CN101870985B - Endo-beta-glucanase gene - Google Patents

Endo-beta-glucanase gene Download PDF

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CN101870985B
CN101870985B CN2010101047280A CN201010104728A CN101870985B CN 101870985 B CN101870985 B CN 101870985B CN 2010101047280 A CN2010101047280 A CN 2010101047280A CN 201010104728 A CN201010104728 A CN 201010104728A CN 101870985 B CN101870985 B CN 101870985B
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beta
gene
inscribe
glucanase
expression cassette
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CN101870985A (en
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金欣
杜风光
夏黎明
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Zhejiang University ZJU
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Abstract

The invention discloses an endo-beta-glucanase gene, which has a base sequence shown by SEQ ID No.2. The invention also discloses an expression cassette containing the endo-beta-glucanase gene, a recombinant carrier and a transformant. When transferred into trichodermareesei, the gene of the invention can be expressed efficiently. Treadmill tests show that the growth speeds of all transformed strains in a culture medium which uses carboxymethylcellulose as a unique carbon source are improved to different extents and that the maximum diameter of transformed daughter colonies is 5 times that of a wild strain.

Description

A kind of inscribe-beta-glucanase gene
Technical field
The present invention relates to biology field, relate in particular to a kind of inscribe-beta-glucanase gene.
Background technology
Mierocrystalline cellulose is the biomass the widest, that standing stock are the abundantest that distribute on the earth, also is the most cheap renewable resources.It can be the general name of the polycomponent enzyme system of glucose with cellulose degradation that cellulase is one type, their synergies, and decomposition of cellulose produces oligosaccharides and cellobiose, finally is hydrolyzed to glucose.Cellulase belongs to glycoside hydrolase, is divided into three types of components traditionally: inscribe-beta-glucanase (endo-1,4-β-D-glucanase, EG, EC 3.2.1.4), CMC enzyme; Circumscribed-beta-glucanase (exo-1,4-β-D-glucanase, EC 3.2.1.91), i.e. cellobiohydrolase, CBH; Beta-glucosidase (β-1,4-D-glucosidase, EC 3.2.1.21) is called for short BG (Fang-yuan, Chao-yin et al.2008).Mostly the bacterial classification of existing production of cellulose enzyme is Trichodermareesei (Trichoderma reesei); These bacterial strains grow rapidly, are easy to cultivate on cellulose matrix; Zymotechnique is ripe; Its weak point is that the ratio vigor of the cellulase (especially inscribe-beta-glucanase) produced is lower, and the consumption of enzyme is big in the practical application, and cost is higher.
The cud anaerobion can secrete the inscribe-beta-glucanase of height ratio vigor, and this quasi-microorganism has been subjected to increasing concern (Wood, Wilson et al.1986 in recent years; Teunissen andOp den Camp 1993).But because anaerobion needs strict culture condition, the speed of growth is slow, and enzyme is single, at present and be not suitable for large-scale production.The cellulose enzyme gene of anaerobic fungi is imported in the Trichodermareesei cell, can learn from other's strong points to offset one's weaknesses, make the leavening property of existing cellulase production bacterial strain obtain improvement.
Make inscribe-beta-glucanase gene of anerobes in the Trichodermareesei cell, obtain effective expression, at first will solve the codon preference problem that exists in the heterologous protein expression.Existing research shows, the common preference GC base of aerobic fungi such as Trichoderma reesei, and the swing position preference G or the C of codeword triplet.Yet, the general preference AT base of the cellulose enzyme gene of cud anaerobion, and high AT sequence is often thought by mistake be to modify or cutoff signal by aerobic fungi, causes albumen not exclusively to be expressed and secretion (Nicholson, Theodorou et al.2005).
According to another bibliographical information; In Trichodermareesei during expressing heterologous albumen; If goal gene and suitable homology linker sequence or catalyzed combination zone (CBD) are formed fusion gene, will more help expression of heterologous genes (Paloheimo, Mantyla et al.2003; Paloheimo, Mantyla et al.2007).
Traditional genetic transforming method to fungi mainly is protoplasm body, electrization and the particle bombardment of PEG mediation; These methods prepare process complicacy, poor repeatability except protoplastis; Time and effort consuming; Problems (Fincham 1989) such as the ubiquity transformation efficiency is low, transformant instability have restricted filamentous fungus and have transformed and Application Research.
Summary of the invention
The invention provides a kind of gene of the inscribe-beta-glucanase of encoding, through this gene is imported Trichodermareesei, this gene can efficiently express in Trichodermareesei.
A kind of inscribe-beta-glucanase gene, its base sequence is shown in SEQ ID NO:2.
Gene of the present invention utilizes codon degeneracy property according to the codon preference of Trichodermareesei (Trichoderma reesei), through information biology software (DNA2.0) inscribe-beta-glucanase gene from the cud anerobes is carried out obtaining after the sequence optimisation.
A kind of above-mentioned inscribe-beta-glucanase expression of gene box that comprises comprises the promotor and the terminator at gene, signal peptide and the two ends of coding inscribe-beta-glucanase.
Preferably, the promotor of said expression cassette and terminator are respectively the promotor (Pcbh1) and the terminator (Tcbh1) of Trichodermareesei (Trichoderma reesei) cellobiohydrolase I gene.
In the Trichodermareesei excretory cellulase protein, cellobiohydrolase CBHI has occupied more than 60% of total protein (Durand, Clanet et al.1988).Therefore, the promotor Pcbh1 of this proteolytic enzyme is considered to strong promoter, the inscribe-beta-glucanase gene after optimizing is packed into express between Pcbh1 and the Tcbh1, can increase expression efficiency.
A kind of recombinant vectors that comprises above-mentioned inscribe-beta-glucanase gene.
Preferably; Described recombinant vectors contains the hygromycin B phosphotransferase expression cassette, and described hygromycin B phosphotransferase expression cassette is made up of the promotor (PtrpC) and the hygromycin B phosphotransferase gene (hygB) of Aspergillus nidulans (Aspergillus nidulans) the tryptophane synthetic gene that connects successively; The base sequence of described hygromycin B phosphotransferase can transform bacterial strain through the HYG screening shown in SEQ ID NO:4.
A kind of transformant that contains above-mentioned inscribe-beta-glucanase gene, host cell is preferably filamentous fungus, is preferably Trichodermareesei (Trichoderma reesei).
Preferably, described transformant prepares through following method:
(1) makes up the inscribe-beta-glucanase expression cassette that connects and composes successively by promotor, signal peptide, inscribe-beta-glucanase gene and terminator;
(2) the hygromycin B phosphotransferase expression cassette is connected with initial carrier, makes recombinant vectors;
(3) inscribe that step (1) is made-beta-glucanase expression cassette is incorporated in the recombinant vectors;
(4) recombinant vectors that step (3) is made changes Trichodermareesei over to through the agrobacterium tumefaciens mediation.
Gene of the present invention can be efficiently expressed after importing Trichodermareesei.In treadmill test, the speed of growth of nearly all conversion bacterial strain on the substratum that with the CMC 99.5 is sole carbon source all has raising in various degree, and wherein maximum transformant colony diameter is 5 times of wild strain.
Description of drawings
Fig. 1 is a pCAMBIA1300 carrier physical map;
Fig. 2 is the structure collection of illustrative plates of reorganization plasmid pUC18-PET;
Fig. 3 is a recombinant plasmid pCB-h collection of illustrative plates;
Fig. 4 is the collection of illustrative plates of recombinant plasmid pCB-hE;
Fig. 5 is for transforming egl in the bacterial strain OptThe PCR checking electrophoretogram of goal gene.
Embodiment
Embodiment 1 inscribe-beta-glucanase gene codon optimized
The codon usage frequency of statistics and calculating Trichodermareesei (T.reesei).As shown in the table.
Figure GSA00000019508200031
Figure GSA00000019508200041
Figure GSA00000019508200051
Coding region (GenBank:U97153.1) in conjunction with inscribe-beta-glucanase original gene; Under the prerequisite that does not change aminoacid sequence; Through new gene order that meets the Trichodermareesei codon preference of information biology software (DNA2.0Tool) design; Called after egl, its base sequence is shown in SEQID NO:1.Adopt ABI3900 type high-throughput dna synthesizer synthetic voluntarily and be cloned into plasmid pUC18, called after pUC18-egl.
The a small amount of rapid extraction of embodiment 2 Trichodermareesei genomic dnas
Trichodermareesei (Trichoderma reesei) coating is inoculated on the potato nutrient agar, cultivates 5-7 days to the spore maturation in 30 ℃.Scrape a small amount of mycelia from flat board and put into the 1.5ml centrifuge tube, add sterilization silica sand and 600 μ l CTAB extracting solutions, several down with the grinding of glass grinding pestle, 65 ℃ of water-bath 1h, the centrifugal 10min of 12000rpm.Get supernatant and add 600 μ l isopropanol precipitatings, the centrifugal 10min of 12000rpm abandons supernatant, 70% washing with alcohol 2 times, and dry back adds 20-30 μ l sterilized water and an amount of RNase, 37 ℃ of insulation 1h.
The prescription of above-mentioned CTAB is: 2% (massfraction) CTAB, 100mM Tris-HCl (pH8.0), 1.4M NaCl, 20mM EDTA.
Linker and CBD sequence that embodiment 3 merges from EGII
(1) genome with Trichodermareesei (T.reesei) is a template, obtains the encoding sequence (GenBank:M19373.1) of EGII gene through pcr amplification, the about 1.4kb of size.
Upstream primer B1, its nucleotide sequence wherein contains the EcoRI restriction enzyme site shown in SEQ ID NO:5.
Downstream primer B2: its nucleotide sequence wherein contains the BamHI restriction enzyme site shown in SEQ ID NO:6, as.
PCR reaction conditions: 94 ℃ of 5min; 94 ℃ of 50s, 64 ℃ of 50s, 70 ℃ of 1min50s, 30 circulations; 72 ℃ of 10min.This PCR product cloning is arrived pUC18 carrier, called after pUC18-eg2 plasmid.
(2) be common template with plasmid pUC18-egl and pUC18-eg2, the linker and the CBD zone of above-mentioned EGII gene are merged with instance 1 said gene egl, obtain a novel inscribe-beta-glucanase gene egl OptThe about 1.3kb of total length.
Design four primers altogether:
Up1: its nucleotide sequence wherein contains restriction enzyme site XhoI shown in SEQ ID NO:7;
Down1: its nucleotide sequence is shown in SEQ ID NO:8;
Up2: its nucleotide sequence is shown in SEQ ID NO:9;
Down2: shown in its nucleotide sequence SEQ ID NO:10, wherein contain restriction enzyme site XbaI;
Concrete steps are following:
With carrying out overlapping extension after four pairs of primers and two kinds of templates mixing.The PCR condition is: 94 ℃ of 5min; 94 ℃ of 50s, 70 ℃ of 1min40s, 30 circulations; 72 ℃ of 10min.
Get above-mentioned PCR product 1 μ l as template, add primer (up1 and down2) and carry out pcr amplification, reaction conditions and preceding same, the base sequence of warp order-checking PCR product is shown in SEQ ID NO:2.It is cloned into the pUC18 carrier, changes glycerol stock DH5a over to and preserve.
The structure of embodiment 4 recombinant plasmid pCB-hE
(1) structure of pCB-h recombinant vectors
Because the promotor of the hygromycin resistance selection markers that has on the carrier pCAMBIA1300 (Fig. 1) is plant promoter CAMV35S Promoter, is not suitable for filamentous fungus and expresses.Therefore must it be left out, make up and insert hygromycin phosphotransferase gene and the promotor thereof that is fit to trichoderma reesei expression.With plasmid pDESTR (GenBank:AB218275.1) is template, obtains the hygromycin resistance expression cassette hph of about 1.4kb through pcr amplification.Wherein, the about 367bp of the promotor (PtrpC) of Aspergillus nidulans tryptophane synthetic gene size, its base sequence shown in SEQ ID NO:3, the big or small about 1055bp of hygromycin phosphotransferase gene (hygB), its base sequence is shown in SEQ ID NO:4.
Upstream primer H1, its nucleotide sequence wherein contains the BstXI restriction enzyme site shown in SEQ ID NO.11;
Downstream primer H2, its nucleotide sequence wherein contains the XhoI restriction enzyme site shown in SEQ ID NO.12.
PCR reaction conditions: 94 ℃ of 5min; 94 ℃ of 50s, 64 ℃ of 50s, 70 ℃ of 1min40s, 30 circulations; 72 ℃ of 10min.
This PCR product cloning is arrived the pUC18 carrier; Through BstXI and XhoI double digestion; Be cloned in the pCAMBIA1300 carrier of BstXI and XhoI double digestion with T4 ligase enzyme (worker is given birth in Shanghai), obtain containing the agriculture bacillus mediated carrier pCB-h (Fig. 3) of hygromycin resistance selection markers.
(2) clone of promotor Pcbh1 (containing signal peptide sequence) and terminator Tcbh1.
Strong promoter (Pcbh1) sequence (Genbank:D86235.1) and signal peptide sequence (Genbank:AY368686.1) according to the Trichodermareesei cellobiohydrolase I that issues; With the Trichodermareesei genomic dna is template; Carry out pcr amplification through upstream primer P1 and downstream primer P2, amplification obtains the Pcbh1 fragment of about 1.6kb size.
Upstream primer P1, its nucleotide sequence wherein contains the BamI restriction enzyme site shown in SEQ ID NO.13;
Downstream primer P2, its nucleotide sequence wherein contains the XhoI restriction enzyme site shown in SEQ ID NO.14.
The PCR reaction conditions is 94 ℃ of 5min; 94 ℃ of 50s, 70 ℃ of 2min30s, 30 circulations; 72 ℃ of 10min.
Gene order according to the terminator Tcbh1 of the cellobiohydrolase I of patent (CN1831131A) issue; Trichodermareesei genomic dna with said extracted is a template; Carry out pcr amplification through upstream primer T1 and downstream primer T2, amplification obtains the Tcbh1 fragment of 1.4kb size.
Upstream primer T1, its nucleotide sequence wherein contains the XbaI enzyme cutting site shown in SEQ ID NO.15;
Downstream primer T2, its nucleotide sequence wherein contains the HindIII restriction enzyme site shown in SEQ ID NO.16.
The PCR reaction conditions is 94 ℃ of 5min; 94 ℃ of 50s, 70 ℃ of 1min30s, 30 circulations; 72 ℃ of 10min.
(3) structure of pCB-hE recombinant expression vector
Respectively with restriction enzyme BamI, XhoI and XbaI, HindIII double digestion Pcbh1 and Tcbh1, with both pUC18 carriers of packing into, obtain carrier pUC18-PT with the T4 ligase enzyme.With XhoI and two respectively pUC18-PT and the egl of cutting of XbaI Opt, among the pUC18-PT that packs into, obtain pUC18-PET (Fig. 2).
At last, with two respectively pUC18-PET and the pCB-h of cutting of restriction enzyme BamHI and HindIII, with pack into the MCS of pCB-h of PET fragment, obtain containing hygromycin selection mark hph and goal gene egl with the T4 ligase enzyme OpAgriculture bacillus mediated carrier pCB-hE (Fig. 4), change glycerol stock DH5 α over to and preserve.
Embodiment 5 adopts the agrobacterium mediation converted technology that goal gene is imported Trichodermareesei
Take freeze-thaw method to change agrobacterium tumefaciens AGL-1 over to recombinant plasmid pCB-hE.The AGL-1 bacterial strain that will contain recombinant plasmid is rule containing on the antibiotic LB substratum, and the picking mono-clonal carries out liquid culture, and 28 ℃, 20-24h; Collect the centrifugal back of thalline and be diluted to OD with the suspension of IM substratum 660=0.15, add Syringylethanone AS 200 μ M, 28 ℃, the 200rpm dark place is cultured to OD660=0.5-0.8;
Wash the spore of Trichodermareesei (ATCC56765) from the PDA inclined-plane of cultivating 4～7d with the 5-8ml sterilized water, cotton is filtered and obtains spore suspension, the blood counting chamber counting.Regulate spore concentration to 10 with sterilized water then 6-10 7/ ml.The spore suspension of getting 150 μ l activatory Agrobacterium bacterium liquid and 150 μ l dilution mixes, and is coated on the nitrocellulose filter of common culture plate, and 48-60h is cultivated in 24-25 ℃ of dark place.Filter membrane is taken off, and counter being taped against on the selectivity PDA flat board that contains 100 μ g/ml HYGs and 200 μ g/ml cephamycins cultivated 3-5d for 28 ℃, observes the formation of transformant.
Above-mentioned IM liquid nutrient medium (1L): 10ml phosphoric acid buffer pH 7.0 (200g/l K 2HPO4,145g/l KH 2PO 4), 20ml magnesium-sodium element solution (30g/l MgSO 47H2O, 15g/lNaCl), 1ml 1%CaCl 22H 2O (w/v), 20ml 20% glucose solution (w/v), 10ml0.01%FeSO 4(w/v), 5ml trace element solution (100mg/l ZnSO47H2O, 100mg/lCuSO 45H 2O, 100mg/l H 3BO 3, 100mg/l MnSO 4H 2O, 100mg/lNa 2MoO 42H 2O), 2.5ml 20%NH 4NO 3(w/v), 40mM MES pH 5.3,0.5%glycerol (w/v), 200 μ M Syringylethanone (AS) (Hooykaas et al., 1979; Mozo andHooykaas, 1991).Add the 15g/l agar powder in the solid medium.
The screening of embodiment 6 Trichodermareesei transformants
Obtain about 120 through HYG resistance PDA plate screening and transform bacterium colony.Replace screening and culturing more than three times through resistant panel and non-resistance flat board, get the stable bacterium colony of phenotype, extract total DNA and carry out PCR checking eglopt gene, process for extracting such as embodiment 2.
DNA with said extracted is a template, utilizes upstream primer up1 and downstream primer down2 to carry out pcr amplification;
PCR condition: 94 ℃ of 5min; 94 ℃ of 50s, 70 ℃ of 1min40s, 30 circulations; 72 ℃ of 10min.Electrophoresis obtains the fragment of the about 1.3kb of size, and the original strain genomic dna then can't increase and obtain the purpose fragment, and is as shown in Figure 5, goal gene egl OptSuccessfully imported in the Trichodermareesei (Trichoderma reesei).Each is transformed bacterium colony isolate three single bacterium colonies again, go down to posterity more than five times not containing on the potato nutrient agar of HYG continuously, carry total DNA once more and carry out PCR checking, egl OptGene is stable existence still.
Embodiment 7 recombinant bacterial strain phenotypic characteristics
Under the aseptic technique environment; With the wild strain is contrast; 50 of picked at random transform bacterial strain, and the bacterium piece that takes the about 0.35cm of diameter with punch tool at the edge of each conversion bacterium colony is to being on the solid-based basal culture medium (MM) of sole carbon source with CMC 99.5 (CMC), in 30 ℃ of cultivation 60h.Observe and write down the growth diameter of each single strain.Every group is provided with three parallel laboratory tests respectively, results averaged, and experimental data is as shown in the table:
Figure GSA00000019508200091
The result shows; Behind the recombinant plasmid pCB-hE importing Trichodermareesei with the present invention's structure; The speed of growth of nearly all conversion bacterial strain on the substratum that with the CMC 99.5 is sole carbon source all has raising in various degree, and wherein maximum transformant colony diameter is 5 times of wild strain.
SEQUENCE LISTING
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< 120>a kind of inscribe-beta-glucanase gene
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ggcttcaacg tcttccgcat ccccaccacc tggaccggcc acttcggcaa cggccccgac 240
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accggcagct acgtcatcct gaacatccac cacgagaact ggaactacgc cttcagcaac 360
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< 213>artificial sequence
<400>9
cacgagctct atgcgcaaca tccccag 27
<210>10
<211>28
<212>DNA
< 213>artificial sequence
<400>10
taatctagat tagggggcgg gggcgacg 28
<210>11
<211>33
<212>DNA
< 213>intestinal bacteria (E.coli)
<400>11
ccaccatgtt gggacgttaa ctgatattga agg 33
<210>12
<211>28
<212>DNA
< 213>intestinal bacteria (E.coli)
<400>12
ctcgagcgtt aactggttcc cggtcggc 28
<210>13
<211>29
<212>DNA
< 213>artificial sequence
<400>13
cgcggatcca attctggaga cggcttgtt 29
<210>14
<211>29
<212>DNA
< 213>artificial sequence
<400>14
ccgctcgagc tccaagtgtt gccatcgta 29
<210>15
<211>31
<212>DNA
< 213>artificial sequence
<400>15
cgctctagat gaacccttac tactctcagt g 31
<210>16
<211>30
<212>DNA
< 213>artificial sequence
<400>16
attaagcttg tcctcggcta cgttgtcatc 30
<210>17
<211>426
<212>PRT
< 213>rumen microorganism (Orpinomyces sp.)
<400>17
Gln Gln Thr Val Trp Gly Gln Cys Gly Gly Ile Gly Trp Ser Gly Pro
1 5 10 15
Thr Asn Cys Ala Pro Gly Ser Ala Cys Ser Thr Leu Asn Pro Tyr Tyr
20 25 30
Ala Gln Cys Ile Pro Gly Ala Thr Thr Ile Thr Thr Ser Thr Arg Pro
35 40 45
Pro Ser Gly Pro Thr Thr Thr Thr Arg Ala Thr Ser Thr Ser Ser Ser
50 55 60
Thr Pro Pro Thr Ser Ser Met Arg Asn Ile Pro Ser Lys Asp Leu Val
65 70 75 80
Lys Glu Leu Asn Ile Gly Trp Asn Leu Gly Asn Ala Leu Asp Ala His
85 90 95
Cys Leu Asp Lys Leu Asp Tyr Asn Lys Asp Gln Leu Ala Ser Glu Thr
100 105 110
Cys Trp Ala Asn Pro Lys Ala Thr Pro Gly Leu Phe Ser Ala Leu Lys
115 120 125
Asn Gln Gly Phe Asn Val Phe Arg Ile Pro Thr Thr Trp Thr Gly His
130 135 140
Phe Gly Asn Gly Pro Asp Tyr Lys Ile Ser Asp Val Trp Met Arg Arg
145 150 155 160
Val His Glu Val Val Asp Tyr Ala Leu Asn Thr Gly Ser Tyr Val Ile
165 170 175
Leu Asn Ile His His Glu Asn Trp Asn Tyr Ala Phe Ser Asn Asn Leu
180 185 190
Gln Lys Ala Lys Pro Ile Leu Ala Ala Ile Trp Lys Gln Ile Ala Ala
195 200 205
Glu Phe Ala Asn Tyr Asp Glu His Leu Ile Phe Glu Gly Met Asn Glu
210 215 220
Pro Arg Lys Val Asp His Pro Asn Glu Trp Asn Gly Gly Asp Gln Glu
225 230 235 240
Gly Trp Asp Phe Val Asn Glu Met Asn Ala Val Phe Leu Gln Thr Val
245 250 255
Arg Ala Ser Gly Gly Asn Asn Ala Ile Arg His Leu Met Ile Pro Thr
260 265 270
Tyr Ala Ala Cys Val Asn Asp Gly Ala Leu Glu Ser Tyr Val Arg Lys
275 280 285
Phe Pro Thr Asn Asp Asn Lys Val Ile Ala Ser Val His Ser Tyr Val
290 295 300
Pro Tyr Asn Phe Ala Leu Asn Thr Gly Ala Gly Ala Glu Lys Thr Phe
305 310 315 320
Gly Ser Thr Ser Asp Ile Glu Trp Ala Met Asn Asn Ile Lys Arg Phe
325 330 335
Leu Val Asp Arg Asn Ile Pro Val Ile Ile Gly Glu Phe Gly Ala Met
340 345 350
Asn Arg Asp Asn Glu Ser Glu Arg Ala Arg Trp Ala Glu Tyr Tyr Ile
355 360 365
Lys Ser Ala Thr Ala Met Gly Val Pro Cys Val Leu Trp Asp Asn Gly
370 375 380
Tyr Thr Gln Gly Thr Gly Glu Leu Phe Gly Val Ile Asp Arg Asn Ser
385 390 395 400
Tyr Arg Ile Ile Phe Gln Gln Phe Ile Asn Gly Leu Met Lys Gly Leu
405 410 415
Gly Gly Lys Lys Thr Val Ala Pro Ala Pro
420 425

Claims (9)

1. inscribe-beta-glucanase gene, it is characterized in that: base sequence is shown in SEQ ID NO:2.
2. inscribe-beta-glucanase expression cassette that comprises the described inscribe of claim 1-beta-glucanase gene.
3. inscribe according to claim 2-beta-glucanase expression cassette is characterized in that: the promotor of inscribe-beta-glucanase expression cassette and terminator are respectively the promotor and the terminator of Trichodermareesei (Trichodermareesei) cellobiohydrolase I gene.
4. recombinant vectors that comprises the described inscribe of claim 1-beta-glucanase gene.
5. recombinant vectors according to claim 4; It is characterized in that: contain the hygromycin B phosphotransferase expression cassette, described hygromycin B phosphotransferase expression cassette is made up of the promotor and the hygromycin B phosphotransferase gene of Aspergillus nidulans (Aspergillus nidulans) the tryptophane synthetic gene that connects successively; The base sequence of described hygromycin B phosphotransferase is shown in SEQ ID NO:4.
6. one kind contains the transformant that right requires 1 described inscribe-beta-glucanase gene.
7. transformant according to claim 6 is characterized in that: host cell is a filamentous fungus.
8. transformant according to claim 7 is characterized in that: host cell is a Trichodermareesei.
9. transformant according to claim 8 prepares through following method:
(1) makes up the inscribe-beta-glucanase expression cassette that connects and composes successively by promotor, signal peptide, inscribe-beta-glucanase gene and terminator;
(2) the hygromycin B phosphotransferase expression cassette is connected with initial carrier, makes recombinant vectors;
(3) inscribe that step (1) is made-beta-glucanase expression cassette is incorporated in the recombinant vectors;
(4) recombinant vectors that step (3) is made changes Trichodermareesei over to through the agrobacterium tumefaciens mediation.
CN2010101047280A 2010-01-29 2010-01-29 Endo-beta-glucanase gene Expired - Fee Related CN101870985B (en)

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CN102268448B (en) * 2011-06-28 2013-03-13 华东理工大学 Expression equipment for expressing heterologous protein in Trichoderma reesei cell, and gene engineering bacteria
CN102304540B (en) * 2011-08-26 2013-10-09 华东理工大学 Expression equipment for expressing exogenous protein by secretion in trichoderma reesei and application of expression equipment
CN102978184B (en) * 2011-09-06 2014-04-30 北京卫诺恩生物科技有限公司 High-specific-activity and wide-pH-range beta-glucanase R-BgluA, and gene and application thereof
CN102978183B (en) * 2011-09-06 2014-04-09 北京卫诺恩生物科技有限公司 Acidic beta-glucanase P-Bglu16A, and gene and application thereof
CN104560921B (en) * 2015-01-29 2017-07-14 东北师范大学 A kind of endoglucanase of acidic beta 1,4 and its application
CN108795781B (en) * 2017-05-03 2020-06-02 中国科学院微生物研究所 Recombinant bacterium for high-yield trichoderma harzianum α -1, 3-glucanase and application thereof
CN109234222A (en) * 2018-10-23 2019-01-18 淮阴师范学院 A kind of trichoderma reesei microbial culture method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
金欣等.厌氧真菌内切–β–葡聚糖酶基因在里氏木霉中的重组与表达.《高校化学工程学报》.2011,第25卷(第4期),637-642. *

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