttt cttgggtatc 1680 catctcgctt cgacgtcgtc cgtgtatctc gagaacgtat ggaactgggt agccgaccac 1740 atcgccgaag aaccgatatc gccgggcggg agcaacatcg ccggaaaggg cggagtgctc 1800 gtcgaagcga ccaaggggac ctggctgcac gcgctgggat cggagcactg gtggctgtac 1860 cagcttaatc tgcgaaaggc gtcgaatgtg ttagtgacga tgttgcaaag cgagaccaac 1920 tatgaccagg gagacaacgc ggtgcaggtg gtgccgcatc cgtggacgcc ggacgtggag 1980 ggatggggcg atccggactt tggctggtgc gcggggcagg cgaacgagaa gaggtgtcga 2040 atggggttcg cgaactacat caatggcgga agcaacattc ggacctacgc cagtgcctcg 2100 tgggcgttct tcagcgggcc gggataccaa gggtgcgcgg ggcagtatca gtgccaacgg 2160 tatatgcatt gggtggagga gacaccggcc aatttgcagg cgtttgggtt gtgctcgaag 2220 gatacgtggg cgacgttgag gttggagaat ggaaccgaga ttgtcacgaa cgaggggttc 2280 accggtcgt ggtctgggtc gggaggcgat gtcggcaggt acactccgga ggcatcg tga 2340 Wherein, atg is a start codon, and tga is a stop codon. 2. The gene glu according to claim 1, whose amino acid composition is: Met Ser Pro Leu Leu Asp Val Ser Ala Ser Val Ser Leu Asn Trp 1 5 10 15 Thr Ser Ala Gln Gly Val Ser Asp Arg Pro Thr Arg Phe Trp Tyr 20 25 30 Ser Ser Ile Asp His Ser Thr Pro Leu Val Arg Gly Phe Ala Pro 35 40 45 Asp Leu Asp Gly Asp Val Asn Tyr Ala Val Phe Lys Ala Val Lys 50 55 60 Pro Gly Asp Gly Ala Ser Ile Gln Thr Ala Ile Asn Ser Gly Thr 65 70 75 Asn Gly Ala Lys Arg His Gly Leu Trp Phe Ala Ser Gln Pro Arg 80 85 90 Val Val Tyr Ile Pro Pro Gly Thr Tyr Glu Ile Ser Glu Thr Ile 95 100 105 Phe Met Asn Thr Asp Thr Val Leu Met Gly Asp Ala Thr Asp Val 110 115 120 Arg Thr Met Ala Ser Pro Met Gln Ala Glu Pro Pro Ile Ile Lys 125 130 135 Ala Ser Ser Asn Phe Ser Gly Asn Gln Thr Leu Ile Ser Gly Gln 140 145 150 Asp Pro Ala Thr Gly Ile Ser Gly Glu Leu Ser Phe Ala Val Ser 155 160 165 Leu Lys Asn Leu Ile Leu Asp Thr Thr Asn Ile Pro Gly Asp Gln 170 175 180 Ala Phe Thr Ala Leu Trp Trp Gly Val Ala Gln Gly Ala Gln Leu 185 190 195 Gln Asn Val Lys Ile Arg Met Ala Pro Ala Ile Asp Gly Glu Gly 200 205 210 His Ser Gly Ile Arg Leu Gly Arg Gly Ser Thr Leu Gly Val Ser 215 220 225 Asp Val Arg Ile Glu Tyr Gly Gln Asn Gly Ile Trp Tyr Asn Gly 230 235 240 His Gln Gln Ala Val Phe Lys Ser Ile Tyr Phe Phe Lys Asn Ala 245 250 255 Val Gly Met Phe Ile Asp Gly Gly Ala Thr Ile Ser Ile Val Asn 260 265 270 Pro Thr Phe Asp Gly Cys Gly Leu Gly Val Tyr His Val Ala Gly 275 280 285 Asn Pro Trp Ile Gly Leu Ile Asp Ala Ile Ser Ile Asn Ser Gly 290 295 300 Thr Thr Leu Lys Thr Thr Asp Trp Pro Asn Tyr Leu Val Glu Asn 305 310 315 Leu Arg Val Ile Ser Gly Lys Thr Glu Asn Ala Val Glu Gly Pro 320 325 330 Gly Asp Phe Val Leu Ala Thr Lys Pro Asn Val Ala Gln Leu Ser 335 340 345 Tyr Ala Asn Thr Val Gly His Asp Pro Ile Tyr Gly Pro Ile Glu 350 355 360 Ala Ala Gln Leu Asn Arg Pro Ser Ser Leu Ala Pro Gly Pro Asp 365 370 375 Gly Arg Tyr Ala Tyr Leu Pro Ala Pro Asn Tyr Ala Glu Leu Ser 380 385 390 Val Gln Asp Phe Leu Asn Val Lys Asp Pro Leu Gln Asn Gly Gly 395 400 405 Cys Leu Val Phe Gly Asp Asn Thr Arg Asp Glu Ser Ser Thr Leu 410 415 420 Asn Ala Ile Leu Arg Leu Ala Ala Arg Gln Asn Lys Ile Ala Tyr 425 430 435 Phe Pro Phe Gly Lys Tyr Arg Val Asp Ser Thr Leu Phe Val Pro 440 445 450 Ser Gly Ser Arg Ile Val Gly Glu Ala Trp Ala Thr Ile Thr Gly 455 460 465 Tyr Gly Pro Phe Phe Thr Asp Ser Ala His Pro Gln Pro Ile Ile 470 475 480 Lys Val Gly Asn Pro Gly Asp Ile Gly Thr Ala His Ile Gln Asp 485 490 495 Met Arg Phe Thr Val Ser Asp Val Leu Pro Gly Ala Ile Ile Leu 500 505 510 Gln Phe Asn Leu Ala Gly Ala Gln Pro Gly Asp Val Ala Ile Trp 515 520 525 Asn Ser Leu Val Thr Val Gly Gly Thr Arg Gly Ala Lys Ala Leu 530 535 540 Thr Asp Lys Cys Val Asn Pro Glu Thr Asp Glu Ala Cys Lys Ala 545 550 555 Ala Phe Leu Gly Ile His Leu Ala Ser Thr Ser Ser Val Tyr Leu 560 565 570 Glu Asn Val Trp Asn Trp Val Ala Asp His Ile Ala Glu Glu Pro 575 580 585 Ile Ser Pro Gly Gly Ser Asn Ile Ala Gly Lys Gly Gly Val Leu 590 595 600 Val Glu Ala Thr Lys Gly Thr Trp Leu His Ala Leu Gly Ser Glu 605 610 615 His Trp Trp Leu Tyr Gln Leu Asn Leu Arg Lys Ala Ser Asn Val 620 625 630 Leu Val Thr Met Leu Gln Ser Glu Thr Asn Tyr Asp Gln Gly Asp 635 640 645 Asn Ala Val Gln Val Val Pro His Pro Trp Thr Pro Asp Val Glu 650 655 660 Gly Trp Gly Asp Pro Asp Phe Gly Trp Cys Ala Gly Gln Ala Asn 665 670 675 Glu Lys Arg Cys Arg Met Gly Phe Ala Asn Tyr Ile Asn Gly Gly 680 685 690 Ser Asn Ile Arg Thr Tyr Ala Ser Ala Ser Trp Ala Phe Phe Ser 695 700 705 Gly Pro Gly Tyr Gln Gly Cys Ala Gly Gln Tyr Gln Cys Gln Arg 710 715 720 Tyr Met His Trp Val Glu Glu Thr Pro Ala Asn Leu Gln Ala Phe 725 730 735 Gly Leu Cys Ser Lys Asp Thr Trp Ala Thr Leu Arg Leu Glu Asn 740 745 750 Gly Thr Glu Ile Val Thr Asn Glu Gly Phe Thr Gly Ser Trp Ser 755 760 765 Gly Ser Gly Gly Asp Val Gly Arg Tyr Thr Pro Glu Ala Ser 770 775 779. 3. A method for cloning endo-β-1,3 glucanase gene glu according to claim 1, characterized in that A) The sequences of the primers used are as follows P1 accggaattcatgtctccattgctggacgt P2 cgtgaattcacgatgcctccggagtgt P3 cccggcggtatgtacacaactcgtggctgggaagcaaacc P4 gttgtgtacataccgccgggaacat P5 gatgctttgatgatgggtggttccgcttgcatgggagg P6 ccacccatcatcaaagcatctcg B) Using the chromosomal DNA of Neurospora crassa AS 3.1604 as a template, a 2432bp nucleotide fragment gluA without the coding signal peptide sequence was amplified by PCR with primers P1 and P2. The PCR amplification conditions were 95°C for 5min; 94°C 30s, 54°C 50s, 72°C 2min 30s, 35 cycles; 72°C 10min; C) The amplified fragment gluA is digested by EcoR I and connected with the same digested pUC18 to obtain the recombinant plasmid pUC-gluA, and the nucleotide sequence determination confirms that the endo-β-1,3 glucanase gene gluA is obtained, wherein The 61bp after the 267th base is the first intron intr1, and the 34bp after the 448th base is the second intron intr2; D) Using the PGR method for exon splicing, removing the two introns in the gene gluA, and designing primers P3, P4, P5 and P6, for the convenience of splicing P3 and P4, the 5' ends of the two pairs of primers P5 and P6 have respectively 20 and 23 bases are complementary; E) Use pUC-gluA as a template, P1 and P3 as primers for PGR, amplify the PCR product P1P3, the PCR amplification conditions are 95°C for 5min; 94°C for 30s, 54°C for 50s, 72°C for 30s, 35 cycles; 72 ℃ 10min; F) Use pUC-gluA as template, P4 and P2 as primers for PGR, and amplify the PCR product P4P2. The PCR amplification conditions are 95°C for 5min; 94°C for 30s, 54°C for 50s, 72°C for 2min, 35 cycles; 72 ℃ 10min; G) Using the mixture of P1P3 and P4P2 as a template, PCR splicing is carried out through primers P1 and P2. The PCR amplification conditions are 95°C for 5min; 94°C for 30s, 54°C for 50s, 72°C for 2min30s, 35 cycles; One containing intr1; H) Use the product obtained in step G) after the removal of the first intron as a template, and then combine P1, P2, P5 and P6, and use the same conditions as E) and F) to obtain the products P1P5 and P6P2 by PCR amplification , and then under the same conditions as G) process, carry out PCR splicing through primers P1 and P2, and remove the second intron intr2; 1) Cloning the glu with the intron removed into pUC18 to obtain the recombinant plasmid pUC-glu and perform sequence determination to confirm that the intron has been correctly removed; J) expression of glu: digest the recombinant plasmid pUC-glu with EcoR I, recover from the gel to obtain the gene glu fragment, clone the gene glu into the EcoR I site of the plasmid pET28a, and obtain the recombinant plasmid pET28a-glu inserted in the forward direction of the gene glu, The recombinant expression plasmid pET28a-glu was transformed into Escherichia coli DE3 to obtain gene recombinant strain EC-Glu.

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