CN108070609B - A method for expressing recombinant proteins using Trichoderma reesei as a host - Google Patents

Abstract

The invention discloses a method for expressing recombinant protein by using Trichoderma reesei as a host. The method for expressing recombinant protein using Trichoderma reesei as a host provided by the present invention comprises the steps of: cloning the expression cassette containing the encoding gene of the target protein to be expressed into the gene whose Protein ID is 68606 in the Trichoderma reesei genome and the non-coding region between the gene with Protein ID 68608. The experiment of the present invention proves that in the cbh1 gene deletion strain, the expression skeleton of the cbh1 gene is integrated into the site ND1, and the expression level of the cbh1 gene is compared with that of the wild strain, and it is found that the recombinant gene is inserted into this site. expression level. The invention further enriches and perfects the technical scheme of using Trichoderma reesei as a host to express recombinant protein efficiently.

Description

A method for expressing recombinant proteins using Trichoderma reesei as a host

technical field

The invention relates to the field of genetic engineering, in particular to a method for expressing recombinant protein using Trichoderma reesei as a host.

Background technique

The filamentous fungus Trichoderma reesei (Trichoderma reesei) is a major industrial cellulase hemicellulase producing strain, and is a food-safe strain certified by the US FDA. Trichoderma reesei has strong protein secretion ability, the amount of exocrine protein of some mutant strains can reach 100g/L, and has a glycosylation system similar to that of higher mammals, so Trichoderma reesei is a very ideal recombinant The protein expression host has been highly valued by internationally renowned enzyme preparation companies (such as Genencor, Novozymes, etc.), and has been successfully used in the expression and production of various drugs, chemical reagents and enzyme preparations. In recent years, with the rapid development of T. reesei genomics and the continuous improvement of its genetic operating system, the use of T. reesei as a host to express recombinant proteins has attracted more and more attention.

It is worth noting that the exogenous DNA fragments in Trichoderma reesei are mostly integrated into the genome by the way of non-homologous end-joining (NHEJ), which is in the form of random insertion. The different insertion sites of the recombinant protein gene expression cassette in the genome will directly affect its expression level. For example, if a recombinant gene is inserted into the regulatory region of a silencer, it may directly lead to its failure to transcribe and fail to express, and if a foreign gene is inserted into the regulatory region of an enhancer, its expression may also be greatly increased. The site of the cellobiohydrolase gene (cbh1) in T. reesei is generally considered to be the site of high gene expression.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for expressing recombinant protein using Trichoderma reesei as a host.

The method for expressing recombinant protein using Trichoderma reesei as a host provided by the present invention may include the steps of: cloning the expression cassette containing the encoding gene of the target protein to be expressed into the Trichoderma reesei genome and being located at a protein ID of 68606. The non-coding region between the gene and the gene with Protein ID 68608.

Wherein, the described "cloning the expression cassette containing the coding gene of the target protein to be expressed into the non-coding region between the gene whose Protein ID is 68606 and the gene whose Protein ID is 68608 in the Trichoderma reesei genome" can be as follows Either:

(a) Replace any segment of the non-coding region between the gene with Protein ID 68606 and the gene with Protein ID 68608 in the Trichoderma reesei genome with the expression cassette containing the gene encoding the target protein to be expressed ;

(b) inserting the expression cassette containing the gene encoding the target protein to be expressed into any non-coding region between the gene with Protein ID 68606 and the gene with Protein ID 68608 in the Trichoderma reesei genome Location.

Further, the nucleotide sequence of the non-coding region located between the gene with Protein ID 68606 and the gene with Protein ID 68608 in the Trichoderma reesei genome is SEQ ID No.1.

In an embodiment of the present invention, the "cloning the expression cassette containing the gene encoding the target protein to be expressed into the Trichoderma reesei genome is located between the gene whose Protein ID is 68606 and the gene whose Protein ID is 68608. The "coding region" is specifically: replacing positions 429-551 of SEQ ID No. 1 in the Trichoderma reesei genome with the expression cassette containing the gene encoding the target protein to be expressed.

As is conventionally understood in the art, in the above method, the expression cassette containing the gene encoding the target protein to be expressed sequentially comprises a promoter from the 5' end to the 3' end, and the expression of the to-be-to-be-expressed protein is promoted by the promoter. The gene encoding the expressed protein of interest, as well as the terminator. Of course, other types of regulatory sequences may also be included as desired.

The term "|expression cassette" is a double-stranded nucleic acid molecule that contains a gene of interest and all regulatory sequences necessary for its expression. The regulatory sequences are capable of directing the coding sequence to express the gene of interest in a suitable host cell under compatible conditions. Such regulatory sequences include, but are not limited to, leader sequences, polyadenylation sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences include a promoter and transcriptional and translational termination signals. In order to introduce specific restriction enzyme sites into the vector for ligation of the regulatory sequences with the coding region of the gene of interest, the regulatory sequences with linkers can be provided. The regulatory sequence may be a suitable promoter sequence, ie, a nucleic acid sequence that is recognized by the host cell in which the nucleic acid sequence is expressed. The promoter sequence contains transcriptional regulatory sequences that mediate the expression of the gene of interest. The promoter can be any nucleic acid sequence that is transcriptionally active in the host cell of choice, including mutated, truncated and hybrid promoters, and can be derived from extracellular or intracellular encoding homologous or heterologous to the host cell Polypeptide gene. The regulatory sequence may also be a suitable transcription termination sequence, ie, a sequence recognized by the host cell to terminate transcription. The termination sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used in the present invention. The regulatory sequence may also be a suitable leader sequence, an untranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' end of the gene of interest. Any leader sequence that is functional in the host cell of choice can be used in the present invention. In these instances, the nucleic acid sequence and regulatory sequences of the gene of interest should be operably linked. The term "operably linked" is defined herein as a conformation in which a regulatory sequence is positioned in a gene of interest such that the regulatory sequence directs expression of the gene of interest.

In one embodiment of the present invention, the promoter is the promoter of the cellobiohydrolase I gene derived from Trichoderma reesei; the terminator is the cellobiohydrolase I derived from Trichoderma reesei the terminator of the gene.

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

Further, the sequence of the promoter of the cellobiohydrolase I gene derived from Trichoderma reesei is specifically positions 1-2078 of SEQ ID No.2 or positions 1939-4016 of SEQ ID No.3. The specific sequence of the terminator of the cellobiohydrolase I gene derived from Trichoderma reesei is positions 3685-5927 of SEQ ID No.2. The specific sequence of the terminator of the cellobiohydrolase II gene derived from Trichoderma reesei is positions 5693-7784 of SEQ ID No.3.

In the above method, the target protein to be expressed is a protein derived from eukaryotes or a protein derived from prokaryotes.

Further, the eukaryotic organisms can be fungi, plants or animals.

Further, in the present invention, the fungus is specifically Aspergillus niger or Trichoderma reesei.

In an embodiment of the present invention, the target protein to be expressed is a lipase derived from Aspergillus niger, and its amino acid sequence is the same as the amino acid sequence encoded by positions 2138-2947 of SEQ ID No.2.

In another embodiment of the present invention, the target protein to be expressed is cellobiohydrolase I derived from Trichoderma reesei, and its amino acid sequence is the same as the one encoded by the 4017-5692 position of SEQ ID No.3. amino acid sequence.

In the above method, the "cloning the expression cassette containing the gene encoding the target protein to be expressed into the non-coding region between the gene with Protein ID 68606 and the gene with Protein ID 68608 in the Trichoderma reesei genome" This can be achieved by introducing a recombinant expression vector into a Trichoderma reesei host; the recombinant expression vector contains the expression cassette and homology arms located upstream and downstream of the expression cassette for site-directed insertion or replacement.

Further, in an embodiment of the present invention, the homology arms are specifically shown in positions 1-1938 (upstream homology arms) and 7785-9710 (downstream homology arms) of SEQ ID No.3 two DNA fragments.

In the present invention, high-throughput screening by flow cytometer is performed on a series of transformants randomly inserted into different positions of the Trichoderma reesei genome by a series of heterologous lipase genes, and gene copy number is performed on the transformants with higher lipase expression. And the identification of the insertion site, finally the insertion site analysis of a high-yielding strain with a single copy of the recombinant protein gene was carried out, and another gene insertion site ND1, which was beneficial to gene expression, was identified in addition to the Trichoderma reesei cbh1 site. . Finally, in the cbh1 gene deletion strain, the expression backbone of the cbh1 gene was integrated into this site, and the expression level of the cbh1 gene was compared with that of the wild strain, and it was found that the recombinant gene was inserted into this site. . The invention further enriches and perfects the technical scheme of using Trichoderma reesei as a host to express recombinant protein efficiently.

Description of drawings

Figure 1 is a schematic diagram of the structure of plasmid pSKLR.

Figure 2 is a schematic diagram of the preparation of protoplasts and the induced expression of red fluorescent protein.

Figure 3 is a schematic diagram of the corresponding relationship between the expression levels of DsRed and lipase.

Figure 4 is a graph showing the results of detection of the expression level of the recombinant lipase gene in strain ND1.

Figure 5 shows the component fragments required for the construction of strain ND1cbh1.

Figure 6 is a graph showing the detection results of the expression level of cbh1 gene in strains TU6, ND1 and ND1cbh1.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Trichoderma reesei QM9414: ATCC 26921.

Trichoderma reesei TU6: ATCC MYA-256, also described 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) 18:71-76", which is publicly available from the applicant, and may only be used to replicate the experiments of the present invention.

Trichoderma reesei N10: described in "Lina Qin, Fu-rong Cai, Xin-ruiDong, Zhen-bang Huang, Yong Tao, Jian-zhong Huang, Zhi-yang Dong. Improved production of heterologous lipase in Trichoderma reesei by RNAi mediated genesilencing of an endogenic highly expressed gene. Bioresour Technol, 2012, 109: 116-122.” The article, which is publicly available from the applicant, can only be used to replicate the experiments of the present invention.

Trichoderma reesei TR1124: It has been deposited in the General Microbiology Center of China Microorganism Culture Collection Management Committee (CGMCC for short) on July 20, 2012. The address is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. Institute of Microbiology, Chinese Academy of Sciences), the deposit registration number is CGMCC No.6384. And recorded in Chinese patent application 201210271826.2.

Trichoderma reesei Tu6Δku70: The construction of this strain is shown in the article "Gene targetingin a nonhomologous end joining deficient Hypocrea jecorina", in this document, the strain is named Δtku70, but later an article was published, the strain was named Tu6Δku70 , see the 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", publicly available from the applicant, available only for The experiments of the present invention were repeated.

The plasmid pSK-Lip is described in "Lina Qin, Fu-rong Cai, Xin-rui Dong, Zhen-bang Huang, Yong Tao, Jian-zhong Huang, Zhi-yang Dong. Improved production of heterologouslipase in Trichoderma reesei by RNAi mediated gene silencing of an endogenic highly expressed gene. Bioresour Technol, 2012, 109: 116-122”, which is available to the public from the applicant, can only be used to replicate the experiments of the present invention.

Plasmid pDsRed-Monomer-N1: Product of TAKARA Company, Cat. No. 632465.

Plasmid pBluescript SK (+): Snapgene, Addgene product.

Embodiment 1, the acquisition and application example of Trichoderma reesei genome insertion site ND1 beneficial to recombinant gene expression

In the present invention, a segment of polypeptide 2A sequence derived from Foot and Mouth Disease Virus (FMDV) is used as a connecting peptide to fuse and express the red fluorescent protein gene DsRed and a lipase gene LipA derived from Aspergillus niger in Trichoderma reesei . The 2A sequence can express the DsRed gene and LipA gene in a ratio of 1:1. The LipA gene will be secreted out of the cell due to the introduction of the signal peptide sequence, and the red fluorescent protein will remain in the cell as a reporter gene as a marker gene for flow. High-throughput screening by cytometry. The fusion expression backbone is randomly integrated into the Trichoderma reesei genome, and transformants with different recombinant protein expression levels will be generated due to different insertion sites and copy numbers. Transformant, the copy number of the transformant was identified and found that the recombinant gene in this strain was a single copy, and then the insertion site of the recombinant gene was analyzed to obtain the flanking sequences at both ends of the insertion site. Finally, in the cbh1 gene deletion strain , integrated the expression backbone of the cbh1 gene into this site, and compared the expression level of the cbh1 gene with the wild strain. It was found that the expression level equivalent to the cbh1 site could be obtained by inserting the recombinant gene into this site.

1. Acquisition of Trichoderma reesei Genome Insertion Site ND1 Facilitating Recombinant Gene Expression

(1) Construction of recombinant plasmid

1. Construction of the fusion expression vector pSKLR of red fluorescent protein and heterologous lipase

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

Using Trichoderma reesei QM9414 genomic DNA as template, Pcbh1-F, Pcbh1-R as primers for PCR amplification, the amplification conditions are: 95 ℃ pre-denaturation 5min, 94 ℃ denaturation 30s, 57 ℃ annealing 30s, 30 cycles of extension at 72°C for 2 min; finally, extension at 72°C for 10 min to obtain the promoter fragment Pcbh1 of the cbh1 gene. Similarly, using Trichoderma reesei QM9414 genomic DNA as the template, Tcbh1-F, Tcbh1-R as primers for PCR amplification, the amplification conditions are: pre-denaturation at 95 °C for 5 min, denaturation at 94 °C for 30 s, and annealing at 60 °C 30 s at 72°C for 2.5min, 30 cycles; the last extension at 72°C for 10min, the terminator fragment Tcbh1 of the cbh1 gene was amplified. Agarose gel electrophoresis was performed to recover the two fragments.

(2) Overlap extension PCR amplification of red fluorescent protein, the fusion fragment L2aR of 2A sequence and lipase gene

The first round of PCR: using plasmid pSK-Lip as template, lipase2a-F, lipase2a-R as primers to amplify the lipase gene fragment containing the 2A sequence and part of the red fluorescent protein gene fragment. Using plasmid pDsRed-Monomer-N1 as template and 2ared-F and 2ared-R as primers, the red fluorescent protein gene containing 2A sequence and part of lipase gene fragment was amplified. The two fragments were subjected to agarose gel electrophoresis and purified using a gel recovery kit.

The second round of PCR: The equimolar mixture of the two PCR products recovered and purified from the gel is used as a template, and lipase2a-F and 2ared-R are used as primers to perform Overlap PCR amplification to obtain the fragment L2AR fused with the two genes. The fragment is cloned into pMD18T -simple vector for sequencing.

(3) Construction of fusion expression vector pSKLR

The plasmid pBluescript SK(+) was double-digested with restriction enzymes SalI and NotI, the Pcbh1 fragment was double-digested with restriction enzymes SalI and EcoRI, and L2aR was double-digested with restriction enzymes EcoRI and SpeI Double-enzyme digestion was performed, and Tcbh1 was double-enzyme digestion with restriction enzymes SpeI and NotI. The digested vector fragment, Pcbh1 fragment, LaR fragment, and Tcbh1 fragment were mixed for four-fragment ligation, and transformed into E. coli Top10, colony The transformant clones were identified by PCR. The primers used were lipase2a-F and 2ared-R. The correct clones were picked and the plasmid was extracted and verified by sequencing to obtain the fusion expression vector pSKLR (see Figure 1 for the structure).

2. Construction of uracil-deficient selection marker vector pSKpyr4

(1) Cloning of Trichoderma reesei pyr4 gene

PCR amplification was carried out with Trichoderma reesei QM9414 genomic DNA as the template and Pyr4-F and Pyr4-R as primers. Extension at 72°C for 2 min, 30 cycles; final extension at 72°C for 10 min. The obtained PCR product was electrophoresed on 1% agarose gel, the target band was recovered by agarose gel electrophoresis kit, and the TA clone was ligated into the pMD18T-Simple vector for sequencing.

(2) Construction of plasmid vector pSKpyr4

The plasmid pBluescript SK(+) and the correctly sequenced pyr4 gene TA cloned plasmid were double-enzyme digested with restriction enzymes ClaI and EcoRI, respectively, and the digested pBluescript SK(+) vector fragment and pyr4 gene fragment were recovered. connect. Transform E. coli Top10, pick the transformants for colony PCR identification, the primers used are Pyr4-F, Pyr4-R, the PCR conditions are the same as before, the plasmid is extracted and verified by sequencing to obtain the complementary plasmid vector pSKpyr4 of the uracil-deficient strain TU6 .

All primers used in the above-mentioned construction of recombinant plasmids are shown in Table 1 for details.

Table 1 Primers used to construct recombinant vectors

(2) Obtainment of Trichoderma reesei transformants fused to express red fluorescent protein and heterologous lipase

1. Protoplast preparation of Trichoderma reesei strain TU6

(1) Take the spores of Trichoderma reesei TU6 on a freshly cultivated slant or plate, wash the spores with an appropriate amount of sterile water to make a spore suspension, and filter through a 200-mesh sieve to remove the residual hyphae. The filtered spore suspension was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of MM medium (2% glucose as the carbon source), and cultured at 28° C. for 13h-14h until the mycelium expanded.

(2) filter the culture solution through a 200-mesh sieve, collect the bacterial cells, wash with sterile water 2-3 times, and finally wash once with a 1.2M MgSO solution, and let the solution flow out naturally _;

(3) Rinse the bacteria on the sieve into a conical flask containing 15 mL of lysing solution (the lysing solution contains 150 mg of Lysing enzyme Sigma, Cat. No. L-1412 and 15 mg of Cellulase R-10, YAKULT, 1.2M MgSO ₄ from Japan), react at 30°C for 1.5h, observe the production of protoplasts under a microscope, and take samples every 10min for observation after 1h;

(4) when protoplasts are produced in a large amount and there is still a large amount of mycelia, add an equal volume of 0.6M sorbitol solution to terminate the reaction, filter with a 200-mesh sieve to remove residual mycelium, and collect protoplast sediments by centrifugation at room temperature 3000rpm for 10min;

(5) Pour off the supernatant along one side of the precipitation, resuspend the protoplast precipitation with 1.0M sorbitol solution, and centrifuge at 3000rpm for 10min at room temperature;

(6) Repeat step (5), discard the supernatant, suspend the protoplasts in 200 μL of 1.0 M sorbitol solution, observe and count with a hemocytometer.

2. Trichoderma reesei transformation of fusion expression vector

(1) The plasmids pSKpyr4 and pSKLR were extracted in large quantities and precipitated overnight with 2 times the volume of absolute ethanol and 1/10 volume of 3.0M sodium acetate (pH 5.2), washed twice with 70% ethanol, and sterilized with ddH Dissolve in ₂ O to make the concentration of each plasmid reach μg level.

(2) The plasmid pSKpyr4 and pSKLR were mixed in a molar ratio of 1:3 to 1:10, and the volume did not exceed 20 μL. The mixture was added to the protoplasts prepared above, mixed gently, and then 50 μL was added to each. PEG4000, mixed again, and placed on ice for 30 min; a control was set, and an equal volume of sterile water was used instead of the DNA mixture for the control.

(3) Add 1 mL of PEG4000 to each of the above tubes, mix well, and place at room temperature for 20 min.

(4) add 1mL 1.0M sorbitol respectively at last, mix again, transfer all protoplast transformation liquids to the 250mL Erlenmeyer flask that 50mL induction medium is housed, and the induction medium formula is to contain 1g in every liter of culture medium Glucose, 1 g glycerol, 20 g lactose, 180 g sorbitol, 0.05 g (NH ₄ ) ₂ SO ₄ , 0.15 g KH ₂ PO ₄ , 0.006 g MgSO ₄ , 0.006 g CaCl ₂ , 0.00005 g FeSO ₄ 7H ₂ O, 0.000016 g _MnSO4.H2O , _0.000014g , _ZnSO4.7H2O , _{0.00002g CoCl2} . The culture condition was 30°C, and the culture was slowly shaken at 100 rpm for 24 h, and then the rotation speed was adjusted to 200 rpm to continue the shaking culture.

(5) After culturing at 30° C. for 3-6 days, collect all mycelia, and prepare protoplasts again for flow cytometry sorting (see FIG. 2 ). In Figure 2, A: Mycelial growth after protoplasts were induced in the regeneration induction medium for 96 hours; B: The induction of DsRed was observed under a fluorescence microscope; C: The newly induced transformants were prepared under an ordinary light microscope The resulting protoplasts; D: The expression of red fluorescent protein after the induced transformants were re-prepared into protoplasts were observed under a fluorescence microscope. It can be seen from Figure 2 that after successfully expressing the recombinant protein, the hyphae are prepared into protoplasts, and the red fluorescence is still clearly expressed, which can be used for flow cytometry sorting.

(3) Flow cytometry screening of transformants

1. Sample preparation: collect all the hyphae of the transformants regenerated under the induction conditions, prepare the protoplast suspension again, and filter through a 400-mesh sieve. The integrity of the protoplast preparation was observed microscopically and counted with a hemocytometer. The protoplast suspension was adjusted to a concentration of 10 ⁷ /mL of suspension, 500 μL. The expression of red fluorescent protein was observed by fluorescence microscope.

2. Debugging of the flow cytometer: Use a FACS Aria flow cytometer (BD FACS Aria, USA) to sort the cells with RFP in the protoplasts. 100 μm nozzle, excitation with 557 nm green light, and sheath fluid pressure of 20 psi. A 100 μm nozzle was selected and ultrasonically cleaned for 1 minute. After starting up, open the main liquid flow breakpoint window, click the main liquid flow, adjust the liquid flow vibration amplitude (Ampl), make the droplet interval value (Gap value) stable, set the debugging parameters to the best possible, and maintain the main liquid flow. Continuity and stability. After the main liquid flow is stable, open the voltage of the side liquid flow window, click Test Sort, and adjust the parameters to make the liquid flow split clearly. Install the four-way sorting device, open the voltage of the side liquid flow window, click Test Sort, close the waste liquid drawer, and adjust the voltage scroll bar of the side liquid flow window so that the deflected sorting liquid flow falls into the corresponding collection tube. Add the Dmpl value actually called in the main flow window to the default window.

3. Four-channel cell sorting: a sorting gate was established with the protoplast of the starting strain TU6 as a negative control. The determination of the sorting gate is based on the following principles: intact protoplasts have a higher ratio of forward scatter (FSC) and side scatter (SSC), and compared with negative controls, RFP-positive cells are in green The optical channel (585nm) has strong emission light. Cells were sorted directly into protoplast regeneration medium (MM+sorbitol).

4. Secondary screening of transformant protoplasts: regenerate the group of protoplasts with the strongest fluorescence intensity sorted above, prepare protoplast suspension again, and perform single cell sorting in a 96-well microplate. Induce regeneration medium.

(4) Corresponding relationship between DsRed and heterologous lipase expression

After single cell sorting in a 96-well microplate, 46 strains of bacteria with different fluorescence intensities were selected to produce spores on a PDA solid plate. The spores were prepared into a spore suspension and inoculated into a seed bottle. The formula of the seed bottle medium was 20 g glucose, 0.05 g (NH ₄ ) ₂ SO ₄ , 0.15 g KH ₂ PO ₄ , 0.006 g MgSO ₄ , 0.006 g per liter of medium. g CaCl ₂ , 0.00005 g FeSO ₄ ·7H ₂ O, 0.000016 g MnSO ₄ ·H ₂ O, 0.000014 g, ZnSO ₄ ·7H ₂ O, 0.00002 g CoCl ₂ . The culture condition is 30°C, after 48 hours of shaking at 200 rpm, the cells are filtered with four layers of gauze, and 1.8 g of the cells are respectively weighed and inoculated into the fermentation medium. The fermentation medium formula is that the carbon source of the seed medium is replaced with lactose. After 96 hours of shaking at 30°C and 200 rpm, the lipase enzyme activity of the supernatant of the fermentation broth was measured, and the fluorescence intensity of the mycelium was measured.

1. Fluorescence assay of red fluorescent protein

Weigh equal weight of mycelium and place it in a 24-well microplate, read the fluorescence value of each transformant with a multi-function fluorescence plate reader, set the excitation light wavelength to 557 nm and the emission light wavelength to 585 nm, using 13× 13 After scanning by multi-point scanning, the average value was taken, and then the fluorescence value of the strain TU6 that did not express fluorescent protein was set to 1, and the relative fluorescence value of the transformant was taken.

2. Determination of lipase activity

The activity of lipase was determined with reference to the national standard GB/T 23535, and the reaction substrate was olive oil emulsion. Lipase activity unit (U) is defined as: under test conditions, 1 μmol of free fatty acid is released from the catalytic substrate per milliliter of enzyme solution per minute, which is defined as one lipase activity unit (U).

The corresponding relationship between the expression levels of DsRed and heterologous lipase is shown in Figure 3. It can be seen from the figure that the expression level of DsRed in the transformants is basically consistent with the activity trend of lipase in vitro. The results show that the use of 2A sequence as a linker Peptide fusion expression of red fluorescent protein and lipase can make these two proteins expressed in equal amounts and exist independently, the lipase with signal peptide can be effectively secreted into the extracellular, while the red fluorescent protein lacking the signal peptide remains in the cell as a reporter Gene high-throughput screening of strains with high lipase expression by flow cytometry.

(5) Gene copy number identification of recombinant genes in recombinant protein high-producing strains

1. Extract the DNA of recombinant protein high-producing strain and control strain N10.

(1) Take recombinant protein high-producing strain and Trichoderma reesei control strain N10, inoculate 10 ⁸ spore suspensions of each strain in MM medium containing 2% glucose and cultivate for 36 hours, then filter and collect through 200 mesh sieves Trichoderma reesei hyphae, pressed dry on filter paper, and ground to powder in liquid nitrogen.

(2) Add 5 mL of DNA extraction buffer per gram of mycelium, shake and mix evenly, then place in a water bath at 37°C for 1 hour, take out every 10 minutes, shake and mix evenly.

(3) An equal volume of phenol:chloroform:isoamyl alcohol (25:24:1, volume ratio) solution was added, mixed upside down and centrifuged at 12000 rpm for 10 min.

(4) Transfer the supernatant to a new centrifuge tube, add 0.5-0.6 times the volume of ice-cold isopropanol, invert and mix, place at -20°C for 30-60min, and then centrifuge at 4°C and 12000rpm 10min.

(5) The precipitate was washed twice with 75% ethanol, dissolved in 50-100 μL of 100 mM Tris-HCl (pH 8.0) containing 20 μg/mL RNase, and stored at -20° C. for later use.

2. Real-time quantitative PCR to identify the copy number of the fusion gene of DsRed and lipase

Using the single-copy gene actin gene as a standard internal reference, the genomic DNAs of N10 and each high-yielding transformant were amplified with primers Actin-F/Actin-R and LipRT-F/LipRT-R (Table 1). All PCR reactions were performed on an ABIPrism 7000 real-time detection system (ABI) instrument using Power SYBR GreenPCR Master Mix (ABI). The system was 25 μL, and the steps used were: 50°C for 2 min; pre-denaturation at 95°C for 10 min, 95°C for 30s, 60°C for 30s, and 72°C for 30s, 40 cycles. Since the copy number of lipase gene in strain N10 is single copy (see 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 Trichodermareesei by RNAi mediated gene silencing of an endogenic highly expressed gene. Bioresour Technol, 2012, 109: 116-122.), therefore, the copy number of the recombinant gene in each transformant was relatively quantified by the 2- ^ΔΔCt method.

(6) Identification of the insertion site in the recombinant protein high-producing strain with a single copy of the recombinant gene

In all transformants, plasmids pSKpyr4 and pSKLR were integrated into the Trichoderma reesei genome by random insertion. In addition to the complete expression cassettes for DsRed and lipase fusion proteins, this plasmid also has a replicon for E. coli Sequence and ampicillin resistance gene. Therefore, we used the plasmid rescue method to identify the insertion site of the gene. In the present invention, insertion site analysis is mainly carried out for a high-yielding strain ND1 whose recombinant protein gene is a single copy, and the specific operation process is as follows:

1. Digest 5-10 μg of genomic DNA with restriction endonuclease SalI. After the digestion is complete, take a water bath at 65°C for 10 minutes to inactivate the restriction endonuclease.

2. Add 1/10 volume of 3M sodium acetate and 2.5 times volume of absolute ethanol to the enzyme digestion system, and precipitate at -20 °C for at least 1 hour.

3. Centrifuge at 4°C for 15 min at 13000 rpm, wash the precipitate twice with 70% ethanol, air dry or vacuum dry, and dissolve with 50-100 μL of sterile ddH ₂ O.

4. In the following ligation system, ligation overnight at 16°C.

5. Treat at 65°C for 10 min to inactivate T4 DNA ligase.

6. Add 1/10 volume of 3M sodium acetate and 2.5 times volume of absolute ethanol, and precipitate at -20°C for at least 1 hour.

7. Centrifuge at 4°C for 15 min at 13000 rpm, wash the pellet twice with 70% ethanol, air dry or vacuum dry, and dissolve the DNA with 5 μL of sterile ddH ₂ O.

8. All DNA dissolved in 5 μL sterile ddH ₂ O was transformed into E. coli XL10-gold competent cells.

9. Pick the clone on the E. coli transformation plate, and use primers Lip-F and Lip-R (Table 1) to carry out colony PCR identification to ensure that the obtained clone contains the lipase gene.

10. Extract the plasmids in the positive clones in the above step 9 and send them to a sequencing company for sequencing, so as to obtain the flanking sequences of the integration sites of the recombinant genes in the genome.

After the screening and identification of the above steps, the strain ND1 was finally obtained.

First, the expression level of the recombinant lipase gene was detected in strain ND1, and the results are shown in Figure 4. In Figure 4, a is the supernatant of the fermentation broth after 120 hours of fermentation under cellulose conditions detected by SDS-PAGE; b is the lipase activity after 120 hours of fermentation under cellulose conditions. Trichoderma reesei strain TR1124 is a high-yielding strain of lipase that integrates the fusion gene of the red fluorescent protein gene and the lipase gene into the cbh1 site. The results in FIG. 4 show that the strain ND1 obtained by high-throughput screening by flow cytometry is also a high-producing strain of lipase, and the lipase gene is not integrated into the cbh1 site in this strain (see below for details).

Second, it can be seen from the results in Table 2 that the copy number of the recombinant lipase gene in the ND1 strain is a single copy.

Table 2 Identification of the copy number of recombinant lipase gene in ND1 strain by Real Time PCR

Third, analysis of the insertion site of the recombinant gene in strain ND1

The insertion site of the recombinant gene in the tested strain ND1 (denoted as ND1 site) is located in the non-coding region (SEQ ID No. 1) between the gene with Protein ID 68606 and the gene with Protein ID 68608.

The sequencing results showed that the recombinant gene lipA-red in strain ND1 specifically replaced the 429-551 position of SEQID No.1 in the Trichoderma reesei genome.

2. Analysis of the effect of insertion site ND1 on recombinant protein expression

The location of the cellobiohydrolase gene cbh1 in the genome is generally considered to be a site of favorable gene expression in the T. reesei genome. In order to compare the ND1 locus identified in step 1 and the cbh1 locus which is more favorable for gene expression. Taking the Trichoderma reesei strain Tu6Δku70 with a very high homologous recombination rate as the starting strain, the lipase gene lipA was integrated into the cbh1 site to replace the cbh1 gene, and the cbh1 gene was integrated into the insertion site ND1 identified in step 1. The expression level of the cbh1 gene in this strain was compared with the control strains Tu6 and ND1. The specific operation steps are as follows:

1. Build the component fragment shown in Figure 5

(1) PCR amplification was performed using plasmid pSKLR as a template and Pcbh1-F and Tcbh1-R (Table 1) as primers, and the amplified fragment was the fragment shown in a in Figure 5 .

The nucleotide sequence of the fragment shown in a in Figure 5 is shown in SEQ ID No. 2, in which the 1-2078th position is the Pcbh1 promoter sequence, the 2138-2947th position is the Lipase gene sequence, and the 2948-2998th position is the 2A Sequence, the 2999-3676 position is the DsRed sequence, and the 3685-5927 position is the Tcbh1 terminator sequence.

(2) using Trichoderma reesei (Trichoderma reesei) QM9414 genomic DNA as a template, primers Flup-F, Fluup-R (Table 1) are primers to carry out PCR amplification, and the amplified fragment is the upstream homology arm of the insertion site Sequence; Take Trichoderma reesei (Trichoderma reesei) QM9414 genomic DNA as template, Pcbh1-2F, cbh1-2R (table 1) are primers to carry out PCR amplification, and the fragment that amplification obtains is the promoter and ORF sequence of cbh1 gene; Take Trichoderma reesei (Trichoderma reesei) QM9414 genomic DNA as template, primer Tcbh2-F and Tcbh2-R (table 1) are primers to carry out PCR amplification, gained fragment is cbh2 gene terminator sequence; Take Trichoderma reesei (Trichoderma reesei) ) QM9414 genomic DNA is a template, primers Fldown-F, Fldown-R (Table 1) are primers to carry out PCR amplification, and the fragment that the amplification obtains is the insert site downstream homology arm sequence; With plasmid pBluescriptSK (+) as template, The primers vec-F and vec-R (Table 1) were used for PCR amplification, and the amplified fragments were vector backbone fragments. The fragments amplified by PCR were recovered by gel, ligated with Gibson cloning kit, and transformed into E. coli XL10-gold competent cells to construct the vector pND1-cbh1. Using plasmid pND1-cbh1 as a template, primers Flup-F and Fldown-R (Table 1) as primers for amplification, the amplified fragment is the component fragment shown in b in Figure 5 .

The nucleotide sequence of the fragment shown in b in Figure 5 is shown in SEQ ID No.3, in which the 1-1938th position is the upstream homology arm sequence, the 1939-4016th position is the Pcbh1 promoter sequence, and the 4017th-5692th position is the cbh1 gene sequence, the 5693-7784th position is the Tcbh1 terminator sequence, and the 7785th-9710th position is the downstream homology arm sequence.

2. After the assembly fragments obtained by the above PCR amplification (that is, the two fragments shown in SEQ ID No. 2 and SEQ ID No. 3) are subjected to PCR purification, and the plasmid pSK-pyr4 is co-transformed into Trichoderma reesei strain Tu6Δku70, The transformation method is as in step one (two).

3. PCR identification of positive transformants: After obtaining the transformants, DNA was extracted, and the primer pair Ocbh1-F/Lip-R (Table 1) was subjected to PCR to identify whether the lipase gene was integrated into the cbh1 site, and the primer pair OND1-F /Vcbh1-R (Table 1) PCR was performed to identify whether the cbh1 gene was integrated into the ND1 site. The identified positive transformants were named ND1cbh1.

The cbh1 expression cassette in strain ND1cbh1 replaces positions 429-551 of SEQ ID No. 1 in the T. reesei genome.

4. Analysis of cbh1 gene expression in the above positive transformants: Inoculate the positive transformants ND1cbh1 and control strains TU6 and ND1 on a PDA plate to produce spores, wash the spores with sterile water to prepare a spore suspension, sieve with 200 mesh Filter, adjust the concentration of the spore suspension to 10 ⁸ /mL, inoculate 1 mL of the spore suspension in a 250 mL Erlenmeyer flask with 50 mL of MM medium (2% glucose as carbon source), and culture at 28°C and 200 rpm for 48 hours. Cells were filtered with gauze, and 1.8 g of wet cells were weighed and transferred to a medium of 50 mL MM+1% microcrystalline cellulose in a 250 mL conical flask. The medium inoculated with strain TU6 needs to be supplemented with 5mM uridine. After culturing in the cellulose-induced medium for 120 hours, SDS-PAGE electrophoresis was used to detect the secretion of CBH1 in the supernatant of the fermentation broth, and the enzymatic activity of CBH1 was measured. The bacterial DNA and RNA were extracted, and the copy number and mRNA level of cbh1 gene were analyzed by real-time fluorescence quantitative PCR.

(1) Identification of the copy number of cbh1 gene in strain ND1cbh1. The hyphae of strains TU6 and ND1cbh1 were collected, ground in liquid nitrogen, and total DNA was extracted. The single-copy gene actin gene was used as a standard internal reference, and primers Actin-F/Actin-R and RTcbh1-F/RTcbh1-R were used respectively (Table 1). Genomic DNA of strains TU6 and ND1cbh1 was amplified. All PCR reactions were performed on an ABI Prism 7000 real-time detection system (ABI) instrument using PowerSYBR Green PCR Master Mix (ABI). The system was 25 μL, and the steps used were: 50°C for 2 min; pre-denaturation at 95°C for 10 min, 95°C for 30s, 60°C for 30s, and 72°C for 30s, 40 cycles. Since the copy number of the cbh1 gene in strain TU6 is a single copy (see Table 3 in the article Genome sequencing and analysis of the biomass ^- degrading fungus Trichoderma reesei (syn. The ^ΔΔCt method was used for relative quantification.

(2) Real Time PCR analysis of mRNA level of cbh1 gene. The hyphae were collected, ground in liquid nitrogen and total RNA was extracted, and a two-step real-time quantitative PCR experiment was performed.

Step 1: Reverse Transcription

Add the following reagents to a 200 μL PCR tube:

Incubate at 65°C for 10min; immediately remove and place on ice for 5min;

Add the following reagents:

The reaction mixture was incubated at 50°C for 1 h; then heated to 85°C for 10 min to inactivate reverse transcriptase.

Step 2: Real Time PCR

All PCR reactions were performed on an ABI Prism 7000 real-time detection system (ABI) instrument using Power SYBR Green PCR Master Mix (ABI). The reaction was performed according to the instructions of ABI Prism 7000. The system was 25 μL. The steps used were: 50°C for 2 min; pre-denaturation at 95°C for 10 min, 95°C for 30s, 60°C for 30s, and 72°C for 30s, 40 cycles. The transcription level of the gene was used as an internal reference for the actin gene.

(3) Determination of the enzymatic activity of cellulosic pond hydrolase (CBH)

Using 4-methylumbelliferyl-D-cellobioside (MUC, Sigma) as a substrate, 15 mg of MUC was weighed, dissolved in 500 μL of DMSO (Sigma), and then transferred to 30 mL of citric acid buffer (pH 4.8 50 mM). Mix 12 μL of appropriately diluted enzyme solution with 200 μL MUC, 25 μL glucose (1M) and 25 μL citrate buffer, this is the experimental group without cellobiose. In the mixed solution, glucose will inhibit BG (β-Glucosidase, β-glucosidase) from degrading MUC, so the measured activity is actually the activity of CBH1 and EG (endoglucosidase). At the same time, a cellobiose-added experimental group was set up: 12 μL enzyme solution, 200 μL MUC, 25 μL glucose, 25 μL (50 mM) cellobiose and 25 μL citrate buffer, and reacted at 50°C for 10 min. The addition of cellobiose inhibited the activity of CBH, so the activity of EG was measured. ₂₅₀ μL _Na2CO3 (1M) was added. Take 100 μL of diluted 11-fold, and measure the absorbance at 370 nm. The OD value of the experimental group without cellobiose was subtracted from the experimental group with cellobiose, which is the more specific activity of CBH. One unit of CBH enzymatic activity was defined as the amount of enzyme required to catalyze the hydrolysis of 1 nmol MUC per second.

5. Results

The identification results of the copy number of cbh1 gene in strain ND1cbh1 are shown in Table 3. It can be seen that the copy number of cbh1 gene in strain ND1cbh1 is close to that of strain TU6, and it is a single copy.

Table 3 Identification of the copy number of recombinant cbh1 gene in ND1cbh1 strain by Real Time PCR

Figure 6 shows the identification results of the expression levels of cbh1 gene in strains Tu6, ND1 and ND1cbh1 strains. In Figure 6, a is the supernatant of the fermentation broth after 120 hours of fermentation under cellulose conditions detected by SDS-PAGE; b is the CBH activity after 120 hours of fermentation under cellulose conditions. It can be seen from the figure that the expression level of the cbh1 gene at the ND1 site is comparable to that of the strain TU6, indicating that the contribution of this site to the expression level of the recombinant gene is comparable to that of the cbh1 gene.

<110> Fujian Normal University

<120> Method for expressing recombinant protein using Trichoderma reesei as host

<130> GNCLN172245

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 1095

<212> DNA

<213> 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 sequences

<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 sequences

<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 tccaggac 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 (7)

1. A method for expressing recombinant protein by using Trichoderma reesei as a host comprises the following steps: cloning an expression cassette containing a coding gene of a target Protein to be expressed into a non-coding region between a gene with Protein ID 68606 and a gene with Protein ID 68608 in a trichoderma reesei genome;

the "cloning of the expression cassette containing the gene encoding the target Protein to be expressed into the non-coding region between the gene with Protein ID 68606 and the gene with Protein ID 68608 in the genome of Trichoderma reesei" is to replace the expression cassette containing the gene encoding the target Protein to be expressed with the 429-551 position of SEQ ID No.1 in the genome of Trichoderma reesei.

2. The method of claim 1, wherein: the expression cassette containing the coding gene of the target protein to be expressed sequentially comprises a promoter, the coding gene of the target protein to be expressed and started to be expressed by the promoter, and a terminator from the 5 'end to the 3' end.

3. The method of claim 2, wherein: the promoter is derived from a cellobiohydrolase I gene of trichoderma reesei; the terminator is derived from the cellobiohydrolase I gene of trichoderma reesei or derived from the cellobiohydrolase II gene of trichoderma reesei.

4. The method of claim 3, wherein: the sequence of the promoter of the cellobiohydrolase I gene derived from the trichoderma reesei is 1-2078 bit of SEQ ID No.2 or 1939-4016 bit of SEQ ID No. 3; the terminator sequence of the cellobiohydrolase I gene derived from the trichoderma reesei is the 3685-5927 th site of SEQ ID No. 2; the terminator of the cellobiohydrolase II gene derived from Trichoderma reesei has the sequence of position 5693-7784 of SEQ ID No. 3.

5. The method according to any one of claims 1-4, wherein: the target protein to be expressed is protein derived from eukaryote or prokaryote.

6. The method of claim 1, wherein: the cloning of the expression cassette containing the coding gene of the target Protein to be expressed into the non-coding region between the gene with the Protein ID 68606 and the gene with the Protein ID 68608 in the trichoderma reesei genome is realized by introducing a recombinant expression vector into trichoderma reesei host bacteria; the recombinant expression vector contains the expression cassette and a homology arm which is positioned at the upstream and downstream of the expression cassette and is used for site-specific insertion or replacement.

7. The method of claim 6, wherein: the homologous arms are two DNA segments shown in the 1 st-1938 th site and the 7785 th-9710 th site of SEQ ID No. 3.

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