CN112094330B

CN112094330B - Polythiodiketopiperazine synthesis related protein and related biological material and application thereof

Info

Publication number: CN112094330B
Application number: CN202011001311.1A
Authority: CN
Inventors: 尹文兵; 刘欢
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2020-09-22
Filing date: 2020-09-22
Publication date: 2022-04-12
Anticipated expiration: 2040-09-22
Also published as: CN112094330A

Abstract

The invention discloses a protein related to synthesis of polythiodiketopiperazine, a related biological material and application thereof. The protein related to synthesis of the polysulfidedione piperazine is named TdaG, is derived from Trichoderma carbonum (Trichoderma hypoxylon), and has an amino acid sequence of the protein of a sequence 2 in a sequence table. The substance for regulating the expression of TdaG and the coding gene thereof can be used for regulating the synthesis of the polythiodiketopiperazine by trichoderma char, such as improving the ability of the trichoderma char to synthesize the polythiodiketopiperazine or endowing the trichoderma char with the ability of synthesizing the polythiodiketopiperazine. The TdaG gene knockout mutant strain of trichoderma reesei obtained by knocking out the TdaG gene of trichoderma reesei can generate polythiodiketopiperazine (a compound with a structural formula as shown in a formula 1) with anti-colon cancer and liver cancer activities. The compound with the structural formula as formula 1 can be used for developing medicaments for resisting colon cancer and liver cancer.

Description

Polythiodiketopiperazine synthesis related protein and related biological material and application thereof

Technical Field

The invention relates to a protein related to synthesis of polythiodiketopiperazine, a related biological material and application thereof.

Background

Trichoderma has the ability to produce secondary metabolites with rich structure and diverse activity, and is an important source for drug discovery. The structure of the polythiodiketopiperazine compound is characterized in that the compound contains a diketopiperazine mother nucleus with a sulfur bridge, and has abundant and diverse biological activities, such as biological activities of resisting tumors, bacteria and Alzheimer's disease. Previous studies have shown that trichoderma reesei can produce secondary metabolites with abundant structures, and the structural formulas of the currently isolated polythiodiketopiperazine compound derivatives trichodermamide a, aspergizine C and trichothecene toxin (harzianum B) are shown as follows:

disclosure of Invention

The technical problem to be solved by the invention is how to prepare the polythiodiketopiperazine with anti-tumor activity.

In order to solve the technical problems, the invention provides a protein related to synthesis of polythiodiketopiperazine, which is named Tdag and is derived from Trichoderma reesei (Trichoderma hypoxylon) and is A1), A2) or A3) as follows:

A1) the amino acid sequence is protein of a sequence 2 in a sequence table;

A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in an amino acid sequence shown in a sequence 2 in a sequence table, has more than 90% of identity with the protein shown in A1), and is related to synthesis of polysulfidediolpiperazine;

A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).

In the above protein, sequence 2 in the sequence table is composed of 497 amino acid residues.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

In the above protein, the protein tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In the above proteins, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above protein, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

Among the above proteins, the Tdag may be derived from Trichoderma reesei (Trichoderma hypoxylon).

Biological materials related to TdaG are also within the scope of the invention.

The TdaG-related biological material provided by the invention is any one of the following B1) -B9):

B1) a nucleic acid molecule encoding TdaG;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2);

B8) a nucleic acid molecule that reduces or inhibits expression of a gene encoding TdaG;

B9) an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line comprising the nucleic acid molecule according to B8).

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In the above materials, the nucleic acid molecule according to B1) may specifically be a gene represented by 1) or 2) below:

1) the coding sequence (CDS) of the coding chain is a cDNA molecule of the 1 st to 1494 th nucleotides in the sequence 1 in the sequence table;

2) the nucleotide is a DNA molecule at 1393-3177 of the sequence 3 in the sequence table.

Sequence 1 in the sequence table is the coding strand of the cDNA gene of TdaG and consists of 1494 nucleotides. The 1393-3177 position of the sequence 3 in the sequence table is the coding strand of the TdaG genome gene, and in the sequence 3, the 1393-1563-position first exon nucleotide sequence, the 1564-1616-position first intron nucleotide sequence, the 1617-1662-position second exon nucleotide sequence, the 1663-1728-position second intron nucleotide sequence, the 1729-2270-position third exon nucleotide sequence, the 2271-2322-position third intron nucleotide sequence, the 2323-2610-position fourth exon nucleotide sequence, the 2611-2669-position fourth intron nucleotide sequence, the 2670-position 2947-position fifth exon nucleotide sequence, the 2948-3008-position fifth intron nucleotide sequence and the 3009-3177-position sixth exon nucleotide sequence.

In the above biological material, the nucleic acid molecule of B8) may be a nucleic acid molecule capable of knocking out a gene encoding TdaG, such as a DNA molecule represented by sequence 4 in the sequence listing.

Wherein, the sequence 4 in the sequence table consists of 5123 nucleotides. In the sequence 4, the 1 st to 1205 th positions are the nucleotide sequences of the upstream homology arms of the TdaG genomic gene (same as the 143 nd-1347 th positions of the sequence 3), the 1261 th-3748 th positions are the nucleotide sequences of the hph gene (hygromycin phosphotransferase gene, hygromycin resistance gene), and the 3910 th-5123 th positions are the nucleotide sequences of the downstream homology arms of the TdaG genomic gene (same as the 2535 th-3748 th positions of the sequence 3).

In the above biological material, the recombinant microorganism containing the nucleic acid molecule of B8) may be a recombinant Trichoderma charratum obtained by introducing a nucleic acid molecule capable of knocking out the gene encoding the protein into Trichoderma charosum (Trichoderma hypoxylon) which does not contain the gene encoding the protein; the DNA molecule shown in the sequence 4 in the sequence table is introduced into the recombinant Trichoderma carbonicola (Trichoderma hypoxylon) to obtain the recombinant Trichoderma carbonicola, and the recombinant Trichoderma carbonicola does not contain the coding gene of the protein. The recombinant trichoderma carbonum is a trichoderma carbonum Tdag gene knockout mutant strain in one embodiment of the invention. The Trichoderma carbonum Tdag gene knockout mutant is a recombinant microorganism obtained by replacing part of the gene (1348-position 2534 in sequence 3) of the Tdag of Trichoderma carbonum (Trichoderma hypoxylon) with a hph gene of which the nucleotide sequence is 1261-position 3748 in sequence 4 in a sequence table and keeping other nucleotides of the Trichoderma carbonum (Trichoderma hypoxylon) unchanged.

In the above-mentioned biological materials, the expression cassette containing a nucleic acid molecule encoding TdaG (TdaG gene expression cassette) according to B2) means a DNA capable of expressing TdaG in a host cell, and the DNA may include not only a promoter which initiates transcription of the TdaG gene but also a terminator which terminates transcription of the TdaG.

In the above biological material, the recombinant microorganism may be specifically yeast, bacteria, algae and fungi.

In order to solve the technical problems, the invention also provides the application of the protein or the related biological material thereof or the substance for regulating and controlling the gene expression in regulating and controlling the synthesis of the polythiodiketopiperazine by trichoderma carbonum; the gene encodes the protein; the structural formula of the polythiodiketopiperazine is shown as formula 1:

the Arabic numerals in the formula 1 are carbon atom epitopes in a chemical structure, and R is hydroxyl or methoxyl. The compound in which the R group is a hydroxyl group is hereinafter referred to as compound 1, and the compound in which the R group is a methoxy group is hereinafter referred to as compound 2.

In the above application, the substance for regulating gene expression may be a substance for regulating at least one of the following 6 kinds of regulation: 1) regulation at the level of transcription of said gene; 2) regulation after transcription of the gene (i.e., regulation of splicing or processing of a primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; 6) post-translational regulation of the gene (i.e., regulation of the activity of a protein translated from the gene).

In the above application, the regulation of gene expression may be inhibition or reduction of the gene expression.

In the above application, the substance for regulating gene expression may be an agent for inhibiting or reducing the gene expression. The agent that inhibits or reduces expression of the gene may be an agent that knocks out the gene. The agent for knocking out the gene may be an agent for knocking out the gene by homologous recombination, an agent for knocking out the gene by CRISPR-Cas 9. The reagent for knocking out the gene by homologous recombination can comprise a DNA molecule shown as a sequence 4 in a sequence table.

In the above application, the agent for inhibiting or reducing the expression of the gene may comprise a polynucleotide targeting the gene, such as siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

In the application, the regulation and control of the synthesis of the polythiodiketopiperazine by the trichoderma char can improve the ability of the trichoderma char to synthesize the polythiodiketopiperazine or endow the trichoderma char to synthesize the polythiodiketopiperazine.

In order to solve the technical problems, the invention also provides a recombinant trichoderma carbonicola capable of producing the polythiodiketopiperazine.

The recombinant trichoderma charred mass producing the polythiodiketopiperazine does not contain the coding gene of the protein.

The recombinant trichoderma carbonicola can be a recombinant bacterium obtained by knocking out a target gene of trichoderma carbonicola, and the target gene coding amino acid sequence is protein of a sequence 1 in a sequence table.

In the recombinant trichoderma charred, the coding region of the coding chain of the target gene is 1393-3177 th site of the sequence 3 in the sequence table.

The application of the recombinant trichoderma carbonicum in the polythiodiketopiperazine also belongs to the protection scope of the invention.

In the application, the recombinant trichoderma reesei is cultured by a culture medium to obtain a fermentation product; obtaining said polythiodiketopiperazine from said fermentation product.

In the above application, the culture medium may be a trichoderma culture medium (a culture medium capable of culturing trichoderma), which may be a solid culture medium, a semi-solid culture medium, or a liquid culture medium. The medium may be a rice medium, a potato dextrose agar medium, or other fungal fermentation media well known to those skilled in the art, and the like. The rice culture medium can be made of rice and water, can also be made of rice, water and inorganic salt, can also be made of rice, water and nitrogen source, and can also be made of rice, water, nitrogen source and inorganic salt. The potato glucose agar medium can be prepared from potato, glucose, agar and water, or from potato, glucose, agar, water and inorganic salt, or from potato, carbon source, agar, water and nitrogen source, or from potato, carbon source, agar, nitrogen source and inorganic salt. The other fungal fermentation media known to those skilled in the art may be prepared from one or more of a fast-acting, slow-acting carbon source, one or more of a fast-acting, slow-acting nitrogen source, water and/or inorganic salts, and the like.

In the above, the rice may be rice or brown rice. The rice or brown rice is a product of rice. The rice refers to a fruit from which rice husk is not removed, and is composed of husk, pericarp, seed coat, endosperm, aleurone layer, endosperm and embryo. The brown rice is the product of rice with husk removed and other parts reserved; rice refers to a product in which only the endosperm is retained and the rest of the rice is completely removed. The carbon source is a nutrient for microbial growth, and is a carbon-containing compound, including quick-acting and slow-acting carbon sources such as saccharides, grease, organic acids, organic acid esters and micromolecular alcohols. The nitrogen source is a substance for providing nitrogen elements required by the nutrition of microorganisms, and comprises quick-acting and slow-acting nitrogen sources such as peanut cake powder, soybean cake powder, yeast powder, peptone, ammonia water, ammonium salt, nitrate and the like.

In the above application, the temperature of the culture can be 25 ℃, and the culture time can be 20 days.

The invention also provides a compound with antitumor activity or a pharmaceutically acceptable salt thereof.

The compound with the anti-tumor activity or the pharmaceutically acceptable salt thereof provided by the invention is the polythiodiketopiperazine, namely the compound with the structural formula 1.

Hereinbefore, the compound of formula 1 includes racemates, enantiomers, diastereomers, tautomers, polymorphs, pseudopolymorphs, amorphous forms, hydrates or solvates.

The compound of formula 1 of the present invention can be used in the form of a pharmaceutically acceptable salt derived from an inorganic acid or an organic acid. The term "pharmaceutically acceptable salt" refers to salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts are described in detail, for example, in s.m. berge, et al, j.pharmaceutical Sciences,1977,66: 1. The salts may be prepared by reacting the free base functionality of the compounds of the present invention with a suitable organic acid.

The application of the compound with the structural formula as formula 1 or the pharmaceutically acceptable salt thereof in preparing the antitumor drug also belongs to the protection scope of the invention.

The invention also provides an anti-tumor and/or anti-tumor cell medicament.

The anti-tumor and/or anti-tumor cell medicine provided by the invention contains a compound with a structural formula 1 or pharmaceutically acceptable salt thereof.

The application of the compound with the structural formula as formula 1 or the pharmaceutically acceptable salt thereof in preparing anti-tumor and/or anti-tumor cell medicines also belongs to the protection scope of the invention.

Above, the tumor may be a solid tumor and/or a non-solid tumor. The solid tumor may be liver cancer or colorectal cancer. The colorectal cancer may be colorectal adenocarcinoma.

Experiments prove that the Trichoderma reesei TdaG gene knockout mutant strain obtained by knocking out the TdaG gene of Trichoderma reesei (Trichoderma hypoxylon) can generate polysulphide diketopiperazine (a compound with a structural formula of 1) with anti-colon cancer and anti-liver cancer activities. Trichoderma reesei (Trichoderma hypoxylon) which is a wild type Trichoderma reesei cannot produce polythiodiketopiperazine with a structural formula of 1, and the Tdag is related protein synthesized by the polythiodiketopiperazine and is related gene synthesized by the polythiodiketopiperazine. The substance for regulating the expression of TdaG and the coding gene thereof can be used for regulating the synthesis of the polythiodiketopiperazine by trichoderma char, such as improving the ability of the trichoderma char to synthesize the polythiodiketopiperazine or endowing the trichoderma char with the ability of synthesizing the polythiodiketopiperazine. In vitro pharmacological activity experiments show that the compound 1 and the compound 2 have obvious inhibitory activity on colon cancer cell DLD1, and the half inhibitory concentrations are as follows: 11.03 mu M and 25 mu M, and the compound 1 and the compound 2 also have obvious inhibitory activity on liver cancer cells HepG2, and the half inhibitory concentrations are respectively as follows: 6.385 μ M and 15.925 μ M. Therefore, the compound with the structural formula as formula 1 can be used for developing medicaments for resisting colon cancer and liver cancer.

Drawings

FIG. 1 shows PCR of TdaG gene knock-out mutants of wild type Trichoderma Taenianum and Trichoderma Taenianum. A and C are designed positions of the primers, and B is an electrophoresis result of PCR amplification products of each primer pair. In the figure, M is a DNA molecular weight standard, WT is a wild type Trichoderma reesei strain, ". DELTA.tdaG" and "mutant" are knocked-out mutants of Trichoderma reesei TdaG gene, P1 is the result of PCR electrophoresis using P1F/R primer pair, P2 is the result of PCR electrophoresis using P2F/R primer pair, P3 is the result of PCR electrophoresis using P3F/R primer pair, 5F is the upstream homology arm of TdaG gene, 3F is the downstream homology arm of TdaG gene, and "tdaG" is TdaG gene (genomic gene).

FIG. 2 shows the LC-MS detection results of the Tdag gene knockout mutant solid fermentation culture of trichoderma carbonicola and the solid fermentation culture of wild trichoderma carbonicola. A is [ M + H ] in liquid chromatography-mass spectrometry detection of wild trichoderma carbonicola solid fermentation culture]⁺An extracted ion flow diagram with peak M/z of 449, and B is [ M + H ] in LC-MS detection of solid fermentation culture of Tdag gene knockout mutant strain of trichoderma carbonum]⁺An extracted ion flow diagram with peak M/z of 449 (an arrow indicates a compound 1), and C is [ M + H ] in LC-MS detection of wild Trichoderma reesei solid fermentation culture]⁺An extracted ion flow diagram with peak M/z of 463, and D is [ M + H ] in LC-MS detection of Trichoderma reesei Tdag gene knockout mutant strain solid fermentation culture]⁺Peak m/z is 463 extracted ion flow diagram (arrow indicates Compound 2).

FIG. 3 is a HR-ESI-MS mass spectrum of Compound 1.

FIG. 4 is a NMR spectrum of Compound 1.

FIG. 5 is a NMR carbon spectrum of Compound 1.

Figure 6 is an HSQC spectrum of compound 1.

Figure 7 is an HMBC map of compound 1.

FIG. 8 is a drawing of Compound 1¹H-¹H COSY map.

FIG. 9 is a HR-ESI-MS mass spectrum of Compound 2.

FIG. 10 is a NMR spectrum of Compound 2.

FIG. 11 is a NMR carbon spectrum of Compound 2.

Figure 12 is an HSQC spectrum of compound 2.

Figure 13 is an HMBC map of compound 2.

FIG. 14 is a drawing of Compound 2¹H-¹H COSY map.

Fig. 15 is a graph of the change in DLD1 cell inhibitory activity of different concentrations of compound 1 and compound 2. In the figure, 1 represents

compound

1, 2 represents compound 2; there are significant differences between processes with different letters.

FIG. 16 shows the change in inhibitory activity of compound 1 and compound 2 at different concentrations on HepG2 cells. In the figure, 1 represents

compound

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The colon cancer cell line DLD1 (human colorectal adenocarcinoma epithelial cells) in the following examples is a product of Guangzhou Saku Biotechnology Co., Ltd, and the culture conditions are as follows: in the presence of 5% CO₂In a humidified atmosphere of (2), cells were cultured at 37 ℃ in RPMI-1640(RPMI-1640+ 10% FBS + 1% P/S) supplemented with 10% FBS and 1% penicillin/streptomycin.

The human hepatoma cell line HepG2 in the following examples is a product of Fenghui Biotech Co., Ltd. The culture conditions were: in the presence of 5% CO₂In a humidified atmosphere of (2), cells were cultured at 37 ℃ in RPMI-1640(RPMI-1640+ 10% FBS + 1% P/S) supplemented with 10% FBS and 1% penicillin/streptomycin.

Trichoderma reesei or Trichoderma longibrachiatum in the following examples are Trichoderma reesei (Trichoderma hypoxylon) (Jingzu Sun, Yunfei Pei, Erweii Li, Wei Li, Kevin D.Hyde, Wen-Bing Yin, Xingzhong Liu.A. new species of Trichoderma hypoxylon hardwoods associated microorganisms. scientific reports.21November 2016) publicly available from the applicant, which was used only for repeating the relevant experiments of the present invention and was not otherwise usable.

Example 1 cloning of Polythiodiketopiperazine Synthesis-related protein and Gene encoding the same

The inventor of the invention separates and clones a polythiodiketopiperazine synthesis related protein from Trichoderma carbonum (Trichoderma hypoxylon), the amino acid sequence of the polythiodiketopiperazine synthesis related protein is shown as a sequence 2 in a sequence table, and the polythiodiketopiperazine synthesis related protein is named as Tdag. The gene encoding TdaG was named TdaG gene (tdaG for short), the nucleotide sequence of cDNA gene of TdaG was sequence 1 in the sequence table, and the nucleotide sequence of coding region of coding strand of genomic gene of TdaG was 1393-3177 th site of sequence 3 in the sequence table. In the sequence 3, the nucleotide sequence of the first exon at the 1393-position 1563, the nucleotide sequence of the first intron at the 1564-position 1616, the nucleotide sequence of the second exon at the 1617-position 1662, the nucleotide sequence of the second intron at the 1663-position 1728, the nucleotide sequence of the third exon at the 1729-position 2270, the nucleotide sequence of the third intron at the 2271-position 2322, the nucleotide sequence of the fourth exon at the 2323-position 2610, the nucleotide sequence of the fourth intron at the 2611-position 2669, the nucleotide sequence of the fifth exon at the 2670-position 2947, the nucleotide sequence of the fifth intron at the 2948-position 3008 and the nucleotide sequence of the sixth exon at the 3009-position 3177. Sequence 3 in the sequence table is a genomic gene containing TdaG of Trichoderma reesei (Trichoderma hypoxylon) and a section of genomic DNA of the upstream and downstream sequences thereof.

The TdaG genome gene is cloned as follows: the genomic DNA of Trichoderma carbonum (Trichoderma hypoxyleon) is taken as a template, F1(5'-atgctaggtctgagtacagtag-3') and R1(5'-ctacatccttcgagaaagtttaac-3') are taken as primers, and PCR amplification is carried out to obtain the genomic gene of TdaG of which the nucleotide sequence of the coding strand is 1393-th and 3177-th positions of the sequence 3 in the sequence table.

The TdaG genome gene and its upstream and downstream fragment cloning method as follows: using the genome DNA of Trichoderma carbonum (Trichoderma hypoxylon) as a template, and using a P2F primer (the nucleotide sequence is 5'-cacagctctctctagtgccg-3', corresponding to the 1 st-20 th site of the sequence 3) and a P3R primer (the nucleotide sequence is 5'-cgtcgtgtaagtgtcagcc-3', corresponding to the R th-3944 th site of the sequence 3) as primers, carrying out PCR amplification to obtain a DNA molecule of which the nucleotide sequence is the sequence 3 in the sequence table. The 1393-3177 position of the sequence 3 in the sequence table is the coding strand of the TdaG genome gene, and in the sequence 3, the 1393-1563-position first exon nucleotide sequence, the 1564-1616-position first intron nucleotide sequence, the 1617-1662-position second exon nucleotide sequence, the 1663-1728-position second intron nucleotide sequence, the 1729-2270-position third exon nucleotide sequence, the 2271-2322-position third intron nucleotide sequence, the 2323-2610-position fourth exon nucleotide sequence, the 2611-2669-position fourth intron nucleotide sequence, the 2670-position 2947-position fifth exon nucleotide sequence, the 2948-3008-position fifth intron nucleotide sequence and the 3009-3177-position sixth exon nucleotide sequence.

The TdaG cDNA gene was cloned as follows: extracting total RNA of Trichoderma carbonum (Trichoderma hypoxylon), performing reverse transcription on an extracted RNA sample according to a program of a first strand cDNA synthesis kit of Tiangen biochemistry company to synthesize first strand cDNA serving as a template of gene cloning, and performing PCR amplification by taking F1(5'-atgctaggtctgagtacagtag-3') and R1(5'-ctacatccttcgagaaagtttaac-3') as primers to obtain a cDNA gene of TdaG with a nucleotide sequence being a sequence 1 in a sequence table.

Example 2 functional verification of Polythiodiketopiperazine Synthesis-related proteins and genes encoding the same

1. Construction of TdaG genome knockout cassette

A genome knockout cassette whose nucleotide sequence is a double-stranded DNA of sequence 4 in the sequence listing, hereinafter referred to as TdaG, was prepared. Wherein, the sequence 4 in the sequence table consists of 5123 nucleotides. In the sequence 4, the 1 st to 1205 th positions are the nucleotide sequences of the upstream homology arm (5F) of the TdaG genomic gene (the same as the 143 nd position 1347 of the sequence 3), the 1261 th position 3748 th position is the nucleotide sequence of the hph gene (hygromycin phosphotransferase gene, hygromycin resistance gene), and the 3910 th position 5123 th position is the nucleotide sequence of the downstream homology arm (3F) of the TdaG genomic gene (the same as the 2535 th position 3748) of the sequence 3.

2. Knocking out TdaG by using PEG-mediated protoplast transformation method to obtain TdaG gene knock-out mutant strain of trichoderma carbonum

2.1 preparation of carbon-cluster trichoderma protoplast:

(1) activating Trichoderma charaeanum (Trichoderma hypoxylon) strains (hereinafter referred to as wild Trichoderma charaeanum) stored on the inclined plane, inoculating wild Trichoderma charaeanum blocks with the diameter of 2mm on a PDA (personal digital assistant) plate placed with cellophane, and culturing for 3 days at 25 ℃;

(2) collecting hyphae in the plate with toothpick to a sterilized 1.5mL centrifuge tube containing steel balls, adding 500 μ L PDB culture medium, and crushing with tissue crusher (60s, 70 Hz);

(3) inoculating the crushed thallus into a PDB liquid culture medium, culturing for 24 hours at 28 ℃ and 200rpm by a shaking table;

(4) the hyphae were collected by filtration using a funnel with Miracloth and disrupted by a tissue disruptor (60s, 70 Hz);

(5) inoculating to TG liquid culture medium, culturing at 28 deg.C and 200rpm for 12 hr;

(6) collecting hyphae with a Miracloth funnel, and washing with N-M to remove residual culture medium;

(7) transferring the hyphae to a 50mL triangular flask, and adding 10mL of filter sterilized enzymolysis liquid;

(8) carrying out enzymolysis at 28 ℃ and 100rpm for 6-8 h;

(9) filtering and collecting the protoplast by using a funnel with Miracloth, and rinsing the residual protoplast by using STC solution;

(10) pouring the filtrate into a 50mL centrifuge tube, and centrifuging at 5000rpm and 4 ℃ for 10min (horizontal rotor);

(11) removing the supernatant, adding 5mL of STC solution, transferring the resuspended protoplast to a 15mL centrifuge tube, centrifuging at 4 ℃ and 5000rpm for 10 min;

(12) and removing the supernatant, and adding a proper amount of STC solution to dilute the protoplast to a certain concentration to obtain the protoplast of the wild trichoderma carbonum.

2.2 PEG-mediated protoplast transformation:

(1) taking a 15mL centrifuge tube, adding 10 μ L of TdaG genome gene knockout box (5 μ g) of

step

1 and 100 μ L of wild Trichoderma carbonum protoplast of step 2.1 by using a pipette, mixing uniformly, and carrying out ice bath for 50 min;

(2) adding 1.25ml of PEG solution, slowly rotating the centrifugal tube to uniformly mix, and standing at room temperature for 30 min;

(3) adding 5mL of STC buffer, mixing uniformly, and inoculating about 1mL of mixed solution for each plate;

(4) immediately adding 5mL of Top SPDA (containing 30 mu g/mL of hygromycin) into each plate, and quickly and uniformly shaking;

(5) after the liquid is sucked dry, the liquid is inversely cultured for 5 to 7 days in an incubator at the temperature of 26 ℃, and the growth state of the thalli is observed;

(6) transferring the grown transformant to PDA containing 60 mug/mL hygromycin, and culturing for 1-2 days at 26 ℃;

(7) and (3) transferring the transformant to PDA platelets, inoculating one transformant to each plate, culturing at 26 ℃ for 2 days, inoculating to a PDB culture medium to extract genome DNA, carrying out PCR by using 3 pairs of primers of a P1F/R primer pair, a P2F/R primer pair and a P3F/R primer pair, carrying out electrophoresis on the obtained PCR product, and verifying the transformant: the P1F/R primer is designed on the TdaG genome gene, a correct TdaG knockout mutant strain cannot amplify a DNA band due to TdaG genome deletion, and a wild type Trichoderma reesei strain can amplify the band; the P1F/R primer pair consisted of a P1F primer (nucleotide sequence 5'-ctaacgcgctggtacgaatg-3', corresponding to position 1525-1544 of sequence 3) and a P1R primer (nucleotide sequence 5'-gcttctcgtgctagattac-3', corresponding to position 2330-2348 of sequence 3). The P2F/R primer pair consists of a P2F primer (nucleotide sequence is 5'-cacagctctctctagtgccg-3', corresponding to position 1-20 of sequence 3) and a P2R primer (nucleotide sequence is 5'-gcctatgcctacagcatcc-3', corresponding to position 1364-1382 of sequence 4), wherein the P2F primer is designed outside the upstream homology arm (5F) of the Tdag genomic gene, the P2R primer is designed inside the hph gene of the screening gene, the gene knockout mutant strain is subjected to homologous exchange, the genomic gene of the Tdag target gene is replaced by the screening gene, so that the Tdag gene knockout mutant strain of trichoderma carbonicola can amplify a band of 4bp (DNA fragment) by using the P2F/152R primer pair, and the wild type trichoderma carbonicola strain cannot amplify the band. The P3F/R primer pair consists of a P3F primer (nucleotide sequence is 5'-CTTGCTGCGGTGGATGGTTC-3', which corresponds to position 3517-3536 of sequence 4) and a P3R primer (nucleotide sequence is 5'-cgtcgtgtaagtgtcagcc-3', which corresponds to position 5'-CTTGCTGCGGTGGATGGTTC-3' -3944 of sequence 3), wherein the P3F primer is designed at the inner side of the hph gene of the screening gene, the P3R primer is designed at the outer side of the downstream homology arm of the Tdag genome gene, the P3F primer is designed at the inner side of the hph gene of the screening gene, a 1803bp band (DNA fragment) can be amplified by using the P3F/R primer pair, and the band cannot be amplified by the wild type Trichoderma reesei strain.

A DNA band cannot be amplified by utilizing the P1F/R primer pair, and a transformant which can amplify a 1524bp band by utilizing the P2F/R primer pair and a 1803bp band by utilizing the P3F/R primer pair is the Trichoderma reesei Tdag gene knockout mutant strain. The PCR verification results of the TdaG gene knockout mutant of the wild Trichoderma reesei strain and Trichoderma reesei are shown in FIG. 1. A is the designed position of the primer, and B is the PCR amplification result of each primer pair. In the figure, WT is a wild type Trichoderma reesei strain, and Δ tdaG is a Trichoderma reesei TdaG gene knock-out mutant strain.

The genomic DNA of the TdaG gene knockout mutant of the wild Trichoderma reesei and Trichoderma reesei was extracted with DNA extraction kits (available from Promega corporation, Inc.)

Genomic DNA Purification Kit, cat # A1120), and the specific steps are completed according to the Kit instructions. Detection of DNA concentration was done quantitatively by Nano drop and agarose gel electrophoresis.

3. Tdag is a protein related to synthesis of polythiodiketopiperazine, and Tdag gene is related to synthesis of polythiodiketopiperazine

Experiments prove that the carbon trichoderma TdaG gene knockout mutant strain in the step 2 can produce the polythiodiketopiperazine with the structural formula of 1, and wild type carbon trichoderma cannot produce the polythiodiketopiperazine with the structural formula of 1, so that TdaG is related protein synthesized by the polythiodiketopiperazine, and TdaG gene is related gene synthesized by the polythiodiketopiperazine.

The Arabic numerals in the formula 1 are carbon atom epitopes in a chemical structure, and R is hydroxyl or methoxyl; the compound in which the R group is a hydroxyl group is hereinafter referred to as compound 1, and the compound in which the R group is a methoxy group is hereinafter referred to as compound 2. The molecular formulas of compound 1 and compound 2 are respectively: c₁₉H₁₆N₂O₇S₂And C₂₀H₁₈N₂O₇S₂. The specific experimental methods and results are as follows:

and (3) respectively culturing the carbon cluster trichoderma Tdag gene knockout mutant strain and the wild carbon cluster trichoderma in the step (2) on a PDA (personal digital assistant) plate at 28 ℃ for 5 days, and then cutting the PDA culture medium and hyphae into fragments as seeds by using a scalpel. Then inoculating the seeds into a 500ml triangular flask filled with a rice culture medium (the 500ml triangular flask is filled with 60g of rice, 90ml of distilled water is added for soaking, and the rice culture medium is obtained by sterilizing at 121 ℃ for 20 minutes), and culturing for 20 days at 25 ℃ to obtain a solid fermentation culture of the TdaG gene knockout mutant strain of trichoderma carbonum and a solid fermentation culture of wild trichoderma carbonum.

Respectively dissolving the Tdag gene knockout mutant solid fermentation culture of trichoderma carbonicola and the solid fermentation culture of wild trichoderma carbonicola in acetonitrile, and detecting whether the target compound is generated or not by using a liquid chromatograph-mass spectrometer (AGILENT 1200HPLC/6520Q-TOFMS liquid chromatography-mass spectrometer). The analysis conditions were: gradient elution from 5% to 100% acetonitrile for 30min (the concentration of the aqueous acetonitrile solution linearly increased from 5% to 100% over 30 min), column washing with 100% aqueous acetonitrile (i.e., acetonitrile) for 5min, and equilibration of the column with 5% aqueous acetonitrile for 5 min.

As a result, as shown in FIG. 2, wild type Trichoderma reesei produced neither Compound 1([ M + H ]]⁺Peak M/z 449) nor does it yield Compound 2([ M + H ]]⁺Peak m/z 463); trichoderma reesei TdaG gene knockout mutant strain produced compound 1([ M + H ]]⁺Peak M/z 449) in turn produces Compound 2([ M + H)]⁺Peak m/z 463).

4. And (3) preparing a compound 1 and a compound 2 by using the Trichoderma reesei Tdag gene knockout mutant strain obtained in the step (2).

And (3) culturing the trichoderma carbonum Tdag gene knockout mutant strain obtained in the step (2) on a PDA (personal digital assistant) plate at 28 ℃ for 5 days, and then cutting the PDA culture medium and hyphae into fragments as seeds by using a scalpel. Then inoculating the seeds into a 500mL triangular flask (the 500mL triangular flask is filled with 60g of rice, 90mL of distilled water is added for soaking, and the rice culture medium is obtained by sterilizing at 121 ℃ for 20 minutes) with a rice culture medium, and culturing for 20 days at 25 ℃ to obtain the solid fermentation culture of the TdaG gene knockout mutant strain of trichoderma carbonum.

And (3) ultrasonically extracting the solid fermentation culture of the Tdag gene knockout mutant strain of trichoderma carbonicola for 3 times by using ethyl acetate, and removing the ethyl acetate by using a rotary evaporator to obtain a fermentation crude extract of the Tdag gene knockout mutant strain of trichoderma carbonicola. The crude fermented extract of the Tdag gene knockout mutant strain of trichoderma carbonum is firstly extracted twice by petroleum ether and acetonitrile with equal volume, and acetonitrile phase is collected for reverse phase column chromatography. The chromatographic column in the reverse phase column chromatography is an octadecyl bonded silica gel open chromatographic column, the specification of the chromatographic column is 5.0 multiplied by 60cm, and the column volume is 1177 mL. The elution procedure used in the reverse phase column chromatography was divided into the following 3 steps: 1) firstly, using 5: 95 acetonitrile-water solution (liquid composed of acetonitrile and water in a volume ratio of acetonitrile to water of 5: 95) was eluted for 3 column volumes; 2) then, using 20: 80 acetonitrile-water solution (liquid composed of acetonitrile and water, in which acetonitrile is presentAnd water in a volume ratio of 20: 80) elute 2 column volumes; 3) reuse 40: 60 acetonitrile-water solution (liquid consisting of acetonitrile and water in a volume ratio of acetonitrile to water of 40: 60) eluted 2 column volumes. And (4) collecting 40: the fraction obtained is eluted with acetonitrile-water solution of 60 and purified by chromatography on sephadex column for the next step: the separation medium used in the Sephadex LH-20 column chromatography is Sephadex LH-20, the specification of the column is 1.5 multiplied by 90cm, the column volume is 159mL, and the mobile phase is methanol solution. The eluted liquid was collected continuously from the start of the elution procedure, 8mL (8 mL/fraction) was collected per tube, and a total of 40 fractions, fractions 1-40, were collected. The fractions 10-15 collected were combined by HPLC to give fraction Fr.3. Fr.3 liquid phase preparation and separation of YMC-C18 to obtain compound 1 (t)_R35.5min), Compound 2 (t)_R41.5 min). The separation column used in the liquid phase preparation of YMC-C18 was YMC-Pack ODS-A, the packing had A particle size of 5 μm, and was purchased from YMC corporation, Japan, and the column had A diameter of 10mm, A height of 250mm, and A column volume of 20 mL. The mobile phase is 35% acetonitrile-water (liquid consisting of acetonitrile and water, the volume ratio of acetonitrile to water in the liquid is 35: 65), the flow rate is 2.5mL/min, and an elution peak with the retention time of 35.5min is collected to obtain a compound 1; the elution peak with retention time of 41.5min was collected to give compound 2.

The structures of compound 1 and compound 2 are identified as formula 1:

in the formula 1, R is hydroxyl or methoxyl; the compound in which the R group is a hydroxyl group is hereinafter referred to as compound 1, and the compound in which the R group is a methoxy group is hereinafter referred to as compound 2. The

compounds

1 and 2 have the following physico-chemical and spectral characteristics:

compound 1: as a pale yellow amorphous powder, UV (MeOH)204nm, 275nm, HR-ESI-MS Mass Spectroscopy (FIG. 3) M/z449.0461[ M + H ]]⁺Calculated value 449.0481; the NMR spectrum and the carbon spectrum are shown in Table 1 and FIGS. 4-5, the HSQC spectrum is shown in FIG. 6, the HMBC spectrum is shown in FIG. 7, and,¹H-¹The H COSY spectrum is shown in figure 8.

TABLE 1 NMR hydrogen and carbon spectra (500M Hz, in DMSO) data for Compound 1

Note: the signal attribution of the table is based on the analysis results of hydrogen spectrum and carbon spectrum.

Compound 2: was a pale yellow amorphous powder, UV (MeOh)204nm, 275nm, HR-ESI-MS Mass Spectroscopy (FIG. 9) M/z 463.0624 [ M + Na ]]⁺Calculated value 463.0628; the NMR spectrum and the carbon spectrum are shown in Table 2 and FIGS. 10-11, the HSQC spectrum is shown in FIG. 12, the HMBC spectrum is shown in FIG. 13, and,¹H-¹The H COSY spectrum is shown in figure 14.

TABLE 2 NMR hydrogen and carbon spectra (500M Hz, in DMSO) data for Compound 2

Note: the table signal is attributed based on the analysis results of hydrogen spectrum and carbon spectrum

2. Determination of antitumor Activity:

the determination method adopts MTT, and the tumor cell strains are respectively a colon cancer cell strain DLD1 (hereinafter referred to as DLD1 cell) and a liver cancer cell strain HepG2 (hereinafter referred to as HepG2 cell).

The operation steps are as follows: pure compound samples, compound 1 and compound 2, prepared in the above examples were accurately weighed and prepared into solutions of desired concentrations using dimethyl sulfoxide (DMSO).

Collecting colon cancer cell strain DLD1 and liver cancer cell strain HepG2 in logarithmic growth phase, making cell suspension, adjusting cell density of each cell to 8000/100 μ L per well by cell counting, inoculating into 96-well plate, culturing at 37 deg.C with RPMI-1640+ 10% FBS + 1% P/S, and culturing at 5% CO₂After overnight incubation in the incubator of (1), the old medium was discarded, and the culture medium containing the drug (Compound 1 or Compound 2) at concentrations of 0, 5, 10, 15 and 20. mu.M (Compound 1 or Compound 2 was added to RPMI-1640+ 10% FBS + 1% P/S)2) were incubated for 24h, the old culture medium was discarded, washed 2 times with PBS, 20 μ L of 5.0mg/mL MTT solution was added to each well, and incubation was continued for 4 h. The culture medium in the plate was discarded, 150. mu.L of DMSO was added to each well to dissolve formazan crystals produced by living cells, the solution was shaken on a shaker for 10min, and the absorbance (OD) of each well was measured on a microplate reader having a wavelength of 570nm₅₇₀ _nm)。

The results of the in vitro anti-tumor activity detection show that: DLD1 and HepG2 cells treated with the above-mentioned different concentrations of Compound 1 and Compound 2 for 24h, the viability of DLD1 and HepG cells was significantly reduced (Table 3-4 and FIGS. 15-16), and IC on DLD1 cells₅₀The values are respectively: 11.03 μ M, 25 μ M; IC on HepG cells₅₀The values are respectively: 6.385 μ M, 15.925 μ M. Indicating that the compound 1 and the compound 2 can obviously inhibit the growth of human colorectal adenocarcinoma cells and human liver cancer cells.

TABLE 3 survival of DLD1 cells treated with different concentrations of Compound 1 and Compound 2 for 24h

TABLE 4 survival of HepG2 cells treated with different concentrations of Compound 1 and Compound 2 for 24h

The experimental results prove that the compound has better inhibitory activity on human colorectal adenocarcinoma cells and human hepatoma cells, can be used as an anti-tumor preparation, and is expected to be applied to the production of related medicaments in the form of any acceptable salt and other derivatives or clinically acceptable auxiliary materials or carriers such as excipient and sustained release agent.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> polythiodiketopiperazine synthesis related protein, related biological material and application thereof

<130> GNCFH202257

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1494

<212> DNA

<213> Trichoderma carbonum (Trichoderma hypoxylon)

<400> 1

atgctaggtc tgagtacagt agaaatattc acggcctcgg ccattctgtt cattgtgtat 60

ttggtcggaa agggcatttg gaggctgtat ctacatcctt tatcacggtt tccaggtccc 120

ccaatcgcag ctctaacgcg ctggtacgaa tgctattttg atgtctatct aggtggacag 180

tatttcaagg agatcgaacg actacattct atctacggcc ctgttatacg catcagccct 240

gaggaattgc atatcaagga ccccgactgg gtgacggagt tatatcccct gggtaataat 300

cggcctcgtg acaaatatgc ttggtttcta tcggagggaa caaacgcaac aagctctgca 360

accgttcaac atcatgtcca tcgccaacgt cgatcaactc tcgctccaaa cttctccaaa 420

cagtccatca tgagcgtaga gaactccatc atccaaccaa gcattcactc gatgtgcgac 480

catctggacg atttcgctcg tgacggcact cctgtcaccg tgggcacggc attcgccagc 540

ttgacggtcg acaccgtttt acagatctgg tatgcctcgt cgccagggca gacccgtcgc 600

tggccctttt ttcctccctg gacagagcca tttccagcac tactaaatgc atcacatctc 660

ctacgacact tccccaaggc tttcttactc ttggctttta tccccaaggc ctactgggac 720

cgcatgcctg ggatatcttt gatatttaaa ctgcaacagg cgtcgtgtaa tctagcacga 780

gaagctatgg tagctgccac caacgacaaa aagccttgtt tgatcagaag actaaaactg 840

agttcgctgc cccctagtga gcgttcgctc cacaggctct cggacgaagg tttcagtttc 900

gtcatggcca gtacagaaag tactgcccag acgttagctg ctatcattta tcatctcgcc 960

gacaacccgg acatcttaac agagctccgc agacagctag ctgatgttca agactcatgg 1020

agtgccctag aagagctccc ttatctacgc tatatagtca tggaagctat gcgtgtgact 1080

gcttctgtga cggggagact gacccgaatt gccacaaatg aaatacttca gtatcaagag 1140

tggcatattc ctcgtggtat accagtcagt atggatcacc atttcactca tttagaccct 1200

gttatttttc cagaaccgca tcgcttcaac ccggaccgtt ggagaatagc gactgagaag 1260

ggcgagtcat ttgagaagta ttttctgccg tttggccgag gaagtcgcat gtgtataggg 1320

attaatctgg ccaaagccat catttatcta accacagcca ccattgtaag acgttatgac 1380

tttgaactgt ttgagacgac tcgagaggac gttgatatcg ttagagacaa cctactaggc 1440

gcagtcaaat ctacatctaa aggagtaaga gttaaacttt ctcgaaggat gtag 1494

<210> 2

<211> 497

<212> PRT

<213> Trichoderma carbonum (Trichoderma hypoxylon)

<400> 2

Met Leu Gly Leu Ser Thr Val Glu Ile Phe Thr Ala Ser Ala Ile Leu

1 5 10 15

Phe Ile Val Tyr Leu Val Gly Lys Gly Ile Trp Arg Leu Tyr Leu His

20 25 30

Pro Leu Ser Arg Phe Pro Gly Pro Pro Ile Ala Ala Leu Thr Arg Trp

35 40 45

Tyr Glu Cys Tyr Phe Asp Val Tyr Leu Gly Gly Gln Tyr Phe Lys Glu

50 55 60

Ile Glu Arg Leu His Ser Ile Tyr Gly Pro Val Ile Arg Ile Ser Pro

65 70 75 80

Glu Glu Leu His Ile Lys Asp Pro Asp Trp Val Thr Glu Leu Tyr Pro

85 90 95

Leu Gly Asn Asn Arg Pro Arg Asp Lys Tyr Ala Trp Phe Leu Ser Glu

100 105 110

Gly Thr Asn Ala Thr Ser Ser Ala Thr Val Gln His His Val His Arg

115 120 125

Gln Arg Arg Ser Thr Leu Ala Pro Asn Phe Ser Lys Gln Ser Ile Met

130 135 140

Ser Val Glu Asn Ser Ile Ile Gln Pro Ser Ile His Ser Met Cys Asp

145 150 155 160

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

165 170 175

Ala Phe Ala Ser Leu Thr Val Asp Thr Val Leu Gln Ile Trp Tyr Ala

180 185 190

Ser Ser Pro Gly Gln Thr Arg Arg Trp Pro Phe Phe Pro Pro Trp Thr

195 200 205

Glu Pro Phe Pro Ala Leu Leu Asn Ala Ser His Leu Leu Arg His Phe

210 215 220

Pro Lys Ala Phe Leu Leu Leu Ala Phe Ile Pro Lys Ala Tyr Trp Asp

225 230 235 240

Arg Met Pro Gly Ile Ser Leu Ile Phe Lys Leu Gln Gln Ala Ser Cys

245 250 255

Asn Leu Ala Arg Glu Ala Met Val Ala Ala Thr Asn Asp Lys Lys Pro

260 265 270

Cys Leu Ile Arg Arg Leu Lys Leu Ser Ser Leu Pro Pro Ser Glu Arg

275 280 285

Ser Leu His Arg Leu Ser Asp Glu Gly Phe Ser Phe Val Met Ala Ser

290 295 300

Thr Glu Ser Thr Ala Gln Thr Leu Ala Ala Ile Ile Tyr His Leu Ala

305 310 315 320

Asp Asn Pro Asp Ile Leu Thr Glu Leu Arg Arg Gln Leu Ala Asp Val

325 330 335

Gln Asp Ser Trp Ser Ala Leu Glu Glu Leu Pro Tyr Leu Arg Tyr Ile

340 345 350

Val Met Glu Ala Met Arg Val Thr Ala Ser Val Thr Gly Arg Leu Thr

355 360 365

Arg Ile Ala Thr Asn Glu Ile Leu Gln Tyr Gln Glu Trp His Ile Pro

370 375 380

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

385 390 395 400

Val Ile Phe Pro Glu Pro His Arg Phe Asn Pro Asp Arg Trp Arg Ile

405 410 415

Ala Thr Glu Lys Gly Glu Ser Phe Glu Lys Tyr Phe Leu Pro Phe Gly

420 425 430

Arg Gly Ser Arg Met Cys Ile Gly Ile Asn Leu Ala Lys Ala Ile Ile

435 440 445

Tyr Leu Thr Thr Ala Thr Ile Val Arg Arg Tyr Asp Phe Glu Leu Phe

450 455 460

Glu Thr Thr Arg Glu Asp Val Asp Ile Val Arg Asp Asn Leu Leu Gly

465 470 475 480

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

485 490 495

Met

<210> 3

<211> 3944

<212> DNA

<213> Trichoderma carbonum (Trichoderma hypoxylon)

<400> 3

cacagctctc tctagtgccg gcactaaaga ttgcaggata cgaacacagt cggcatctgt 60

atgattatgg agactgtgct tcataaagta ggcctttgcg tcgatgacag tttggggttc 120

aaaaaaattg tgctgctgga atcggactcg aggtgcaatc tggttatctg gacctaggga 180

tgggggagga aagagatcct gtactaggaa ggagagattg gggaagtgct gtatatgtta 240

gcctagtaga atagagagat aagggctata tctagtacct ttgccaagaa aactgagaca 300

tggcctacac ctccgccaat atctaccaaa gtaccgtttc ccaaggaggt ccaatcataa 360

cactcacaaa gactggtcat agattcttca tcaactaaaa gaaggggcca ttgttagcaa 420

gagtttattt gccaaattcc gatgttggcc actcacatgc tgtcgcgcct tgcattgctt 480

tgccaaatcg atccgcgcgg tccgaatgct tctggtagaa atcgtatatg gataggccga 540

atctggtaga gaatggtgtt acatctgctc gatgctcgta tgggcttttt ttaattgcct 600

ctgccgtact ggaggcggct tggtacatct cgtcaagcct actccatatt agcgtaatat 660

cattttctcg gacgatgtca actgactgag cagctagggc acattgagat gactgatcct 720

gtgccaataa cttagatctg gaagtgtgta cgaaaacctc ctcttctggt tcggtgaaaa 780

ctccgtgcaa agtgagcaag cgcatcaccc gaccggctcg gtcttcatct atacctgcac 840

gcttagataa ttccgataaa gaaaggcctt caagcaacgg aatgtggtca aataaatgaa 900

attcgaaagc cacttggaat gcagccaaat catagtgcga gagttgtagc tggcgaaaaa 960

agttttgggg ggaaacgact tgtcgcaaga gctcttcagt actattctgc aaatcaatcg 1020

tcagggagtc ccaaccggcg ttcttgcact ggctcgtttc tccgcctgtt gcttctagtt 1080

ctgcgacaag agactgagtt atgcgttgaa ttttatcggc taattggagg atctgatccc 1140

gagagttaga cgccatctcg ttttgcgtgg cagtagatgc aagcacaaat tcgggcagta 1200

aatgcagtcc aatgcgaacg agcctcttag tttgttttgt attgtatttt ttgttggggt 1260

gtatggcttc atctatccgt tcggcgaccg ctgtttcacc ggtggccgac actgactagt 1320

gataaagctg tggcgttagt cgagaggtaa cccaagctaa tggatctagt aactattcat 1380

gatcaggaaa atatgctagg tctgagtaca gtagaaatat tcacggcctc ggccattctg 1440

ttcattgtgt atttggtcgg aaagggcatt tggaggctgt atctacatcc tttatcacgg 1500

tttccaggtc ccccaatcgc agctctaacg cgctggtacg aatgctattt tgatgtctat 1560

ctagtaagaa ttaagcgccc taaagttatt ttaatattct gattaactat ttatagggtg 1620

gacagtattt caaggagatc gaacgactac attctatcta cggtatgata atagggtaac 1680

ctgaactcag cttcggcata taactgaccg ttacatacac gttgaaaggc cctgttatac 1740

gcatcagccc tgaggaattg catatcaagg accccgactg ggtgacggag ttatatcccc 1800

tgggtaataa tcggcctcgt gacaaatatg cttggtttct atcggaggga acaaacgcaa 1860

caagctctgc aaccgttcaa catcatgtcc atcgccaacg tcgatcaact ctcgctccaa 1920

acttctccaa acagtccatc atgagcgtag agaactccat catccaacca agcattcact 1980

cgatgtgcga ccatctggac gatttcgctc gtgacggcac tcctgtcacc gtgggcacgg 2040

cattcgccag cttgacggtc gacaccgttt tacagatctg gtatgcctcg tcgccagggc 2100

agacccgtcg ctggcccttt tttcctccct ggacagagcc atttccagca ctactaaatg 2160

catcacatct cctacgacac ttccccaagg ctttcttact cttggctttt atccccaagg 2220

cctactggga ccgcatgcct gggatatctt tgatatttaa actgcaacag gttcgatatc 2280

ttcaaaatgg atccgagaat acgagtttaa ctaggttgat aggcgtcgtg taatctagca 2340

cgagaagcta tggtagctgc caccaacgac aaaaagcctt gtttgatcag aagactaaaa 2400

ctgagttcgc tgccccctag tgagcgttcg ctccacaggc tctcggacga aggtttcagt 2460

ttcgtcatgg ccagtacaga aagtactgcc cagacgttag ctgctatcat ttatcatctc 2520

gccgacaacc cggacatctt aacagagctc cgcagacagc tagctgatgt tcaagactca 2580

tggagtgccc tagaagagct cccttatcta gtaagtcttc tttctgacat cttctatggg 2640

ttgaattgct cacgaattct tctcaatagc gctatatagt catggaagct atgcgtgtga 2700

ctgcttctgt gacggggaga ctgacccgaa ttgccacaaa tgaaatactt cagtatcaag 2760

agtggcatat tcctcgtggt ataccagtca gtatggatca ccatttcact catttagacc 2820

ctgttatttt tccagaaccg catcgcttca acccggaccg ttggagaata gcgactgaga 2880

agggcgagtc atttgagaag tattttctgc cgtttggccg aggaagtcgc atgtgtatag 2940

ggattaagta ggtgcaggaa ttctattatg aatctatgct ggtagatccg aagttgacat 3000

ttatctagtc tggccaaagc catcatttat ctaaccacag ccaccattgt aagacgttat 3060

gactttgaac tgtttgagac gactcgagag gacgttgata tcgttagaga caacctacta 3120

ggcgcagtca aatctacatc taaaggagta agagttaaac tttctcgaag gatgtagagg 3180

aaggtaaaga gctattaaat gagcatgtac tctgtggata caggtctatt gaataatggg 3240

ttaatatctt tatgtccaga gcagttcgtt ctacttcata gagatgaagg tatgacccct 3300

tactctgcat gtatccaaag tgtccaccaa cagatcgaaa tgatgcctac tctttagttt 3360

aagaaacctc acctacgggt catgacaaga taatgattga ggatctacaa atatgtattt 3420

gagaatctct ggctgtatat taagagatag aacaaaacgt gatatgaggt accaatggct 3480

tcaaaatgct aaaaggcaga agaggcttct ccatattctt catcttgagc ataatatcca 3540

aaaagaacgg cagtgaaaaa gcaaaccagc agattaccca aatgcgtccc aaccattgct 3600

tccaaagcct ggggctttga ggagagcagc tgatagagtc atagatgtat cggatcaaat 3660

cttctgtggt tatggcagca gcttgaagca tgaagaattg ggaaaagcca atgctccgct 3720

cactaaagga tatgttggat ggtgctggaa taatatagat agctgcacta tgcatgattc 3780

cactgataga aaaagacagc catagccgaa agtatgtccg aaaaaggctt ggtggcaagc 3840

aaagtagatc tgccgctgct ttggtataca cattgaccgt ctgataaaga ttagttgcga 3900

gtgtctcgaa actgcgggga agttaggctg acacttacac gacg 3944

<210> 4

<211> 5123

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

cggactcgag gtgcaatctg gttatctgga cctagggatg ggggaggaaa gagatcctgt 60

actaggaagg agagattggg gaagtgctgt atatgttagc ctagtagaat agagagataa 120

gggctatatc tagtaccttt gccaagaaaa ctgagacatg gcctacacct ccgccaatat 180

ctaccaaagt accgtttccc aaggaggtcc aatcataaca ctcacaaaga ctggtcatag 240

attcttcatc aactaaaaga aggggccatt gttagcaaga gtttatttgc caaattccga 300

tgttggccac tcacatgctg tcgcgccttg cattgctttg ccaaatcgat ccgcgcggtc 360

cgaatgcttc tggtagaaat cgtatatgga taggccgaat ctggtagaga atggtgttac 420

atctgctcga tgctcgtatg ggcttttttt aattgcctct gccgtactgg aggcggcttg 480

gtacatctcg tcaagcctac tccatattag cgtaatatca ttttctcgga cgatgtcaac 540

tgactgagca gctagggcac attgagatga ctgatcctgt gccaataact tagatctgga 600

agtgtgtacg aaaacctcct cttctggttc ggtgaaaact ccgtgcaaag tgagcaagcg 660

catcacccga ccggctcggt cttcatctat acctgcacgc ttagataatt ccgataaaga 720

aaggccttca agcaacggaa tgtggtcaaa taaatgaaat tcgaaagcca cttggaatgc 780

agccaaatca tagtgcgaga gttgtagctg gcgaaaaaag ttttgggggg aaacgacttg 840

tcgcaagagc tcttcagtac tattctgcaa atcaatcgtc agggagtccc aaccggcgtt 900

cttgcactgg ctcgtttctc cgcctgttgc ttctagttct gcgacaagag actgagttat 960

gcgttgaatt ttatcggcta attggaggat ctgatcccga gagttagacg ccatctcgtt 1020

ttgcgtggca gtagatgcaa gcacaaattc gggcagtaaa tgcagtccaa tgcgaacgag 1080

cctcttagtt tgttttgtat tgtatttttt gttggggtgt atggcttcat ctatccgttc 1140

ggcgaccgct gtttcaccgg tggccgacac tgactagtga taaagctgtg gcgttagtcg 1200

agaggggtgg cggccgctct agaactagtg gatcccccgg gctgcaggaa ttcgatatca 1260

agctttaatg cggtagttta tcacagttaa attgctaacg cagtcaggca ccgtgtatga 1320

aatctaacaa tgcgctcatc gtcatcctcg gcaccgtcac cctggatgct gtaggcatag 1380

gcttggttat gccggtactg ccgggcctct tgcgggatat cgtccattcc gacagcatcg 1440

ccagtcacta tggcgtgctg ctagcgctat atgcgttgat gcaatttcta tgcgcacccg 1500

ttctcggagc actgtccgac cgctttggcc gccgcccagt cctgctcgct tcgctacttg 1560

gagccactat cgactacgcg atcatggcga ccacacccgt cctgtggatc tgcacccaat 1620

cggcaggcac gggcggcgat ctccaatctg cgggatcagt cagatcaccc gagtgcgtgg 1680

gcatgacaat cgtgccctgg ggaccaacac aatccagaag ggcctgaatc actgcgaccg 1740

gccctcccgc gacccagccg agcgagctta gcgaactgtg gacgagaact gtgccaccaa 1800

gcgtaaggcc gttctctcgc atttgccttg ctaggctcgc gcgagttgct ggctgaggcg 1860

ttctcgaaat cagctcttgt tcggtcggca tctactctat tcctttgccc tcggacgagt 1920

gctggggcgt cggtttccac tatcggcgag tacttctaca cagccatcgg tccagacggc 1980

cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg gctccggatc ggacgattgc 2040

gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg ccgtcaacca agctctgata 2100

gagttggtca agaccaatgc ggagcatata cgcccggagc cgcggcgatc ctgcaagctc 2160

cggatgcctc cgctcgaagt agcgcgtctg ctgctccata caagccaacc acggcctcca 2220

gaagaagatg ttggcgacct cgtattggga atccccgaac atcgcctcgc tccagtcaat 2280

gaccgctgtt atgcggccat tgtccgtcag gacattgttg gagccgaaat ccgcgtgcac 2340

gaggtgccgg acttcggggc agtcctcggc ccaaagcatc agctcatcga gagcctgcgc 2400

gacggacgca ctgacggtgt cgtccatcac agtttgccag tgatacacat ggggatcagc 2460

aatcgcgcat atgaaatcac gccatgtagt gtattgaccg attccttgcg gtccgaatgg 2520

gccgaacccg ctcgtctggc taagatcggc cgcagcgatc gcatccatgg cctccgcgac 2580

cggctgcaga acagcgggca gttcggtttc aggcaggtct tgcaacgtga caccctgtgc 2640

acggcgggag atgcaatagg tcaggctctc gctgaattcc ccaatgtcaa gcacttccgg 2700

aatcgggagc gcggccgatg caaagtgccg ataaacataa cgatctttgt agaaaccatc 2760

ggcgcagcta tttacccgca ggacatatcc acgccctcct acatcgaagc tgaaagcacg 2820

agattcttcg ccctccgaga gctgcatcag gtcggagacg ctgtcgaact tttcgatcag 2880

aaacttctcg acagacgtcg cggtgagttc aggtggatca aagggcaggc tgctgccctc 2940

tttcttgtcg gaggactcgt caaagtaggc cgtcttgccc gagaggtgga cgccgggggt 3000

gacgggtacg ttggtgatgt cgggatggag tgcagggtga gagctttctt gggaagcgat 3060

tgaaggtcgc tcgggcttgc cgtctgtggt atctgtaggg gcgctcatgg cgattgtagg 3120

ctgagcacct gttgcactat atcaaagggt tagatatcga taagctccga gggctggtaa 3180

gggcagaagg gttgcgactg ggtcatttga ggtggtatgg ggtattggca ggcaggagaa 3240

gacggttgac aagacagacg acacgaggca cgctgcaggg cacgagacgt cactctggtg 3300

ctatctctgt ggtggtgata tggtcgagat agcggacaga gagagggagg cgcgcggtgg 3360

tggggacgta ccaaggacta tgacactggc gcggggcaga ttgtgcaggt cacaggaggg 3420

caaaccagag ggtctgggag gaggagaggg agtcaacaga ggcggcagat gtgttagaag 3480

ctcacagaag gccgacgagg gcaatgggga agaaaacttg ctgcggtgga tggttcatcc 3540

ccacaaccac ggcttacttg agccggggga ccagggcaca catggctctg ggctgctgca 3600

ttcccattcc catcgtggtc gagctacaaa gctcgccgcc agcttgaccg ctctgcagag 3660

gtttgctggg ccggtcaaca tgatgtcagg ccattttcat atggcaatgc gcaggtgggc 3720

cttgacatgt gcagccggtg gagcggcgtc gacctcgagg gggggcccgg tacccagctt 3780

ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc 3840

tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 3900

taaagcctgc atcttaacag agctccgcag acagctagct gatgttcaag actcatggag 3960

tgccctagaa gagctccctt atctagtaag tcttctttct gacatcttct atgggttgaa 4020

ttgctcacga attcttctca atagcgctat atagtcatgg aagctatgcg tgtgactgct 4080

tctgtgacgg ggagactgac ccgaattgcc acaaatgaaa tacttcagta tcaagagtgg 4140

catattcctc gtggtatacc agtcagtatg gatcaccatt tcactcattt agaccctgtt 4200

atttttccag aaccgcatcg cttcaacccg gaccgttgga gaatagcgac tgagaagggc 4260

gagtcatttg agaagtattt tctgccgttt ggccgaggaa gtcgcatgtg tatagggatt 4320

aagtaggtgc aggaattcta ttatgaatct atgctggtag atccgaagtt gacatttatc 4380

tagtctggcc aaagccatca tttatctaac cacagccacc attgtaagac gttatgactt 4440

tgaactgttt gagacgactc gagaggacgt tgatatcgtt agagacaacc tactaggcgc 4500

agtcaaatct acatctaaag gagtaagagt taaactttct cgaaggatgt agaggaaggt 4560

aaagagctat taaatgagca tgtactctgt ggatacaggt ctattgaata atgggttaat 4620

atctttatgt ccagagcagt tcgttctact tcatagagat gaaggtatga ccccttactc 4680

tgcatgtatc caaagtgtcc accaacagat cgaaatgatg cctactcttt agtttaagaa 4740

acctcaccta cgggtcatga caagataatg attgaggatc tacaaatatg tatttgagaa 4800

tctctggctg tatattaaga gatagaacaa aacgtgatat gaggtaccaa tggcttcaaa 4860

atgctaaaag gcagaagagg cttctccata ttcttcatct tgagcataat atccaaaaag 4920

aacggcagtg aaaaagcaaa ccagcagatt acccaaatgc gtcccaacca ttgcttccaa 4980

agcctggggc tttgaggaga gcagctgata gagtcataga tgtatcggat caaatcttct 5040

gtggttatgg cagcagcttg aagcatgaag aattgggaaa agccaatgct ccgctcacta 5100

aaggatatgt tggatggtgc tgg 5123

Claims

1. The protein is the protein of A1) or A2) as follows:

A1) the amino acid sequence is protein of a sequence 2 in a sequence table;

A2) a1) at the N-terminus or/and C-terminus.

2. The biomaterial related to the protein of claim 1, which is any one of the following B1) -B6):

B1) a nucleic acid molecule encoding the protein of claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B5) a nucleic acid molecule that reduces or inhibits expression of a gene encoding the protein of claim 1;

B6) an expression cassette, a recombinant vector or a recombinant microorganism comprising the nucleic acid molecule according to B5).

3. The biomaterial of claim 2, wherein: B1) the nucleic acid molecule is a coding gene of the protein shown in the following 1) or 2):

1) the coding sequence of the coding chain is a cDNA molecule of the 1 st to 1494 th nucleotides in the sequence 1 in the sequence table,

2) the coding sequence of the coding strand is a DNA molecule at the 1393-3177 th site of the sequence 3 in the sequence table;

B5) the nucleic acid molecule is a nucleic acid molecule capable of knocking out a coding gene of the protein;

the recombinant microorganism containing the nucleic acid molecule B5) is recombinant Trichoderma charratum obtained by introducing the nucleic acid molecule capable of knocking out the coding gene of the protein into Trichoderma charratum (Trichoderma hypoxylon), wherein the recombinant Trichoderma charratum does not contain the coding gene of the protein.

4. Use of a nucleic acid molecule that reduces or inhibits expression of a gene encoding a protein according to claim 1 in the preparation of a polythiodiketopiperazine having the formula 1:

in the formula 1, R is hydroxyl or methoxyl.

5. The recombinant trichoderma carbonicola is a recombinant bacterium obtained by knocking out a target gene of trichoderma carbonicola, and the target gene coding amino acid sequence is protein of a sequence 1 in a sequence table.

6. The use of recombinant trichoderma reesei of claim 5 in the preparation of a polythiodiketopiperazine having the structural formula 1:

in the formula 1, R is hydroxyl or methoxyl.

7. The application of polythiodiketopiperazine or pharmaceutically acceptable salt thereof in preparing anti-tumor and/or anti-tumor cell drugs, wherein the structural formula of the polythiodiketopiperazine is shown in formula 1:

in the formula 1, R is hydroxyl or methoxyl.