CN114350700A - A kind of Saccharomyces cerevisiae vector and its construction method and application - Google Patents

Abstract

The invention discloses a saccharomyces cerevisiae vector and a construction method and application thereof, wherein the construction method of the saccharomyces cerevisiae vector comprises the following steps: a PGBKT7 yeast two-hybrid vector is taken as a basic framework, and a 35S HYG sequence fragment, a bacterial plasmid transformant related element sequence, a CEN6-ARSH4 sequence fragment and an MCS sequence fragment are sequentially recombined to obtain a PGBKT7-Hyg-pVS-CEN6-ARSH4-MCS recombinant vector, namely the Saccharomyces cerevisiae vector for plant artificial chromosome assembly. The saccharomyces cerevisiae vector constructed by the invention can be used for assembling related large segments of plant artificial chromosomes so as to meet the requirements of large segment sequence synthesis and assembly in plant artificial chromosome experiments and functional verification in plants, and has practical research value and definite application prospect.

Description

A kind of Saccharomyces cerevisiae vector and its construction method and application

technical field

The invention relates to the technical field of gene recombination, in particular to a Saccharomyces cerevisiae vector and a construction method and application thereof.

Background technique

Following the discovery of the DNA double helix and the human genome sequencing project, synthetic biology, marked by the design and synthesis of genomes, has triggered the third biotechnology revolution. Synthetic biology is the study of artificial biological systems based on genetic engineering and engineering methods of systems biology, from gene fragments, DNA molecules, gene regulatory networks and signal transduction pathways to the artificial design and synthesis of cells. If gene sequencing is "reading" genes, then synthetic biology is "writing" genes. Advances in recent years have shown that synthetic biology has the potential to drive technological innovation in diverse fields of application including biocomputing, biomaterials, electronic interfaces, therapeutic gene editing, multiplex diagnostics and cell recording, third-generation biorefinery, and biotherapy Wait. At present, major breakthroughs have been made in the artificial synthesis of eukaryotic chromosomes. Some teams have successively realized the artificial synthesis of yeast chromosomes and the artificial synthesis of yeast eukaryotic cells containing only a single chromosome. However, no breakthrough has been made in the artificial synthesis of chromosomes in higher eukaryotes.

Therefore, the existing technology still needs to be improved and developed.

SUMMARY OF THE INVENTION

In view of the above-mentioned deficiencies of the prior art, the purpose of the present invention is to provide a method for constructing a Saccharomyces cerevisiae vector that can be used for the assembly of large fragments related to plant artificial chromosomes.

The technical scheme of the present invention is as follows:

A method for constructing a Saccharomyces cerevisiae vector, comprising the steps:

Provide PGBKT7 yeast two-hybrid vector;

The PGBKT7 yeast two-hybrid vector is linearized and homologously recombined with the 35S:HYG sequence fragment by enzyme cutting to obtain a PGBKT7-Hyg recombinant vector, and the nucleotide sequence of the 35S:HYG sequence fragment is shown in SEQ ID NO.1 ;

The PGBKT7-Hyg recombinant vector is homologously recombined with the bacterial plasmid transformant-related element sequence by enzyme cutting and linearization to obtain the PGBKT7-Hyg-pVS recombinant vector;

The PGBKT7-Hyg-pVS recombinant vector is homologously recombined with the CEN6-ARSH4 sequence fragment by enzyme cutting and linearization to obtain the PGBKT7-Hyg-pVS-CEN6-ARSH4 recombinant vector, the nucleotide sequence of the CEN6-ARSH4 sequence fragment As shown in SEQ ID NO.2;

The PGBKT7-Hyg-pVS-CEN6-ARSH4 recombinant vector is linearized and homologously recombined with the MCS sequence fragment by enzyme cutting to obtain the PGBKT7-Hyg-pVS-CEN6-ARSH4-MCS recombinant vector, that is, the Saccharomyces cerevisiae vector is constructed and obtained , the nucleotide sequence of the MCS sequence fragment is shown in SEQ ID NO.3.

The construction method of the Saccharomyces cerevisiae vector, wherein, the bacterial plasmid transformant-related element sequence includes PVS1 StaA sequence, PVS1-RepA sequence and PVS1 OriV sequence, wherein, the nucleotide sequence of the PVS1 StaA sequence is as SEQ ID NO. .4, the nucleotide sequence of the PVS1-RepA sequence is shown in SEQ ID NO.5, and the nucleotide sequence of the PVS1OriV sequence is shown in SEQ ID NO.6.

The construction method of the Saccharomyces cerevisiae vector, wherein, the preparation of the 35S:HYG sequence fragment comprises the steps:

Take pMDC83 plasmid as template, adopt upstream primer whose nucleotide sequence is as shown in SEQ ID NO.7 and the downstream primer whose nucleotide sequence is as shown in SEQ ID NO.8 to carry out PCR amplification to obtain the 35S:HYG sequence Fragment.

The construction method of the Saccharomyces cerevisiae vector, wherein, the preparation of the bacterial plasmid transformant-related element sequence comprises the steps:

Take pMDC83 plasmid as template, adopt the upstream primer whose nucleotide sequence is as shown in SEQ ID NO.9 and the downstream primer whose nucleotide sequence is as shown in SEQ ID NO.10 to carry out PCR amplification to obtain the bacterial plasmid transformant Related element sequences.

The construction method of the Saccharomyces cerevisiae vector, wherein, the preparation of the CEN6-ARSH4 sequence fragment comprises the steps:

Taking the yeast genome as a template, using the upstream primer whose nucleotide sequence is shown in SEQ ID NO.11 and the downstream primer whose nucleotide sequence is shown in SEQ ID NO.12 to carry out PCR amplification to obtain a CEN6 sequence fragment;

Taking the yeast genome as a template, using the upstream primer whose nucleotide sequence is shown in SEQ ID NO.13 and the downstream primer whose nucleotide sequence is shown in SEQ ID NO.14 for PCR amplification, the ARSH4 sequence fragment is obtained;

Taking the mixture of the CEN6 sequence fragment and the ARSH4 sequence fragment as a template, using the upstream primer whose nucleotide sequence is shown in SEQ ID NO.15 and the downstream primer whose nucleotide sequence is shown in SEQ ID NO.16 to carry out PCR amplification , to obtain the CEN6-ARSH4 sequence fragment.

The construction method of the Saccharomyces cerevisiae vector, wherein, the preparation of the MCS sequence fragment comprises the steps:

Taking PEG101-MCS as a template, using the upstream primer whose nucleotide sequence is shown in SEQ ID NO.17 and the downstream primer whose nucleotide sequence is shown in SEQ ID NO.18 to carry out PCR amplification to obtain the MCS sequence fragment .

A Saccharomyces cerevisiae vector, which is prepared by the construction method of the Saccharomyces cerevisiae vector of the present invention.

An application of a Saccharomyces cerevisiae vector, wherein the Saccharomyces cerevisiae vector of the present invention is used to assemble a large segment of artificially synthesized plant chromosomes.

The application of the Saccharomyces cerevisiae vector, wherein, the assembly of the Saccharomyces cerevisiae for artificially synthesizing large fragments of plant chromosomes comprises the steps:

Design an artificially synthesized plant chromosome-related fragment ACEN2, the nucleotide sequence of which is shown in SEQ ID NO.19;

The artificially synthesized plant chromosome-related fragment ACEN2 is subjected to Kpn I single digestion to obtain an ACEN2 digestion product;

The ACEN2 enzyme cleavage product is cyclized to obtain an ACEN2 cyclization product;

performing rolling circle amplification on the ACEN2 cyclization product to obtain an ACEN2 rolling circle amplification product;

Homologously recombine the Saccharomyces cerevisiae vector with the ACEN2-linker sequence fragment to obtain a PACB-MCS-ACEN2-linker recombinant vector, and the nucleotide sequence of the ACEN2-linker sequence fragment is shown in SEQ ID NO.20;

The PACB-MCS-ACEN2-linker recombinant vector was linearized by enzyme digestion and then transformed into yeast with the ACEN2 rolling circle amplification product, and after culturing in a Trp-deficient medium, the expression of PACB-MCS-ACEN2-linker and Positive recombinant yeast for ACEN2 rolling circle amplification product;

The positive yeast genome was electroporated into E. coli, and the recombinants of PACB-MCS-ACEN2-linker and ACEN2 rolling circle amplification products were propagated.

Beneficial effects: The present invention takes the PGBKT7 yeast two-hybrid vector as the basic skeleton, and successively recombines the 35S:HYG sequence fragment, the bacterial plasmid transformant-related element sequence, the CEN6-ARSH4 sequence fragment and the MCS sequence fragment, and successfully constructs a 35S:HYG sequence fragment that can be used in plants. The Saccharomyces cerevisiae vector assembled with artificial chromosome-related large fragments can meet the needs of large fragment sequence synthesis and assembly in plant artificial chromosome experiments and functional verification in plants. The invention has practical research value and clear application prospects.

Description of drawings

Fig. 1 is the flow chart of the construction method of a kind of Saccharomyces cerevisiae vector of the present invention.

Figure 2 is a schematic diagram of the constructed PGBKT7-Hyg-pVS-CEN6-ARSH4-MCS recombinant vector.

Figure 3 is a schematic diagram of the constructed PACB-MCS-ACEN2-linker recombinant vector.

Figure 4 shows the positive yeast colonies obtained by homologous recombination of PACB-MCS-ACEN2 and the target fragment in yeast.

Figure 5 shows the positive colonies of Escherichia coli after electrotransformation of yeast genome after PACB-MCS-ACEN2 homologous recombination.

Figure 6 shows the identification diagram of the restriction enzyme digestion of the plasmid after PACB-MCS-ACEN2 recombination, wherein the bands from left to right are: the first bar is a 15Kb marker, and the second bar is a PACB-MCS-ACEN2-linker vector restriction enzyme digestion For the control, the third line is a recombinant plasmid containing a target fragment of about 7.5 kb, and the fourth to the 14th line are all recombinant plasmids containing a smaller target fragment.

Detailed ways

The present invention provides a Saccharomyces cerevisiae vector and a construction method and application thereof. In order to make the purpose, technical scheme and effect of the present invention clearer and clearer, the present invention is further described below in detail. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention.

Saccharomyces cerevisiae is the first eukaryotic organism whose whole genome has been sequenced. It has the advantages of short growth cycle, strong fermentation ability, easy large-scale culture, and rich nutrients such as a variety of proteins, amino acids, vitamins, and biologically active substances. It has always been the main object of basic and applied research. Saccharomyces cerevisiae has the same structure as animal and plant cells which are both eukaryotes and is easy to cultivate. Therefore, it is very important to construct the corresponding yeast vector for the study of artificial chromosome synthesis in plants. Saccharomyces cerevisiae large fragment homologous recombination vector usually needs to contain the following elements: first, it needs to contain at least one yeast selectable marker gene; The centromeric sequence; thirdly, it needs to contain the relevant elements (BACcassette) required for bacterial propagation; fourthly, it needs to contain the selectable marker gene required for bacterial propagation, such as Kana; fifthly, it needs to be used in plants Screened marker genes, such as HYGR, Kana, etc. Sixth, it needs to contain multiple cloning sites, which are required for subsequent related experiments using the recombinant vector.

The PGBKT7 yeast two-hybrid vector is a fusion protein specially designed to express the GAL4 DNA binding domain (DNA-BD, AA 1-147) and the bait protein. The fusion protein is expressed at high levels under the ADH1 promoter. Contains c-Myc epitope tag. In order to promote in vitro transcription, a T7 promoter was introduced between the tags, the vector carrying the Kan resistance gene, which can be used for E. coli screening, and the TRP1 nutritional marker gene for yeast screening; compared with strains carrying other DNA-BD binding domain vectors, pGBKT7 has higher transformation efficiency. However, the PGBKT7 yeast two-hybrid vector cannot be directly used for large fragment assembly of plant artificial chromosomes.

Based on this, the invention provides a method for constructing a Saccharomyces cerevisiae carrier, as shown in Figure 1, which comprises the steps:

S10. Provide PGBKT7 yeast two-hybrid vector;

S20, after the described PGBKT7 yeast two-hybrid vector is linearized with 35S: HYG sequence fragment homologous recombination, obtain PGBKT7-Hyg recombinant vector, the nucleotide sequence of described 35S: HYG sequence fragment is such as SEQ ID NO. 1 shown;

S30, the PGBKT7-Hyg recombinant vector is subjected to homologous recombination with the bacterial plasmid transformant-related element sequence after linearization by enzyme cutting to obtain the PGBKT7-Hyg-pVS recombinant vector;

S40, homologous recombination with the CEN6-ARSH4 sequence fragment after the PGBKT7-Hyg-pVS recombinant vector is linearized by enzyme cutting to obtain the PGBKT7-Hyg-pVS-CEN6-ARSH4 recombinant vector, the core of the CEN6-ARSH4 sequence fragment The nucleotide sequence is shown in SEQ ID NO.2;

S50, homologously recombine the PGBKT7-Hyg-pVS-CEN6-ARSH4 recombinant vector with the MCS sequence fragment after linearization by enzyme cleavage to obtain the PGBKT7-Hyg-pVS-CEN6-ARSH4-MCS recombinant vector, that is, construct and obtain the Saccharomyces cerevisiae vector, the nucleotide sequence of the MCS sequence fragment is shown in SEQ ID NO.3.

The present invention utilizes the homologous recombination ability of yeast itself, takes the PGBKT7 yeast two-hybrid vector as the basic framework, and successively recombines the 35S:HYG sequence fragment, the bacterial plasmid transformant related element sequence, the CEN6-ARSH4 sequence fragment and the MCS sequence fragment, and successfully constructs the A Saccharomyces cerevisiae vector that can be used for the assembly of large fragments related to plant artificial chromosomes is developed to meet the needs of large fragment sequence synthesis and assembly in plant artificial chromosome experiments and functional verification in plants. The invention has practical research value and clear application prospects. .

The present invention will be further described in detail below through specific examples. In the present invention, the involved carriers, reagents and kits are conventional commercially available products, or can be obtained by conventional technical means in the art.

Example 1

Design and synthesis of oligonucleotide chains and acquisition of target fragments

1. The preparation of the 35S:HYG sequence fragment comprises the steps: using the pMDC83 plasmid as a template, using an upstream primer whose nucleotide sequence is shown in SEQ ID NO.7 and a downstream primer whose nucleotide sequence is shown in SEQ ID NO.8 PCR amplification is performed to obtain the 35S:HYG sequence fragment, which is a hygromycin resistance gene required for plant screening.

2. The relevant element sequences of bacterial plasmid transformants include PVS1 StaA sequence, PVS1-RepA sequence and PVS1OriV sequence, wherein, the nucleotide sequence of the PVS1 StaA sequence is shown in SEQ ID NO. The nucleotide sequence is shown in SEQ ID NO.5, and the nucleotide sequence of the PVS1 OriV sequence is shown in SEQ ID NO.6. The preparation of the relevant element sequence of the bacterial plasmid transformant comprises the steps of: using the pMDC83 plasmid as a template, using the upstream primer whose nucleotide sequence is shown in SEQ ID NO.9 and the nucleotide sequence shown in SEQ ID NO.10 The downstream primer is subjected to PCR amplification to obtain the relevant element sequence of the bacterial plasmid transformant.

3. The preparation of the CEN6-ARSH4 sequence fragment includes the steps: using the yeast genome as a template, using an upstream primer whose nucleotide sequence is shown in SEQ ID NO.11 and a downstream primer whose nucleotide sequence is shown in SEQ ID NO.12 PCR amplification was performed to obtain a CEN6 sequence fragment, the CEN6 sequence fragment was a yeast chromosome 6 centromere-related sequence; using the yeast genome as a template, the upstream primer and nuclear nucleotide sequence shown in SEQ ID NO.13 were used. The downstream primer of the nucleotide sequence shown in SEQ ID NO.14 is subjected to PCR amplification to obtain an ARSH4 sequence fragment, which is a sequence related to the origin of replication of the yeast genome; the CEN6 sequence fragment and the ARSH4 sequence fragment are used for PCR amplification. The mixture is a template, and the upstream primer with the nucleotide sequence shown in SEQ ID NO.15 and the downstream primer with the nucleotide sequence shown in SEQ ID NO.16 are used for PCR amplification to obtain the CEN6-ARSH4 sequence fragment .

4. The preparation of the MCS sequence fragment includes the steps: using PEG101-MCS as a template, using the upstream primer whose nucleotide sequence is shown in SEQ ID NO.17 and the downstream primer whose nucleotide sequence is shown in SEQ ID NO.18 to carry out PCR Amplify to obtain the MCS sequence fragment.

Example 2

Enzymatic digestion of PGBKT7 yeast two-hybrid vector and introduction of target nucleotide fragments by in vitro homologous recombination ligation

1. Double-enzyme digestion of the PGBKT7 yeast two-hybrid vector with restriction enzymes Pvu I/EcoR I, and agarose gel electrophoresis to recover and purify large fragments; according to the relevant steps in the instructions of Novozan ClonExpress Ultra One Step Cloning Kit, carry out The PGBKT7 and 35S:HYG sequence fragments after Pvu I/EcoR I digestion were subjected to homologous recombination to obtain the PGBKT7-Hyg recombinant vector; the PGBKT7-Hyg recombinant vector was transformed into E. Sangon Bioengineering (Shanghai) Co., Ltd. performed sequencing, and the correct sequenced PGBKT7-Hyg recombinant vector was used for further vector transformation.

2. The PGBKT7-Hyg recombinant vector obtained in 1 was single-enzyme digested and linearized with the restriction enzyme BstB I, and the digested product was recovered and purified by agarose gel electrophoresis; In the step, the PGBKT7-Hyg after BstB I digestion is subjected to homologous recombination with the above-mentioned pVS sequence fragment, thereby obtaining a PGBKT7-Hyg-pVS recombinant vector. The recombinant product was transformed into Escherichia coli, cultured, and monoclonal identified. The positive monoclonal colonies were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing. The correctly sequenced PGBKT7-Hyg-pVS recombinant vector was used for further vector transformation.

3. The PGBKT7-Hyg-pVS recombinant vector obtained in 2 was linearized by single restriction endonuclease Avr II, and the digested product was recovered and purified by agarose gel electrophoresis; according to the instructions of Novozan ClonExpress Ultra One StepCloning Kit In the relevant steps, the PGBKT7-Hyg-pVS after Avr II digestion was subjected to homologous recombination with the above-mentioned CEN6-ARSH4 sequence fragment, thereby obtaining the PGBKT7-Hyg-pVS-CEN6-ARSH4 recombinant vector. The recombinant product was transformed into E. coli, cultured, and monoclonal identified. The positive monoclonal colonies were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing. The correct PGBKT7-Hyg-pVS-CEN6-ARSH4 recombinant vector was used for further vectors. Retrofit.

4. The PGBKT7-Hyg-pVS-CEN6-ARSH4 recombinant vector obtained in 3 was linearized by single restriction endonuclease EcoR I, and the digested product was recovered and purified by agarose gel electrophoresis. According to the relevant steps in the manual of Novozan ClonExpress Ultra OneStep Cloning Kit, carry out the homologous recombination of PGBKT7-Hyg-pVS-CEN6-ARSH4 and the above-mentioned MCS sequence fragment after EcoR I digestion to obtain PGBKT7-Hyg-pVS-CEN6- ARSH4-MCS. The recombinant product was transformed into E. coli, cultured, and monoclonal identified. The positive monoclonal colonies were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing. The correct PGBKT7-Hyg-pVS-CEN6-ARSH4-MCS recombinant vector, namely PACB -MCS vector (shown in Figure 2) was used for subsequent experiments.

Example 3

An application of a Saccharomyces cerevisiae carrier is provided, and the Saccharomyces cerevisiae of the present invention is used for the assembly of artificially synthesized plant chromosome large fragments, which comprises the following steps:

1. Design an artificially synthesized plant chromosome-related fragment ACEN2, the nucleotide sequence of which is shown in SEQ ID NO.19;

2. Kpn I single digestion was performed on the artificially synthesized plant chromosome-related fragment ACEN2, and the ACEN2 digestion product was recovered and purified by agarose gel electrophoresis. Cyclization to obtain ACEN2 cyclization product;

3. Using the ACEN2 cyclization product in the above 2 as a template, perform rolling circle amplification on the ACEN2 cyclization product according to the relevant steps of the RCA rolling circle amplification kit of Xinhai Genetic Testing Co., Ltd. to obtain an ACEN2 rolling circle amplification product ;

4. Double-enzyme digestion of the PACB-MCS vector with restriction enzymes Stu I/Sac I, and agarose gel electrophoresis to recover and purify large fragments; according to the relevant steps in the Novozan ClonExpress Ultra One Step Cloning Kit instructions, carry out Stu The PACB-MCS and ACEN2-linker sequence fragments after I/Sac I digestion were subjected to homologous recombination to obtain the PACB-MCS-ACEN2-linker recombinant vector (as shown in Figure 3); the recombinant product was transformed into E. coli, cultured, Monoclonal identification, positive monoclonal colonies were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing, and the correct PACB-MCS-ACEN2-linker recombinant vector was sequenced for subsequent experiments. The nucleotides of the ACEN2-linker sequence fragment were The sequence is shown in SEQ ID NO.20;

5. Double-enzyme digestion of the PACB-MCS-ACEN2-linker recombinant vector with restriction enzymes Apa I/EcoR I, and agarose gel electrophoresis to recover and purify the large fragment, which is the linearized PACB-MCS-ACEN2-linker . The linearized PACB-MCS-ACEN2-linker and the ACEN2 rolling circle amplification product in the above 3 were transformed into yeast and cultured in Trp-deficient medium for 48 h to obtain the expression of PACB-MCS-ACEN2-linker and ACEN2 rolling circle amplification. The positive yeast genome of the amplified product is shown in Figure 4;

6. Transform the positive yeast genome into DH5α Escherichia coli by electric shock, and multiply the recombinants of PACB-MCS-ACEN2-linker and ACEN2 rolling circle amplification products, as shown in Figure 5;

7. Pick the Escherichia coli single clone in the above 6, carry out propagation and plasmid extraction. The extracted plasmid was identified by StuI/Sac I double-enzyme digestion, and a target vector containing a recombinant fragment of about 7.5 kb was successfully screened for subsequent experiments, as shown in Figure 6.

It should be understood that the application of the present invention is not limited to the above examples. For those of ordinary skill in the art, improvements or transformations can be made according to the above descriptions, and all these improvements and transformations should belong to the protection scope of the appended claims of the present invention.

sequence listing

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<400> 5

ttaggaaccg gcggatgctt cgccctcgat caggttgcgg tagcgcatga ctaggatcgg 60

gccagcctgc cccgcctcct ccttcaaatc gtactccggc aggtcatttg acccgatcag 120

cttgcgcacg gtgaaacaga acttcttgaa ctctccggcg ctgccactgc gttcgtagat 180

cgtcttgaac aaccatctgg cttctgcctt gcctgcggcg cggcgtgcca ggcggtagag 240

aaaacggccg atgccgggat cgatcaaaaa gtaatcgggg tgaaccgtca gcacgtccgg 300

gttcttgcct tctgtgatct cgcggtacat ccaatcagct agctcgatct cgatgtactc 360

cggccgcccg gtttcgctct ttacgatctt gtagcggcta atcaaggctt caccctcgga 720

taccgtcacc aggcggccgt tcttggcctt cttcgtacgc tgcatggcaa cgtgcgtggt 780

gtttaaccga atgcaggttt ctaccaggtc gtctttctgc tttccgccat cggctcgccg 840

gcagaacttg agtacgtccg caacgtgtgg acggaacacg cggccgggct tgtctccctt 900

cccttcccgg tatcggttca tggattcggt tagatgggaa accgccatca gtaccaggtc 960

gtaatcccac acactggcca tgccggccgg ccctgcggaa acctctacgt gcccgtctgg 1020

aagctcgtag cggatcacct cgccagctcg tcggtcacgc ttcgacagac ggaaaacggc 1080

cacgtccatg atgctgcgac tatcgcgggt gcccacgtca tagagcatcg gaacgaaaaa 1140

atctggttgc tcgtcgccct tgggcggctt cctaatcgac ggcgcaccgg ctgccggcgg 1200

ttgccgggat tctttgcgga ttcgatcagc ggccgcttgc cacgattcac cggggcgtgc 1260

ttctgcctcg atgcgttgcc gctgggcggc ctgcgcggcc ttcaacttct ccaccaggtc 1320

atcacccagc gccgcgccga tttgtaccgg gccggatggt ttgcgacc 1368

<210> 6

<211> 195

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 6

tatgcacagg ccaggcgggt tttaagagtt ttaataagtt ttaaagagtt ttaggcggaa 60

aaatcgcctt ttttctcttt tatatcagtc acttacatgt gtgaccggtt cccaatgtac 120

ggctttgggt tcccaatgta cgggttccgg ttcccaatgt acggctttgg gttcccaatg 180

tacgtgctat ccaca 195

<210> 7

<211> 55

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 7

gcgcaactgt tgggaagggc gatcgcacat tgcggacgtt tttaatgtac tgaat 55

<210> 8

<211> 52

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 8

gcaggtcgac ggatccccgg gaattccttt ccagtcggga aacctgtcgt gc 52

<210> 9

<211> 51

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 9

gttgggcgtc gcttggtcgg tcatttcgtg agtgagctga taccgctcgc c 51

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 10

gagcgggact ctggggttcg aaacagcttg cgtcatgcgg tcgctgcgta t 51

<210> 11

<211> 53

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 11

ctttatatgt ttataattgt acatatgatc gccaacaaat actacctttt atc 53

<210> 12

<211> 55

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 12

ttcagttagc ctccccctag gataatatat tgcagaaaat gtgctagtaa tattg 55

<210> 13

<211> 55

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 13

ttcagttagc ctccccctag gataatatat tgcagaaaat gtgctagtaa tattg 55

<210> 14

<211> 53

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 14

gataaaaggt agtatttgtt ggcgatcata tgtacaatta taaacatata aag 53

<210> 15

<211> 55

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 15

ttcagttagc ctccccctag gataatatat tgcagaaaat gtgctagtaa tattg 55

<210> 16

<211> 48

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 16

gctggagttc ttcgcccacc gatcgcttgc ctgtaactta cacgcgcc 48

<210> 17

<211> 43

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 17

caggtttccc gactggaaag gtttgccccg gagattacaa tgg 43

<210> 18

<211> 43

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 18

cggatccccg ggacgcgtct gatagtttaa actgaaggcg gga 43

<210> 19

<211> 913

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 19

ggtaccatca agtcatattc gactccaaaa cactaaccaa ccttcttctt gcttctcaaa 60

gctttcatgg tgtagccaaa gtccatatga gtctttggct ttgtgtcttc taacaaggaa 120

acactactta gcttttaaga tcgggttgcg gtttaagttg ttatactcaa tcatacacat 180

gacatcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 240

aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 300

atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 360

tacgccaagc ttgtcagcgt tgaactgcgt gatgcggatc aacaggtggt tgcaactgga 720

caaggcacta gcgggacttt gcaagtggtg aatccgcacc tctggcaacc gggtgaaggt 780

tatctctatg aactgtgcgt cacagccaaa agccagacag agtgtgatat ctacccgctt 840

cgcgtcggca tccggtcagt ggcagtgaag ggcgaacagt tcctgattaa ccacaaaccg 900

ttctactggt acc 913

<210> 20

<211> 742

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 20

ggtaccatca agtcatattc gactccaaaa cactaaccaa ccttcttctt gcttctcaaa 60

gctttcatgg tgtagccaaa gtccatatga gtctttggct ttgtgtcttc taacaaggaa 120

acactactta gcttttaaga tcgggttgcg gtttaagttg ttatactcaa tcatacacat 180

gacatcagct ggcacgacag gtttcccgac gaattcgggc ccgcggatca acaggtggtt 240

gcaactggac aaggcactag cgggactttg caagtggtga atccgcacct ctggcaaccg 300

ggtgaaggtt atctctatga actgtgcgtc acagccaaaa gccagacaga gtgtgatatc 360

tacccgcttc gcgtcggcat ccggtcagtg gcagtgaagg gcgaacagtt cctgattaac 720

cacaaaccgt tctactggta cc 742

Claims (9)

1. A method for constructing a yeast vector, comprising the steps of:

providing a PGBKT7 yeast two-hybrid vector;

carrying out enzyme digestion linearization on the PGBKT7 yeast two-hybrid vector, and then carrying out homologous recombination with a 35S-HYG sequence fragment to obtain a PGBKT7-Hyg recombinant vector, wherein the nucleotide sequence of the 35S-HYG sequence fragment is shown in SEQ ID NO. 1;

carrying out enzyme digestion linearization on the PGBKT7-Hyg recombinant vector, and then carrying out homologous recombination on the recombinant vector and a related element sequence of a bacterial plasmid transformant to obtain a PGBKT7-Hyg-pVS recombinant vector;

carrying out enzyme digestion linearization on the PGBKT7-Hyg-pVS recombinant vector, and then carrying out homologous recombination on the PGBKT7-Hyg-pVS recombinant vector and a CEN6-ARSH4 sequence fragment to obtain a PGBKT7-Hyg-pVS-CEN6-ARSH4 recombinant vector, wherein the nucleotide sequence of the CEN6-ARSH4 sequence fragment is shown in SEQ ID NO. 2;

and (3) carrying out enzyme digestion linearization on the PGBKT7-Hyg-pVS-CEN6-ARSH4 recombinant vector, and then carrying out homologous recombination on the recombinant vector and the MCS sequence fragment to obtain a PGBKT7-Hyg-pVS-CEN6-ARSH4-MCS recombinant vector, namely constructing the saccharomyces cerevisiae, wherein the nucleotide sequence of the MCS sequence fragment is shown in SEQ ID NO. 3.

2. The construction method of the saccharomyces cerevisiae vector according to claim 1, wherein the sequences of the elements related to the bacterial plasmid transformants comprise a PVS1 StaA sequence, a PVS1-RepA sequence and a PVS1OriV sequence, wherein the nucleotide sequence of the PVS1 StaA sequence is shown in SEQ ID No.4, the nucleotide sequence of the PVS1-RepA sequence is shown in SEQ ID No.5, and the nucleotide sequence of the PVS1OriV sequence is shown in SEQ ID No. 6.

3. The construction method of the Saccharomyces cerevisiae vector according to claim 1, wherein the preparation of the 35S: HYG sequence fragment comprises the steps of:

and (3) carrying out PCR amplification by using a pMDC83 plasmid as a template and adopting an upstream primer with a nucleotide sequence shown as SEQ ID NO.7 and a downstream primer with a nucleotide sequence shown as SEQ ID NO.8 to obtain the 35S: HYG sequence fragment.

4. The method for constructing the Saccharomyces cerevisiae vector according to claim 1, wherein the preparation of the related element sequence of the bacterial plasmid transformant comprises the steps of:

and (3) carrying out PCR amplification by using a pMDC83 plasmid as a template and adopting an upstream primer with a nucleotide sequence shown as SEQ ID NO.9 and a downstream primer with a nucleotide sequence shown as SEQ ID NO.10 to obtain a related element sequence of the bacterial plasmid transformant.

5. The construction method of the Saccharomyces cerevisiae vector according to claim 1, wherein the CEN6-ARSH4 sequence fragment is prepared by the following steps:

taking a yeast genome as a template, and carrying out PCR amplification by adopting an upstream primer with a nucleotide sequence shown as SEQ ID NO.11 and a downstream primer with a nucleotide sequence shown as SEQ ID NO.12 to obtain a CEN6 sequence fragment;

taking a yeast genome as a template, and carrying out PCR amplification by adopting an upstream primer with a nucleotide sequence shown as SEQ ID NO.13 and a downstream primer with a nucleotide sequence shown as SEQ ID NO.14 to obtain an ARSH4 sequence fragment;

and (2) performing PCR amplification by using the mixture of the CEN6 sequence fragment and the ARSH4 sequence fragment as a template and adopting an upstream primer with a nucleotide sequence shown as SEQ ID NO.15 and a downstream primer with a nucleotide sequence shown as SEQ ID NO.16 to obtain the CEN6-ARSH4 sequence fragment.

6. The method for constructing the Saccharomyces cerevisiae vector of claim 1, wherein the preparation of the MCS sequence fragment comprises the steps of:

taking PEG101-MCS as a template, and carrying out PCR amplification by adopting an upstream primer with a nucleotide sequence shown as SEQ ID NO.17 and a downstream primer with a nucleotide sequence shown as SEQ ID NO.18 to obtain the MCS sequence fragment.

7. A Saccharomyces cerevisiae vector, which is characterized by being prepared by the construction method of the Saccharomyces cerevisiae vector of any one of claims 1-6.

8. Use of a Saccharomyces cerevisiae vector according to claim 7 for the assembly of large fragments of synthetic plant chromosomes.

9. The use of the Saccharomyces cerevisiae vector according to claim 8, wherein the Saccharomyces cerevisiae vector used for the assembly of large synthetic plant chromosome segments comprises the steps of:

designing an artificially synthesized plant chromosome related fragment ACEN2, wherein the nucleotide sequence of the fragment is shown in SEQ ID NO. 19;

carrying out Kpn I single enzyme digestion on the artificially synthesized plant chromosome related fragment ACEN2 to obtain an ACEN2 enzyme digestion product;

cyclizing the enzyme-cut product of ACEN2 to obtain a cyclized product of ACEN 2;

performing rolling circle amplification on the ACEN2 cyclization product to obtain an ACEN2 rolling circle amplification product;

carrying out homologous recombination on the saccharomyces cerevisiae vector and an ACEN2-linker sequence fragment to obtain a PACB-MCS-ACEN2-linker recombinant vector, wherein the nucleotide sequence of the ACEN2-linker sequence fragment is shown as SEQ ID NO. 20;

converting the PACB-MCS-ACEN2-linker recombinant vector and the ACEN2 rolling circle amplification product into yeast, and culturing the yeast in a Trp defect culture medium to obtain positive recombinant yeast expressing the PACB-MCS-ACEN2-linker and the ACEN2 rolling circle amplification product;

and (3) transforming the positive yeast genome into escherichia coli by electric shock, and carrying out propagation of a recombinant of PACB-MCS-ACEN2-linker and ACEN2 rolling circle amplification products.

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