CN108070609A - By the use of trichoderma reesei as the method for host expresses recombinant protein - Google Patents

Abstract

The invention discloses a kind of by the use of trichoderma reesei as the method for host expresses recombinant protein.It is provided by the present invention by the use of trichoderma reesei as the method for host expresses recombinant protein, include the following steps：The expression cassette of encoding gene containing destination protein to be expressed is cloned into the noncoding region being located in trichoderma reesei genome between the gene that Protein ID are 68606 and the gene that Protein ID are 68608.The present invention experiment prove in cbh1 gene-deleted strains, by the expression backbone integration angle of striking ND1 of cbh1 genes, and compared with wild strain cbh1 genes expression, find recombination being inserted into the site, can obtain and the comparable expression in cbh1 sites.The present invention further enriches perfect by the use of trichoderma reesei as the technical solution of host's highly effective expressing recombinant protein.

Description

By the use of trichoderma reesei as the method for host expresses recombinant protein

Technical field

The present invention relates to genetic engineering fields, and in particular to a kind of by the use of trichoderma reesei as host expresses recombinant protein Method.

Background technology

Filamentous fungus T. reesei (Trichoderma reesei) is industrial main cellulase hemicellulase Bacterial strain is produced, is the aliment security level bacterial strain of U.S. FDA certification.Trichoderma reesei has powerful protein secretion ability, some prominent The exocrine protein amount of mutant has the glycosylation system similar to higher mammal up to 100g/L, therefore Richter scale wood Mould is a kind of ideal expression of recombinant proteins host, obtain internationally famous enzyme preparation company (such as Genencor, Novozymes etc.) great attention, and be successfully applied to the expression and production of multi-medicament, chemical reagent and enzyme preparation.Closely Nian Lai with the fast development of trichoderma reesei genomics and its constantly improve of genetic operating system, is made using trichoderma reesei It is also increasingly taken seriously for host expresses recombinant protein.

It is worth noting that exogenous dna fragment is mostly with the approach NHEJ of non-homologous end joining in trichoderma reesei (nonhomologous end-joining) is integrated into genome, in the form of radom insertion.The gene expression frame of recombinant protein Different insertion points in genome can directly influence its expression.For example, if recombination is inserted into silencer Control region may directly result in it and cannot transcribe and express failure, if foreign gene is inserted into the regulatory region of enhancer May increase substantially its expression quantity.The site of cellobiose hydrolase gene (cbh1) is typically considered in trichoderma reesei The site of gene efficient expression.

The content of the invention

The object of the present invention is to provide a kind of by the use of trichoderma reesei as the method for host expresses recombinant protein.

It is provided by the present invention by the use of trichoderma reesei as the method for host expresses recombinant protein, it may include following steps： The expression cassette of encoding gene containing destination protein to be expressed is cloned into trichoderma reesei genome and is located at Protein ID Noncoding region between the gene that gene and Protein ID for 68606 are 68608.

Wherein, it is described " expression cassette of the encoding gene containing destination protein to be expressed to be cloned into trichoderma reesei gene The noncoding region being located in group between the gene that Protein ID are 68606 and the gene that Protein ID are 68608 " can be such as Under it is any：

(a) the trichoderma reesei genome is substituted with the expression cassette of the encoding gene containing destination protein to be expressed In be located at Protein ID be 68606 gene and Protein ID be 68608 gene between noncoding region arbitrary piece Section；

(b) expression cassette of the encoding gene containing destination protein to be expressed is inserted into the trichoderma reesei gene It is located at the arbitrary of the noncoding region between the gene that Protein ID are 68606 and the gene that Protein ID are 68608 in group Position.

Further, the gene and Protein ID that Protein ID are 68606 are located in the trichoderma reesei genome The nucleotides sequence of noncoding region between 68608 gene is classified as SEQ ID No.1.

In an embodiment of the present invention, it is described " to clone the expression cassette of the encoding gene containing destination protein to be expressed It is located at into trichoderma reesei genome between the gene that Protein ID are 68606 and the gene that Protein ID are 68608 Noncoding region " is specially：The expression cassette of the encoding gene containing destination protein to be expressed is substituted into the trichoderma reesei 429-551 of SEQ ID No.1 in genome.

Such as the routine understanding of this field, in the above-mentioned methods, the encoding gene containing destination protein to be expressed Expression cassette from 5 ' end to 3 ' end successively comprising promoter, by the promoter start expression the destination protein to be expressed Encoding gene and terminator.Certainly, other kinds of regulating and controlling sequence can also be included as needed.

Term " | expression cassette " it is the double-strandednucleic acid point for including all regulating and controlling sequences necessary to target gene and its expression Son.The regulating and controlling sequence can instruct coded sequence to express target gene in suitable host cell under its consistency condition.Institute It states regulating and controlling sequence to include but not limited to, targeting sequencing, polyadenylation sequence, promoter, signal sequence and transcription terminator. Bottom line, regulating and controlling sequence will include the termination signal of promoter and transcription and translation.In order to import the specific limitation of carrier Property enzyme site can provide regulating and controlling sequence with joint so that the code area of regulating and controlling sequence and target gene is attached.It adjusts It can be suitable promoter sequence to control sequence, you can be expressed the nucleotide sequence of the host cell identification of nucleotide sequence.Start Subsequence contains the transcription regulating nucleotide sequence of mediation destination gene expression.Promoter can have transcription to live in selected host cell Property any nucleotide sequence, including mutation, truncated and heterozygosis promoter, can derive from coding and host cell it is homologous or The gene of heterologous extracellular or intracellular polypeptide.Regulating and controlling sequence can also be suitable transcription terminator, can be by host cell It identifies to terminate one section of sequence of transcription.Terminator sequence is operatively connected in 3 ' ends of the nucleotide sequence of coded polypeptide. Any terminator that can be functioned in selected host cell may be used to the present invention.Before regulating and controlling sequence can also be suitable Sequence is led, i.e., mRNA non-translational regions highly important to the translation of host cell.Targeting sequencing is operatively connectable to target gene 5 ' ends.Any targeting sequencing that can be functioned in selected host cell is used equally for the present invention.In these examples, Together with should the nucleotide sequence of target gene be operatively connected with regulating and controlling sequence.Term " being operatively connected " is defined as in the text Such a conformation, wherein regulating and controlling sequence are located at the appropriate location of target gene, so that regulating and controlling sequence instructs the table of target gene It reaches.

In one embodiment of the invention, the promoter is the cellobiohydrolase I bases from trichoderma reesei The promoter of cause；The terminator is the terminator of the cellobiohydrolase I gene from trichoderma reesei.

In another embodiment of the present invention, the promoter is the cellobiohydrolase I from trichoderma reesei The promoter of gene；The terminator is the terminator of the cellobiohydrolase II genes from trichoderma reesei.

Further, the sequence of the promoter of the cellobiohydrolase I gene from trichoderma reesei is specially 1939-4016 of the 1-2078 or SEQ ID No.3 of SEQ ID No.2.The fiber from trichoderma reesei The sequence of the terminator of disaccharide-hydrolysing enzymes I gene is specially 3685-5927 of SEQ ID No.2.It is described to derive from Richter scale The sequence of the terminator of the cellobiohydrolase II genes of trichoderma is specially 5693-7784 of SEQ ID No.3.

In the above-mentioned methods, the destination protein to be expressed is to be given birth to from Eukaryotic albumen or from protokaryon The albumen of object.

Further, the eucaryote can be fungi, plant or animal.

Further, the fungi is specially aspergillus niger or trichoderma reesei in the present invention.

In one embodiment of the invention, the destination protein to be expressed is the lipase from aspergillus niger, Amino acid sequence is the same as the amino acid sequence of the 2138-2947 coding gained of SEQ ID No.2.

In another embodiment of the present invention, the destination protein to be expressed is the fiber two from trichoderma reesei Glycosylhydrolase I, amino acid sequence is the same as the amino acid sequence of the 4017-5692 coding gained of SEQ ID No.3.

In the above-mentioned methods, it is described " expression cassette of the encoding gene containing destination protein to be expressed to be cloned into Richter scale The non-coding being located in reesei gene group between the gene that Protein ID are 68606 and the gene that Protein ID are 68608 Area " can be realized by importing recombinant expression carrier into T. reesei host；Containing described on the recombinant expression carrier Expression cassette and the homology arm positioned at the expression cassette upstream and downstream for pinpointing insertion or replacing.

Further, in one embodiment of the invention, the homology arm is specially the 1-1938 of SEQ ID No.3 Two segment DNA segments shown in position (upstream homology arm) and 7785-9710 (downstream homology arm).

The present invention is by turning a series of heterologous lipase gene radom insertions to trichoderma reesei genome different position Beggar carries out the high flux screening of flow cytometer, and carries out gene copy number to the higher transformant of lipase expression quantity and insert The identification of angle of striking is finally that the superior strain singly copied carries out insertion point analysis to a plant weight histone gene, identifies Another in addition to trichoderma reesei cbh1 sites is conducive to the gene insertion site ND1 of gene expression.Finally in cbh1 bases Because in gene-deleted strain, the expression backbone integration of cbh1 genes is entered into the site, and compared with wild strain cbh1 genes expression water It is flat, it finds recombination being inserted into the site, can obtain and the comparable expression in cbh1 sites.The present invention further enriches It is perfect by the use of trichoderma reesei as the technical solution of host's highly effective expressing recombinant protein.

Description of the drawings

Fig. 1 is the structure diagram of plasmid pSKLR.

Fig. 2 is the preparation of protoplast and the abduction delivering schematic diagram of red fluorescent protein.

Fig. 3 is the correspondence schematic diagram of red fluorescent protein and lipase expression quantity.

Fig. 4 is expression testing result figure of the restructuring lipase gene in bacterial strain ND1.

Fig. 5 is the structure required component segments of bacterial strain ND1cbh1.

Fig. 6 is expression testing result figure of the cbh1 genes in bacterial strain TU6, ND1 and ND1cbh1 bacterial strain.

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Trichoderma reesei (Trichoderma reesei) QM9414：ATCC 26921.

Trichoderma reesei (Trichoderma reesei) TU6：ATCC MYA-256, while be recorded in " E Gruber, j.Visser C.P.Kubicek,L.H.de Graaff.The development of a heterologous transformation system for the cellulolytic fungus Trichoderma reesei based on a pyrG-negative mutant strain.Curr Genet(1990)18:A 71-76 " texts, the public can be at applicant It obtains, can only be used to repeat present invention experiment use.

Trichoderma reesei (Trichoderma reesei) N10：It is recorded in " Lina Qin, Fu-rong Cai, Xin-rui Dong,Zhen-bang Huang,Yong Tao,Jian-zhong Huang,Zhi-yang Dong.Improved production of heterologous lipase in Trichoderma reesei by RNAi mediated gene silencing of an endogenic highly expressed gene.Bioresour Technol,2012,109: A 116-122. " texts, the public can obtain at applicant, can only be used to repeat present invention experiment use.

Trichoderma reesei (Trichoderma reesei) TR1124：China Microbiological is preserved on July 20th, 2012 (abbreviation CGMCC, address are culture presevation administration committee common micro-organisms center：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Number, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC No.6384.And it is recorded in Chinese patent application 201210271826.2。

Trichoderma reesei (Trichoderma reesei) Tu6 Δs ku70：The structure of the bacterial strain is shown in " Gene targeting An in a nonhomologous end joining deficient Hypocrea jecorina " texts, in the document, bacterium Strain is named as Δ tku70, but has article to deliver later, which is named as Tu6 Δ ku70, sees article " A novel major facilitator transporter TrSTR1 is essential for pentose utilization and involved in xylanase induction in Trichoderma reesei,Biochemical and biophysical research communications,2015,460(3):663-669 ", the public can obtain at applicant , it can only be used to repeat present invention experiment use.

Plasmid pSK-Lip be recorded in " Lina Qin, Fu-rong Cai, Xin-rui Dong, Zhen-bang Huang, Yong Tao,Jian-zhong Huang,Zhi-yang Dong.Improved production of heterologous lipase in Trichoderma reesei by RNAi mediated gene silencing of an endogenic highly expressed gene.Bioresour Technol,2012,109:A 116-122 " texts, the public can be from applicant Place obtains, and can only be used to repetition present invention experiment and uses.

Plasmid pDsRed-Monomer-N1：For TAKARA Products, article No.：632465.

Plasmid pBluescript SK (+)：For Snapgene, Addgene products.

Embodiment 1, beneficial to recombinant gene expression trichoderma reesei genomic insertion site ND1 acquisition and application example

The present invention utilizes one section in foot and mouth disease virus Foot and mouth disease virus (FMDV) Polypeptide 2A sequences are as connection peptide by a red fluorescent protein gene DsRed and lipase gene LipA from aspergillus niger Amalgamation and expression is carried out in trichoderma reesei.2A sequences can make DsRed genes and LipA genes be in 1：1 ratio is expressed, LipA genes will be secreted into extracellularly because introducing signal peptide sequence, and red fluorescent protein will stay in cell as reporter gene The interior high flux screening that flow cytometer is used for as marker gene.Amalgamation and expression skeleton random integration enters trichoderma reesei gene Group because the difference of insertion point and copy number can generate the different transformant of expression of recombinant proteins amount, passes through fluidic cell Instrument screening obtains the higher transformant of a plant weight histone expression quantity, and identification is carried out to the copy number of this plant of transformant and finds the bacterium Recombination copies to be single in strain, and the flank that analysis obtains insertion point both ends has then been carried out to the insertion point of recombination The expression backbone integration of cbh1 genes finally in cbh1 gene-deleted strains, is entered the site by sequence, and compared with wild strain The expression of cbh1 genes finds recombination being inserted into the site, can obtain and the comparable expression in cbh1 sites.

First, beneficial to recombinant gene expression trichoderma reesei genomic insertion site ND1 acquisition

(1) structure of recombinant plasmid

1st, the structure of red fluorescent protein and heterologous lipase fusion expression vector pSKLR

(1) promoter and terminator of PCR amplification cellobiohydrolase I (CBH1) gene

Using trichoderma reesei (Trichoderma reesei), QM9414 genomic DNAs is templates, and Pcbh1-F, Pcbh1-R are Primer carries out PCR amplification, and amplification condition is：95 DEG C of pre-degeneration 5min, 94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C extend 2min, 30 Xun Huans；Last 72 DEG C of extensions extension 10min, amplification obtain the promoter fragment Pcbh1 of cbh1 genes.Equally, with Trichoderma reesei (Trichoderma reesei) QM9414 genomic DNAs are template, and Tcbh1-F, Tcbh1-R carry out for primer PCR amplification, amplification condition are：95 DEG C of pre-degeneration 5min, 94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 2.5min, 30 Xun Huan；Last 72 DEG C of extensions extension 10min, amplification obtain the termination sub-piece Tcbh1 of cbh1 genes.Agarose gel electrophoresis, Glue recycling is carried out to two segments respectively.

(2) Overlap extension PCR amplification red fluorescent protein, 2A sequences merge segment L2aR with lipase gene

First round PCR：Using plasmid pSK-Lip as template, lipase2a-F, lipase2a-R contain 2A sequences for primer amplification The lipase gene segment of row and part red fluorescent protein genetic fragment.Using plasmid pDsRed-Monomer-N1 as template, 2ared-F, 2ared-R are the red fluorescent protein gene that primer amplification contains 2A sequences and partial fat enzyme gene segment.It will The two segments are purified into row agarose gel electrophoresis blend compounds QIAquick Gel Extraction Kit.

Second wheel PCR：Two PCR product equimolars of glue recovery purifying are mixed as template, lipase2a-F, 2ared-R is that primer carries out the segment L2AR that OverlapPCR amplifications can obtain two Gene Fusions, which is cloned into It is sequenced in pMD18T-simple carriers.

(3) structure of fusion expression vector pSKLR

Double digestion is carried out to plasmid pBluescript SK (+) with restriction enzyme SalI and NotI, with restricted interior Enzyme cutting SalI and EcoRI is double digested to the progress of Pcbh1 segments, and L2aR is carried out with restriction enzyme EcoRI and SpeI It is double digested, Tcbh1 is carried out with restriction enzyme SpeI and NotI it is double digested, by the carrier segments digested, Pcbh1 segments, LaR segments, the mixing of Tcbh1 segments carry out four segment connections, convert Escherichia coli Top10, and bacterium colony PCR identifications turn Beggar clones, and the primer is lipase2a-F and 2ared-R, and extracting plasmid is correctly cloned in picking identification, and is tested through sequencing Up to fusion expression vector pSKLR after card is correct (structure is shown in Fig. 1).

2nd, the structure of uracil-deficient selection markers carrier pSKpyr4

(1) clone of trichoderma reesei pyr4 genes

Using trichoderma reesei (Trichoderma reesei), QM9414 genomic DNAs is templates, and Pyr4-F, Pyr4-R are to draw Object carries out PCR amplification, and amplification condition is：95 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 30s, 62 DEG C of annealing 40s, 72 DEG C of extension 2min, 30 Xun Huans；Last 72 DEG C of extensions extension 10min.By gained PCR product in 1% agarose gel electrophoresis, Ago-Gel electricity Kit of swimming recycling purpose band, TA clones are connected into pMD18T-Simple carriers, are sequenced.

(2) structure of plasmid vector pSKpyr4

Respectively with restriction enzyme ClaI and EcoRI to plasmid pBluescript SK (+) and the correct pyr4 of sequencing Gene TA cloned plasmids carry out double digestion, pBluescript SK (+) carrier segments and pyr4 gene pieces of recycling digestion processing Section, and be attached.Conversion Escherichia coli Top10, the progress bacterium colony PCR identifications of picking transformant, the primer Pyr4-F, Pyr4-R, PCR condition are the same, extract plasmid and after sequence verification is correct up to the complementary plasmid of uracil-deficient bacterial strain TU6 Carrier pSKpyr4.

The whole primers arrived used in the process of above-mentioned construction recombination plasmid are referring specifically to table 1.

Table 1 is used to build the primer of recombinant vector

(2) acquisition of the trichoderma reesei transformant of amalgamation and expression red fluorescent protein and heterologous lipase

1st, prepared by the protoplast of Li's Trichoderma strains TU6

(1) spore of trichoderma reesei (Trichoderma reesei) TU6 on the inclined-plane or tablet of fresh cultured is taken, is used Spore suspension is made in appropriate amounts of sterilized water washing spore, and 200 mesh sieve are filtered to remove remaining mycelia.The spore suspension of filtering is connect Kind is into the 500mL triangular flasks equipped with 100mL MM culture mediums (2% glucose is carbon source), 28 DEG C of culture 13h-14h, until mycelia Stretching, extension.

(2) culture solution through 200 mesh sieve is filtered, collects thalline, sterile water washing 2-3 times, finally with the MgSO of 1.2M₄ Solution washed once, and solution is allowed to flow to end naturally；

(3) thalline on sieve is flushed in the triangular flask equipped with 15mL lysates to (lysate is splitting containing 150mg Enzyme Lysing enzyme Sigma, Cat.No.L-1412 and 15mg cellulase Cellulase R-10, YAKULT are solved, The 1.2M MgSO of Japan₄), 30 DEG C of reaction 1.5h, the situation that micro- Microscopic observation protoplast generates, every 10min after 1h Sampling observation is once；

(4) in the presence of protoplast largely generates and still there are a large amount of mycelia, the sorbitol solution of isometric 0.6M is added Reaction is terminated, 200 mesh sieve are filtered to remove remaining mycelia, and room temperature 3000rpm centrifugations 10min collects protoplast pellet；

(5) supernatant is removed along precipitation one side, protoplast pellet is resuspended with 1.0M sorbitol solutions, room temperature 3000rpm Centrifuge 10min；

(6) step (5) is repeated, supernatant is abandoned and protoplast is suspended in 200 μ L 1.0M sorbitol solutions, blood cell plate meter Number device is observed and counted.

2nd, the trichoderma reesei conversion of fusion expression vector

(1) plasmid pSKpyr4 and pSKLR are subjected to largely extracting and with the absolute ethyl alcohols of 2 times of volumes and 1/10 volume 3.0M sodium acetates (pH5.2) overnight precipitation, is washed 2 times with 70% ethyl alcohol, with sterilizing ddH₂O dissolves, and each plasmid concentration is made to reach μ G grades.

(2) plasmid pSKpyr4 and pSKLR in molar ratio 1:3 to 1:10 ratio mixing, volume is no more than 20 μ L, by this Mixed liquor is added in the protoplast of above-mentioned preparation, gently mixing, then respectively adds in 50 μ L PEG4000 thereto, is mixed again It is even, 30min is placed on ice；If control, control replaces DNA mixed liquors with isometric sterile water.

(3) 1mL PEG4000 are respectively added in into above-mentioned pipe again, mixing is placed at room temperature for 20min.

(4) last each to add in 1mL 1.0M sorbierites again, mixing, all protoplast transformation liquid is transferred to again In 250mL triangular flasks equipped with 50mL inducing cultures, Fiber differentiation based formulas is glucose containing 1g in every liter of culture medium, and 1g is sweet Oil, 20g lactose, 180g sorbierites, 0.05g (NH₄)₂SO₄, 0.15g KH₂PO₄, 0.006g MgSO₄, 0.006g CaCl₂, 0.00005g FeSO₄·7H₂O, 0.000016g MnSO₄·H₂O, 0.000014g, ZnSO₄·7H₂O, 0.00002g CoCl₂。 Condition of culture is 30 DEG C, and rotating speed is adjusted to 200rpm afterwards for 24 hours and continues shake culture by the slow shake cultures of 100rpm.

After (5) 30 DEG C are cultivated 3-6 days, all mycelia are collected, protoplast is prepared again and carries out selected by flow cytometry apoptosis With (see Fig. 2).In Fig. 2, A：Protoplast induces the mycelial growth situation after 96h in inducing culture is regenerated；B：Fluorescence is shown The induction situation of micro- Microscopic observation red fluorescent protein；C：It observes under ordinary optical microscope and again prepares the transformant of induction Into protoplast；D：The transformant of induction is prepared into red fluorescent protein after protoplast by fluorescence microscopy Microscopic observation again Expression.From Figure 2 it can be seen that after successful expression recombinant protein, mycelia is prepared into protoplast, red fluorescence still has substantially Expression, available for selected by flow cytometry apoptosis.

(3) the flow cytometer screening of transformant

1st, sample preparation：All transformants regenerated mycelia under inductive condition is collected, protoplast is prepared into again and hangs Liquid, the filtering of 400 mesh sieve.The integrality of microscopy observation protoplast preparation is simultaneously counted with blood counting chamber.By plasm It is 10 that body suspension, which is adjusted to concentration,⁷The suspension of/mL, 500 μ L.Fluorescence microscope red fluorescent protein expression.

2nd, flow cell sorter is debugged：It is sorted with FACS Aria flow cytometers (U.S. BD FACS Aria) primary Cell with RFP in plastid.100 μm of nozzles, the excitation of 557nm green lights, sheath hydraulic coupling are 20psi.Select 100 μm of nozzles, ultrasound Cleaning 1 minute.After start, main liquid stream breakpoint window is opened, clicks on main liquid stream, liquid stream Oscillation Amplitude (Ampl) is adjusted, makes drop Spacing value (Gap values) is stablized, and tuning parameter is set as far as possible to most preferably, keeps the stability of main liquid stream.Main liquid stream After stabilization, opening side flow stream window voltage clicks on Test Sort, and adjusting parameter makes liquid stream beam splitting clear.The sorting of four tunnels is installed Device, opening side flow stream window voltage click on Test Sort, pack up waste liquid drawer, adjust the voltage scroll bar of side liquid stream window, The sorting liquid stream of deflection is made to fall into corresponding collecting pipe.The Dmpl values actually recalled in main liquid stream window are added to default window In.

3rd, four-way cell sorting：Reject gate is established as negative control using the protoplast of starting strain TU6.Reject gate Determine based on following principle：Complete protoplast have higher forward angle (forward scatter, FSC) and Lateral angle (side scatter, SSC) ratio, compared with negative control, RFP positive cells have by force in green channel (585nm) Strong transmitting light.Cell is directly sorted into protoplast regeneration culture medium (MM+ sorbierites).

4th, the postsearch screening of transformant protoplast：By one group of most strong protoplast of the fluorescence intensity being sorted into above into Row regeneration, prepares protoplast suspension again, carries out 96 hole microwell plate individual cells sortings, regeneration induction training is packed into microwell plate Support base.

(4) correspondence of red fluorescent protein and heterologous lipase expression quantity measures

After 96 hole microwell plate individual cells sortings, choose 46 plants of variant bacterium of fluorescence intensity and produced on PDA solid plates Spore, the formula of PDA solid mediums is 200g containing potato, glucose 20g, agar 20g in every liter of culture medium.Spore is prepared into Spore suspension is inoculated with seed bottle, and the formula of seed bottle culture medium is glucose containing 20g in every liter of culture medium, 0.05g (NH₄)₂SO₄, 0.15g KH₂PO₄, 0.006g MgSO₄, 0.006g CaCl₂, 0.00005g FeSO₄·7H₂O, 0.000016g MnSO₄· H₂O, 0.000014g, ZnSO₄·7H₂O, 0.00002g CoCl₂.Condition of culture is 30 DEG C, after 200rpm shake cultures 48h, four Layer filtered through gauze thalline, weighs 1.8g thalline and is seeded in fermentation medium, fermentative medium formula is seed culture medium respectively Carbon source be changed to lactose, 30 DEG C, after 200rpm shake cultures 96h, measure the lipase activity of fermented liquid supernatant, and right Mycelium fluorescence intensity is measured.

1st, the fluoremetry of red fluorescent protein

The mycelium for the weights such as weighing is placed in 24 hole microwell plates, and the glimmering of each transformant is read with Multifunction fluorescent plate reader Light value, setting excitation light wave a length of 557nm, wavelength of transmitted light 585nm are scanned by the way of 13 × 13 multi-point scannings After take its average value, the fluorescent value for the bacterial strain TU6 for not expressing fluorescin is then set to 1, transformant takes relative fluorescence.

2nd, the determination of activity of lipase

With reference to national standard GB/T 23535, reaction substrate is olive oil emulsion for the determination of activity of lipase.Lipase activity Unit (U) is defined as：Under test conditions, every milliliter of enzyme solution catalysis substrate per minute releases the free fatty of 1 μm of ol, fixed Justice is a lipase activity unit of force (U).

The correspondence of red fluorescent protein and heterologous lipase expression quantity is shown in Fig. 3, as seen from the figure, transformant it is red The activity trend of the expression of color fluorescin and external lipase coincide substantially, the results showed that, by the use of 2A sequences as company Peptide amalgamation and expression red fluorescent protein and lipase are connect, the two albumen equivalent can be made to express and be individually present, there is signal The lipase of peptide can effectively be secreted into it is extracellular, and the red fluorescent protein for lacking signal peptide stay in intracellular as reporter gene into The bacterial strain of the high expression of row flow cytometer high flux screening lipase.

(5) gene copy number of recombination is identified in recombinant protein superior strain

1st, recombinant protein superior strain and the DNA of control strain N10 are extracted.

(1) recombinant protein superior strain and trichoderma reesei (Trichoderma reesei) control strain N10 are taken, by each bacterium Strain 10⁸Spore suspension be inoculated in the MM culture mediums containing 2% glucose cultivate 36h after, 200 mesh sieve be collected by filtration Richter scale wood Mould silk, press dry on filter paper, and liquid nitrogen grinding is to powdered.

(2) every gram of mycelia adds in 5mL DNA extraction buffers, and vibration mixing is taken out after 37 DEG C of water-bath 1h every 10min Vibrate mixing once.

(3) isometric phenol is added in：Chloroform：Isoamyl alcohol (25：24：1, volume ratio) solution, turn upside down mixing, 12000rpm centrifuges 10min.

(4) supernatant is transferred in a new centrifuge tube, adds in the isopropanol of the ice precooling of 0.5-0.6 times of volume and overturned Mixing places 30-60min in -20 DEG C, and then at 4 DEG C, 12000rpm centrifuges 10min.

(5) precipitation is washed twice with 75% ethyl alcohol, is dissolved in the 100mM Tris-HCl that 50-100 μ L contain 20 μ g/mL RNase (pH8.0) in, -20 DEG C save backup.

2nd, the copy number of real-time fluorescence quantitative PCR identification red fluorescent protein and lipase fusion

Using single copy gene actin genes as standard internal reference, respectively with primer Actin-F/Actin-R, LipRT-F/ LipRT-R (table 1) expands the genomic DNA of N10 and each high yield transformant.All PCR are reacted in ABI It is carried out on Prism 7000real-time detection system (ABI) instrument, reagent is Power SYBR Green PCR Master Mix(ABI).System is 25 μ L, the step of using for：50℃2min；95 DEG C of pre-degenerations 10min, 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 40 Xun Huans.Since the copy number of lipase gene in bacterial strain N10 is copied to be single (referring to article： Lina Qin,Fu-rong Cai,Xin-rui Dong,Zhen-bang Huang,Yong Tao,Jian-zhong Huang, Zhi-yang Dong.Improved production of heterologous lipase in Trichoderma reesei by RNAi mediated gene silencing of an endogenic highly expressed gene.Bioresour Technol,2012,109:116-122.), therefore, in each transformant the copy number of recombination with 2^-ΔΔCtMethod relative quantification.

(6) recombination list copy recombinant protein superior strain in insertion point identification

In all transformants, plasmid pSKpyr4 and pSKLR are integrated into trichoderma reesei genome in a manner of radom insertion In, this plasmid also has in addition to containing red fluorescent protein and the complete expression cassette of fatty enzyme fusion proteins for big The duplication subsequence of enterobacteria and ampicillin resistance.Therefore we using plasmid rescue method to the insertion point of gene into Row identification.Superior strain ND1 in the present invention mainly for a plant weight histone gene singly to copy carries out insertion point analysis, Specific operation process is as follows：

1st, the genomic DNA of 5-10 μ g is digested with restriction enzyme SalI, after digestion is complete, 65 DEG C of water-bath 10min, Inactivate restriction enzyme.

2nd, the 3M sodium acetates of 1/10 volume and the absolute ethyl alcohol of 2.5 times of volumes are added in digestion system, -20 DEG C are precipitated to Few more than 1h.

3rd, 13000rpm, 4 DEG C of centrifugation 15min, precipitation are washed twice with 70% ethyl alcohol, and naturally dry or vacuum are drained, and are used The sterile ddH of 50-100 μ L₂O dissolves.

4th, in following linked system, 16 DEG C of connections are overnight.

5th, 10min are handled to inactivate T4DNA ligases for 65 DEG C.

6th, the 3M sodium acetates of 1/10 volume and the absolute ethyl alcohol of 2.5 times of volumes, -20 DEG C of precipitation at least more than 1h are added in.

7th, 13000rpm, 4 DEG C of centrifugation 15min, precipitation are washed twice with 70% ethyl alcohol, and naturally dry or vacuum are drained, with 5 The sterile ddH of μ L₂O dissolving DNAs.

8th, by the 5 sterile ddH of μ L₂The DNA of O dissolvings is totally converted Escherichia coli XL10-gold competent cells.

9th, the clone on picking Escherichia coli conversion tablet carries out bacterium colony PCR mirror using primer Lip-F, Lip-R (table 1) It is fixed, contain lipase gene in clone to ensure.

10th, extract the plasmid in above step 9 in positive colony and sequencing company is sent to be sequenced, so as to obtain restructuring base Because of the flanking sequence of the integration site in genome.

It is final to obtain bacterial strain ND1 by the screening and identification of above steps.

First, the expression that recombinant lipase gene is carried out to bacterial strain ND1 detects, and the results are shown in Figure 4.In Fig. 4, a For SDS-PAGE detect cellulose under the conditions of fermentation 120 it is small when after fermented liquid supernatant；B is small for fermentation 120 under the conditions of cellulose When after lipase active.Li's Trichoderma strains TR1124 is one plant of fusion by red fluorescent protein gene and lipase gene Gene integration enters the lipase superior strain in cbh1 sites.Fig. 4's the result shows that, obtained by flow cytometer high flux screening Bacterial strain ND1 be also one plant of lipase superior strain, and in the bacterial strain lipase gene be not integrated into cbh1 sites (in detail It sees below).

Second, by the result of table 2 as it can be seen that copy number of the recombinant lipase gene in ND1 bacterial strains copies to be single.

2 Real Time PCR of table identify copy number of the recombinant lipase gene in ND1 bacterial strains

Recombination insertion point is analyzed in 3rd, bacterial strain ND1

Recombination insertion point (insertion point is denoted as ND1 sites) is located at Protein in bacterial strain ND1 after testing Noncoding region (SEQ ID No.1) between the gene that the gene and Protein ID that ID is 68606 are 68608.

Sequencing result is shown：Recombination lipA-red is specifically instead of SEQ in trichoderma reesei genome in bacterial strain ND1 429-551 of ID No.1.

2nd, the analysis that insertion point ND1 influences expression of recombinant proteins

Positions of the cellobiose hydrolase gene cbh1 in genome, which is typically considered in trichoderma reesei genome, to be had Beneficial to the site of gene expression.For the ND1 sites that comparison step one is identified and cbh1 sites, which is more advantageous to base The expression of cause.Using the high Li's Trichoderma strains Tu6 Δs ku70 of homologous recombination rate as starting strain, lipase gene lipA is integrated Enter cbh1 sites and replace cbh1 genes, while cbh1 gene integrations are entered into the insertion point ND1 that step 1 identified, detection should The expression of cbh1 genes and compared with control strain Tu6 and ND1 in bacterial strain.Concrete operation step is as follows：

1st, component segment as shown in Figure 5 is built

(1) using plasmid pSKLR as template, Pcbh1-F, Tcbh1-R (table 1) carry out PCR amplification for primer, expand Segment is segment shown in a in Fig. 5.

The nucleotide sequence of segment shown in a is as shown in SEQ ID No.2 in Fig. 5, wherein 1-2078 start for Pcbh1 Subsequence, 2138-2947 are Lipase gene orders, and 2948-2998 are 2A sequences, and 2999-3676 are DsRed sequences, 3685-5927 are Tcbh1 terminator sequences.

(2) using trichoderma reesei (Trichoderma reesei) QM9414 genomic DNAs template, primers F lup-F, Flup-R (table 1) carries out PCR amplification for primer, and the segment expanded is insertion point upstream homology arm sequence；With Richter scale wood Mould (Trichoderma reesei) QM9414 genomic DNAs are template, and Pcbh1-2F, cbh1-2R (table 1) carry out for primer PCR amplification, the segment expanded are the promoter of cbh1 genes and ORF sequences；With trichoderma reesei (Trichoderma Reesei) QM9414 genomic DNAs are template, and primer Tcbh2-F and Tcbh2-R (table 1) carry out PCR amplification, gained for primer Segment is cbh2 gene termination sequences；Using trichoderma reesei (Trichoderma reesei) QM9414 genomic DNAs template, Primers F ldown-F, Fldown-R (table 1) carry out PCR amplification for primer, and the segment expanded is homologous for insertion point downstream Arm sequence；With plasmid pBluescriptSK (+) for template, primer vec-F, vec-R (table 1) carry out PCR amplification, and amplification obtains Segment be vector backbone segment.These segments that PCR amplification is obtained carry out glue recycling, using Gibson Cloning Kits into Row connection, and convert Escherichia coli XL10-gold competent cells, carrier construction pND1-cbh1.Using plasmid pND1-cbh1 as Template, primers F lup-F, Fldown-R (table 1) are expanded for primer, and the segment expanded is the group shown in b in Fig. 5 Part segment.

The nucleotide sequence of segment shown in b is as shown in SEQ ID No.3 in Fig. 5, wherein 1-1938 homologous for upstream Arm sequence, 1939-4016 be Pcbh1 promoter sequences, 4017-5692 be cbh1 gene orders, 5693-7784 Position is Tcbh1 terminator sequences, and 7785-9710 are downstream homology arm sequence.

2nd, component segment (two pieces i.e. shown in SEQ ID No.2 and SEQ ID No.3 obtained more than PCR amplification Section) carry out PCR after purification, with plasmid pSK-pyr4 cotransformation Li's Trichoderma strains Tu6 Δ ku70, method for transformation such as step 1 (2).

3rd, PCR identifies positive transformant：After obtaining transformant, DNA is extracted, primer pair Ocbh1-F/Lip-R (table 1) is carried out PCR identifies whether lipase gene is integrated into cbh1 sites with this, and primer pair OND1-F/Vcbh1-R (table 1) carries out PCR identifications Whether cbh1 genes are integrated into ND1 sites.Identified positive transformant is named as ND1cbh1.

Cbh1 expression cassettes are instead of the 429-551 of SEQ ID No.1 in trichoderma reesei genome in bacterial strain ND1cbh1 Position.

4th, in above positive transformants cbh1 gene expression amounts analysis：By positive transformant ND1cbh1 and control bacterium Strain TU6 and ND1 is inoculated on PDA plate and produces spore, washes by spore with sterile water and is prepared into spore suspension, 200 mesh sieve mistakes Filter, the concentration of spore suspension are adjusted to 10⁸/ mL, in the 250mL triangles equipped with 50mL MM culture mediums (2% glucose is carbon source) 1mL spore suspensions are inoculated in bottle, 28 DEG C, after 200rpm cultivates 48h, four layers of filtered through gauze thalline weigh the switching of 1.8g wet thallus Into the culture medium of the 50mL MM+1% microcrystalline celluloses of 250mL triangular flasks.Wherein need to add in the culture medium of inoculating strain TU6 Add the uridine of 5mM, after cultivating 120h in cellulose inducing culture, in their fermented liquid supernatant of SDS-PAGE electrophoresis detections CBH1 secretes situation, and surveys the enzyme activity of CBH1.And thallus DNA and RNA are extracted, real-time fluorescence quantitative PCR analysis cbh1 genes Copy number and its mRNA level in-site.

(1) identification of cbh1 genes copy number in bacterial strain ND1cbh1.Collect the mycelia of bacterial strain TU6 and ND1cbh1, liquid It is ground in nitrogen and extracts total DNA, using single copy gene actin genes as standard internal reference, respectively with primer Actin-F/ Actin-R, RTcbh1-F/RTcbh1-R (table 1) expand the genomic DNA of bacterial strain TU6 and ND1cbh1.All PCR Reaction carries out on ABI Prism 7000 real-time detection system (ABI) instrument, reagent Power SYBR Green PCR Master Mix(ABI).System is 25 μ L, the step of using for：50℃2min；95 DEG C of pre-degenerations 10min, 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 40 Xun Huans.Since the copy number of cbh1 genes in bacterial strain TU6 copies to be single (referring to article Genome sequencing and analysis of the biomass-degrading fungus Table 3 in Trichoderma reesei (syn.Hypocrea jecorina)), therefore, gene copies in ND1cbh1 bacterial strains Shellfish number is with 2^-ΔΔCtMethod carries out relative quantification.

(2) Real Time PCR analyze the mRNA level in-site of cbh1 genes.Mycelia is collected, ground in liquid nitrogen and is extracted is total RNA carries out realtime fluorescent quantitative PCR experiment using two-step method.

The first step：Reverse transcription

Following reagent is added in 200 μ L PCR pipes：

65 DEG C of incubation 10min；It takes out immediately and is put in 5min on ice；

Add in following reagent：

Reaction mixture is incubated 1h in 50 DEG C；85 DEG C of 10min are warming up to again to inactivate reverse transcriptase.

Second step：Real Time PCR

All PCR are reacted on ABI Prism 7000real-time detection system (ABI) instrument It carries out, reagent is Power SYBR Green PCR Master Mix (ABI).Reaction is used with reference to ABI Prism 7000 and said Bright, system is 25 μ L, the step of using for：50℃2min；95 DEG C of pre-degenerations 10min, 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 40 Xun Huans.The transcriptional level of gene is using actin genes as internal reference.

(3) enzyme activity determination of two pool hydrolase (CBH) of fiber

Make substrate with 4-methylumbelliferyl-D-cellobioside (MUC, Sigma), weigh 15mg MUC, 500 μ L DMSO (Sigma) are dissolved in, then are transferred into 30mL citrate buffer solutions (pH4.8 50mM).12 μ L are suitably dilute Enzyme solution and 200 μ L MUC, 25 μ l glucose (1M) and the mixing of 25 μ L citrate buffer solutions released, this is tested to be not added with cellobiose Group.In mixed solution, glucose can inhibit BG (β-Glucosidase, beta-glucosidase) degradation MUC, it is therefore possible for the detected that The active actual activity for CBH1 and EG (endoglucosidase endoglucanases).It sets simultaneously plus cellobiose is tested Group：12 μ L enzyme solutions and 200 μ L MUC, 25 μ L glucoses, 25 μ L (50mM) cellobioses and 25 μ L citrate buffer solutions, in 50 DEG C React 10min.Add in cellobiose can inhibit the activity of CBH, therefore measure be EG activity.Add in 250 μ L Na₂CO₃ (1M).100 μ L is taken to dilute 11 times, absorbance is measured in 370nm.Cellobiose experimental group OD values will be not added with to subtract plus cellobiose Experimental group is the more special activity of CBH.The CBH enzyme activity of one unit is defined as required for catalysis 1nmol MUC hydrolysis per second Enzyme amount.

5th, result

The qualification result of cbh1 genes copy number in bacterial strain ND1cbh1 is as shown in table 3, it is seen that：Cbh1 genes are in bacterial strain Copy number in ND1cbh1 is approached with bacterial strain TU6, for single copy.

Copy number of 3 Real Time PCR of the table identification restructuring cbh1 genes in ND1cbh1 bacterial strains

Expression qualification result of the cbh1 genes in bacterial strain Tu6, ND1 and ND1cbh1 bacterial strain is as shown in Figure 6.Fig. 6 In, a be SDS-PAGE detect cellulose under the conditions of fermentation 120 it is small when after fermented liquid supernatant；B be cellulose under the conditions of ferment 120 it is small when after CBH activity.As seen from the figure：Expression of the cbh1 genes in ND1 sites is suitable with bacterial strain TU6, illustrates the position Contribution of the point to recombinating gene expression dose is suitable with cbh1 genes.

<110>Fujian Normal University

<120>By the use of trichoderma reesei as the method for host expresses recombinant protein

<130> GNCLN172245

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 1095

<212> DNA

<213>Trichoderma reesei（Trichoderma reesei）

<400> 1

gagagcaatg aactatctct tgggatggct gtgatgacag gctgctccgt atgataggta 60

cagtatgtac gaagaagtac tactattaca attcttcatc aaggctgatt gttgctaggt 120

acacaaaggc cattactcag agcgaagccc aattgcccat cccccataga tcctctcttg 180

agaagaatga aatggatcag cattgacctc aactcccatg atccagtcaa cggctccaaa 240

caagtggcac aatggcccaa tccgtccaag tttttagggt cctcttcaac tatccccgag 300

tccaccacaa ctttcttagc aaataatatc ttcacgataa cacacacaca cagccctaaa 360

ttctctcatg gctaaaatca cacgcccgga taccaccgga tcatttcccg cccggctttt 420

ggccgttgaa tgtttgcttc caactgccga catcgccgtc actatgattg tatcgagagc 480

cgatcaattg gcccttaccc ctcctttttt aaatgtgacg gttggtgaga atccggtcga 540

ctataaattg ggatgaaaat cacgcagaag tcaatgctcg tttctcttcc ttttcacatc 600

tcgttcatcg ttcttccacc aagaaagaat caaaagtcaa ctcaatcgta acctatccaa 660

ataaaagtat tcagtgatat acttcacacc ccccctccaa ccccctctct ccaatgcatc 720

tgacacacag caggccaaat accacactct cctgtcggta caacctcgtc cgtcgccatg 780

gcgtccttcg gtggtagtcc gtccagacgg ggttctgtct cctcatcttc aggtaatgtg 840

tatcgcatga gccggtgccc gggctccccc ccttcctttg tcccacttca ctcctctccc 900

ctcgtctctg cttgtcttta caaacaagga aacacaccca atgagatgga gaaggtgatt 960

gtgtggaaat aaggaagagg gggctcgggc tgagaatata cggccctgaa cttgatctgg 1020

ataataccag cgaaaggatc atgctcctcc cccttttcaa tcccgtccaa atccctaacc 1080

cttgtaggcc catca 1095

<210> 2

<211> 5927

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 2

gacttccgcc cagagctgaa ggtcgcacaa ccgcatgata tagggtcggc aacggcaaaa 60

aagcacgtgg ctcaccgaaa agcaagatgt ttgcgatcta acatccagga acctggatac 120

atccatcatc acgcacgacc actttgatct gctggtaaac tcgtattcgc cctaaaccga 180

agtgcgtggt aaatctacac gtgggcccct ttcggtatac tgcgtgtgtc ttctctaggt 240

gccattcttt tcccttcctc tagtgttgaa ttgtttgtgt tggagtccga gctgtaacta 300

cctctgaatc tctggagaat ggtggactaa cgactaccgt gcacctgcat catgtatata 360

atagtgatcc tgagaagggg ggtttggagc aatgtgggac tttgatggtc atcaaacaaa 420

gaacgaagac gcctcttttg caaagttttg tttcggctac ggtgaagaac tggatacttg 480

ttgtgtcttc tgtgtatttt tgtggcaaca agaggccaga gacaatctat tcaaacacca 540

agcttgctct tttgagctac aagaacctgt ggggtatata tctagagttg tgaagtcggt 600

aatcccgctg tatagtaata cgagtcgcat ctaaatactc cgaagctgct gcgaacccgg 660

agaatcgaga tgtgctggaa agcttctagc gagcggctaa attagcatga aaggctatga 720

gaaattctgg agacggcttg ttgaatcatg gcgttccatt cttcgacaag caaagcgttc 780

cgtcgcagta gcaggcactc attcccgaaa aaactcggag attcctaagt agcgatggaa 840

ccggaataat ataataggca atacattgag ttgcctcgac ggttgcaatg caggggtact 900

gagcttggac ataactgttc cgtaccccac ctcttctcaa cctttggcgt ttccctgatt 960

cagcgtaccc gtacaagtcg taatcactat taacccagac tgaccggacg tgttttgccc 1020

ttcatttgga gaaataatgt cattgcgatg tgtaatttgc ctgcttgacc gactggggct 1080

gttcgaagcc cgaatgtagg attgttatcc gaactctgct cgtagaggca tgttgtgaat 1140

ctgtgtcggg caggacacgc ctcgaaggtt cacggcaagg gaaaccaccg atagcagtgt 1200

ctagtagcaa cctgtaaagc cgcaatgcag catcactgga aaatacaaac caatggctaa 1260

aagtacataa gttaatgcct aaagaagtca tataccagcg gctaataatt gtacaatcaa 1320

gtggctaaac gtaccgtaat ttgccaacgg cttgtggggt tgcagaagca acggcaaagc 1380

cccacttccc cacgtttgtt tcttcactca gtccaatctc agctggtgat cccccaattg 1440

ggtcgcttgt ttgttccggt gaagtgaaag aagacagagg taagaatgtc tgactcggag 1500

cgttttgcat acaaccaagg gcagtgatgg aagacagtga aatgttgaca ttcaaggagt 1560

atttagccag ggatgcttga gtgtatcgtg taaggaggtt tgtctgccga tacgacgaat 1620

actgtatagt cacttctgat gaagtggtcc atattgaaat gtaagtcggc actgaacagg 1680

caaaagattg agttgaaact gcctaagatc tcgggccctc gggccttcgg cctttgggtg 1740

tacatgtttg tgctccgggc aaatgcaaag tgtggtagga tcgaacacac tgctgccttt 1800

accaagcagc tgagggtatg tgataggcaa atgttcaggg gccactgcat ggtttcgaat 1860

agaaagagaa gcttagccaa gaacaatagc cgataaagat agcctcatta aacggaatga 1920

gctagtaggc aaagtcagcg aatgtgtata tataaaggtt cgaggtccgt gcctccctca 1980

tgctctcccc atctactcat caactcagat cctccaggag acttgtacac catcttttga 2040

ggcacagaaa cccaatagtc aaccgcggac tgcgcatcat gtatcggaag ttggccgtca 2100

tctcggcctt cttggccaca gctcgtgctg aattccgagt gtctcgactt ccacgttgga 2160

tgagctgcaa ttgttcgcgc aatggtctgc cgcagcttat tgctcgaaca atatcgactc 2220

ggacgactcc aacgtgacat gcacggccga cgcctgtcca tcagtcgagg aagcgagcac 2280

caagatgctg ctggagtttg atttgacaaa taactttgga ggcacagccg gtttcctggc 2340

cgcggacaac accaacaagc ggctcgtggt cgccttccga ggcagtagca ccatcaagaa 2400

ctggattgct gatctcggct tcatcctgca agataacgat gacctctgta ctggctgcaa 2460

ggttcacact ggattctgga aggcatggga agccgctgca gacaatctga cgagcaagat 2520

caagtccgcg atgagcacgt actcaggcta taccctctac ttcaccgggc acagcttggg 2580

cggcgcattg gctacgctgg gagcaacggt cttgcgaaat gacggttata gcgttgaact 2640

gtacacctat ggatgtcctc gagtcggaaa ctatgcgctg gccgagcaca tcaccagcca 2700

gggatcagga gcgaacttcc gcgttacaca cttgaacgac atcgtccccc ggttgccacc 2760

catggacttt ggattcagcc agccaagtcc agaatactgg atcaccagtg gcaccggagc 2820

cagtgtcacg gcgtcggata ttgaactcat cgagggaatc aattcgacgg cggggaatgc 2880

aggcgaagca acggtggacg ttttggctca cttgtggtac tttttcgcga tttccgagtg 2940

cctgctaaac ttcgacctcc tcaagctcgc cggcgacgtc gagtccaacc ccggccccat 3000

ggacaacacc gaggacgtca tcaaggagtt catgcagttc aaggtgcgca tggagggctc 3060

cgtgaacggc cactacttcg agatcgaggg cgagggcgag ggcaagccct acgagggcac 3120

ccagaccgcc aagctgcagg tgaccaaggg cggccccctg cccttcgcct gggacatcct 3180

gtccccccag ttccagtacg gctccaaggc ctacgtgaag caccccgccg acatccccga 3240

ctacatgaag ctgtccttcc ccgagggctt cacctgggag cgctccatga acttcgagga 3300

cggcggcgtg gtggaggtgc agcaggactc ctccctgcag gacggcacct tcatctacaa 3360

ggtgaagttc aagggcgtga acttccccgc cgacggcccc gtaatgcaga agaagactgc 3420

cggctgggag ccctccaccg agaagctgta cccccaggac ggcgtgctga agggcgagat 3480

ctcccacgcc ctgaagctga aggacggcgg ccactacacc tgcgacttca agaccgtgta 3540

caaggccaag aagcccgtgc agctgcccgg caaccactac gtggactcca agctggacat 3600

caccaaccac aacgaggact acaccgtggt ggagcagtac gagcacgccg aggcccgcca 3660

ctccggctcc cagtagacta gtccagctcc gtggcgaaag cctgacgcac cggtagattc 3720

ttggtgagcc cgtatcatga cggcggcggg agctacatgg ccccgggtga tttatttttt 3780

ttgtatctac ttctgaccct tttcaaatat acggtcaact catctttcac tggagatgcg 3840

gcctgcttgg tattgcgatg ttgtcagctt ggcaaattgt ggctttcgaa aacacaaaac 3900

gattccttag tagccatgca ttttaagata acggaataga agaaagagga aattaaaaaa 3960

aaaaaaaaaa caaacatccc gttcataacc cgtagaatcg ccgctcttcg tgtatcccag 4020

taccacggca aaggtatttc atgatcgttc aatgttgata ttgttcccgc cagtatggct 4080

ccacccccat ctccgcgaat ctcctcttct cgaacgcggt agtggcgcgc caattggtaa 4140

tgacccatag ggagacaaac agcataatag caacagtgga aattagtggc gcaataattg 4200

agaacacagt gagaccatag ctggcggcct ggaaagcact gttggagacc aacttgtccg 4260

ttgcgaggcc aacttgcatt gctgtcaaga cgatgacaac gtagccgagg accgtcacaa 4320

gggacgcaaa gttgtcgcgg atgaggtctc cgtagatggc atagccggca atccgagagt 4380

agcctctcaa caggtggcct tttcgaaacc ggtaaacctt gttcagacgt cctagccgca 4440

gctcaccgta ccagtatcga ggattgacgg cagaatagca gtggctctcc aggatttgac 4500

tggacaaaat cttccagtat tcccaggtca cagtgtctgg cagaagtccc ttctcgcgtg 4560

cgagtcgaaa gtcgctatag tgcgcaatga gagcacagta ggagaatagg aacccgcgag 4620

cacattgttc aatctccaca tgaattggat gactgctggg cagaatgtgc tgcctccaaa 4680

atcctgcgtc caacagatac tctggcaggg gcttcagatg aatgcctctg ggcccccaga 4740

taagatgcag ctctggattc tcggttacga tgatatcgcg agagagcacg agttggtgat 4800

ggaggggacg aggaggcata ggtcggccgc aggcccataa ccagtcttgc acagcattga 4860

tcttcctcac gaggagctcc tgatgcagaa actcctccat gttgctgatt gggttgagaa 4920

tttcatcgct cctggatcgt atggttgctg gcaagaccct gcttaaccgt gccgtgtcat 4980

ggtcatctct ggtggcttcg tcgctggcct gtctttgcaa ttcgacagca aatggtggag 5040

atctctctat cgtgacagtc atggtagcga tagctaggtg tcgttgcacg cacataggcc 5100

gaaatgcgaa gtggaaagaa tttcccggcg cggaatgaag tctcgtcatt ttgtactcgt 5160

actcgacacc tccaccgaag tgttaagaat ggatccacga tgccaaaaag cttgttcatt 5220

tcggctagcc cgtgatcctg gcgcttctag ggctgaaact gtgttgttaa tgtattattg 5280

gctgtgtaac tgacttgaat ggggaatgag gagcgcgatg gattcgcttg catgtcccct 5340

ggccaagacg agccgctttg gcggtttgtg attcgaaggt gtgtcagcgg aggcgccagg 5400

gcaacacgca ctgagccagc caacatgcat tgctgccgac atgaatagac acgcgccgag 5460

cagacatagg agacgtgttg actgtaaaaa ttctactgaa tattagcacg catggtctca 5520

ataagagcaa taggaatgct tgccaatcat aagtacgtat gtgctttttc ctgcaaatgg 5580

tacgtacgga cagttcatgt tgtctgtcat cccccactca ggctctcatg atcattttat 5640

gggactgggg ttttgctgac tgaatggatt cagccgcacg aaacaaattg ggggccatgc 5700

agaagggaag cccccccagc cccctgttca taatttgtta agagtcggag agctgcctag 5760

tatgaagcag caattgataa cgttgacttt gcgcatgagc tctgaagccg ggcatatgta 5820

tcacgtttct gcctagagcc gcacgggacc caagaagctc ttgtcataag gtatttatga 5880

gtgttcagct gccaacgctg gttctacttt ggctcaaccg catccca 5927

<210> 3

<211> 9710

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 3

gacacggtag acaagcgaat cgaaacttta ttgttttgtt cttgcgttct ccctaagtga 60

tgacgaagga ggtgtagcac tgaaagtggc agagccacgt acaggagaga acagctcgta 120

cctgatgaac tgctgtctca gattgaatat catacagctc gtggcattga agctccatag 180

atatgctagc gataagcagg tacagtgagg acacggcttg atcgtggctt gagaatcacc 240

aaggacagtg gaacggagtg gctgctttgg tgattccgac caactgttca cgagaccagc 300

tcggatgccc gggccaacga atcaaccctc cgtaagatgg agccacgatc gtataaccct 360

cgctctcaaa cagatgttaa tatttaggcg gaattgtaaa ctcgggggcc ctggagaggc 420

cgcaactggg cttcatgggc agaacagcgg cgccatgccg gcatcccaga agtggaatgg 480

gcggatcgga gctggtgcgc ggggggaagt aagctgggtg gcaggccctt cggatagctg 540

ggagctgtct caactactag aggagcttag ctttggcagc tcttgataac tgtgacagct 600

attcgccgtt tctcgtagtg ttcctggact gtgtctgaga gctgtcatcc cctcatcacc 660

acttacatgg acgaacagtt gtgtgaatga gcgcaatcat ccacctccgt tctccatctt 720

catcatcagc caacatgtcc agctcaacgc ccacgcggcg cctcatcggc ctctcgacca 780

aaatgtactt ttctctccag caaacaagag acttcacaaa cagcttctta tccaagctgg 840

cagacgtccc gatagagctc ctctcccaca ttgacatctt catcatcccc gacttcgtct 900

ccctgaccac cacaatcgcc cagcttaagt ccagctccgt tgccatttgg accggcgctc 960

aagactgcca ctgggaagac caaggcgcat tcaccggcga agtcagcccc tccgtgttgc 1020

gcagcgcagg cgtcaagctc gtggaagtcg gacatgcaga gcgcaggagc atctttggcg 1080

aggacgacac catggttgcg aataaggctg ccgctgtaag tcgcaatggc atgataccgc 1140

ttgtgtgcat tggagaaaag actcaaggcg acgtgcatat cgcggttgag gagtgtcggg 1200

tgcaggttga tgctgtcatg aatgctgtgc ccgccgaggc agaggtcatt ttggcgtatg 1260

agcctgtatg ggcgattggt gccagtcaac ctgctggaga gaagcacatc ctcgatgttg 1320

ttgctgggat cagagggttg gagtccgttg ggaggagggc ggggacgacg agggttttgt 1380

atggggggag tgccgggccg gggctctacg agaagttgaa gagcggactg gacggattgt 1440

tcctggggcg gttcggacac gatccggagc agtttgtcaa gacgatacag gaagtagctg 1500

aagcgtgagg gagagcaatg aactatctct tgggatggct gtgatgacag gctgctccgt 1560

atgataggta cagtatgtac gaagaagtac tactattaca attcttcatc aaggctgatt 1620

gttgctaggt acacaaaggc cattactcag agcgaagccc aattgcccat cccccataga 1680

tcctctcttg agaagaatga aatggatcag cattgacctc aactcccatg atccagtcaa 1740

cggctccaaa caagtggcac aatggcccaa tccgtccaag tttttagggt cctcttcaac 1800

tatccccgag tccaccacaa ctttcttagc aaataatatc ttcacgataa cacacacaca 1860

cagccctaaa ttctctcatg gctaaaatca cacgcccgga taccaccgga tcatttcccg 1920

cccggctttt ggccgttgga cttccgccca gagctgaagg tcgcacaacc gcatgatata 1980

gggtcggcaa cggcaaaaaa gcacgtggct caccgaaaag caagatgttt gcgatctaac 2040

atccaggaac ctggatacat ccatcatcac gcacgaccac tttgatctgc tggtaaactc 2100

gtattcgccc taaaccgaag tgcgtggtaa atctacacgt gggccccttt cggtatactg 2160

cgtgtgtctt ctctaggtgc cattcttttc ccttcctcta gtgttgaatt gtttgtgttg 2220

gagtccgagc tgtaactacc tctgaatctc tggagaatgg tggactaacg actaccgtgc 2280

acctgcatca tgtatataat agtgatcctg agaagggggg tttggagcaa tgtgggactt 2340

tgatggtcat caaacaaaga acgaagacgc ctcttttgca aagttttgtt tcggctacgg 2400

tgaagaactg gatacttgtt gtgtcttctg tgtatttttg tggcaacaag aggccagaga 2460

caatctattc aaacaccaag cttgctcttt tgagctacaa gaacctgtgg ggtatatatc 2520

tagagttgtg aagtcggtaa tcccgctgta tagtaatacg agtcgcatct aaatactccg 2580

aagctgctgc gaacccggag aatcgagatg tgctggaaag cttctagcga gcggctaaat 2640

tagcatgaaa ggctatgaga aattctggag acggcttgtt gaatcatggc gttccattct 2700

tcgacaagca aagcgttccg tcgcagtagc aggcactcat tcccgaaaaa actcggagat 2760

tcctaagtag cgatggaacc ggaataatat aataggcaat acattgagtt gcctcgacgg 2820

ttgcaatgca ggggtactga gcttggacat aactgttccg taccccacct cttctcaacc 2880

tttggcgttt ccctgattca gcgtacccgt acaagtcgta atcactatta acccagactg 2940

accggacgtg ttttgccctt catttggaga aataatgtca ttgcgatgtg taatttgcct 3000

gcttgaccga ctggggctgt tcgaagcccg aatgtaggat tgttatccga actctgctcg 3060

tagaggcatg ttgtgaatct gtgtcgggca ggacacgcct cgaaggttca cggcaaggga 3120

aaccaccgat agcagtgtct agtagcaacc tgtaaagccg caatgcagca tcactggaaa 3180

atacaaacca atggctaaaa gtacataagt taatgcctaa agaagtcata taccagcggc 3240

taataattgt acaatcaagt ggctaaacgt accgtaattt gccaacggct tgtggggttg 3300

cagaagcaac ggcaaagccc cacttcccca cgtttgtttc ttcactcagt ccaatctcag 3360

ctggtgatcc cccaattggg tcgcttgttt gttccggtga agtgaaagaa gacagaggta 3420

agaatgtctg actcggagcg ttttgcatac aaccaagggc agtgatggaa gacagtgaaa 3480

tgttgacatt caaggagtat ttagccaggg atgcttgagt gtatcgtgta aggaggtttg 3540

tctgccgata cgacgaatac tgtatagtca cttctgatga agtggtccat attgaaatgt 3600

aagtcggcac tgaacaggca aaagattgag ttgaaactgc ctaagatctc gggccctcgg 3660

gccttcggcc tttgggtgta catgtttgtg ctccgggcaa atgcaaagtg tggtaggatc 3720

gaacacactg ctgcctttac caagcagctg agggtatgtg ataggcaaat gttcaggggc 3780

cactgcatgg tttcgaatag aaagagaagc ttagccaaga acaatagccg ataaagatag 3840

cctcattaaa cggaatgagc tagtaggcaa agtcagcgaa tgtgtatata taaaggttcg 3900

aggtccgtgc ctccctcatg ctctccccat ctactcatca actcagatcc tccaggagac 3960

ttgtacacca tcttttgagg cacagaaacc caatagtcaa ccgcggactg cgcatcatgt 4020

atcggaagtt ggccgtcatc tcggccttct tggccacagc tcgtgctcag tcggcctgca 4080

ctctccaatc ggagactcac ccgcctctga catggcagaa atgctcgtct ggtggcacgt 4140

gcactcaaca gacaggctcc gtggtcatcg acgccaactg gcgctggact cacgctacga 4200

acagcagcac gaactgctac gatggcaaca cttggagctc gaccctatgt cctgacaacg 4260

agacctgcgc gaagaactgc tgtctggacg gtgccgccta cgcgtccacg tacggagtta 4320

ccacgagcgg taacagcctc tccattggct ttgtcaccca gtctgcgcag aagaacgttg 4380

gcgctcgcct ttaccttatg gcgagcgaca cgacctacca ggaattcacc ctgcttggca 4440

acgagttctc tttcgatgtt gatgtttcgc agctgccgta agtgacttac catgaacccc 4500

tgacgctatc ttcttgttgg ctcccagctg actggccaat tcaaggtgcg gcttgaacgg 4560

agctctctac ttcgtgtcca tggacgcgga tggtggcgtg agcaagtatc ccaccaacac 4620

cgctggcgcc aagtacggca cggggtactg tgacagccag tgtccccgcg atctgaagtt 4680

catcaatggc caggccaacg ttgagggctg ggagccgtca tccaacaacg cgaacacggg 4740

cattggagga cacggaagct gctgctctga gatggatatc tgggaggcca actccatctc 4800

cgaggctctt accccccacc cttgcacgac tgtcggccag gagatctgcg agggtgatgg 4860

gtgcggcgga acttactccg ataacagata tggcggcact tgcgatcccg atggctgcga 4920

ctggaaccca taccgcctgg gcaacaccag cttctacggc cctggctcaa gctttaccct 4980

cgataccacc aagaaattga ccgttgtcac ccagttcgag acgtcgggtg ccatcaaccg 5040

atactatgtc cagaatggcg tcactttcca gcagcccaac gccgagcttg gtagttactc 5100

tggcaacgag ctcaacgatg attactgcac agctgaggag gcagaattcg gcggatcctc 5160

tttctcagac aagggcggcc tgactcagtt caagaaggct acctctggcg gcatggttct 5220

ggtcatgagt ctgtgggatg atgtgagttt gatggacaaa catgcgcgtt gacaaagagt 5280

caagcagctg actgagatgt tacagtacta cgccaacatg ctgtggctgg actccaccta 5340

cccgacaaac gagacctcct ccacacccgg tgccgtgcgc ggaagctgct ccaccagctc 5400

cggtgtccct gctcaggtcg aatctcagtc tcccaacgcc aaggtcacct tctccaacat 5460

caagttcgga cccattggca gcaccggcaa ccctagcggc ggcaaccctc ccggcggaaa 5520

cccgcctggc accaccacca cccgccgccc agccactacc actggaagct ctcccggacc 5580

tacccagtct cactacggcc agtgcggcgg tattggctac agcggcccca cggtctgcgc 5640

cagcggcaca acttgccagg tcctgaaccc ttactactct cagtgcctgt aaagcgttga 5700

cttgcctctg gtctgtccag acgggggcac gatagaatgc gggcacgcag ggagctcgta 5760

gacattgggc ttaatatata agacatgcta tgttgtatct acattagcaa atgacaaaca 5820

aatgaaaaag aacttatcaa gcactgtacc aaggaagctc attatgcgtc tggcgggttc 5880

aaatgatccg tgataggtta tgccagctga ttgtttgccc gcagggttga caccaccagg 5940

ggataatggc cactttcatc tgaatcaagg acaggagccg ttgatgattt ccagatatcc 6000

tatcttcatc gctagtacta ataacaagta agcaaacagc cagctacact cgtacacacc 6060

gctcatgaaa acataagaca aagctcaagc catgtcaagc accaccccat cgtacacgta 6120

catcttctac caatctgtcc agatgccggc tacgtcagcc tccaacccat tgcgatacaa 6180

tgacggatgt cgtatagacc atggcaacgg ctcccaccac cattgtcact gcgtccaaag 6240

tcttctccca cctgctctca gcaacgccct tgaaatgcag gaatgccgga tacatgtaga 6300

ccaatggaat gcaggcaaaa gatcccgtca gcgccacaaa cttatccaaa tcacttgctc 6360

ccaccaccgc cacgccgatg catgccacaa tggtcaaggc ccggaggccg ttcttcttcc 6420

acttgattgc cgcgctcttc ttaccagtgg cgcgctctcc gaaaatcgac gtttccaata 6480

tgcggatcgc ggggaacagc tgcacaggct cgccagccag tacggccagc gagtagagaa 6540

actggactgc attgacgaga ggcgagtcct ggggaaaatt ggagatgatt tgaatcttgg 6600

tgtgttctcc aaacgtcgca tagcagagtg cgccgactga tgtgaagatg gccgtgatga 6660

ggagcatgac aaagtagagg aggcccttga actgatgagg cttcttcata ctcgactgaa 6720

ttggcaggat caggccaatg ccctcaaacg tgaagatcgc cgagcccaga gtcaggggaa 6780

aatcggaggg gttgaagagc ttgacgcttg gctccatacc gtgcctgacc aggcttcgaa 6840

tgtcaaaata ccagatgtat acgagtccaa tgaggatgaa ggcgtccgcc accagagcaa 6900

ccggacccag cttggagatg ttgcggatcc aggctaacgg aatgagaggg aggaactgaa 6960

gcgcaattag gcccgacacc ccgaaatgac caaggccgtt ggtgacagcg tcgaggaagg 7020

agaagagatt ttcagcagtg aagataatgc ctgagcagac gaagcccagc tgagaaatgg 7080

caatggaggc gagaatcaag cttcgaaacc ggggtccaac gacggcctcg ccaatgtctc 7140

catagccacc gccgtacttg tggcggcagt ccagaagtaa gcggaaacag aagcaattga 7200

ccagtgacac ggagatgagc gccagtgacg aaaagaggat acctccgttg cggaatgcct 7260

tgggcaggaa caggatgcct gtgccgatga aagccttgag cagcgtgaag aaggtcttga 7320

ctgtgctcgc gtcaccctgg cgggcaagac ggcgggagct tctccggcga ccagcactcg 7380

gtcgcgcaag cagcggacgc cgctcgtcgg cggcttcttc cgcctcatca gtgacggcag 7440

actcgtcatc gtcgtccgta tcgtcgagcg cttcgccggc aaagctgccg tagaggtcga 7500

ggaagtcaac gaacgtgttg gccacaatca tgccgctctg ctgcctctgt cgagctcggc 7560

gccgcaggtg ctgccgcctg aaaccgccgg gctcgagctg ctcagagtgc gtcaactcgc 7620

ctgccggact caggtcgggc gagcggcggt gagagaaggt attggatcgg cggagagagc 7680

ctgctttggc cttgatcttg aacatgtcgc gatggatgtc gccgccctga agcttgagcg 7740

aggactcgag cgtctcgagg gtcggcgttg cgtcgctgct cccggatgaa aatcacgcag 7800

aagtcaatgc tcgtttctct tccttttcac atctcgttca tcgttcttcc accaagaaag 7860

aatcaaaagt caactcaatc gtaacctatc caaataaaag tattcagtga tatacttcac 7920

accccccctc caaccccctc tctccaatgc atctgacaca cagcaggcca aataccacac 7980

tctcctgtcg gtacaacctc gtccgtcgcc atggcgtcct tcggtggtag tccgtccaga 8040

cggggttctg tctcctcatc ttcaggtaat gtgtatcgca tgagccggtg cccgggctcc 8100

cccccttcct ttgtcccact tcactcctct cccctcgtct ctgcttgtct ttacaaacaa 8160

ggaaacacac ccaatgagat ggagaaggtg attgtgtgga aataaggaag agggggctcg 8220

ggctgagaat atacggccct gaacttgatc tggataatac cagcgaaagg atcatgctcc 8280

tccccctttt caatcccgtc caaatcccta acccttgtag gcccatcatg agccctcccg 8340

ccgccgtctc gcccccccag cgaaccgctg aactcgtcac cccgtccaag atggccgttg 8400

cccagccgca gcagcacctc gaggcccagg ccaagtccgt ctcggacatg ttcggccagt 8460

gggactcgtt caccttctcg cccatccgcg agtcccaggt gtcgcgcgcc atgacccgcc 8520

gctacttcga ggacctcgac cgctacgccg agtccgacat tgtcatcatc ggcgccggct 8580

cctgcggcct cagcaccgcc tacgtcctcg gcacccagcg cccggacctc aagattgcca 8640

tcatcgaggc ctccgtctcc cccggcggcg gtgcctggct gggcggccag ctcttctccg 8700

ccatggtcat gcgcaagcct gccgatgcct tcctccgcga gattggcgtc ccgtacgagg 8760

acgagggcaa ctacgtcgtc gtcaagcacg ccgccctctt cacctccacc atcatggcca 8820

aggtgctgca gctacccaac gtcaagctct tcaacgccac ctgcgtcgaa gacctcatca 8880

cccgcccctc tgccgagggc gtgcgcatcg ccggtgtcgt caccaactgg accctcgtct 8940

ccatgcacca cgacgaccag tcctgcatgg accccaacac catcaacgcg cccctcgtca 9000

tctccaccac cggccacgac ggccccatgg gtgccttctg cgtcaagcgc cttgtcagca 9060

tgggccgcat cgagaagctt ggcggcatgc gcggcctcga catgagcagg gctgaggatg 9120

ccattgtcaa gaacacccgt gaggttgttc ccggcctgat tgtcggagga atggagctgt 9180

ctgagattga cggagccaac cgcatgggta tgttccctct ttgaatctcc ctctcgcaag 9240

aacatgtatt aacctgtgtg tgtgtacagg tcctaccttt ggtgccatgg ccctcagcgg 9300

tgtcaaggct gccgaggagg ctctcaaggt ctttgaggcc cgccgcaagg agaatgctca 9360

gtaaatgagc tctgtgacct tgttccgtca ttcatttgat agtgtacttg catgactggt 9420

cactcatttg atgtaaagca gtaaaagtac aaaagaccaa aaaaaagcat gatcacgctg 9480

cgaaccagat tgaataaact gcagcatatt actcaagata atcgttctca ttccatccgt 9540

gatcttctga tttggttcct gtaccctggt caggtaggta taggaaacgt attccacagt 9600

aaaacaagca cagcacttgc ctctgttgga tcggcacctc agaagagatt gccatgaaga 9660

cgcactagaa gaaacaagtc gacgtcgtgg caagctcatg cttgagcacg 9710

Claims (10)

1. it is a kind of by the use of trichoderma reesei as the method for host expresses recombinant protein, include the following steps：It will be containing to be expressed The expression cassette of the encoding gene of destination protein be cloned into trichoderma reesei genome be located at Protein ID be 68606 gene and Protein ID are the noncoding region between 68608 gene.

2. according to the method described in claim 1, it is characterized in that：It is described " by the coding base containing destination protein to be expressed The expression cassette of cause is cloned into trichoderma reesei genome is positioned at the Protein ID genes for being 68606 and Protein ID Noncoding region between 68608 gene " is following any：

(a) the trichoderma reesei genome middle position is substituted with the expression cassette of the encoding gene containing destination protein to be expressed The arbitrary segment of noncoding region between the gene that the Protein ID genes for being 68606 and Protein ID are 68608；

(b) expression cassette of the encoding gene containing destination protein to be expressed is inserted into the trichoderma reesei genome Any position of noncoding region between the gene that the Protein ID genes for being 68606 and Protein ID are 68608.

3. method according to claim 1 or 2, it is characterised in that：It is located at Protein in the trichoderma reesei genome The nucleotides sequence of noncoding region between the gene that the gene and Protein ID that ID is 68606 are 68608 is classified as SEQ ID No.1。

4. according to the method described in claim 3, it is characterized in that：It is described " by the coding base containing destination protein to be expressed The expression cassette of cause is cloned into trichoderma reesei genome is positioned at the Protein ID genes for being 68606 and Protein ID Noncoding region between 68608 gene " is to substitute the expression cassette of the encoding gene containing destination protein to be expressed 429-551 of SEQ ID No.1 in the trichoderma reesei genome.

5. according to any method in claim 1-4, it is characterised in that：The volume containing destination protein to be expressed The expression cassette of code gene starts the mesh to be expressed of expression from 5 ' ends to 3 ' ends comprising promoter, by the promoter successively Albumen encoding gene and terminator.

6. according to the method described in claim 5, it is characterized in that：The promoter is the cellobiose from trichoderma reesei The promoter of hydrolase I gene；The terminator be from trichoderma reesei cellobiohydrolase I gene terminator or From the terminator of the cellobiohydrolase II genes of trichoderma reesei.

7. according to the method described in claim 6, it is characterized in that：The cellobiohydrolase I from trichoderma reesei The sequence of the promoter of gene is 1939-4016 of the 1-2078 or SEQ ID No.3 of SEQ ID No.2；It is described The 3685-5927 that sequence from the terminator of the cellobiohydrolase I gene of trichoderma reesei is SEQ ID No.2 Position；The sequence of the terminator of the cellobiohydrolase II genes from trichoderma reesei is the of SEQ ID No.3 5693-7784.

8. according to any method in claim 1-7, it is characterised in that：The destination protein to be expressed be from Eucaryote or the albumen of prokaryotes.

9. according to any method in claim 1-8, it is characterised in that：It is described " destination protein to be expressed to be contained Encoding gene expression cassette be cloned into trichoderma reesei genome be located at Protein ID be 68606 gene and Protein ID is the noncoding region between 68608 gene " it is to be realized by importing recombinant expression carrier into T. reesei host； It is inserted into or replaces containing the expression cassette and positioned at the expression cassette upstream and downstream for fixed point on the recombinant expression carrier Homology arm.

10. according to the method described in claim 9, it is characterized in that：The homology arm is 1-1938 of SEQ ID No.3 With two segment DNA segments shown in 7785-9710.