CN105154466B - A kind of double gene coexpression recombinant vector and its construction method and the application in Blakeslea trispora - Google Patents

Abstract

The invention discloses a kind of double gene coexpression recombinant vectors, and gene expression frame sequence is carried in starting vectorsyn, the gene expression frame sequencesynContain：Terminator-multiple cloning sites-two-way startup subsequence PCarB-TA clones prepare sequence-terminator, the gene expression frame sequencesynNucleotide sequence such as SEQ.ID.NO:Shown in 1；Hygromycin gene is also carried in the starting vectorhygR, the hygromycin genehygRNucleotide sequence such as SEQ.ID.NO:2；A double gene coexpression recombinant vector pPMPT specifically is constructed, to contain gene expression frame sequence in starting vector pATZsynAnd hygromycin genehygR, nucleotide sequence such as SEQ.ID.NO:Shown in 3, while the construction method for also disclosing the carrier and the application in filamentous fungi Blakeslea trispora.Recombinant vector provided by the invention can add two genes simultaneously on a plasmid, and can be integrated into filamentous fungi genome by agriculture bacillus mediated, be inserted into single copy, realize double gene coexpression, inheritance stability.

Description

A double-gene co-expression recombinant vector and its construction method and in B. trispora Applications

technical field

The invention relates to the field of gene technology, in particular to a dual-gene co-expression recombinant vector and its construction method and application in the filamentous fungus B. trispora.

Background technique

Conventional transgenics mostly use a single target gene to improve individual traits of organisms, but the transformation of a single target gene cannot meet the needs of biological improvement, especially the genetic modification of some metabolic pathways or quantitative traits. With the deepening of genetic engineering and molecular biology research, multi-gene transformation research came into being and developed rapidly.

The multi-gene co-expression system for biological genetic transformation usually includes a multi-plasmid co-transformation system and a single vector expression cassette system for multiple genes. In the multi-plasmid co-transformation system, multiple foreign genes are respectively cloned into different resistance vectors, and multiple antibiotics are added at the same time after co-transformation to realize the co-expression of multiple genes. However, this system can only achieve the co-expression of a few genes. In addition, when co-expressing, the compatibility of multiple expression plasmids needs to be considered, and the operation is cumbersome, and the transformation efficiency is not high. It can be seen that a single vector expressing multiple gene expression cassette systems has more research and application value.

The gene expression carrier refers to the carrier that implements the target gene and ensures that the target gene reaches the recipient cell and can be expressed. The composition of the gene expression carrier is constructed on the basis of the carrier. The necessary component of the gene expression carrier is the promoter +target gene+terminator+marker gene. A multi-gene expression vector can be used for multiple gene insertion, allowing multiple target genes to reach and express in recipient cells.

The construction of gene expression vector is the core of genetic engineering, which is the process of combining the target gene with the carrier, in fact, the process of recombining DNA from different sources. Usually based on the theory of molecular genetics and modern methods of molecular biology and microbiology, genes from different sources are used to construct hybrid DNA molecules in vitro according to a pre-designed blueprint, and then introduced into living cells to change the biological origin. Some genetic traits, acquisition of new varieties, production of new products. If a plasmid is used as a carrier in this process, it is first necessary to cut the plasmid with a certain restriction enzyme to make a gap in the plasmid and expose the sticky end; then use the same restriction enzyme to cut the target gene to produce the same sticky end (part Restriction endonucleases can cut out blunt ends, which have the same effect); insert the excised fragment of the target gene into the nick of the plasmid, first base complementary pairing, the two sticky ends are anastomosed together, and the bases Form hydrogen bonds, and then add an appropriate amount of DNA ligase to catalyze the formation of phosphodiester bonds between the two DNA strands, thereby linking adjacent deoxyribose nucleic acids to form a recombinant DNA molecule. For example, the human insulin gene is combined with the plasmid DNA molecule in Escherichia coli to form a recombinant DNA molecule (also called a recombinant plasmid) through this method.

At present, most of the expression vectors constructed in the field of genetic engineering are prokaryotic and plant expression vectors. For example, PUC-T is a frequently used prokaryotic expression vector, but it is not suitable for eukaryotes, and commonly used plant expression vectors such as PBI121, PBI221, etc. The use in fungi is also subject to certain limitations. In addition, when a technician wants to express an exogenous gene, he usually needs to select a suitable restriction site for insertion of the exogenous gene by means of an restriction enzyme connection so that the transformed host can be expressed. However, in the prior art, there are few studies on the construction of expression vectors for filamentous fungi in this field, especially for the construction of gene expression vectors for B. trispora.

Blakeslea trispora belongs to the order Peronospora, Albiraceae, and its Latin name is Blakeslea trispora. It is currently the main strain for large-scale industrial production of natural β-carotene. The bacteria often need to be genetically modified during use, and the process is basically: (1) amplify the required insert, (2) clone the T vector for enzyme digestion, (3) construct the vector by enzyme digestion and ligation . For example, PCAMBIAI1303 is the commonly used vector for modifying B. trispora, and the promoter used is CaMV35Spromoter. Since the plasmid contains only one multiple cloning site, the PCR product cannot be directly used for ligation. As another example, Li Jigang et al. studied the improvement of filamentous fungal transformation vectors (Henan Agricultural Sciences, 2013, Issue 05), and reported the construction process of pATZ and pTBZ. The improved vectors contained resistance markers and multiple cloning sites by restriction enzyme ligation point, but if you want to achieve the expression of foreign genes, you also need to add promoters, terminators and corresponding target genes by enzyme-cut ligation, and it is difficult to find a suitable enzyme-cut site when inserting the target gene point, and some enzymes have low activity, which will bring great trouble to the operation. For another example, CN1759174A discloses a method for genetically modifying Brailletella organisms, corresponding organisms and uses thereof. The pBinAHygR vector is constructed using the pBinA vector, which contains hygromycin resistance and a The multiple cloning site, select the appropriate enzyme cutting site, insert any gene at the multiple cloning site by enzyme cutting and ligation, construct the vector to be expressed, and then use the Agrobacterium-mediated method to transform the three spora At least one cell in B. boulardii undergoes homokaryon transformation through mutagenesis, so that one or more cells can obtain the same genetic traits, so that they can be inherited stably. However, it is limited to the addition of a single gene. If you want to To express multiple genes, multiple promoters need to be added, which requires multiple connections with foreign vectors or co-transformation, which is not only cumbersome and complicated, but also easily leads to inconsistent gene expression and antisense inactivation And genetic instability; in addition, it also causes certain difficulties to insert long fragments of target genes containing multiple restriction sites.

Contents of the invention

The purpose of the present invention is to provide a double-gene co-expression recombinant vector and its construction method and application in B. trispora to solve the existing expression vector needs to carry out multiple exogenous The integration of genes is cumbersome to operate, as well as problems such as inconsistent gene expression, antisense inactivation, and genetic instability.

The purpose of the present invention is achieved through the following technical solutions:

A double-gene co-expression recombinant vector, with the initial vector as the backbone carrying a complete gene expression cassette sequence syn, the sequence syn contains: terminator sequence (Ter) - multiple cloning site (MCS) - bidirectional promoter sequence PCarB —TA clone preparation sequence—terminator sequence (Ter), the nucleotide sequence of the gene syn is shown in SEQ ID NO:1;

The molecular weights of the terminator (Ter), the multiple cloning site (MCS), the bidirectional promoter PCarB, the TA cloning site, and the terminator (Ter) are 559bp, 43bp, 610bp, 28bp, and 559bp in sequence. The sequence SEQ.ID.NO:1 is arranged from front to back.

The initial vector is any one of pATZ, pPZP100, pPZP101, pPZP102, pPZP111, pPZP112, pPZP121, pPZP122, pPZP200, pPZP201, pPZP202, pPZP211, pPZP212, pPZP221 or pPZP222;

The present invention constructs a recombinant vector for expressing one or two exogenous genes in a filamentous fungus. By adding multiple cloning sites on one side of the bidirectional promoter and adding TA clones on the other side, the introduction of exogenous genes can It is more convenient and fast; the application experiment in the filamentous fungus B. trispora has proved that two target genes can be added to the recombinant vector at the same time, and can be integrated into the genome of B. trispora through the mediation of Agrobacterium. Copy insertion, stable inheritance.

The double-gene co-expression recombinant vector provided by the present invention also carries the hygromycin resistance gene hygR in the vector, which can be used as a marker gene for recombinant selection. The nucleotide sequence of the hygromycin resistance gene hygR is as follows: Shown in SEQ.ID.NO:2.

The present invention also provides a dual-gene co-expression recombinant vector pPMPT, which carries the gene expression cassette sequence syn with the initial vector pATZ as the backbone, and uses the hygromycin resistance gene hygR as a selection marker gene, and the gene expression cassette sequence syn Contains: terminator sequence (Ter)—multiple cloning site (MCS)—bidirectional promoter sequence PCarB—TA clone preparation sequence—terminator sequence (Ter), the nucleotide sequence of which is shown in SEQ ID NO:1; The nucleotide sequence of the hygromycin resistance gene hygR is shown in SEQ.ID.NO:2; its plasmid map is:

The nucleotide sequence of the recombinant vector pPMPT is shown in SEQ ID NO:3.

The construction method of the double-gene co-expression recombinant vector pPMPT provided by the invention comprises the following steps:

(a) Artificially synthesize a complete gene expression cassette sequence syn according to the nucleotide sequence shown in SEQ ID NO:1, and clone it into the vector pMD19-T to obtain the plasmid pMD19-T-syn;

(b) amplify the hygromycin resistance gene hygR, and clone it into the vector pMD19-T to obtain the plasmid pMD19-T-hygR;

(c) Digest the plasmid pMD19-T-hygR and the plasmid pMD19-T-syn respectively with SalI restriction endonuclease, recover the two fragments of the digested product pMD19-T-hygR and syn, and ligate overnight at 16°C, Get pMD19-T-hygR-syn;

(d) Digest pMD19-T-hygR-syn and initial vector pATZ with BssHII and HindIII restriction endonucleases respectively, recover the hygR-syn fragment of the digested product and the remaining T-DNA-containing fragment after digestion of plasmid pATZ Part, the two recovered fragments were ligated overnight at 16°C, and the ligated product was transformed into Escherichia coli DH5α to obtain the plasmid pATZ-hygR-syn, which was named as the recombinant vector pPMPT.

The build process is shown in Figure 15.

The complete gene expression cassette sequence syn in the recombinant vector pPMPT disclosed by the present invention is a synthetic sequence, which includes: terminator sequence (Ter)—multiple cloning site (MCS)—bidirectional promoter sequence PCarB—TA clone preparation sequence (AhdI, SpeI, AhdI)—terminator sequence (Ter), when the sequence is synthesized, a SalI restriction site is added to the 5 ^' end of the upstream primer, and a HindIII and SalI restriction site is added to the 3 ^' end of the downstream primer in sequence.

Both the beginning and the end of the terminator sequence are from the trpC terminator in Aspergillus nidulans, which has a termination function in filamentous fungi and plants. Its size is 559bp, and it is located at the beginning and end of the gene expression frame sequence syn. The information in the final constructed vector sequence SEQ ID NO:3 is:

Ter 9034-9592 559 misc feature

Ter 10274-10832 559 misc feature

The restriction endonucleases contained in its multiple cloning site (MCS) are XbaI, BstEII, SacI, KpnI, SmaI, MfeI, BglII in sequence, and the exogenous gene fragments to be expressed can be added according to the needs by enzyme cutting and ligation. Since the front and back ends contain corresponding promoters and terminators, it can be directly expressed if the insertion is in the forward direction; the size of the cloning site (MCS) is 43bp, and it is located at the 560th to 602nd position in the complete gene expression frame sequence syn bit, the sequence information in the final constructed vector is:

MCS 9593-9635 43 misc feature

The sequence source of its bidirectional promoter PCarB is the promoter sequence of phytoene dehydrogenase gene (carB) and lycopene cyclase gene (carRA) in B. trispora. The promoter has a bidirectional start function and can start the expression of its upstream and downstream gene sequences. Its molecular weight is 610bp, and it is located at the 603rd to 1212th position in the gene syn. The sequence information in the final constructed vector is as follows:

PCarB 9636-10245 610 misc feature

Its TA clone preparation sequence, the preparation of the TA clone in this vector is directly prepared by enzyme digestion, its molecular weight is 28bp, and it is in the 1213th to 1240th position in the gene syn, and the sequence information in the final constructed vector is:

TA clone 10246-10273 28 misc feature

The above base fragment was artificially synthesized and named the gene expression frame sequence syn. In the present invention, the artificial fragment was synthesized by Jinweizhi Biotechnology Co., Ltd. and carried by the puc57 vector, namely the plasmid puc57-syn.

For the construction method of the initial vector pATZ in step (d) of the present invention, see "Improvement of Transformation Vectors for Filamentous Fungi" (Li Jigang et al., "Journal of Henan Agricultural Sciences", 2013, 42(5): 110-113).

The present invention verifies the applicability of the constructed recombinant vector pPMPT in B. trispora. The exogenous gene neoR was introduced into the multiple cloning site (MCS) of plasmid pPMPT by BglII and XbaI, and the exogenous gene crtZ was introduced into the TA cloning site of plasmid pPMPT by TA cloning, and the constructed plasmid was transformed by the method mediated by Agrobacterium B. trispora was used to verify the normal expression and stable inheritance of exogenous genes integrated into the genome. Passage the transformant on the hygromycin resistance plate for 4-5 times, pick the B. trispora grown on the final resistance plate for DNA extraction, and amplify the hygromycin resistance gene and the inserted foreign gene neoR and crtZ, verify whether the exogenous gene is integrated into the genome of B. trispora, and then pass the neomycin resistance test to detect whether the exogenous gene neomycin resistance gene (neoR) is expressed; Pull the mold and extract the pigment substances in the fermentation product for HPLC analysis to detect whether the exogenous gene crtZ is expressed. The crtZ gene can express β-carotene hydroxylase, which can further convert β-carotene, the final product of B. trispora, into zeaxanthin. Through HPLC analysis, the presence of zeaxanthin was detected in the fermentation product of the B. trispora transformant. Therefore, we obtained an engineering strain of B. trispora that is resistant to neomycin and accumulates zeaxanthin, and can be stably inherited through asexual spores, which proves that the recombinant vector can transfer two target genes into B. trispora The purpose and use of foreign gene co-expression.

The carrier constructed by the present invention is a transformation carried out on the basis of a binary Ti carrier pATZ. The sequence of the carrier pATZ is analyzed. First, a complete gene expression cassette sequence syn is artificially synthesized. This synthetic sequence includes a terminator sequence ( Ter)—multiple cloning site (MCS)—bidirectional promoter sequence PCarB—TA cloning preparation sequence—terminator sequence (Ter), using the method of enzyme digestion and ligation, insert the syn sequence and the hygromycin resistance expression cassette into two Between the T-DNA regions, the recombinant vector pPMPT was finally constructed. Since the vector contains a bidirectional promoter, TA cloning and multiple cloning sites are respectively added on both sides of the bidirectional promoter, so that no matter at the TA cloning site or the multiple cloning site, exogenous expression gene fragments can be added, and the vector contains Transformation of filamentous fungi, especially B. trispora, after homokaryotic conversion, can easily achieve the expression of a single exogenous gene or two related or unrelated exogenous genes in filamentous fungi at the same time. The process of finding an appropriate promoter and adding it. Simultaneously the present invention also has the following advantages:

1. The recombinant vector constructed by the present invention overcomes the shortcomings of tedious work such as adding a specific site or adding a promoter before the second gene when realizing double-gene co-expression in eukaryotic genes;

2. It can avoid the occurrence of gene trans-inactivation caused by homologous recombination caused by the simultaneous addition of similar promoters;

3. The recombinant vector contains multiple cloning sites, which can be inserted into different gene fragments by enzyme-cut ligation, making the assembly of the expression cassette very convenient;

4. The vector can be digested by AhdI endonuclease, and the vector can generate TA cloning sites, so the PCR product can be directly connected, especially for some foreign genes containing more restriction endonuclease sites Fragments, often can not find a suitable enzyme cutting site when performing enzyme digestion and connection, this T-containing vector is more important and more convenient to operate;

5. The vector contains two T-DNA borders, which can be transformed using the Agrobacterium-mediated method to improve the transformation efficiency, and the hygromycin resistance contained can be used to screen positive clones.

It has been proved by experiments that the recombinant vector constructed by the present invention is inserted into the coding region sequences of the neoR gene and the crtZ gene, and after being transformed into B. trispora, the B. trispora has neomycin resistance and can express zeaxanthin. Mold engineering strain, and its genetic stability is good, which proves that the recombinant vector pPMPT has very good applicability for the introduction of foreign genes to B. trispora. At the same time, this vector also has application potential in other filamentous fungi .

Description of drawings

Figure 1 is the enzyme digestion and recovery map of the artificially synthesized complete gene expression cassette sequence syn; in the figure, M is 500bp DNALadder (Beijing Xiyang Huizhi), and 1 is syn.

Figure 2 is the amplified electrophoresis pattern of the hygR expression cassette of the hygromycin resistance gene; in the figure, M ₁ is DL5,000 DNA Maker (Dalian Bao Biology), and 6 is the entire expression cassette of hygR.

Figure 3 is the verification of the positive clone amplification detection of the expression cassette of the hygromycin resistance gene hygR; in the figure, M ₂ is a 500bp DNALadder (Beijing Xiyang Huizhi), and 3 is the entire expression cassette of hygR.

Figure 4 is the electrophoretic pattern of forward-linked pMD19-T-hygR-syn verified by amplification; in the figure, M ₃ is DL5,000 DNA Maker (Dalian Bao Biology), and 4 and 5 are hygR-syn fragments.

Figure 5 is the electrophoretic pattern of the positive clone pATZ-hygR-syn verified by amplification; in the figure, M ₄ is DL5,000 DNAMaker (Dalian Bao Biology), and 7 is the hygR-syn fragment.

Figure 6 is the gel electrophoresis pattern of neomycin resistance gene neoR amplification; in the figure, M ₅ is DL5,000 DNA Maker (Dalian Bao Biology), 8 and 9 are neomycin resistance gene neoR without promoter, The length is 816bp.

Figure 7 is the electrophoretic pattern of the pPMPT plasmid and the T vector containing neomycin resistance gene neoR with BglII and XbaI for double enzyme digestion and recovery detection; M ₆ in the figure is a 500bp DNA Ladder (Beijing Xiyang Huizhi), 10 The fragment obtained by digestion with pPMPT is 11161bp in length, and 11 is a neoR fragment with a length of 816bp.

Figure 8 is the electrophoresis image of the positive amplification verification of the neomycin resistance gene neoR in pPMPT-neoR; in the figure, M ₇ is DL5,000 DNA Maker (Dalian Bao Biology), 12 and 13 are both neoR, and the length is 816bp.

Figure 9 is the amplified electrophoresis image of the crtZ gene in Erwinia erdophila; in the figure, M ₈ is DL5,000 DNA Maker (Dalian Baobiology), 14 and 15 are both crtZ genes, and the length is 528bp.

Figure 10 is the electrophoresis diagram of the ligation amplification verification of the crtZ gene in pPMPT- _neoR -crtZ; in the figure, M9 is DL5,000DNA Maker (Dalian Bao Biology), and 16 is the crtZ gene, with a length of 528bp.

Figure 11 is the amplification verification diagram of the forward connection fragment in pPMPT-neoR-crtZ amplified with verification primers; M ₁₀ in the figure is DL5,000DNA Maker (Dalian Baobiology), and 17, 18 and 19 are all forward connections , with a length of 1971bp.

Figure 12 is the electrophoresis image of post-transformation DNA amplification verification; in the figure, M ₁₁ is DL5,000 DNA Maker (Dalian Bao Biology), 20 is positive transformation, and the length is 1971bp.

Figure 13 is a high performance liquid chromatogram of zeaxanthin standard substance.

Figure 14 is a high performance liquid chromatogram of a sample of zeaxanthin to be tested.

Fig. 15 is a flow chart of the construction of a double-gene co-expression recombinant vector.

Detailed ways

The following examples are used to further describe the present invention in detail, but do not limit the present invention in any form.

Technical terms in the present invention:

Vector: refers to a DNA molecule used to introduce foreign DNA and, if desired, propagate said foreign DNA in cells (see also "Vector" in Rompp Lexikon chemie CD ROM version 2.0, Stuttgart/New York: Georg Thieme Verlag 1999 ) in the present application is also meant to include plasmids, cosmids and other molecules of the same purpose.

Binary Ti vector: refers to a shuttle plasmid that contains two independent regions of T-DNA and can replicate in Escherichia coli and Agrobacterium.

Expression: Refers to the transfer of genetic information from DNA to RNA to gene products (here preferably carotenoids).

Genetic information: Nucleic acids that are preferably introduced into B. trispora and lead to genetic modification of B. trispora, ie, listed such as those that result in increased or decreased enzyme activity compared to the starting organism.

Wild type: Refers to the corresponding non-genetically modified B. trispora starting organism.

Organism: Refers to the starting organism (wild type) of B. trispora, the genetically modified organism of B. trispora, or both. Insertion: Refers to the addition of exogenous nucleic acid into a vector.

Trans-inactivation: Trans-inactivation is mainly due to the gene silencing caused by the interaction of the silencing site with homologous sequences and the DNA of other sites. The research of Vaucheret et al. shows that the promoter region only needs 90 bp sequence Homologous, can make trans-inactivation between two genes.

Integration: refers to the connection of two non-homologous genes, such as the random insertion of foreign fragments added to the vector into the recipient's genome.

Example 1 Construction of double-gene co-expression vector

(1) Synthetic gene expression cassette sequence syn: terminator sequence (Ter) - multiple cloning site (MCS) - bidirectional promoter sequence PCarB - TA clone preparation sequence - terminator sequence (Ter), which is added upstream when synthesizing the sequence SalⅠ restriction site, and HindⅢ and SalⅠ restriction sites are added downstream.

Both the beginning and the end of the terminator (Ter) sequences are from trpC terminator in Aspergillus nidulans, which have termination function in filamentous fungi and plants, both of which are 559bp in size, located at the beginning and end of the sequence syn. The information in the final constructed vector sequence SEQ ID NO:3 is:

Ter 9034-9592 559 misc feature

Ter 10274-10832 559 misc feature

The restriction endonucleases contained in its multiple cloning site (MCS) are XbaI, BstEII, SacI, KpnI, SmaI, MfeI, BglII in sequence, and the fragments to be expressed can be added according to the needs by enzyme cutting and ligation. The end contains the corresponding promoter and terminator, so if the insertion is in the forward direction, it can be directly expressed; its size is 43bp, and it is from the 560th to the 602nd in the sequence syn. The sequence information in the final constructed vector is:

MCS 9593-9635 43 misc feature

The source of its bidirectional promoter (PCarB) sequence is the promoter sequence of phytoene dehydrogenase gene (carB) and lycopene cyclase gene (carRA) in B. trispora. The promoter has a bidirectional start function and can start the expression of its upstream and downstream gene sequences. Its molecular weight is 610bp, and it is from the 603rd to the 1212th position in the sequence syn. The sequence information in the final constructed vector is as follows:

Bidirectional promoter PCarB 9636-10245 610 misc feature

Its TA clone preparation sequence, the preparation of the TA clone in this vector is directly prepared by enzyme digestion, the sequence includes two AhdI restriction sites, and a SpeI restriction site is introduced between the two AhdI sites. Benefits of the SpeI enzyme cutting site: ① SpeI digestion can prevent circularization caused by incomplete digestion of the AhdI site in the vector. ②A protective base is provided between the two AhdⅠ restriction sites to facilitate the digestion of AhdⅠ, thereby forming a highly efficient T-terminal. The plasmid can be digested with AhdI to produce a T-terminal, and the PCR product with polyA can be added directly. Its molecular weight is 28bp, and it is located at the 1213th to 1240th position in the gene syn. The sequence information in the final constructed vector is as follows:

TA cloning site 10246-10273 28 misc feature

The above base fragment is artificially synthesized into the sequence syn. In the present invention, the artificial fragment is synthesized by Jinweizhi Biotechnology Co., Ltd. and carried by the pMD19-T vector, namely the plasmid pMD19-T-syn.

After the synthesized plasmid pMD19-T-syn was digested with SalI, the detection map of the synthesis sequence digested and recovered is shown in Fig. 1 .

(2) Amplify the hygromycin resistance gene by using the constructed Ti vector pATZ: design primers (F1, R1) to amplify the expression cassette containing hygromycin, and the hygromycin primers contain the restriction site BssHII and enzyme The hygromycin resistance gene hygR with a size of 2120 bp was amplified by cutting site SalI. The electrophoretic pattern is shown in FIG. 2 , and the sequence is shown in SEQ ID NO:2.

Among them, the construction method of the Ti vector pATZ is described in the article "Improvement of Transformation Vectors for Filamentous Fungi" (Li Jigang. 2013).

The primer sequences were designed as:

F1: GTCGACGTCTTCTACTATAACTTCCTC

R1: GCGCGCTTTCTTCCTAATGGAGAT

The hygromycin resistance gene (hygR) amplification system is as follows:

Amplification conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s; annealing at 57°C for 30 s; extension at 72°C for 3 min, 30 cycles; extension at 72°C for 5 min. PCR products were detected by 1.5% agarose gel electrophoresis. The amplified product contains TtrpC, HygR and PtrpC, the sequence of which is shown in SEQ ID NO:2.

The sequence information of this sequence in the final constructed vector is:

TtrpC 6908-7466 559 misc feature

HygR 7641-8666 1026 misc feature

/gene = aph(4)-Ia

/product=aminoglycoside phosphotransferase from E.Coli

/note = confers resistance to hygR romycin

/translation

＝MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE

341amino acids＝38.0kDa

PtrpC 8671-9028 358 misc feature

(3) Ligate the hygromycin resistance gene hygR amplified in step (2) with the pMD-19-T (simple) vector in a constant temperature metal bath at 16°C overnight; screen positive clones. The positive clones were amplified with M13 primers to obtain pMD19-T-hygR, and its amplified electrophoresis pattern is shown in Figure 3 .

(4) Digest the positive cloned plasmid pMD19-T-hygR screened in step (3) and the plasmid pMD19-T-syn in step (1) respectively with SalI restriction endonuclease, and recover the digested product pMD19- Two fragments of T-hygR and syn, and pMD19-T-hygR and syn were ligated overnight at 16°C; and primers (F3, R3) were designed to amplify and verify that the positively connected clone was selected, as shown in Figure 4, to obtain pMD19 -T-hygR-syn.

The primer sequences are:

F3: GCACTCTTTGCTGCTTGGA

R3: GCTACTTACACACAGGACATC

The forward connection amplification system is as follows:

Amplification conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s; annealing at 57°C for 30 s; extension at 72°C for 4 min, 30 cycles; extension at 72°C for 5 min. PCR products were detected by 1.5% agarose gel electrophoresis.

(5) The pATZ plasmid and the forward-connected clone pMD19-T-hygR-syn obtained in step (4) were digested with BssHII and HindIII, respectively, and the digested product was recovered hygR-syn, and the rest of the plasmid pATZ was digested with For the part of T-DNA, connect the recovered two fragments overnight at 16°C, amplify with the primers (F3, R3) in step (4) to verify whether the connection is correct, screen positive clones, and obtain the positive clone plasmid pATZ-hygR- syn is the double gene co-expression recombinant vector pPMPT. The amplification procedure for screening positive clones is as in step (4), and the amplification electrophoresis results are shown in FIG. 5 .

Application of embodiment 2 recombinant vector

In this embodiment, the vector pPMPT constructed in Example 1 is inserted into two genes: gene neoR (see SEQ ID NO: 4) and gene crtZ (see SEQ ID NO: 5); after transformation, verify whether the transformation is successful and stable inheritance , it can be subcultured for 4-5 times on the neomycin resistance (neoR) plate, pick the B. trispora grown on the final resistance plate for DNA extraction, and amplify the hygromycin contained in the plasmid pPMPT The resistance gene and the inserted neoR gene and crtZ gene were verified.

A neomycin resistance gene neoR and crtZ gene with an autonomous replicator but lacking its own promoter were amplified in the pet-28a plasmid and Erwinia erdophila, respectively, and the crtZ gene can express β-carotene hydroxylase , the final product β-carotene of B. trispora was converted into zeaxanthin, and these two genes were inserted into the constructed plasmid pPMPT to obtain the pPMPT-neoR-crtZ plasmid, which was transformed by Agrobacterium-mediated transformation. The pigment was extracted from the B. trispora grown on the neomycin-resistant plate, and detected by HPLC, it was found that zeaxanthin was expressed, and a neomycin-resistant and zeaxanthin-expressed B. trispora strain was obtained. And the performance is stable, and can be stably inherited through spore asexual reproduction, which proves the function of the plasmid.

The specific process is as follows:

(1) Determination of the concentration of cefotaxime sodium required to inhibit the growth of Agrobacterium

Draw 100 μL of Agrobacterium cultured overnight, spread it on the cefotaxime sodium-resistant plate with concentration gradients of 0, 50, 100, 150, and 200 μg/mL, do three parallels for each gradient, and cultivate for three days to find out, with cephalosporin As the concentration of sodium thioxime increased, the growth of Agrobacterium also became weak, and when it reached 50μg/mL, the Agrobacterium basically did not grow.

(2) Determination of the screening concentration for B. trispora resistance to neomycin and cefotaxime sodium

Make a spore suspension of B. trispora, and spread 200 μL on neomycin and cefotaxime sodium-resistant plates with different concentration gradients. , 100, 150, 200 μg/mL, observed the growth of B. trispora, and found that when the concentration of neomycin reached 200 μg/mL, B. trispora grew completely, and when the concentration of cefotaxime sodium reached 200 μg/mL, it still grew Good, so the determined final neomycin screening concentration is 200 μg/mL and cefotaxime sodium concentration is 100 μg/mL.

(3) Construction of pPMPT-neoR plasmid

First, the neomycin resistance gene was amplified from pet-28a, and the amplification map is shown in FIG. 6 .

When designing primers, add restriction enzyme sites BglII and XbaI to the upstream and downstream ends respectively, and the sequence of the amplification primers is:

F4: AGATCTATGAGCCATATTCAACGGG

R4: TCTAGATTAGAAAAACTCATCGAGCA

The neomycin resistance gene (neoR) amplification system is as follows:

Amplification conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s; annealing at 58°C for 30 s; extension at 72°C for 1 min, 30 cycles; extension at 72°C for 5 min. PCR products were detected by 1.5% agarose gel electrophoresis.

The amplified neomycin resistance gene was connected to the T vector overnight at a certain ratio, transformed into Escherichia coli, and clones were picked for verification.

The positive clones were cultured overnight, the plasmids were extracted and digested, and the plasmids and pPMPT plasmids were digested with bglII and XbaI respectively. The enzyme digestion recovery pattern is shown in Figure 7.

According to a certain ratio, the recovered products were ligated overnight, and chloramphenicol was used as a resistance selection marker to pick positive clones, and neomycin primers were used for amplification verification.

The neomycin resistance gene (neoR) amplification system is as follows:

Amplification conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s; annealing at 57°C to 60°C for 30 s; extension at 72°C for 1 min, 30 cycles; extension at 72°C for 5 min. PCR products were detected by 1.5% agarose gel electrophoresis. The amplification map is shown in Figure 8.

(4) Construction of pPMPT-neoR-crtZ plasmid

To directly amplify the crtZ sequence, the designed primer sequences are as follows:

F5: 5'-AAGCTTATGTTGTGGATTTGG-3'

R5: 5'-AAGCTTAGGATCCTTACTTCCCG-3'

The amplification system of the crtZ gene is as follows:

Amplification conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s; annealing at 57°C to 60°C for 30 s; extension at 72°C for 1 min, 30 cycles; extension at 72°C for 5 min. PCR products were detected by 1.5% agarose gel electrophoresis. The amplification map is shown in Figure 9.

The pPMPT-neoR plasmid constructed in (3) was digested with AhdI to form a linear T-terminus, directly connected to the amplified product of the crtZ gene overnight, transformed into Escherichia coli, and chloramphenicol was used as a resistance selection marker to obtain The pPMPT-neoR-crtZ plasmid was amplified with primers (F5 and R5) to detect positive clones.

The crtZ gene (crtZ) amplification system and amplification procedure are as above. Its detection spectrum is shown in Figure 10.

Use the neoR downstream primer and the crtZ gene downstream primer to amplify the positive clones, verify and screen the forward and reverse connections of the crtZ gene. The amplification detection map is shown in Figure 11.

The forward connection amplification system is as follows:

Amplification conditions: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds; annealing at 57°C to 60°C for 30 seconds; extension at 72°C for 2 minutes, 30 cycles; extension at 72°C for 5 minutes. PCR products were detected by 1.5% agarose gel electrophoresis.

Transform the constructed pPMPT-neoR-crtZ plasmid into Agrobacterium, and use the Agrobacterium-mediated method to transform B. trispora. The resistance selection marker used in the transformation is a neomycin resistance selection marker. For the positive transformant grown on the resistance plate, the DNA was extracted, and the neomycin resistance gene was amplified to detect whether the transformation was successful.

The amplification detection map is shown in Figure 12.

The transformed B. trispora was fermented for about 7 days, and the fermentation product was subjected to a series of treatments such as freeze-drying, grinding, and extraction to obtain the desired pigment, and the presence of zeaxanthin was found through HPLC detection.

The HPLC chromatograms of zeaxanthin standard substance and samples to be tested are shown in Figure 13 and Figure 14.

The neoR and crtZ genes inserted above can be replaced with any desired gene to obtain the target product, thereby realizing the function of the plasmid. It can be seen that the present invention can express two or more genes at the same time without adding a promoter and integrating multiple exogenous genes. Stablize.

The TA cloning site in the present invention can be prepared by selectively replacing the two AhdⅠ restriction sites with XcmⅠ, BciⅥ, BmrⅠ, HphⅠ, Hpy188Ⅰ, HpyCH4Ⅲ, MboⅡ, and MnlⅠ restriction sites when transforming different vectors T-terminus for direct ligation of PCR products.

SEQUENCE LISTING

<110> Hebei University

<120> A Double-gene Co-expression Recombinant Vector and Its Construction Method and Application

<130>

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 1799

<212>DNA

<213> gene expression cassette sequence syn

<400> 1

aaagaaggat tacctctaaa caagtgtacc tgtgcattct gggtaaacga ctcataggag 60

agttgtaaaa aagtttcggc cggcgtattg ggtgttacgg agcattcact aggcaaccat 120

ggttactatt gtataccatc ttagtaggaa tgatttcgag gtttatacct acgatgaatg 180

tgtgtcctgt aggcttgaga gttcaaggaa gaaacatgca attatctttg cgaacccagg 240

gctggtgacg gaattttcat agtcaagcta tcagagtaaa gaagaggagc atgtcaaagt 300

acaattagag acaaatatat agtcgcgtgg agccaagagc ggattcctca gtctcgtagg 360

tctcttgacg accgttgatc tgcttgatct cgtctcccga aaatgaaaat agctctgcta 420

agctattctt ctcttcgccg gagcctgaag gcgttactag gttgcagtca atgcattaat 480

gcattgcaga tgagctgtat ctggaagagg taaacccgaa aacgcgtttt attcttgttg 540

acatggagct attaaatcat ctagaggtca ccgagctcgg taccccccggg caattgagat 600

ctgagattaa aatagataag gaaaagaaag tgaaaagaaa ttcggaagca tggcacattc 660

ttctttttat aaatacatgc ctgactttct ttttccatcg atatgatata tgcatatgat 720

agatatacaa gcaatcttct tcaaggagtt tgaaattttg tcctccagga gcaaaaaaaa 780

gttttttttt atacatgttt gtacacaaga atagttacca atttgctttg gtcttacgtg 840

ctgcaagttt atatcgtttt caatttcttt gtctttacat tttctttgtc ctttatcttt 900

cctcatttag tctttgggag aattaggaaa agggagcgga aaggtaagaa atgcttgcgt 960

attttactaa ttcggcaaac atccaatttg gcaaacagca gcctgtgcaa cgctctcgag 1020

atgacagtat ctttgattac actctaaatc tcgatgaccc gaccaaaaag agcgaacaaa 1080

gaaataatct tgtgcattcg aatatgatgg aagatttttt cccccttatt ctaaatgttg 1140

acatagcgtg tatgttatat aaacaaaaag aaattgtaca aactttcttt tcttctcttt 1200

ttattttc tcgactctgg gtcactagtg accccagagtc actaaattat cgaggtacag 1260

ttgttcttat tttgcgcaaa agcccaaatg gagaaggtct atgtcgagta gacgttacgt 1320

aattacgtaa ctgacgttgg atcattgcgg aagtccgagg ccgcttctct tcttatcgaa 1380

tcgtctcgat aaaagtaaaa gccctctgct ctagttcgtc tagttgccag cagttctctg 1440

gatgctctga ctccttaggc gagaaccgag gtgcgctgat atataaacag agattaacat 1500

gaaactgtac gaggagaaga aatgagacta tcgaactgat acttttaagg cagtggtcgg 1560

gacccaagcg tttctattaa cgtacaaaga aggaacttga gagttcggat gtcctgtgtg 1620

taagtagcat ccatatttgg agctttagta aggatgattc taccatatgt tatcattggt 1680

accaacggat cacttacgag gcattgtggg ttatgcggcc ggctttgaaa aaatgttgag 1740

aggatactca gcaaatgggt ccttacgtgtc catgtgaaca aatctccatt aggaagaaa 1799

<210> 2

<211> 2120

<212>DNA

<213> hygromycin resistance gene hygR

<400> 2

cgcgcaaaga aggattacct ctaaacaagt gtacctgtgc attctgggta aacgactcat 60

aggagagttg taaaaaagtt tcggccggcg tattgggtgt tacggagcat tcactaggca 120

accatggtta ctattgtata ccatcttagt aggaatgatt tcgaggtta tacctacgat 180

gaatgtgtgt cctgtaggct tgagagttca aggaagaaac atgcaattat ctttgcgaac 240

ccagggctgg tgacggaatt ttcatagtca agctatcaga gtaaagaaga ggagcatgtc 300

aaagtacaat tagagacaaa tatatagtcg cgtggagcca agagcggatt cctcagtctc 360

gtaggtctct tgacgaccgt tgatctgctt gatctcgtct cccgaaaatg aaaatagctc 420

tgctaagcta ttcttctctt cgccggagcc tgaaggcgtt actaggttgc agtcaatgca 480

ttaatgcatt gcagatgagc tgtatctgga agaggtaaac ccgaaaacgc gttttattct 540

tgttgacatg gagctattaa atcactagaa ggcactcttt gctgcttgga caaatgaacg 600

tatcttatcg agatcctgaa caccatttgt ctcaactccg gagctgacat cgacaccaac 660

gatcttatat ccagattcgt caagctgttt gatgatttca gtaacgttaa gtggatccgg 720

tcggcatcta ctctattcct ttgccctcgg acgagtgctg gggcgtcggt ttccactatc 780

ggcgagtact tctacacagc catcggtcca gacggccgcg cttctgcggg cgatttgtgt 840

acgcccgaca gtcccggctc cggatcggac gattgcgtcg catcgaccct gcgcccaagc 900

tgcatcatcg aaattgccgt caaccaagct ctgatagagt tggtcaagac caatgcggag 960

catatacgcc cggagccgcg gcgatcctgc aagctccgga tgcctccgct cgaagtagcg 1020

cgtctgctgc tccatacaag ccaaccacgg cctccagaag aagatgttgg cgacctcgta 1080

ttgggaatcc ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc ggccattgtc 1140

cgtcaggaca ttgttggagc cgaaatccgc gtgcacgagg tgccggactt cggggcagtc 1200

ctcggcccaa agcatcagct catcgagagc ctgcgcgacg gacgcactga cggtgtcgtc 1260

catcacagtt tgccagtgat acacatgggg atcagcaatc gcgcatatga aatcacgcca 1320

tgtagtgtat tgaccgattc cttgcggtcc gaatgggccg aacccgctcg tctggctaag 1380

atcggccgca gcgatcgcat ccatggcctc cgcgaccggc tgcagaacag cgggcagttc 1440

ggtttcaggc aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc aataggtcag 1500

gctctcgctg aattccccaa tgtcaagcac ttccggaatc gggagcgcgg ccgatgcaaa 1560

gtgccgataa acataacgat ctttgtagaa accatcggcg cagctattta cccgcaggac 1620

atatccacgc cctcctacat cgaagctgaa agcacgagat tcttcgccct ccgagagctg 1680

catcaggtcg gagacgctgt cgaacttttc gatcagaaac ttctcgacag acgtcgcggt 1740

gagttcaggc tttttcatat cgatgcttgg gtagaatagg taagtcagat tgaatctgaa 1800

ataaagggag gaagggcgaa cttaagaagg tatgaccggg tcgttcactt accttgcttg 1860

acaaacgcac aagttatcgt gcaccaagca gcagatgata ataatgtcct cgttcctgtc 1920

tgctaataag agtcacactt cgagcgccgc cgctactgct tacaagtggg ctgatctgac 1980

cagttgccta aatgaaccat cttgtcaaac gacacaaatt ttgtgatccg cctggacgac 2040

taaaccaaaa tagcattgat gtgttgacct cactagctc cagccaagcc caaaaatgct 2100

ccttcaatat catcttctgg 2120

<210> 3

<211> 11198

<212> DNA

<213> pPMPT

<400> 3

agtactttga tccaacccct ccgctgctat agtgcagtcg gcttctgacg ttcagtgcag 60

ccgtcttctg aaaacgacat gtcgcacaag tcctaagtta cgcgacaggc tgccgccctg 120

cccttttcct ggcgttttct tgtcgcgtgt tttagtcgca taaagtagaa tacttgcgac 180

tagaaccgga gacattacgc catgaacaag agcgccgccg ctggcctgct gggctatgcc 240

cgcgtcagca ccgacgacca ggacttgacc aaccaacggg ccgaactgca cgcggccggc 300

tgcaccaagc tgttttccga gaagatcacc ggcaccaggc gcgaccgccc ggagctggcc 360

aggatgcttg accacctacg ccctggcgac gttgtgacag tgaccaggct agaccgcctg 420

gcccgcagca cccgcgacct actggacatt gccgagcgca tccaggaggc cggcgcgggc 480

ctgcgtagcc tggcagagcc gtgggccgac accaccacgc cggccggccg catggtgttg 540

accgtgttcg ccggcattgc cgagttcgag cgttccctaa tcatcgaccg cacccggagc 600

gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc cccgccctac cctcaccccg 660

gcacagatcg cgcacgcccg cgagctgatc gaccaggaag gccgcaccgt gaaagaggcg 720

gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg cacttgagcg cagcgaggaa 780

gtgacgccca ccgaggccag gcggcgcggt gccttccgtg aggacgcatt gaccgaggcc 840

gacgccctgg cggccgccga gaatgaacgc caagaggaac aagcatgaaa ccgcaccagg 900

acggccagga cgaaccgttt ttcattaccg aagagatcga ggcggagatg atcgcggccg 960

ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcg gctgcatgaa atcctggccg 1020

gtttgtctga tgccaagctg gcggcctggc cggccagctt ggccgctgaa gaaaccgagc 1080

gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag cttgcgtcat gcggtcgctg 1140

cgtatatgat gcgatgagta aataaacaaa tacgcaaggg gaacgcatga aggttatcgc 1200

tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc gcaacccatc tagcccgcgc 1260

cctgcaactc gccggggccg atgttctgtt agtcgattcc gatccccagg gcagtgcccg 1320

cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt gtcggcatcg accgcccgac 1380

gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc gtagtgatcg acggagcgcc 1440

ccaggcggcg gacttggctg tgtccgcgat caaggcagcc gacttcgtgc tgattccggt 1500

gcagccaagc ccttacgaca tatgggccac cgccgacctg gtggagctgg ttaagcagcg 1560

cattgaggtc acggatggaa ggctacaagc ggcctttgtc gtgtcgcggg cgatcaaagg 1620

cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg tacgagctgc ccattcttga 1680

gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc gccgccggca caaccgttct 1740

tgaatcagaa cccgagggcg acgctgcccg cgaggtccag gcgctggccg ctgaaattaa 1800

atcaaaactc atttgagtta atgaggtaaa gagaaaatga gcaaaagcac aaacacgcta 1860

agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa cgttggccag cctggcagac 1920

acgccagcca tgaagcgggt caactttcag ttgccggcgg aggatcacac caagctgaag 1980

atgtacgcgg tacgccaagg caagaccatt accgagctgc tatctgaata catcgcgcag 2040

ctaccagagt aaatgagcaa atgaataaat gagtagatga attttagcgg ctaaaggagg 2100

cggcatggaa aatcaagaac aaccaggcac cgacgccgtg gaatgcccca tgtgtggagg 2160

aacgggcggt tggccaggcg taagcggctg ggttgtctgc cggccctgca atggcactgg 2220

aacccccaag cccgaggaat cggcgtgagc ggtcgcaaac catccggccc ggtacaaatc 2280

ggcgcggcgc tgggtgatga cctggtggag aagttgaagg ccgcgcaggc cgcccagcgg 2340

caacgcatcg aggcagaagc acgccccggt gaatcgtggc aagcggccgc tgatcgaatc 2400

cgcaaagaat cccggcaacc gccggcagcc ggtgcgccgt cgattaggaa gccgcccaag 2460

ggcgacgagc aaccagattt tttcgttccg atgctctatg acgtgggcac ccgcgatagt 2520

cgcagcatca tggacgtggc cgttttccgt ctgtcgaagc gtgaccgacg agctggcgag 2580

gtgatccgct acgagcttcc agacgggcac gtagaggttt ccgcagggcc ggccggcatg 2640

gccagtgtgt gggattacga cctggtactg atggcggttt cccatctaac cgaatccatg 2700

aaccgatacc gggaagggaa gggagacaag cccggccgcg tgttccgtcc acacgttgcg 2760

gacgtactca agttctgccg gcgagccgat ggcggaaagc agaaagacga cctggtagaa 2820

acctgcattc ggttaaacac cacgcacgtt gccatgcagc gtacgaagaa ggccaagaac 2880

ggccgcctgg tgacggtatc cgagggtgaa gccttgatta gccgctacaa gatcgtaaag 2940

agcgaaaccg ggcggccgga gtacatcgag atcgagctag ctgattggat gtaccgcgag 3000

atcacagaag gcaagaaccc ggacgtgctg acggttcacc ccgattactt tttgatcgat 3060

cccggcatcg gccgttttct ctaccgcctg gcacgccgcg ccgcaggcaa ggcagaagcc 3120

agatggttgt tcaagacgat ctacgaacgc agtggcagcg ccggagagtt caagaagttc 3180

tgtttcaccg tgcgcaagct gatcgggtca aatgacctgc cggagtacga tttgaaggag 3240

gaggcggggc aggctggccc gatcctagtc atgcgctacc gcaacctgat cgagggcgaa 3300

gcatccgccg gttcctaatg tacggagcag atgctagggc aaattgccct agcaggggaa 3360

aaaggtcgaa aaggtctctt tcctgtggat agcacgtaca ttgggaaccc aaagccgtac 3420

attgggaacc ggaacccgta cattgggaac ccaaagccgt attgggaa ccggtcacac 3480

atgtaagtga ctgatataaa agagaaaaaa ggcgattttt ccgcctaaaa ctctttaaaa 3540

ctttattaaaa ctcttaaaac ccgcctggcc tgtgcataac tgtctggcca gcgcacagcc 3600

gaagagctgc aaaaagcgcc tacccttcgg tcgctgcgct ccctacgccc cgccgcttcg 3660

cgtcggccta tcgcggccgc tggccgctca aaaatggctg gcctacggcc aggcaatcta 3720

ccagggcgcg gacaagccgc gccgtcgcca ctcgaccgcc ggcgcccaca tcaaggcacc 3780

ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac 3840

ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc 3900

gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta 3960

tactggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt 4020

gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg 4080

ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 4140

gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 4200

ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 4260

cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 4320

ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 4380

accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 4440

catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 4500

gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 4560

tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 4620

agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 4680

actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 4740

gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 4800

aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 4860

gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gcatgatata 4920

tctcccaatt tgtgtagggc ttattatgca cgcttaaaaa taataaaagc agacttgacc 4980

tgatagtttg gctgtgagca attatgtgct tagtgcatct aatcgcttga gttaacgccg 5040

gcgaagcggc gtcggcttga acgaatttct agctagagga tcgcaccaat aactgcctta 5100

aaaaaattac gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct 5160

gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac 5220

cttgtcgcct tgcgtataat atttgcccat tgtgaaaacg ggggcgaaga agttgtccat 5280

attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa 5340

catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc 5400

ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga 5460

aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac 5520

cagctcaccg tctttcattg ccatacggaa ctccggatga gcattcatca ggcgggcaag 5580

aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc 5640

cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc 5700

aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt 5760

ctccatgatg tttaactttg ttttagggcg actgccctgc tgcgtaacat cgttgctgct 5820

ccataacatc aaacatcgac ccacggcgta acgcgcttgc tgcttggatg cccgaggcat 5880

agactgtacc ccaaaaaaac atgtcataac aagaagccat gaaaaccgcc actgcgccgt 5940

taccaccgct gcgttcggtc aaggttctgg accacgttgcg tgacggcagt tacgctactt 6000

gcattacagc ttacgaaccg aacgaggctt atgtccactg ggttcgtgcc cgaattgatc 6060

acaggcagca acgctctgtc atcgttacaa tcaacatgct accctccgcg agatcatccg 6120

tgtttcaaac ccggcagctt agttgccgtt cttccgaata gcatcggtaa catgagcaaa 6180

gtctgccgcc ttacaacggc tctcccgctg acgccgtccc ggactgatgg gctgcctgta 6240

tcgagtggtg attttgtgcc gagctgccgg tcggggagct gttggctggc tggtggcagg 6300

atatattgtg gtgtaaacaa attgacgctt agacaactta ataacacatt gcggacgttt 6360

ttaatgtact gaattaacgc cgaattaatt cgggggatct atagggactt taggtgatct 6420

ggattttagt actggatttt ggttttagga attagaaatt ttatgatag aagtatttta 6480

caaatacaaa tacatactaa gggtttctta tatgctcaac acgtgagcga aaccctataa 6540

gaaccctaat tcccttatct gggaactact cacacattat tatggagaaa ctcgatgtcg 6600

atcgactcta gctagaggat cgatccgaac cccagagtcc cgctcagaag aactcgtcaa 6660

gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga 6720

agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt 6780

cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat 6840

tttccaccat gatattcggc aagcaggcat cgccatgtgt cacgacgaga tcctcgccgt 6900

cgggcatgcg cgcaaagaag gattacctct aaacaagtgt acctgtgcat tctgggtaaa 6960

cgactcatag gagagttgta aaaaagtttc ggccggcgta ttgggtgtta cggagcattc 7020

actaggcaac catggttact attgtatacc atcttagtag gaatgatttc gaggtttata 7080

cctacgatga atgtgtgtcc tgtaggcttg agagttcaag gaagaaacat gcaattatct 7140

ttgcgaaccc agggctggtg acggaatttt catagtcaag ctatcagagt aaagaagagg 7200

agcatgtcaa agtacaatta gagacaaata tatagtcgcg tggagccaag agcggattcc 7260

tcagtctcgt aggtctcttg acgaccgttg atctgcttga tctcgtctcc cgaaaatgaa 7320

aatagctctg ctaagctatt cttctcttcg ccggagcctg aaggcgttac taggttgcag 7380

tcaatgcatt aatgcattgc agatgagctg tatctggaag aggtaaaccc gaaaacgcgt 7440

tttattcttg ttgacatgga gctattaaat cactagaagg cactctttgc tgcttggaca 7500

aatgaacgta tcttatcgag atcctgaaca ccatttgtct caactccgga gctgacatcg 7560

acaccaacga tcttatatcc agattcgtca agctgtttga tgatttcagt aacgttaagt 7620

ggatccggtc ggcatctact ctattccttt gccctcggac gagtgctggg gcgtcggttt 7680

ccactatcgg cgagtacttc tacacagcca tcggtccaga cggccgcgct tctgcgggcg 7740

atttgtgtac gcccgacagt cccggctccg gatcggacga ttgcgtcgca tcgaccctgc 7800

gcccaagctg catcatcgaa attgccgtca accaagctct gatagagttg gtcaagacca 7860

atgcggagca tatacgcccg gagccgcggc gatcctgcaa gctccggatg cctccgctcg 7920

aagtagcgcg tctgctgctc catacaagcc aaccacggcc tccagaagaa gatgttggcg 7980

acctcgtatt gggaatcccc gaacatcgcc tcgctccagt caatgaccgc tgttatgcgg 8040

ccattgtccg tcaggacatt gttggagccg aaatccgcgt gcacgaggtg ccggacttcg 8100

gggcagtcct cggcccaaag catcagctca tcgagagcct gcgcgacgga cgcactgacg 8160

gtgtcgtcca tcacagtttg ccagtgatac acatggggat cagcaatcgc gcatatgaaa 8220

tcacgccatg tagtgtattg accgattcct tgcggtccga atgggccgaa cccgctcgtc 8280

tggctaagat cggccgcagc gatcgcatcc atggcctccg cgaccggctg cagaacagcg 8340

ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg ggagatgcaa 8400

taggtcaggc tctcgctgaa ttccccaatg tcaagcactt ccggaatcgg gagcgcggcc 8460

gatgcaaagt gccgataaac ataacgatct ttgtagaaac catcggcgca gctatttacc 8520

cgcaggacat atccacgccc tcctacatcg aagctgaaag cacgagattc ttcgccctcc 8580

gagagctgca tcaggtcgga gacgctgtcg aacttttcga tcagaaactt ctcgacagac 8640

gtcgcggtga gttcaggctt tttcatatcg atgcttgggt agaataggta agtcagattg 8700

aatctgaaat aaagggagga agggcgaact taagaaggta tgaccgggtc gttcacttac 8760

cttgcttgac aaacgcacaa gttatcgtgc accaagcagc agatgataat aatgtcctcg 8820

ttcctgtctg ctaataagag tcacacttcg agcgccgccg ctactgctta caagtgggct 8880

gatctgacca gttgcctaaa tgaaccatct tgtcaaacga cacaaatttt gtgatccgcc 8940

tggacgacta aaccaaaata gcattgatgt gttgacctcc actagctcca gccaagccca 9000

aaaatgctcc ttcaatatca tcttctggtc gacaaagaag gattacctct aaacaagtgt 9060

acctgtgcat tctgggtaaa cgactcatag gagagttgta aaaaagtttc ggccggcgta 9120

ttgggtgtta cggagcattc actaggcaac catggttact attgtatacc atcttagtag 9180

gaatgatttc gaggtttata cctacgatga atgtgtgtcc tgtaggcttg agagttcaag 9240

gaagaaacat gcaattatct ttgcgaaccc agggctggtg acggaatttt catagtcaag 9300

ctatcagagt aaagaagagg agcatgtcaa agtacaatta gagacaaata tatagtcgcg 9360

tggagccaag agcggattcc tcagtctcgt aggtctcttg acgaccgttg atctgcttga 9420

tctcgtctcc cgaaaatgaa aatagctctg ctaagctatt cttctcttcg ccggagcctg 9480

aaggcgttac taggttgcag tcaatgcatt aatgcattgc agatgagctg tatctggaag 9540

aggtaaaccc gaaaacgcgt tttattcttg ttgacatgga gctattaaat catctagagg 9600

tcaccgagct cggtaccccc gggcaattga gatctgagat taaaatagat aaggaaaaga 9660

aagtgaaaag aaattcggaa gcatggcaca ttcttctttt tataaataca tgcctgactt 9720

tctttttcca tcgatatgat atatgcatat gatagatata caagcaatct tcttcaagga 9780

gtttgaaatt ttgtcctcca ggagcaaaaa aaagtttttt tttatacatg tttgtacaca 9840

agaatagtta ccaatttgct ttggtcttac gtgctgcaag tttatatcgt tttcaatttc 9900

tttgtcttta cattttcttt gtcctttatc tttcctcatt tagtctttgg gagaattagg 9960

aaaagggagc ggaaaggtaa gaaatgcttg cgtattttac taattcggca aacatccaat 10020

ttggcaaaca gcagcctgtg caacgctctc gagatgacag tatctttgat tacactctaa 10080

atctcgatga cccgaccaaa aagagcgaac aaagaaataa tcttgtgcat tcgaatatga 10140

tggaagattt tttccccctt attctaaatg ttgacatagc gtgtatgtta tataaacaaa 10200

aagaaattgt acaaactttc ttttcttctc tttttatttt atctcgactc tgggtcacta 10260

gtgacccaga gtcactaaat tatcgaggta cagttgttct tattttgcgc aaaagcccaa 10320

atggagaagg tctatgtcga gtagacgtta cgtaattacg taactgacgt tggatcattg 10380

cggaagtccg aggccgcttc tcttcttatc gaatcgtctc gataaaagta aaagccctct 10440

gctctagttc gtctagttgc cagcagttct ctggatgctc tgactcctta ggcgagaacc 10500

gaggtgcgct gatatataaa cagagattaa catgaaactg tacgaggaga agaaatgaga 10560

ctatcgaact gatactttta aggcagtggt cgggacccaa gcgtttctat taacgtacaa 10620

agaaggaact tgagagttcg gatgtcctgt gtgtaagtag catccatatt tggagcttta 10680

gtaaggatga ttctaccata tgttatcatt ggtaccaacg gatcacttac gaggcattgt 10740

gggttatgcg gccggctttg aaaaaatgtt gagaggatac tcagcaaatg ggtcttacgt 10800

gtccatgtga acaaatctcc attaggaaga aaaagcttgg cactggccgt cgttttacaa 10860

cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct 10920

ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc 10980

agcctgaatg gcgaatgcta gagcagcttg agcttggatc agattgtcgt ttcccgcctt 11040

cagtttaaac tatcagtgtt tgacaggata tattggcggg taaacctaag agaaaagagc 11100

gtttataga ataacggata tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg 11160

tatgtgcatg ccaaccacag ggttcccctc gggatcaa 11198

<210> 4

<211> 528

<212>DNA

<213> Gene crtZ

<400> 4

atgttgtgga tttggaatgc cctgatcgtt ttcgttaccg tgattggcat ggaagtgatt 60

gctgcactgg cacacaaata catcatgcac ggctggggtt ggggatggca tctttcacat 120

catgaaccgc gtaaaggtgc gtttgaagtt aacgatcttt atgccgtggt ttttgctgca 180

ttatcgatcc tgctgatta tctgggcagt acaggaatgt ggccgctcca gtggattggc 240

gcaggtatga cggcgtatgg attackctat tttatggtgc acgacgggct ggtgcatcaa 300

cgttggccat tccgctatat tccacgcaag ggctacctca aacggttgta tatggcgcac 360

cgtatgcatc acgccgtcag gggcaaagaa ggttgtgttt cttttggctt cctctatgcg 420

ccgcccctgt caaaacttca ggcgacgctc cgggaaagac atggcgctag agcgggcgct 480

gccagagatg cgcagggcgg ggaggatgag cccgcatccg ggaagtaa 528

<210> 5

<211> 816

<212>DNA

<213> gene neoR

<400> 5

atgagccata ttcaacggga aacgtcttgc tctaggccgc gattaaattc caacatggat 60

gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc 120

tatcgattgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc 180

gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct 240

cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg 300

atccccggga aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt 360

gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct 420

tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg 480

gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa 540

gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca 600

cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc 660

ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct 720

ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa 780

ttgcagtttc atttgatgct cgatgagtttttctaa 816

Claims (8)

1. a kind of double gene coexpression recombinant vector, which is characterized in that carry gene expression frame sequence by skeleton of starting vector Syn is arranged, the sequence syn contains：It is prepared by terminator sequence-multiple cloning sites-two-way startup subsequence PCarB-TA clones Sequence-terminator sequence, the nucleotide sequence such as SEQ ID NO of the sequence syn:Shown in 1.

2. double gene coexpression recombinant vector according to claim 1, which is characterized in that the starting vector be pATZ, pPZP100、pPZP101、pPZP102、pPZP111、pPZP112、pPZP121、pPZP122、pPZP200、pPZP201、 Any one in pPZP202, pPZP211, pPZP212, pPZP221 or pPZP222.

3. double gene coexpression recombinant vector according to claim 1, which is characterized in that also taken in the recombinant vector With hygromycin gene hygR, the nucleotide sequence such as SEQ ID NO of the hygromycin gene hygR:2.

4. a kind of double gene coexpression recombinant vector pPMPT, which is characterized in that carry gene using starting vector pATZ as skeleton Expression cassette sequence syn, and using hygromycin gene hygR as riddled basins, the gene expression frame sequence syn contains Have：Terminator sequence-multiple cloning sites-two-way startup subsequence PCarB-TA clones prepare sequence-terminator sequence, Nucleotide sequence such as SEQ ID NO:Shown in 1；The nucleotide sequence of the hygromycin gene hygR such as SEQ ID NO:2 institutes Show；

Its structural formula is：

5. double gene coexpression recombinant vector pPMPT according to claim 4, which is characterized in that recombinant vector pPMPT's Nucleotide sequence such as SEQ ID NO:Shown in 3.

6. the construction method of the double gene coexpression recombinant vector pPMPT described in a kind of claim 4 or 5, which is characterized in that packet Include following steps：

(a) according to such as SEQ ID NO:Nucleotide sequence synthetic gene expression cassette sequence syn shown in 1, and it is cloned in load In body pMD19-T, plasmid pMD19-T-syn is obtained；

(b) the expressed intact frame of amplification hygromycin gene hygR, is cloned in carrier pMD19-T, obtains plasmid pMD19-T-hygR；

(c) the plasmid pMD19-T-hygR and plasmid pMD19-T-syn are subjected to digestion with I restriction enzymes of Sal respectively, Digestion products two segments of pMD19-T-hygR and syn are recycled, 16 DEG C of connections overnight, obtain pMD19-T-hygR-syn；

(d) by pMD19-T-hygR-syn and starting vector pATZ respectively with III restriction enzymes double zyme of BssH II and Hind It cuts, the remaining part containing T-DNA after digestion products hygR-syn, pATZ digestion is recycled, by two segments of recycling 16 It is connected overnight at DEG C, obtains pATZ-hygR-syn, be named as recombinant vector pPMPT.

7. target gene is transferred to filamentous fungi using the double gene coexpression recombinant vector described in claim 1,2,3,4 or 5 The middle application for realizing expression.

8. target gene is transferred to three spore Bradleys using the double gene coexpression recombinant vector described in claim 1,2,3,4 or 5 The mould middle application for realizing expression.