阅读说明：本技术 用于快速筛选重组菌株的重组表达载体及应用 (Recombinant expression vector for rapidly screening recombinant strain and application thereof ) 是由 姚斌 苏小运 高飞 罗会颖 黄火清 柏映国 王苑 涂涛 王亚茹 孟昆 于 2018-07-27 设计创作，主要内容包括：本发明属于农业生物技术领域,具体涉及一种用于快速筛选高表达菌株的重组表达载体和快速筛选高表达菌株的方法。本发明将一个外源的红色荧光蛋白和烟曲霉细胞表面蛋白定位信号融合表达,将该融合基因(DsRed-AfMP1)整合到里氏木霉基因组中,从而构建里氏木霉表面展示红色荧光蛋白的菌株。将表面展示红色荧光蛋白的里氏木霉菌株通过流式细胞仪分选,可以快速分离出有益于纤维素酶活提高的基因变化。(The invention belongs to the technical field of agricultural biology, and particularly relates to a recombinant expression vector for rapidly screening high-expression strains and a method for rapidly screening the high-expression strains. According to the invention, an exogenous red fluorescent protein and an Aspergillus fumigatus cell surface protein positioning signal are fused and expressed, and the fusion gene (DsRed-AfMP1) is integrated into a Trichoderma reesei genome, so that a strain for displaying the red fluorescent protein on the surface of Trichoderma reesei is constructed. The Trichoderma reesei strain with the surface displaying the red fluorescent protein is sorted by a flow cytometer, and the gene change beneficial to improving the cellulase activity can be quickly separated.)

1. A recombinant expression vector of Trichoderma reesei for rapidly screening high-expression cellulase, which is characterized in that the recombinant expression vector comprises the following components in sequence from upstream to downstream in a gene expression cassette: the kit comprises a cbh1 promoter, a red fluorescent protein gene DsRed, a cell surface protein anchoring signal peptide gene AfMp1 and a cbh1 terminator, wherein the nucleotide sequence of the DsRed gene is shown as SEQ ID No.2, and the nucleotide sequence of the AfMp1 gene is shown as SEQ ID No. 3.

2. A recombinant Trichoderma reesei comprising the recombinant expression vector for Trichoderma reesei highly expressing cellulase to be screened according to claim 1.

3. The use of the recombinant expression vector for rapidly screening Trichoderma reesei highly expressing cellulase as set forth in claim 1.

4. A method for rapidly screening recombinant Trichoderma reesei, which comprises the following steps:

(1) introducing a gene expression cassette containing an element cbh1 promoter, a red fluorescent protein gene DsRed, a cell surface protein anchoring signal peptide gene AfMp1 and a cbh1 terminator into a plasmid to obtain a recombinant expression vector, wherein the nucleotide sequence of the DsRed gene is shown as SEQ ID No.2, and the nucleotide sequence of the AfMp1 gene is shown as SEQ ID No. 3;

(2) transforming host cells by using the recombinant expression vector constructed in the step (1) to obtain a recombinant strain;

(3) culturing the recombinant strain, and inducing the red fluorescent protein to be expressed on the surface of the recombinant strain;

(4) recombinant strains displaying red fluorescence were screened.

5. The method for rapid screening of recombinant Trichoderma reesei according to claim 4, wherein in step (4), the recombinant strain whose surface exhibits red fluorescence is screened by flow cytometry.

6. The method for rapidly screening trichoderma reesei with high cellulase expression and high enzyme activity is characterized by comprising the following steps of:

(1) introducing a gene expression cassette containing an element cbh1 promoter, a red fluorescent protein gene DsRed, a cell surface protein anchoring signal peptide gene AfMp1 and a cbh1 terminator into a plasmid to obtain a recombinant expression vector, wherein the nucleotide sequence of the DsRed gene is shown as SEQ ID No.2, and the nucleotide sequence of the AfMp1 gene is shown as SEQ ID No. 3;

(2) transforming host cells by using the recombinant expression vector constructed in the step (1) to obtain recombinant trichoderma reesei;

(3) introducing genes related to a protein secretion pathway into the recombinant trichoderma reesei obtained in the step (2), or randomly inserting vectors or genes into the recombinant trichoderma reesei obtained in the step (2) to obtain a mutant library of the recombinant trichoderma reesei;

(4) screening recombinant trichoderma reesei with the surface displaying strong red fluorescence by using a flow cytometer;

(5) and (4) determining the cellulase activity of the recombinant trichoderma reesei obtained in the step (4) to obtain the recombinant trichoderma reesei with improved cellulase activity.

7. The method for rapidly screening Trichoderma reesei that highly expresses cellulase and has high enzyme activity according to claim 6, wherein in the step (3), the genes related to protein secretion pathway include bip1 gene, hac1 gene, ftt1 gene, sso2 gene, sar1 gene, ypt1 gene.

8. The method for rapidly screening Trichoderma reesei with high cellulase expression and high enzyme activity according to claim 6, wherein in the step (3), Trichoderma reesei is transformed by Agrobacterium tumefaciens to obtain Trichoderma reesei mutant library.

9. The method for rapidly screening the protein secretion pathway related gene highly related to improving the cellulase activity is characterized by comprising the following steps of:

(1) constructing a recombinant expression vector of a DsRed-AfMP1 fusion gene containing a red fluorescent protein gene and a cell surface protein anchoring signal peptide gene, wherein the nucleotide sequence of the DsRed gene is shown as SEQ ID NO.2, and the nucleotide sequence of the AfMp1 gene is shown as SEQ ID NO. 3;

(2) transforming a strain by using the recombinant expression vector constructed in the step (1) to obtain a recombinant strain;

(3) introducing a target gene to be screened into the recombinant strain obtained in the step (2), or randomly inserting a vector or a gene into the recombinant strain obtained in the step (2) to obtain a mutant library of the recombinant strain;

(4) screening the recombinant strain with the surface displaying red fluorescence by using a flow cytometer;

(5) determining the cellulase activity of the recombinant strain with the surface displaying red fluorescence screened in the step (4) to obtain the recombinant strain with improved cellulase activity;

(6) and determining the exogenous target gene expressed in the recombinant strain with the improved cellulase activity or the interfered endogenous gene, thereby obtaining the protein secretion pathway related gene related to the improvement of the cellulase activity.

10. The method for rapidly screening the genes related to the protein secretion pathway with high correlation to the enzymatic activity of cellulase according to claim 9, wherein the recombinant strain is trichoderma reesei, and preferably, the genes related to the protein secretion pathway comprise bip1 gene and hac1 gene.

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to a recombinant expression vector for rapidly screening recombinant strains and application thereof.

Background

The plant cell wall mainly comprises cellulose (cellulose), hemicellulose (hemicellulose) and lignin (lignin), wherein the cellulose is used as a main component for constructing the cell wall and is a natural linear macromolecular polymer formed by connecting 8,000-10,000 glucose through β -l, 4-glycosidic bond, cellobiose is used as a basic unit of the natural linear macromolecular polymer, and the complex cellulase enzyme system synergy is required for degrading cellulose polysaccharide in the plant cell wall into fermentable simple oligosaccharide and monosaccharide such as glucose.

Trichoderma reesei, one of the major filamentous fungi, has a strong ability to secrete cellulase enzymes. In industry, the yield of cellulase produced by the modified trichoderma reesei can reach more than 100g/L, so the modified trichoderma reesei has important application value in degrading plant cell wall polysaccharide and related applications.

The cellulase system of trichoderma reesei includes two exocellulases, five endocellulases, one β -glucosidase and three lytic polysaccharide monooxygenases (LPMOs, a member of the original GH61 family).

With the continuous optimization of a trichoderma reesei genetic operation system, the transformation efficiency can basically meet the industrial requirements, but when a large number of transformants are obtained, how to screen the transformants which efficiently express the target protein becomes the key of the problem. The trichoderma reesei is a filamentous fungus and is different from a common single pure line bacterial colony, and the multi-core bacterial colony of the trichoderma reesei is a bacterial colony structure formed by winding multi-core hyphae. In order to obtain pure line transformants on transformation plates with high false positive background, the mononucleospores can only be separated through a tedious monospore separation process, so that the genetic modification period is too long, and further, the screening of strains with high expression quantity is time-consuming and labor-consuming.

Flow cytometry (Flow cytometry) is a technique for rapid quantitative analysis and sorting of individual cells or other biological particles, etc. in a liquid stream. The flow cytometer can analyze tens of thousands of cells at high speed, simultaneously measure a plurality of cell characteristic parameters from one cell, perform qualitative or quantitative analysis, and has the characteristics of high speed, high precision, good accuracy and the like. However, the current flow detection technology can only detect the change of the regulatory factor acting on the transcription stage, but cannot detect the change of the regulatory factor at the later stage of the transcription stage. The Trichoderma reesei cellulase performs secretory expression instead of intracellular expression after a plurality of key links such as transcription, translation, transport, glycosylation modification and the like. Therefore, the current flow detection technology which can only detect the change of the transcription level can not meet the requirement of screening the cellulase with improved enzyme activity.

Disclosure of Invention

In order to solve the problem that trichoderma reesei transformants with improved enzyme activity cannot be rapidly screened in the prior art, the application provides a recombinant expression vector for rapidly screening recombinant strains, and rapid screening and application.

The invention aims to provide a recombinant expression vector for rapidly screening high-expression strains.

It is still another object of the present invention to provide a recombinant strain containing the above recombinant expression vector.

The invention further aims to provide a method for rapidly screening trichoderma reesei with high cellulase expression.

The invention further aims to provide a method for rapidly screening trichoderma reesei with high cellulase expression and high enzyme activity.

According to the embodiment of the invention, the recombinant expression vector for rapidly screening the high-expression strain comprises the following components in sequence from upstream to downstream in a gene expression cassette of the recombinant expression vector: the kit comprises a cbh1 promoter, a red fluorescent protein gene DsRed, a cell surface protein anchoring signal peptide gene AfMp1 and a cbh1 terminator, wherein the nucleotide sequence of the DsRed gene is shown as SEQ ID No.2, and the nucleotide sequence of the AfMp1 gene is shown as SEQ ID No. 3.

The nucleotide sequence of the cbh1 promoter is shown in SEQ ID NO. 1:

Tcaacctttggcgtttccctgattcagcgtacccgtacaagtcgtaatcactattaacccagactgaccggacgtgttttgcccttcatttggagaaataatgtcattgcgatgtgtaatttgcctgcttgaccgactggggctgttcgaagcccgaatgtaggattgttatccgaactctgctcgtagaggcatgttgtgaatctgtgtcgggcaggacacgcctcgaaggttcacggcaagggaaaccaccgatagcagtgtctagtagcaacctgtaaagccgcaatgcagcatcactggaaaatacaaaccaatggctaaaagtacataagttaatgcctaaagaagtcatataccagcggctaataattgtacaatcaagtggctaaacgtaccgtaatttgccaacggcttgtggggttgcagaagcaacggcaaagccccacttccccacgtttgtttcttcactcagtccaatctcagctggtgatcccccaattgggtcgcttgtttgttccggtgaagtgaaagaagacagaggtaagaatgtctgactcggagcgttttgcatacaacc

aagggcagtgatggaagacagtgaaatgttgacattcaaggagtatttagccagggatgcttgagtgtatcgtgtaaggaggtttgtctgccgatacgacgaatactgtatagtcacttctgatgaagtggtccatattgaaatgtaagtcggcactgaacaggcaaaagattgagttgaaactgcctaagatctcgggccctcgggccttcggcctttgggtgtacatgtttgtgctccgggcaaatgcaaagtgtggtaggatcgaacacactgctgcctttaccaagcagctgagggtatgtgataggcaaatgttcaggggccactgcatggtttcgaatagaaagagaagcttagccaagaacaatagccgataaagatagcctcattaaacggaatgagctagtaggcaaagtcagcgaatgtgtatatataaaggttcgaggtccgtgcctccctcatgctctccccatctactcatcaactcagatcctccaggagacttgtacaccatcttttgaggcacagaaacccaatagtcaaccgcggactgcgcatcatgtatcggaagttggccgtcatctcggccttcttggccacagctcgtgct

the nucleotide sequence of the DsRed gene is shown in SEQ ID NO. 2:

ggcggctccggctccggctccggctccagcactggtactgccactgcctccaccagcaccaacctcctcAAGactggcgccgccagcaaggagcacttcagctactccctcggcggtgccgtcgtcgcggccgccatcgccgtcgctctctaa

the nucleotide sequence of the AfMp1 gene is shown in SEQ ID NO. 3:

Atggacaacaccgaggacgtcatcaaggagttcatgcagttcaaggtgcgcatggagggctccgtgaacggccactacttcgagatcgagggcgagggcgagggcaagccctacgagggcacccagaccgccaagctgcaggtgaccaagggcggccccctgcccttcgcctgggacatcctgtccccccagttccagtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacatgaagctgtccttccccgagggcttcacctgggagcgctccatgaacttcgaggacggcggcgtggtggaggtgcagcaggactcctccctgcaggacggcaccttcatctacaaggtgaagttcaagggcgtgaacttccccgccgacggccccgtaatgcagaagaagactgccggctgggagccctccaccgagaagctgtacccccaggacggcgtgctgaagggcgagatctcccacgccctgaagctgaaggacggcggccactacacctgcgacttcaagaccgtgtacaaggccaagaagcccgtgcagctgcccggcaaccactacgtggactccaagctggacatcaccaaccacaacgaggactacaccgtggtggggcagtacgagcacgccgaggcccgccactccggctcccag

the nucleotide sequence of the cbh1 terminator is shown in SEQ ID NO. 4:

Agctccgtggcgaaagcctgacgcaccggtagattcttggtgagcccgtatcatgacggcggcgggagctacatggccccgggtgatttattttttttgtatctacttctgacccttttcaaatatacggtcaactcatctttcactggagatgcggcctgcttggtattgcgatgttgtcagcttggcaaattgtggctttcgaaaacacaaaacgattccttagtagccatgcattttaagataacggaatagaagaaagaggaaattaaaaaaaaaaaaaaaacaaacatcccgttcataacccgtagaatcgccgctcttcgtgtatcccagtaccacggcaaaggtatttcatgatcgttcaatgttgatattgttcccgccagtatggctccacccccatctccgcgaatctcctcttctcgaacgcggtagtggcgcgccaattggtaatgacccatagggagacaaacagcataatagcaacagtggaaatt

the invention also provides a recombinant strain of the recombinant expression vector for rapidly screening the high-expression strain, wherein the recombinant strain is preferably Trichoderma reesei.

The invention also provides a method for rapidly screening the recombinant trichoderma reesei, which comprises the following steps:

(1) introducing a gene expression cassette containing an element cbh1 promoter, a red fluorescent protein gene DsRed, a cell surface protein anchoring signal peptide gene AfMp1 and a cbh1 terminator into a plasmid to obtain a recombinant expression vector, wherein the nucleotide sequence of the DsRed gene is shown as SEQ ID No.2, and the nucleotide sequence of the AfMp1 gene is shown as SEQ ID No. 3;

(2) transforming host cells by using the recombinant expression vector constructed in the step (1) to obtain a recombinant strain;

(3) culturing the recombinant strain, and inducing the red fluorescent protein to be expressed on the surface of the recombinant strain;

(4) recombinant strains displaying red fluorescence were screened.

According to the specific embodiment of the invention, the recombinant strain with the surface displaying red fluorescence is screened by a flow cytometer to obtain the recombinant strain.

The invention also provides a method for rapidly screening trichoderma reesei with high cellulase expression and high enzyme activity, which is characterized by comprising the following steps:

(1) introducing a gene expression cassette containing an element cbh1 promoter, a red fluorescent protein gene DsRed, a cell surface protein anchoring signal peptide gene AfMp1 and a cbh1 terminator into a plasmid to obtain a recombinant expression vector, wherein the nucleotide sequence of the DsRed gene is shown as SEQ ID No.2, and the nucleotide sequence of the AfMp1 gene is shown as SEQ ID No. 3;

(2) transforming host cells by using the recombinant expression vector constructed in the step (1) to obtain recombinant trichoderma reesei;

(3) introducing genes related to a protein secretion pathway into the recombinant trichoderma reesei obtained in the step (2), or randomly inserting vectors or genes into the recombinant trichoderma reesei obtained in the step (2) to obtain a mutant library of the recombinant trichoderma reesei;

(4) screening recombinant trichoderma reesei with the surface displaying strong red fluorescence by using a flow cytometer;

(5) and (4) determining the cellulase activity of the recombinant trichoderma reesei screened in the step (4) to obtain the recombinant trichoderma reesei with improved cellulase activity.

According to the embodiment of the invention, in the method for rapidly screening the protein secretion pathway-related genes related to the improvement of the cellulase activity, in the step (3), the protein secretion pathway-related genes include a bip1 gene, a hac1 gene, a ftt1 gene, a sso2 gene, a sar1 gene and a ypt1 gene.

The nucleotide sequence of the bip1 gene is shown as SEQ ID NO. 5:

ataaagtggcccatcgtcacctctcggcttcaactcgagtttttccctttttccttttcttcttcttcttcaaacaaagataccccccctcaacccggtgcaaccagcctggtccgaggacaaaacacgatagagctcgctgcgttggatcgctgcgcgtcttctcttgttctctctctcttttcttctctgcaacgcttataactctttttgcgcggggcatctgggaaaaccgtttcttcacacatctcttcttccacaatggctcgttcacggagctccctggccctcgggctgggcctgctctgctggatcacgctgctcttcgctcctctggcgtttgtcggaaaggccaatgccgcgagcgacgacgcggacaactacggcactgttatcggaattgtaagtcgactgacggcagcaaccccgccattttcttggtgttgatgctcaggcagccctgctaacacgcttctcctccgcccaggatctcggaactacctacagctgcgtcggtgtgatgcagaagggcaaggttgagattctcgtcaacgaccagggtaaccgaatcactccctcctacgtggcctttaccgacgaggagcgtctggttggcgattccgccaagaaccaggccgccgccaaccccaccaacaccgtctacgatgtcaagtcagttctaccgccctgttggcttctattgtataagtggacaattagctaactgttgtcacaggcgattgattggccgcaaattcgacgagaaggagatccaggccgacatcaagcacttcccctacaaggtcattgagaagaacggcaagcccgtcgtccaggtccaggtcaacggccagaagaagcagtt cactcccgaggagatttctgccatgattcttggcaagatgaaggaggttgccgagtcgtacctgggcaagaaggttacccacgccgtcgtcaccgtccctgcctacttcaacgtgagtcttttccccgaaattcctcgaggattccaagagccatctgctaacagcccgataggacaaccagcgacaggccaccaaggacgccggtaccattgccggcttgaacgttctccgaatcgtcaacgaacccaccgctgccgctatcgcctatggtctggacaagaccgacggtgagcgccagatcattgtctacgatctcggtggtggtacctttgatgtttctctcctgtccattgacaatggcgtcttcgaggtcttggctaccgccggtgacacccaccttggtggtgaggactttgaccagcgcattatcaactacctggccaaggcctacaacaagaagaacaacgtcgacatctccaaggacctcaaggccatgggcaagctcaagcgtgaagccgaaaaggccaagcgtaccctctcttcccagatgagcactcgtatcgaaatcgaggccttcttcgagggcaacgacttctccgagactctcacccgggccaagttcgaggagctcaacatggacctcttcaagaagaccctgaagcctgtcgagcaggttctcaaggacgccaacgtcaagaagagcgaggttgacgacatcgttctggtcggcggttccacccgtatccccaaggttcagtctcttatcgaggagtactttaacggcaagaaggcttccaagggtatcaaccccgacgaggctgttgctttcggtgccgccgtccaggccggtgtcctttctggtgaggaaggtaccgatgacattgttctcatggacgtcaaccccctgactctcggtatcgagaccactggcggagtcatgaccaagctcattccccgcaacacccccatccccactcgcaagagccagatcttctcgactgctgccgataaccagcccgtcgtcctgatccaggtcttcgagggtgagcgttccatgaccaaggacaacaacctcctgggcaagttcgagcttaccggcattcctcctgccccccgcggtgtcccccagattgaggtttccttcgagttggatgccaacggtatcctcaaggtctccgctcacgacaagggcaccggcaagcaggagtccatcaccatcaccaacgacaagggccgtctcacccaggaggagattgaccgcatggttgccgaggccgagaagttcgccgaggaggacaaggctacccgtgagcgcatcgaggcccgtaacggtcttgagaactacgccttcagcctgaagaaccaggtcaatgacgaggagggcctcggcggcaagattgacgaggaggacaaggagactgtaagttgaagcgatccatcactgctttctgatgcggacatgtcacactaacacttgaccagattcttgacgccgtcaaggaggctaccgagtggctcgaggagaacggcgccgacgccactaccgaggactttgaggagcagaaggagaagctgtccaacgtcgcctaccccatcacctccaagatgtaccagggtgctggtggctccgaggacgatggcgacttccacgacgaattgtaaaaaattaaaaaaagggaaattattgatgcatagatacttattagaggaaccaaagaagttcccaggtgttatcgtcggttatgacgcggatgtgttttcagtcttgtaaagttcgaatgcagctctgagtgtagtagatgcataaatgaatc

hac1 gene has a nucleotide sequence shown in SEQ ID NO. 6:

ATGGCGTTCCAGCAGTCGTCTCCCCTCGTCAAGTTTGAGGCCTCTCCCGCCGAATCCTTCCTCTCCGCCCCCGGCGACAACTTCACATCCCTCTTCGCCGACTCAACACCCTCAACACTTAACCCTCGGGACATGATGACCCCTGACAGCGTCGCCGACATCGACTCTCGCCTGTCCGTCATCCCCGAATCACAGGACGCGGAAGATGACGAATCACACTCCACATCCGCTACCGCACCCTCTACCTCAGAAAAGAAGCCCGTCAAGAAGAGGAAATCATGGGGCCAGGTTCTTCCTGAGCCCAAGACCAACCTCCCTCCTCGAAAACGTGCAAAGACGGAAGATGAAAAGGAGCAGCGCCGCGTCGAGCGTGTTCTCCGCAACCGCCGCGCCGCGCAGTCCTCGCGCGAGCGCAAGAGGCTCGAGGTCGAGGCTCTCGAGAAGCGCAACAAGGAGCTCGAGACGCTCCTCATCAACGTCCAGAAGACCAACCTGATCCTCGTCGAGGAGCTCAACCGCTTCCGACGCAGCTCAGGCGTCGTCACCCGCTCGTCCTCCCCCCTCGACTCTCTCCAGGACAGCATCACTCTCTCCCAGCAACTCTTTGGCTCGCGGGATGGCCAAACCATGTCCAACCCCGAGCAGTCCTTGATGGACCAGATCATGAGATCTGCCGCTAACCCTACCGTTAACCCGGCCTCTCTTTCCCCCTCCCTCCCCCCCATCTCGGACAAGGAGTTCCAGACCAAGGAGGAGGACGAGGAACAGGCCGACGAAGATGAAGAGATGGAGCAGACATGGCACGAGACCAAAGAAGCCGCCGCCGCCAAGGAGAAGAACAGCAAGCAGTCCCGCGTCTCCACTGATTCGACACAACGTCCTGCAGTGTCAATCGGTGGAGATGCCGCTGTCCCTGTCTTCTCAGACGACGCCGGCGCAAACTGCCTTGGCCTGGACCCTGTTCATCAGGATGATGGTCCTTTCAGCATCGGCCATTCTTTCGGCCTGTCAGCGGCCCTTGATGCAGATCGCTATCTCCTCGAAAGCCAACTTCTCGCTTCGCCCAACGCCTCAACTGTTGACGACGATTATCTGGCTGGTGACTCTGCCGCCTGCTTCACGAATCCTCTCCCCTCCGACTACGACTTCGACATCAACGACTTCCTCACAGACGACGCAAACCACGCCGCCTATGACATTGTGGCAGCGAGCAACTATGCCGCTGCGGACCGCGAGCTCGACCTCGAGATCCACGACCCTGAGAATCAGATCCCTTCGCGACATTCTATCCAGCAGCCCCAGTCTGGCGCGTCCTCTCATGGATGCGACGATGGCGGCATTGCGGTTGGTGTCTGA

according to the embodiment of the present invention, in the step (3) of the method for rapidly screening a strain highly expressing a gene associated with a protein secretory pathway, a gene mutant library of a recombinant strain is constructed using an Agrobacterium tumefaciens-mediated recombinant strain.

According to the specific embodiment of the invention, in the step (3), the recombinant vector is screened, wherein a Ti plasmid is used as a backbone plasmid, the Ti plasmid is connected with pry4 gene, and the nucleotide sequence of pry4 gene is shown as SEQ ID NO 7:

gactgacccccccggttgggcccctcgtcccgtctccaacagagcaccaccagacaaagacccctgcccgcgcgaatccagacccccccagcaattccgggcctcgttgatcctcctctactgtagttgtacatacatacctaccgactgcattgcattggtacagctgcaggcacttccaggcacggccaccaaattgcagcggcccttgcttgcttgcttggttcgagaactaggatctgtgtcttttgccttgccttgtcttgtctgggttcctgctcgtctgcggcaatcggaacgccgccagtgcggtgccaagcaaaccagccaaggtaggtacctaccactaggcttcttttctcgttgtctcactctctcttttcctctttgtcctctcttatccccatcttttctctctctctctgctcctttcctaaccacttccctacctttctctttttccttttcttgtcatctccatcttggctgacgaaaaaggtctgactgggtaggtattatctggcagacttgtgtgtatcattcaccctatttctgcttcatagtacatgtactgtacctgaacggctcaaccgctatttacgactcttatttttttgtggcgttggtcacgtttgccagctgttgtccgtctttctagggctcctcaaacttgacctgaccgagctccctttctggacccggtgggcttcact tccagctgctgagcgacctgagccgaacatcctcagtccttgtccagcgcaattcattttctttccttttctttttttttattcctttctttacttttattctctctttttctcctcttcctcttcttcttctttctcctcctcctccatatcctcactctcgtctccctcattactaccctctcggctcctcaggtccaccaaccctcccgcacccaaacctctgccgctgaaacccattcggtggtcgccgttttttttttttttttctcacccccaaagtcgcaatatcgggtatcgccgccggcattgaatcgccttctccgctagcatcgactactgctgctctgctctcgttgccagcgctgctccctagaattttgaccaggggacgagcccgacattaaagcaactccctcgcctcgagacgactcggatcgcacgaaattctcccaatcgccgacagttcctactcctcttcctcccgcacggctgtcgcgcttccaacgtcattcgcacagcagaattgtgccatctctctcttttttttccccccctctaaaccgccacaacggcaccctaagggttaaactatccaaccagccgcagcctcagcctctctcagcctcatcagccatggcaccacacccgacgctcaaggccaccttcgcggccaggagcgagacggcgacgcacccgctgacggcttacctgttcaagctcatggacctcaaggcgtccaacctgtgcctgagcgccgacgtgccgacagcgcgcgagctgctgtacctggccgacaagattggcccgtcgattgtcgtgctcaagacgcactacgacatggtctcgggctgggacttccacccggagacgggcacgggagcccagctggcgtcgctggcgcgcaagcacggcttcctcatcttcgaggaccgcaagtttggcgacattggccacaccgtcgagctgcagtacacgggcgggtcggcgcgcatcatcgactgggcgcacattgtcaacgtcaacatggtgcccggcaaggcgtcggtggcctcgctggcccagggcgccaagcgctggctcgagcgctacccctgcgaggtcaagacgtccgtcaccgtcggcacgcccaccatggactcgtttgacgacgacgccgactccagggacgccgagcccgccggcgccgtcaacggcatgggctccattggcgtcctggacaagcccatccactcgaaccggtccggcgacggccgcaagggcagcatcgtctccatcaccaccgtcacccagcagtacgagtccgtctcctcgccccggttaacaaaggccatcgccgagggcgacgagtcgctcttcccgggcatcgaggaggcgccgctgagccgcggcctcctgatcctcgcccaaatgtccagccagggcaacttcatgaacaaggagtacacgcaggcctgcgtcgaggccgcccgggagcacaaggactttgtcatgggcttcatctcgcaggagacgctcaacaccgagcccgacgatgcctttatccacatgacgcccggctgccagctgccccccgaagacgaggaccagcagaccaacggatcggtcggtggagacggccagggccagcagtacaacacgccgcacaagctgattggcatcgccggtagcgacattgccattgtgggccggggcatcctcaaggcctcagaccccgtagaggaggcagagcggtaccgatcagcagcgtggaaagcctacaccgagaggctgctgcgataggggagggaagggaagaaagaagtaaagaaaggcatttagcaagaagggggaaaagggagggaggacaaacggagctgagaaagagctcttgtccaaagcccggcatcatagaatgcagctgtatttaggcgacctctttttccatcttgtcgatttttgttatgacgtaccagttgggatgatggatgattgtaccccagctgcgattgatgtgtatctttgcatgcaacaacacgcgatggcggaggcgaactgcacattggaaggttcatatatggtcctgacatatctggtggatctggaagcatggaattgtatttttgatttggcatttgcttttgcgcgtggagggaacatatcaccctcgggcatttttcatttggtaggatggtttggatgcagttgatcgataagcttgatatcg

the screening marker gene is pyr4, and is a gene of a nutrition selective marker gene orotidine-5' -monophosphate decarboxylase.

The invention also provides a method for rapidly screening the protein secretion pathway related genes which are highly related to improving the enzyme activity of the cellulase, which comprises the following steps:

(1) constructing a recombinant expression vector of a DsRed-AfMP1 fusion gene containing a red fluorescent protein gene and a cell surface protein anchoring signal peptide gene, wherein the nucleotide sequence of the DsRed gene is shown as SEQ ID NO.2, and the nucleotide sequence of the AfMp1 gene is shown as SEQ ID NO. 3;

(2) transforming a strain by using the recombinant expression vector constructed in the step (1) to obtain a recombinant strain;

(3) introducing a target gene to be screened into the recombinant strain obtained in the step (2), or randomly inserting a vector or a gene into the recombinant strain obtained in the step (2) to obtain a mutant library of the recombinant strain;

(4) screening the recombinant strain with the surface displaying red fluorescence by using a flow cytometer;

(5) determining the cellulase activity of the recombinant strain with the surface displaying red fluorescence screened in the step (4) to obtain the recombinant strain with improved cellulase activity;

(6) and determining the exogenous target gene expressed in the recombinant strain with the improved cellulase activity or the interfered endogenous gene, thereby obtaining the protein secretion pathway related gene related to the improvement of the cellulase activity.

The invention has the beneficial effects that:

the invention constructs a recombinant expression vector containing a fusion gene expression cassette, wherein the expression cassette is sequentially connected with a cbh1 promoter, a cbh1 gene signal peptide sequence, a red fluorescent protein gene, an Aspergillus fumigatus-derived MP1 anchored protein signal peptide sequence and a cbh1 gene terminator from upstream to downstream. The recombinant expression vector can induce the surface of a strain to display fluorescence, thereby being beneficial to rapid screening.

The invention provides a method for rapidly screening recombinant trichoderma reesei for expressing cellulase, which is characterized in that the expression capacity of the cellulase is identified according to the red fluorescence intensity of the recombinant trichoderma reesei for expressing the cellulase, and the higher the red fluorescence intensity is, the stronger the ability of the recombinant trichoderma reesei for expressing the cellulase is. To correlate expression of DsRed with expression of t. CBH1 is cellulase with the highest expression level in Trichoderma reesei, and accounts for 50-60% of secreted protein. Therefore, the DsRed gene is connected with the downstream of a strong induction CBH1 promoter, the red depth of the DsRed gene is positively correlated with the induction degree of the CBH1 promoter, and then the DsRed gene is coupled with a flow cytometry to screen the Trichoderma reesei cellulase high-yield strain in high throughput. Experiments prove that the red fluorescent protein with the signal peptide can be effectively anchored on cell walls, and the higher the red fluorescence intensity is, the higher the cellulase expression capability of the recombinant trichoderma reesei is. The fluorescence intensity of the red fluorescent protein can well represent the expression quantity of the cellulase, and the fluorescence intensity of the red fluorescent protein on the cell wall is in positive correlation with the secretion quantity of the cellulase in the fermentation liquid.

Drawings

FIG. 1 shows a Trichoderma reesei colony, and the left image shows SUS 2: starting strain, right panel SUS 4: a positive transformant transformed with the plasmid pDsRed-AfMP 1;

FIG. 2 shows Trichoderma reesei hyphae under a fluorescence microscope, and the left image is Trichoderma reesei hyphae displaying red fluorescent protein on the surface; the right panel shows Trichoderma reesei mycelia observed in a fluorescence microscope under bright field;

FIG. 3 is a flow cytometry plot of secretion pathway-associated plasmid transformants;

FIG. 4 shows the colony PCR results of secretion pathway-related transformants by flow screening, in which 4-1 is hac1 colony PCR, 4-2 is bip1 colony PCR, 4-3 is ypt1 colony PCR, 4-4 is sso2 colony PCR, 4-5 is sar1 colony PCR, and 4-6 is ftt1 colony PCR;

FIG. 5 is a graph showing the result of EG enzyme activity measurement of shake flask fermentation broth of starting strains and secretion pathway related gene transformants;

FIG. 6 is a copy number identification chart of transformants of genes related to the modified secretory pathway;

FIG. 7 shows the results of colony PCR to verify the transformation of Ti-pyr4 plasmid into Agrobacterium;

FIG. 8 is a diagram of flow cytometry for random insertion of Agrobacterium into a mutant library;

FIG. 9 shows the results of PCR verification of flow-screened colonies of Agrobacterium randomly inserted into transformants;

FIG. 10 shows the results of the enzyme activity measurement of Agrobacterium randomly inserted transformants.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

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

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The components of the MM medium described in the examples below include: (NH)₄)₂SO₄，5.0g/L；KH₂PO₄，15.0g/L；MgSO₄·7H₂O，0.6g/L；CaCl₂·2H₂O，0.6g/L；CoCl₂·6H₂O，0.0037g/L；FeSO₄·7H₂O，0.005g/L；ZnSO₄·7H₂O，0.0014g/L；MnSO₄·H₂O, 0.0016 g/L; glucose (or carbon source such as Avicel), 20 g/L; the solvent used is water.