CN112626092A

CN112626092A - Application of NAT gene as screening marker in genetic transformation of chlamydomonas

Info

Publication number: CN112626092A
Application number: CN201910903429.4A
Authority: CN
Inventors: 潘俊敏; 杨新佳; 彭佳琳
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2019-09-24
Filing date: 2019-09-24
Publication date: 2021-04-09
Anticipated expiration: 2039-09-24
Also published as: CN112626092B

Abstract

The invention discloses application of NAT gene as a screening marker in genetic transformation of Chlamydomonas. The invention firstly discloses the application of NAT gene as a screening marker in genetic transformation of Chlamydomonas and/or preparation of expression strains of target genes of Chlamydomonas and/or preparation of transgenic Chlamydomonas strains. The invention further discloses a DNA molecule shown in the sequence 1 in the sequence table. The invention takes the Nolserine N-acetyltransferase gene as the screening marker of the chlamydomonas genetic transformation and/or the preparation of target gene expression strains of chlamydomonas and/or the preparation of transgenic chlamydomonas strains, is simple and convenient, has good repeatability, can be effectively applied to the field of chlamydomonas gene modification, and further promotes the more economical and efficient production of recombinant protein by the chlamydomonas as a biological factory for medical treatment, health care and aquaculture disease prevention and treatment.

Description

Application of NAT gene as screening marker in genetic transformation of chlamydomonas

Technical Field

The invention relates to the field of biotechnology, in particular to application of NAT gene as a screening marker in genetic transformation of chlamydomonas

Background

Chlamydomonas is a model organism, and a hot research on Chlamydomonas is being developed into an emerging plant for producing recombinant proteins with high commercial value. If the water is expressed to produce biological disease-resistant protein in chlamydomonas, the disease of aquaculture industry can be reduced after the algae is eaten by aquatic organisms, and the economic benefit is improved. Therefore, high level expression of recombinant proteins and simultaneous expression of multiple recombinant proteins in chlamydomonas are currently hot spots. However, the screening of target gene expression strains of Chlamydomonas is greatly limited by the number of resistance markers that are currently available, such as only paromomycin, hygromycin, bleomycin and zeocin which are currently commonly used.

Therefore, the development of new resistance markers for the study of chlamydomonas is crucial to driving chlamydomonas to protein production plants.

Disclosure of Invention

The invention aims to solve the technical problem of obtaining the resistance marker suitable for genetic transformation and screening of chlamydomonas.

In order to solve the technical problems, the invention firstly provides the application of the Nolsporin-N-acetyltransferase gene (NAT gene) as a screening marker in the genetic transformation of Chlamydomonas and/or the preparation of expression strains of target genes of Chlamydomonas and/or the preparation of transgenic Chlamydomonas strains.

In the above application, the nourseothricin-N-acetyltransferase gene is a DNA molecule of any one of the following 1) -5):

1) a DNA molecule shown as a sequence 1 in a sequence table;

2) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;

3) a DNA molecule shown as a sequence 2in a sequence table;

4) the coding region is a DNA molecule shown as a sequence 2in a sequence table;

5) a DNA molecule which hybridizes under stringent conditions with a DNA molecule as defined in 1) or 2) or 3) or 4) and which encodes a nourseothricin-N-acetyltransferase.

Wherein the sequence 1 is obtained by codon optimization of a sequence shown in the sequence 2, and the DNA molecules shown in the

sequences

1 and 2 can express nourseothricin-N-acetyltransferase with an amino acid sequence shown as a sequence 3 in the sequence table.

There are many DNA molecules encoding nourseothricin-N-acetyltransferase according to the degeneracy of the codon, and those skilled in the art can design many sequences encoding nourseothricin-N-acetyltransferase according to the nourseothricin-N-acetyltransferase gene sequence shown in SEQ ID No. 1 or SEQ ID No. 2, which can be used as selection marker, and all fall within the protection scope of the present invention.

The application of the biological material related to the nourseothricin-N-acetyltransferase gene in the preparation of the chlamydomonas target gene expression strain and/or the preparation of transgenic chlamydomonas strain is also within the protection scope of the invention.

In the above application, the biomaterial is any one of the following:

C1) an expression cassette containing the above-mentioned nourseothricin-N-acetyltransferase gene;

C2) a recombinant vector comprising the above-mentioned nourseothricin-N-acetyltransferase gene or a recombinant vector comprising the expression cassette according to C1);

C3) a recombinant microorganism containing the above-mentioned nourseothricin-N-acetyltransferase gene, a recombinant microorganism containing the expression cassette described in C1), or a recombinant microorganism containing the recombinant vector described in C2);

C4) a transgenic Chlamydomonas cell line containing the nourseothricin-N-acetyltransferase gene, a transgenic Chlamydomonas cell line containing the expression cassette described in C1), or a transgenic Chlamydomonas cell line containing the recombinant vector described in C2).

In the above-mentioned biomaterials, the expression cassette described in C1) means a DNA molecule capable of expressing nourseothricin-N-acetyltransferase in a host cell, and the DNA molecule may include not only a promoter for initiating transcription of nourseothricin-N-acetyltransferase gene but also a terminator for terminating transcription of nourseothricin-N-acetyltransferase. Further, the expression cassette may also include an enhancer sequence.

Specifically, the expression cassette C1) can be HR-NAT-HA, the sequence of the expression cassette is shown as sequence 4 in the sequence table, wherein, the 1 st to 509 th positions of the sequence 4 are sequences of promoter HSP70a/RBCS2, the 518 th to 662 th positions of the sequence 4 are sequences of enhancer RBCS2intron1, the 679 th to 1248 th positions of the sequence 4 are sequences of NAT genes, the 1255 th to 1380 th positions of the sequence 4 are sequences of HA, and the 1400 th to 1632 th positions of the sequence 4 are sequences of terminator RBCS 2; C1) the expression cassette can also be HR-NAT, and the sequence of the expression cassette is obtained by removing 1255 th to 1380 th positions of the sequence 4 (namely removing the sequence of HA).

According to the invention, the expression cassette C1) can also contain a target gene and a 2A sequence shown as a sequence 7, wherein the 2A sequence, the target gene and the NAT gene share a promoter and a terminator, and the 2A sequence is positioned between the NAT gene and the target gene.

For the above use, C2) the recombinant vector may comprise a DNA molecule encoding a nourseothricin-N-acetyltransferase. The recombinant vector containing the nourseothricin-N-acetyltransferase coding gene or the nourseothricin-N-acetyltransferase coding gene expression cassette can be constructed using an existing common molecular cloning vector or a chlamydomonas expression vector. The molecular cloning vector used in the invention is ZT4-blunt vector (Chun alliance, Beijing), the Chlamydomonas expression vector is PSAD-HA (plasmid PSAD-IFT54-HA (hygromycin)⁺) Digested) and pBR25 (digested from plasmid pBR 25-sfGFP-TUA).

The recombinant vector can be pHR-NAT-HA, pPSAD-IFT54-HA (NAT)⁺) Or pHR-NAT-2A-IFT 54-HA. Wherein the pHR-NAT-HA comprises an expression cassette HR-NAT-HA of NAT; the pPSAD-IFT54-HA (NAT)⁺) There are two expression cassettes, namely the NAT gene expression cassette: HR-NAT and gene of interest IFT54 expression cassette: PSAD-IFT 54-HA; the pHR-NAT-2A-IFT54-HA HAs an expression box HR-NAT-2A-IFT54-HA containing a target gene IFT54, a NAT gene and a 2A sequence.

In the above application, the recombinant microorganism of C3) can be yeast, bacteria, algae and fungi.

The invention further provides a DNA molecule.

The DNA molecule of the present invention is any one of the following 1) to 5):

1) a DNA molecule shown as a sequence 1 in a sequence table;

3) a DNA molecule shown as a sequence 2in a sequence table;

Biological materials related to the above DNA molecules are also within the scope of the present invention.

The biological material in the invention is any one of the following materials:

C1) an expression cassette containing the above DNA molecule;

C2) a recombinant vector containing the DNA molecule or a recombinant vector containing the expression cassette described in C1);

C3) a recombinant microorganism containing the above DNA molecule, or a recombinant microorganism containing the expression cassette described in C1), or a recombinant microorganism containing the recombinant vector described in C2);

C4) a transgenic chlamydomonas cell line containing the DNA molecule, or a transgenic chlamydomonas cell line containing the expression cassette described in C1), or a transgenic chlamydomonas cell line containing the recombinant vector described in C2).

Specifically, the expression cassette described in C1) may be HR-NAT-HA, and the sequence of the expression cassette is represented by sequence 4 in the sequence table, wherein positions 1 to 509 of sequence 4 are sequences of promoter HSP70a/RBCS2, positions 518 to 662 of sequence 4 are sequences of enhancer RBCS2intron1, positions 679 to 1248 of sequence 4 are sequences of the above DNA molecule (NAT gene), positions 1255 to 1380 of sequence 4 are sequences of HA, positions 1400 to 1632 of sequence 4 are sequences of terminator RBCS 2; C1) the expression cassette can also be HR-NAT, and the sequence of the expression cassette is obtained by removing 1255 th to 1380 th positions of the sequence 4 (namely removing the sequence of HA).

The expression cassette of C1) in the invention can further contain a target gene and a 2A sequence shown as sequence 7, wherein the 2A sequence, the target gene and the DNA molecule (NAT gene) share a promoter and a terminator, and the 2A sequence is positioned between the DNA molecule (NAT gene) and the target gene.

In the above-mentioned biomaterial, C2) the recombinant vector may contain a DNA molecule encoding nourseothricin-N-acetyltransferase. The recombinant vector containing the nourseothricin-N-acetyltransferase coding gene or the nourseothricin-N-acetyltransferase coding gene expression cassette can be constructed using an existing common molecular cloning vector or a chlamydomonas expression vector. The molecular cloning vector used in the invention is ZT4-blunt vector (Chun alliance, Beijing), the Chlamydomonas expression vector is PSAD-HA (plasmid PSAD-IFT54-HA (hygromycin)⁺) Digested) and pBR25 (digested from plasmid pBR 25-sfGFP-TUA).

The recombinant vector can be pHR-NAT-HA, pPSAD-IFT54-HA (NAT)⁺) Or pHR-NAT-2A-IFT 54-HA. Wherein the pHR-NAT-HA comprises an expression cassette HR-NAT-HA of NAT; the pPSAD-IFT54-HA (NAT)⁺) There are two expression cassettes, namely the NAT gene expression cassette: HR-NAT and gene of interest IFT54 expression cassette: PSAD-IFT 54-HA; the pHR-NAT-2A-IFT54-HA HAs an expression cassette containing a target gene IFT54, a NAT gene and a 2A sequence: HR-NAT-2A-IFT 54-HA.

In the above biological material, C3) the recombinant microorganism may be yeast, bacteria, algae and fungi.

The invention discovers that the Nolserine can be used for resistance screening of Chlamydomonas for the first time, and further shows that the Nolserine has no cross reaction with other aminoglycoside antibiotics such as paromomycin and hygromycin. The expression cassette HR-NAT-HA of the Nolsporin N-acetyltransferase gene (NAT gene) is constructed, and the Nolsporin resistance can be endowed to Chlamydomonas by transferring the expression cassette HR-NAT-HA into Chlamydomonas. On the basis, the invention further integrates the NAT gene expression cassette into an expression vector to form a plasmid 2(pPSAD-IFT54-HA (NAT)⁺) Successfully screening target gene expression strains, and successfully obtaining expression strains expressing IFT54-HA protein. Meanwhile, the invention also constructs plasmid 3(pHR-NAT-2A-IFT54-HA) with higher screening efficiency on the basis. Therefore, the Nolserine can be used as an excellent antibiotic for genetic transformation and screening of Chlamydomonas, namely, the Nolserine N-acetyltransferase gene (NAT gene) is used as a screening marker of a Chlamydomonas target gene expression strain. This is achieved byThe plasmids are added with proper enzyme cutting sites at both sides of each component, such as a promoter, a terminator, a label and a target gene, and other scholars can flexibly modify the plasmids according to self requirements. In addition, the total length of the NAT gene expression box is only 1498bp, the transformation efficiency of the NAT gene expression box in the chlamydomonas is similar to the currently most effective paromomycin resistance gene expression box and hygromycin resistance gene expression box, and the NAT gene expression box is an ideal insertion fragment for constructing a chlamydomonas mutant library and can be applied to screening chlamydomonas mutants.

In conclusion, the method for screening the chlamydomonas target gene expression strain by using the nourseothricin-N-acetyltransferase gene as the screening marker is simple, convenient and good in repeatability, can be effectively applied to the field of chlamydomonas gene modification, and further promotes the chlamydomonas to be used as a biological factory to produce recombinant protein more economically and efficiently for medical treatment, health care and aquaculture disease prevention and treatment.

Drawings

FIG. 1 shows the growth of wild type Chlamydomonas cells in culture media at various concentrations of nourseothricin.

FIG. 2 shows the growth of wild type Chlamydomonas (WT), Chlamydomonas with paromomycin resistance gene (paro), Chlamydomonas with hygromycin resistance gene (hyg), and Chlamydomonas with paromomycin and hygromycin resistance genes (paro/hyg) on non-resistant TAP plates, TAP plates with nourseothricin, TAP plates with paromomycin, and TAP plates with hygromycin.

FIG. 3 shows a comparison of the DNA sequences of codon-optimized NAT genes and non-optimized NAT genes and their corresponding amino acid sequences.

A in FIG. 4 is a schematic structural diagram of NAT gene expression cassette HR-NAT-HA; b is the single algae colony condition grown on the plate containing (+)/not containing (-) norserine after the chlamydomonas is transformed into an expression cassette HR-NAT-HA by electric shock; c is the immunoblot to examine the protein expression of NAT-HA in several transformants randomly selected on the above-mentioned nourseothricin plates.

In FIG. 5, A is a schematic structural diagram of an expression cassette of plasmid 2; b Monocoystal colonies were grown on Nolsemiin plates after electroporation into plasmid 2in paromomycin resistant ift54 mutants. The IFT54-HA expression levels of Wild Type (WT), paromomycin-resistant IFT54 mutant (IFT54), and 12 IFT54-HA expression strains (IFT 54:: IFT54-HA) obtained by screening with nourseothricin were examined using HA and IFT54 antibodies, respectively. Meanwhile, CDPK3 antibody is used to ensure that the protein loading amount is consistent when different algae strains are electrophoresed; c is the flagella character of Wild Type (WT), IFT54 mutant (IFT54) with paromomycin resistance and IFT54-HA expression strain (IFT 54:: IFT54-HA) obtained by utilizing a differential interference microscope to detect; d Single algal colonies were grown on Nolsemiin plates after electroporation into plasmid 3 in a strain with paromomycin/hygromycin double resistance (paro/hyg). The IFT54-HA expression levels of Wild Type (WT), strain having paromomycin/hygromycin double resistance (paro/hyg), and 7 IFT54-HA expression strains (paro/hvg:: IFT54-HA) obtained by nourseothricin screening were examined using HA and IFT54 antibodies, respectively. At the same time, the CDPK3 antibody is used to ensure that the protein loading amount is consistent when different algae strains are electrophoresed.

In FIG. 6, A is a schematic structural diagram of an expression cassette of plasmid 3; b Monocoystal colonies were grown on Nolsemiin plates after electroporation into plasmid 3 in paromomycin resistant ift54 mutants. IFT54 antibody was used to detect the IFT54-HA expression levels of Wild Type (WT), IFT54 mutant (IFT54) having paromomycin resistance, and 10 IFT54-HA expression strains (IFT 54:: NAT-2A-IFT54-HA) obtained by screening with nourseothricin. And simultaneously, the CDPK3 antibody is used for ensuring that the protein loading amount is consistent when different strains are electrophoresed.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The following media formulations were used in the examples as follows:

wherein, the formula of the trace elements is as follows: 1g/L H₃BO₃，1g/L ZnSO₄·7H₂O，0.303g/L MnSO₄·H₂O，0.2g/L CoCl₂·6H₂O，0.2g/L Na₂MoO₄·2H₂O，0.0625g/L CuSO₄·5H₂O。

TAP plate: 15g of agar per 1L of the medium was added to the above TAP liquid medium.

Wherein the stock Solution formulas of Solution I, Solution II and Solution III are as follows:

solution I (salt Solution): 2g/L CaCl₂·2H₂O，15g/L NH₄Cl，4g/L MgSO₄·7H₂O。

Solution II (phosphate buffer): 288g/L K₂HPO4·3H₂O，144g/L KH₂PO4。

Solution III (trace elements): ZnSO₄·7H₂O 22g 100ml，H₃BO₃ 11.4g 200ml，MnCl₂·4H₂O 5.06g 50ml，CoCl₂·6H₂O 1.61g 50ml，CuSO₄·5H₂O 1.57g 50ml，(NH4)₆Mo₇O₂₄ 1.10g 50ml，FeSO₄·7H₂O 4.99g 50ml。

Dissolving the above chemicals with corresponding volume of distilled water respectively, mixing well, heating to boil, adding EDTA solution (50g dissolved in 250ml distilled water), and making the solution green. To complete dissolution and the temperature was reduced to 70 ℃, 85ml of pre-heated 20% KOH solution (20g dissolved in 100ml distilled water) was added. Standing for 1-2 weeks, shaking once a day to obtain a solution which slowly changes from green to purple, filtering to remove precipitate, and storing at 4 deg.C.

The antibodies used in the following examples are as follows:

wherein, the antibody IFT54 is a fusion protein of 123 rd-384 th amino acid of bacteria expression and purification IFT54 and his label, and is used as an antigen to immunize rabbits to obtain multi-antiserum. The amino acid sequence at position 123-384 of IFT54 is shown in sequence 8.

The antibody CDPK3 is a protein obtained by bacterial expression and purification of CDPK3 with 1 st-202 th amino acids fused with his tags, and is used as antigen to immunize rabbits to obtain multi-antiserum. The amino acid sequence from position 1 to 202 of CDPK3 is shown in SEQ ID NO 9.

EXAMPLE 1 determination of the optimal concentration of Nolserine for Chlamydomonas and a proof test for the mutual compatibility of Nolserine with the other two antibiotics

Determining the optimal concentration of Nolserine for Chlamydomonas

In order to find out the optimum concentration capable of killing chlamydomonas cells, the chlamydomonas cells are inoculated into culture media of nourseothricin with different concentrations, and the growth condition of the chlamydomonas cells is observed, and the specific steps are as follows:

1. preparing culture medium of nourseothricin with different concentrations

100mg of nourseothricin was dissolved in 10ml of ultrapure water, and the solution was sterilized by filtration through a 0.22 μm filter to obtain a stock solution of nourseothricin at 10 mg/ml. Mu.l of the above mother liquors were added to 500ml of TAP containing 1.5% agar to give TAP plates with nourseothricin concentrations of 0, 2.5, 5, 10 and 40. mu.g/ml, respectively.

2. Inoculating wild Chlamydomonas into R liquid culture medium, culturing for 3 days until its concentration reaches 3 × 10⁶When the strain is cultured per ml, transferring the strain into M liquid culture medium, and continuing culturing for 2 days to make the concentration reach 2 × 10⁶One per ml. Centrifuging at 2000rpm for 3min to enrich Chlamydomonas to 1 × 10⁸One per ml. 3 drops of each Chlamydomonas suspension (1X 10 per drop) were inoculated onto TAP plates at norselysin concentrations of 0, 2.5, 5, 10 and 40. mu.g/ml⁶Wild type chlamydomonas), cultured at 23 ℃ for 4 days under 14h/10h light/dark conditions, and photographed on day 4 to record the experimental results.

The results showed that a concentration of 5. mu.g/mL of nourseothricin in the medium was sufficient to completely inhibit the growth of wild-type Chlamydomonas cells (FIG. 1).

Secondly, verifying that the nourseothricin and the other two antibiotics can be mutually compatible

In order to detect the presence of cross-resistance of paromomycin, hygromycin and nourseothricin in chlamydomonas, whether cells have resistance to nourseothricin due to the presence of paromomycin and hygromycin resistance genes separately or simultaneously is detected. The method comprises the following specific steps:

TAP plates containing paromomycin (10. mu.g/mL), hygromycin (20. mu.g/mL) and nourseothricin (10. mu.g/mL) were prepared as described in example 1 for the preparation of medium with varying concentrations of nourseothricin.

Wild type Chlamydomonas (WT), Chlamydomonas having paromomycin resistance gene (paro), Chlamydomonas having hygromycin resistance gene (hyg), and Chlamydomonas having paromomycin and hygromycin resistance gene (paro/hyg) were cultured and resuspended in the same manner as the wild type Chlamydomonas in example 1. Each strain was inoculated in 3 drops (each drop contained 1X 10)⁶Chlamydomonas) were incubated on non-resistant TAP plates and TAP plates containing paromomycin (10. mu.g/mL), hygromycin (20. mu.g/mL) and nourseothricin (10. mu.g/mL) at 23 ℃ under 14h/10h light/dark conditions for 4 days, and the results were recorded by photography. The results showed that 10. mu.g/ml of nourseothricin was sufficient to kill the above algal strains with different resistance genes within 4 days (FIG. 2), indicating that paromomycin, hygromycin and nourseothricin could work independently at the same time without interfering with each other.

Example 2 introduction of Nolsethricin resistance Gene (i.e., Nolsethricin N-acetyltransferase gene, nourseothricin N-acetyl transferase, hereinafter abbreviated as NAT gene) into Chlamydomonas so that Chlamydomonas has Nolsethricin resistance

Firstly, constructing a chlamydomonas expression plasmid 1 of an NAT gene (shown as a sequence 1 in a sequence table) containing optimized codons (figure 3): pHR-NAT-HA. The specific steps for constructing the plasmid are as follows:

taking a plasmid pBR25-sfGFP-TUA shown in the sequence 5 as a template, and adopting F1 and R1 primers to carry out PCR amplification to obtain PCR amplificationThe product fragment HSP70a/RBCS2 promoter-RBCS 2intron 1; using NAT gene containing optimized codon as template, adopting F2 and R2 primer to make PCR amplification to obtain PCR amplification product fragment NAT; the plasmid PSAD-IFT54-HA (hygromycin) shown as a sequence 6⁺) Performing PCR amplification by using F3 and R3 primers as a template to obtain a PCR amplification product fragment HA; and carrying out PCR amplification by using a plasmid pBR25-sfGFP-TUA as a template and adopting F4 and R4 primers to obtain a PCR amplification product fragment RBCS2 terminator.

F1：5′-CTCGAGGACGGCGGGGAGCTC-3′

R1：5′-GTCGTCCAGGGTGGTGCCCATCATATGCATGGCCATCCT-3′

F2：5′-ATGGGCACCACCCTGGA-3′

R2：5′-CGCTAGCGACGCCATAGATCTGGGGCAGGGCATGCTCATGTAC-3′

F3：5′-AGATCTATGGCGTCGCTAGCG-3′

R3：5′-GGAATTCGGATCTCCTAGGTTAGGC-3′

F4：5′-ACCTAGGAGATCCGAATTCCCGCTCCGTGTAAATGGAG-3′

R4：5′-GGTACCCGCTTCAAATACGC-3′。

Four-segment overlap PCR is carried out to obtain a complete PCR amplification product segment HSP70a/RBCS2 promoter-RBCS 2intron1-NAT-HA-RBCS 2terminator (namely, an NAT gene expression box HR-NAT-HA), which is detailed in a sequence 4 and A in a picture 4, wherein the 1 st to 509 th positions of the sequence 4 are sequences of a promoter HSP70a/RBCS2, the 518 th to 662 th positions of the sequence 4 are sequences of an enhancer RBCS2intron1, the 679 th to 1248 th positions of the sequence 4 are sequences of an NAT gene, the 1255 th to 1380 th positions of the sequence 4 are sequences of HA, and the 1400 th to 2 nd positions of the sequence 4 are sequences of a terminator RBCS 163 2. Further, the fragment was ligated to ZT4-blunt vector (Zhuang, Beijing) by ordinary blunt-end ligation, and constructed as plasmid 1: pHR-NAT-HA.

Second, obtaining expression algal strains

1. The plasmid 1 is transformed into wild chlamydomonas (WT) cells by an electric transfer technology, and the specific steps are as follows:

(1) culturing chlamydomonas cells: picking wild Chlamydomonas cells into TAP liquid culture medium, and air-blowing culturing for 3-4And (5) day. When the cell concentration of Chlamydomonas reaches 5X 10⁶At cell/mL, Chlamydomonas cells were diluted and transferred to shake flasks containing fresh TAP liquid medium to an initial concentration of 1X 10⁶Continuously culturing for 20h with continuous light to make the cell concentration of Chlamydomonas reach 3 × 10⁶one/mL, the transformation can begin.

(2) Cleaning an electric shock cup: the cuvette stored in 95% alcohol was removed before transformation, rinsed three times with absolute ethanol in a clean bench, and blown dry before use.

(3) Enrichment of chlamydomonas cells: in a super clean bench, after washing the cells once with pre-cooled TAP liquid medium +60mM Sorbitol, 2000rpm, 3min enriched the Chlamydomonas cells to 2X 10⁸And each cuvette is added with 250ul of the chlamydomonas cells and 50ng of linearized plasmid 1, mixed evenly and placed on ice for 10min (if the algae strain cells are clustered, a proper amount of autolysin is added to treat the cells into single cells before rinsing).

(4) Electric shock: the light and light bullet uniform electric shock cup is put into an electric shock instrument BTX ECM630 for electric shock, and the parameters are 800V, 1575 omega and 50 uF. Immediately after electric shock, the cells were transferred to ice and allowed to stand for 10 min.

(5) And (3) recovering: transferring the chlamydomonas cells in the electric shock cup to a 50ml sterilization tube in which 10ml TAP liquid culture medium is added in advance, and recovering for 8 hours in a dark place on a slow shaking table.

(6) Coating a plate: and (3) centrifugally enriching the chlamydomonas cells recovered for 8 hours, coating each tube on 2 TAP plates containing 10 mu g/ml nourseothricin, inversely placing the tubes on a photoperiod for culturing for 6-9 days, and detecting after the monoclonal chlamydomonas grows out.

Plasmid 1 is integrated into the chlamydomonas genome and stable inheritance is achieved by the above method. In 3 independent replicates, an average of 528 monoclonal algal strains per 50ng linearized plasmid 1 on TAP plates containing 10. mu.g/ml nourseothricin (shown in FIG. 4B) were produced.

2. Screening of expression Strain

Randomly picked 7 monoclonal algae strains were subjected to immunoblot detection using an HA antibody, and the results showed that the 7 monoclonal algae strains (denoted as "NAT-HA transformations" in the figure) each expressed NAT-HA (shown as C in FIG. 4), while wild-type Chlamydomonas (WT) did not express NAT-HA. It was shown that the NAT gene expression cassette in plasmid 1 can produce a biologically active nourseothricin resistance protein, rendering chlamydomonas nourseothricin resistant.

The immunoblotting procedure was as follows:

(1) preparation of protein samples

Picking out the monoclonal Chlamydomonas to TAP flat plate, culturing for 5-7 days, picking it to 24-well plate, and enlarging culturing. Adding 1.5ml of M culture medium into each hole of the 24-hole plate, and shaking for 1 day to obtain chlamydomonas cells; collecting Chlamydomonas cells in M culture medium, centrifuging at 14000rpm for 1min, discarding supernatant, quickly freezing in liquid nitrogen, and storing in-80 deg.C refrigerator for a long time.

② the Chlamydomonas cells were taken out from the liquid nitrogen, placed on ice, and 75. mu.l of buffer A (50mM Tris-HCl (pH 7.5), 10mM MgCl) was rapidly added₂1mM EDTA, 1mM DTT. Subpackaging, storing at-20 ℃, adding protease inhibitor cOmplete, EDTA-free, Cat No.: 04693132001, proteasome inhibitor MG-132, cat #: s2619), blow even. Then 25ul of 4 × loading buffer (200mM Tris-HCl (pH 6.8), 80g/L SDS, 2g/L bromophenol blue, 61.7g/L DTT and 40% glycerol are added, and after subpackaging, the mixture is stored at the temperature of 20 ℃ below zero) and is evenly blown, the mixture is placed in boiling water for boiling for 10min, and is centrifuged at 14000rpm for 10min, and the supernatant is taken out to be a new EP tube, thus obtaining the prepared protein sample.

(2) SDS-PAGE gel electrophoresis and membrane transfer

Firstly, glue preparation: the concentration of the separation gel is selected according to the size of the protein of interest. Sequentially adding each component of the SDS-PAGE gel according to the formula, uniformly mixing, injecting the mixture between two glass plates, and rapidly adding distilled water for sealing. Standing at room temperature for 40min, sucking out distilled water, preparing upper layer concentrated gel according to the same method, mixing, injecting into an insertion comb, standing for 15min, and removing the comb after complete solidification.

Sample adding: and clamping the prepared gel on an inner core device of an electrophoresis tank, putting the gel into the electrophoresis tank, and pouring a new prepared electrophoresis solution to ensure that the gel does not pass through the anode and the cathode of the electrophoresis tank. Mu.l protein sample and 5. mu.l protein marker were added to each well and blank wells were filled with 1 × loadingbuffer.

③ electrophoresis: and setting a constant voltage of 80V, after the bromophenol blue migrates to the separation gel and the marker is unfolded, changing the constant voltage of 120V until the bromophenol blue migrates to the tail end of the gel, and stopping electrophoresis.

Fourthly, transferring the film: the sandwich is clamped in a membrane transferring solution, a sponge, three layers of filter paper, gel, a PVDF membrane (methanol activation is soaked in advance), three layers of filter paper and sponge are sequentially placed from a black surface to a white surface, the sandwich is clamped and placed in a membrane transferring groove, precooled membrane transferring buffer solution is poured in, an ice box and a rotor are placed at the same time, and the mixture is continuously stirred on a magnetic stirrer, so that protein degradation caused by overheating is prevented. Setting constant voltage of 100V, limiting maximum current of 360mA, and rotating the membrane for 60 min.

(3) Antibody incubation

Sealing: after the membrane transfer is finished, the PVDF membrane is taken out and placed in TBST buffer solution containing 5% skimmed milk powder to be incubated for 1h in a shaking table at room temperature.

Second, first resistance: the blocking solution was decanted, a primary antibody solution prepared with TBST buffer containing 3% skimmed milk powder was added, and the mixture was incubated for 1h on a shaker at room temperature.

Fourthly, secondary antibody: the primary antibody was decanted, TBST was added and shaken on a shaker for 5min, decanted off, and fresh TBST was added and the process repeated three times. Subsequently, a secondary antibody solution prepared in TBST buffer containing 3% skimmed milk powder was added and incubated for 60min at room temperature in a shaker.

Chemiluminescence: rinsing the PVDF membrane on a shaking table for three times by using TBST buffer solution, turning on a chemiluminescence imager, placing the membrane in the center of the luminoscope after the temperature of a camera is reduced to about-40 ℃, mixing luminous liquid A and B in an equal volume of 1: 1, uniformly dripping the luminous liquid on the PVDF membrane, starting reaction, and automatically recording and storing the presented strips by a machine.

Example 3 screening of Chlamydomonas target Gene-expressing strains by Using NAT Gene as a screening marker

Firstly, constructing a plasmid 2: pPSAD-IFT54-HA (NAT)⁺)

Since the HA tag is not required when the NAT gene is used as a selection marker, the HA tag of the expression cassette HR-NAT-HA of the NAT gene in example 1 is removed by molecular cloning to be changed into the expression cassette HR-NAT. The expression cassette is then integrated into a chlamydomonas target gene expression plasmid to construct plasmid 2: pPSAD-IFT54-HA (N)AT⁺) In plasmid 2, there are two expression cassettes which do not interfere with each other, and expression cassette 1 is NAT gene expression cassette: HR-NAT, expression cassette 2 is the expression cassette of the target protein IFT 54: the PSAD-IFT54-HA comprises the following specific steps:

(1) with the plasmid PSAD-IFT54-HA (hygromycin)⁺) Performing PCR amplification on the template by adopting F5 and R5 primers to obtain a PCR amplification product fragment IFT 54-HA; with the plasmid PSAD-IFT54-HA (hygromycin)⁺) Carrying out PCR amplification on the template by adopting F6 and R6 primers to obtain a PCR amplification product fragment PSAD terminator; carrying out PCR amplification by using a plasmid pHR-NAT-HA template and F7 and R7 primers to obtain a PCR amplification product fragment HSP70a/RBCS2 promoter-RBCS 2intron 1-NAT; carrying out PCR amplification by using a plasmid pHR-NAT-HA template and F8 and R8 primers to obtain a PCR amplification product fragment RBCS2 terminator; and carrying out PCR amplification by using a plasmid PSAD-IFT54-HA (hygromycin +) template and F9 and R9 primers to obtain a PCR amplification product fragment vector.

The primer sequences are as follows:

F5：5′-TACTCACAACAAGCCGATATCATGTGCGATAACTGGCAGGC-3′；

R5：5′-GTCCAGCTGCTGCCAGATATCGGATCTCCTAGGTTA-3′；

F6：5′-GATATCTGGCAGCAGCTGGAC-3′；

R6：5′-GAGCTCCCCGCCGTCCTCGAGCACCGCGGTGGAGCT-3′；

F7：5′-CTCGAGGACGGCGGGGAGCTCG-3′；

R7：5′-CTTAGGGGCAGGGCATG-3′；

F8：5′-GCATGCCCTGCCCCTAAGGAATTCCCGCTCCGTGTAAAT-3′；

R8：5′-CACTAAAGGGAACAAAAGCTGGTACCCGCTTCAAATACGC-3′；

F9：5′-GATATCGGCTTGTTGTGAGTAGCAGTG-3′；

R9：5′-AGCTTTTGTTCCCTTTAGTG-3′。

(2) obtaining a complete PCR amplification product fragment IFT54-HA-PSAD terminator-HSP70a/RBCS2 promoter-RBCS 2intron 1-NAT-RBCS 2terminator by pairwise overlap PCR of four fragments (IFT54-HA, PSAD terminator, HSP70a/RBCS2 promoter-RBCS 2intron1-NAT and RBCS2 terminator), and then carrying out the fragmentHomologous recombination with fragment vector yielded plasmid 2: pPSAD-IFT54-HA (NAT)⁺) The expression cassette is shown as A in FIG. 5.

Second, obtaining target gene expression strain of Chlamydomonas

Linearized plasmid 2 was integrated into the paromomycin-resistant IFT54 mutant using the "electrotransformation technique" of example 2 (literature: Zhu X, Liang Y, Gao F, Pan J. IFT54 regulations IFT20 stability but not actual viral infection for tubulin transport along with mutagenesis. cell Mol Life Sci.2017; 74 (18): 3425-37.). 192 monoclonal algal strains were randomly selected from among the colonies grown on the nourseothricin-resistant plate, and the expression level of IFT54-HA was examined by immunoblotting in example 2 using HA, IFT54 and CDPK3 antibodies (CDPK3 antibody was used to ensure that the protein loading levels of the different algal strains were consistent during electrophoresis), and it was revealed that 12 of these algal strains (shown as "IFT 54:: IFT 54-HA" in the figure) expressed IFT54-HA and that the expression level of IFT54-HA protein of these 12 algal strains was equivalent to the expression level of IFT54 protein of wild type Chlamydomonas (shown as "WT" in the figure) (B in FIG. 5). The screening probability of IFT54-HA expression strain was 6.3% (i.e., 192 algal strains were screened, and 12 of them had IFT54-HA expression). Further, the wild type (denoted by "WT" in the figure), paromomycin-resistant IFT54 mutant (denoted by "IFT 54" in the figure), and 12 IFT 54-HA-expressing strains (denoted by "IFT 54:: IFT 54-HA", only one strain) were cultured in accordance with the Chlamydomonas culture method in example 1 and tested for flagella trait by differential interference microscopy (C in FIG. 5). The paromomycin resistant IFT54 mutant failed to flagella due to deletion of the IFT54 protein, clumped together in liquid and stood still, while expression of IFT54-HA therein restored the IFT54 mutant to the wild type chlamydomonas phenotype, single in liquid, free swimming, with two flagella of equal length of around 12 μm. Therefore, plasmid 2 can not only express protein with high efficiency and accuracy, but also express in paromomycin resistant strain.

Further, the linearized plasmid 2 was integrated into the strain wdr92 with dual resistance to paromomycin/hygromycin using the "electrotransformation technique" of example 2: : WDR92-YFP (the Chlamydomonas itself expresses IFT54 protein) (literature: Liu G, Wang L, Pan J. Chlamydomonas WDR92 in association with R2TP-like complex and multiple DNAs AFs to regulated circulation protein assembly. J. Mol Cell biol.2018.). From among the monoclonals grown on the nourseothricin-resistant plates, 116 monoclonal algal strains were randomly selected and tested for IFT54-HA expression levels by "immunoblotting" in example 2 using HA, IFT54 and CDPK3 antibodies, respectively, and it was found that 7 of the monoclonal algal strains (shown as "paro/hyg:: IFT 54-HA") among them were tested for IFT54-HA expression, while the wild type Chlamydomonas (shown as "WT") and the strain wdr92 having paromomycin/hygromycin dual resistance: : neither WDR92-YFP (shown as "paro/hyg") expressed IFT54-HA (D in FIG. 5). The screening probability of IFT54-HA expression strain was 6.03% (i.e., 116 algal strains were screened, 7 of them had IFT54-HA expression). Therefore, the plasmid 2 can not only express protein efficiently and accurately, but also express in paromomycin/hygromycin double-resistant algal strains.

Thus, plasmid 2 can express the protein of interest in Chlamydomonas without interference from other screening markers currently available.

Example 4 plasmid constructed by inserting 2A sequence shown in SEQ ID No. 7 between NAT gene and target gene can greatly improve the screening efficiency of target gene expression strain

Firstly, constructing a plasmid 3: pHR-NAT-2A-IFT54-HA

Construction of plasmid 3: pHR-NAT-2A-IFT54-HA, 2A being located between NAT and IFT54-HA in plasmid 3 (shown as A in FIG. 6), the detailed procedure was as follows:

(1) carrying out PCR amplification by taking plasmid pHR-NAT-HA as a template and adopting F10 and R10 primers to obtain a PCR amplification product fragment HSP70a/RBCS2 promoter-RBCS 2intron 1-NAT; carrying out PCR amplification by using a plasmid pBR25-sfGFP-TUA template and F11 and R11 primers to obtain a PCR amplification product fragment 2A (shown as a sequence 7); with the plasmid PSAD-IFT54-HA (hygromycin)⁺) Carrying out PCR amplification on the template by adopting F12 and R12 primers to obtain a PCR amplification product fragment IFT 54; carrying out PCR amplification by using a plasmid PSAD-IFT54-HA (hygromycin +) template and F3 and R13 primers to obtain a PCR amplification product fragment HA. The primer sequences are as follows:

F10：5′-ACCGCGGTGGCGGCCGCTCTAGACGGCGGGGAGCTC-3′；

R10：5′-GTTCAGGGTCTGCTTCACGGGGCAGGGCATGCTCATGTAC-3′；

F11：5′-GTGAAGCAGACCCTGAAC-3′；

R11：5′-GGGGCCCGGGTTGCT-3′；

F12：5′-AGCAACCCGGGCCCCATGTGCGATAACTGGCAGGC-3′；

R12：5′-CGCTAGCGACGCCATAGATCTCCGCCCAGCGGTTGC-3′：

R13：5′-ATTTACACGGAGCGGGAATTCCGCGGATCTCCTAGGTTA-3′。

(2) the plasmid pBR25-sfGFP-TUA was double-digested with restriction enzymes EcoRI and XbaI to obtain the vector pBR 25.

(3) A complete PCR amplification product fragment RSP70a/RBCS2 promoter-RBCS 2intron 1-NAT-2A-IFT54-HA is obtained by four-fragment pairwise overlap PCR. The above fragments and the vector were subjected to homologous recombination to obtain plasmid pHR-NAT-2A-IFT54-HA, the expression cassette of which is shown in A of FIG. 6.

Second, obtaining target gene expression strain of Chlamydomonas

Linearized plasmid 3 was integrated into the paromomycin-resistant ift54 mutant using the "electrotransformation technique" of example 2. From the colonies grown on the nourseothricin-resistant plate, 204 monoclonal algal strains were randomly selected, and the IFT54-HA expression levels of the 204 algal strains were examined by immunoblotting in example 2 using IFT54 and CDPK3 antibodies (CDPK3 antibody was used to ensure consistent protein loading levels when different algal strains were electrophoresed), and it was revealed that 122 of these 204 algal strains (shown as "IFT 54:: NAT-2A-IFT 54-HA", only 10 of these strains are shown) expressed IFT54-HA, and that the IFT54-HA protein expression levels of these 10 algal strains were equivalent to the IFT54 protein expression level of wild type Chlamydomonas (shown as "WT" in FIG. 6) (B). The screening efficiency of IFT54-HA expression strain was 59.8% (i.e., 204 selected algal strains, of which 122 had IFT54-HA expression), which was about 9.5 times that of example 3. Furthermore, the flagella trait of the 122 strain was observed by the method for observing the flagella trait of Chlamydomonas in example 3, and it was found that the phenotype of wild type Chlamydomonas was also restored. Therefore, the plasmid 3 not only can express protein efficiently and accurately, but also can obviously improve the screening efficiency of target gene expression strains.

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

SEQUENCE LISTING

<110> Qinghua university

Application of <120> NAT gene as screening marker in genetic transformation of chlamydomonas

<130> GNCFY196079

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 573

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgggcacca ccctggacga caccgcgtac cgctaccgca ccagcgtgcc cggcgacgcc 60

gaggcgatcg aggcgctgga cggcagcttc accaccgaca ccgtgttccg cgtgaccgcc 120

accggcgacg gcttcaccct gcgcgaggtg cccgtggacc cccccctgac caaggtgttc 180

cccgacgacg agtccgacga cgagagcgac gacggcgagg acggcgaccc cgacagccgc 240

accttcgtgg cgtacggcga cgacggcgac ctggccggct tcgtggtcgt gagctacagc 300

ggctggaacc gccgcctgac cgtggaggac atcgaggtgg cgcccgagca ccgcggccac 360

ggcgtgggcc gcgccctgat gggcctggcc accgagttcg cgcgcgagcg cggcgcgggc 420

cacctgtggc tggaggtgac caacgtgaac gcccccgcga tccacgccta ccgccgcatg 480

ggcttcaccc tgtgcggcct ggacaccgcc ctgtacgacg gcaccgcgag cgacggcgag 540

caggccctgt acatgagcat gccctgcccc taa 573

<210> 2

<211> 573

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgggcacca ccctggacga cacggcttac cggtaccgca ccagtgtccc gggggacgcc 60

gaggccatcg aggcactgga tgggtccttc accaccgaca ccgtcttccg cgtcaccgcc 120

accggggacg gcttcaccct gcgggaggtg ccggtggacc cgcccctgac caaggtgttc 180

cccgacgacg aatcggacga cgaatcggac gacggggagg acggcgaccc ggactcccgg 240

acgttcgtcg cgtacgggga cgacggcgac ctggcgggct tcgtggtcgt ctcgtactcc 300

ggctggaacc gccggctgac cgtcgaggac atcgaggtcg ccccggagca ccgggggcac 360

ggggtcgggc gcgcgttgat ggggctcgcg acggagttcg cccgcgagcg gggcgccggg 420

cacctctggc tggaggtcac caacgtcaac gcaccggcga tccacgcgta ccggcggatg 480

gggttcaccc tctgcggcct ggacaccgcc ctgtacgacg gcaccgcctc ggacggcgag 540

caggcgctct acatgagcat gccctgcccc taa 573

<210> 3

<211> 190

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Gly Thr Thr Leu Asp Asp Thr Ala Tyr Arg Tyr Arg Thr Ser Val

1 5 10 15

Pro Gly Asp Ala Glu Ala Ile Glu Ala Leu Asp Gly Ser Phe Thr Thr

20 25 30

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

35 40 45

Glu Val Pro Val Asp Pro Pro Leu Thr Lys Val Phe Pro Asp Asp Glu

50 55 60

Ser Asp Asp Glu Ser Asp Asp Gly Glu Asp Gly Asp Pro Asp Ser Arg

65 70 75 80

Thr Phe Val Ala Tyr Gly Asp Asp Gly Asp Leu Ala Gly Phe Val Val

85 90 95

Val Ser Tyr Ser Gly Trp Asn Arg Arg Leu Thr Val Glu Asp Ile Glu

100 105 110

Val Ala Pro Glu His Arg Gly His Gly Val Gly Arg Ala Leu Met Gly

115 120 125

Leu Ala Thr Glu Phe Ala Arg Glu Arg Gly Ala Gly His Leu Trp Leu

130 135 140

Glu Val Thr Asn Val Asn Ala Pro Ala Ile His Ala Tyr Arg Arg Met

145 150 155 160

Gly Phe Thr Leu Cys Gly Leu Asp Thr Ala Leu Tyr Asp Gly Thr Ala

165 170 175

Ser Asp Gly Glu Gln Ala Leu Tyr Met Ser Met Pro Cys Pro

180 185 190

<210> 4

<211> 1632

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gacggcgggg agctcgctga ggcttgacat gattggtgcg tatgtttgta tgaagctaca 60

ggactgattt ggcgggctat gagggcgggg gaagctctgg aagggccgcg atggggcgcg 120

cggcgtccag aaggcgccat acggcccgct ggcggcaccc atccggtata aaagcccgcg 180

accccgaacg gtgacctcca ctttcagcga caaacgagca cttatacata cgcgactatt 240

ctgccgctat acataaccac tcagctagct taagatccca tcaagcttgc atgccgggcg 300

cgccagaagg agcgcagcca aaccaggatg atgtttgatg gggtatttga gcacttgcaa 360

cccttatccg gaagccccct ggcccacaaa ggctaggcgc caatgcaagc agttcgcatg 420

cagcccctgg agcggtgccc tcctgataaa ccggccaggg ggcctatgtt ctttactttt 480

ttacaagaga agtcactcaa catcttaaaa tggccaggtg agtcgacgag caagcccggc 540

ggatcaggca gcgtgcttgc agatttgact tgcaacgccc gcattgtgtc gacgaaggct 600

tttggctcct ctgtcgctgt ctcaagcagc atctaaccct gcgtcgccgt ttccatttgc 660

aggatggcca tgcatatgat gggcaccacc ctggacgaca ccgcgtaccg ctaccgcacc 720

agcgtgcccg gcgacgccga ggcgatcgag gcgctggacg gcagcttcac caccgacacc 780

gtgttccgcg tgaccgccac cggcgacggc ttcaccctgc gcgaggtgcc cgtggacccc 840

cccctgacca aggtgttccc cgacgacgag tccgacgacg agagcgacga cggcgaggac 900

ggcgaccccg acagccgcac cttcgtggcg tacggcgacg acggcgacct ggccggcttc 960

gtggtcgtga gctacagcgg ctggaaccgc cgcctgaccg tggaggacat cgaggtggcg 1020

cccgagcacc gcggccacgg cgtgggccgc gccctgatgg gcctggccac cgagttcgcg 1080

cgcgagcgcg gcgcgggcca cctgtggctg gaggtgacca acgtgaacgc ccccgcgatc 1140

cacgcctacc gccgcatggg cttcaccctg tgcggcctgg acaccgccct gtacgacggc 1200

accgcgagcg acggcgagca ggccctgtac atgagcatgc cctgccccag atctatggcg 1260

tcgctagcgg atccgtcgcg atacccctac gacgtgcccg actacgccta cccctacgac 1320

gtgcccgact acgccgatcg atccggaccg tacccctacg acgtgcccga ctacgcctaa 1380

cctaggagat ccgaattccc gctccgtgta aatggaggcg ctcgttgatc tgagccttgc 1440

cccctgacga acggcggtgg atggaagata ctgctctcaa gtgctgaagc ggtagcttag 1500

ctccccgttt cgtgctgatc agtctttttc aacacgtaaa aagcggagga gttttgcaat 1560

tttgttggtt gtaacgatcc tccgttgatt ttggcctctt tctccatggg cgggctgggc 1620

gtatttgaag cg 1632

<210> 5

<211> 6485

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gaattcccgc tccgtgtaaa tggaggcgct cgttgatctg agccttgccc cctgacgaac 60

ggcggtggat ggaagatact gctctcaagt gctgaagcgg tagcttagct ccccgtttcg 120

tgctgatcag tctttttcaa cacgtaaaaa gcggaggagt tttgcaattt tgttggttgt 180

aacgatcctc cgttgatttt ggcctctttc tccatgggcg ggctgggcgt atttgaagcg 240

ggtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca 300

acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc 360

tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg 420

cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 480

ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 540

cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 600

ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 660

tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 720

gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 780

ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 840

gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt 900

ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 960

aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 1020

aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 1080

cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 1140

ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 1200

cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 1260

ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 1320

gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 1380

gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 1440

acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 1500

cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 1560

acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 1620

ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 1680

ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 1740

gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 1800

atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 1860

ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 1920

attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 1980

ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 2040

aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 2100

aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 2160

ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg 2220

tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 2280

ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 2340

cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 2400

agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 2460

gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 2520

ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 2580

tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 2640

tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 2700

cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 2760

tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 2820

gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc 2880

agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg 2940

agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg 3000

tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc 3060

aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctccaccg cggtggcggc 3120

cgctctagac ggcggggagc tcgctgaggc ttgacatgat tggtgcgtat gtttgtatga 3180

agctacagga ctgatttggc gggctatgag ggcgggggaa gctctggaag ggccgcgatg 3240

gggcgcgcgg cgtccagaag gcgccatacg gcccgctggc ggcacccatc cggtataaaa 3300

gcccgcgacc ccgaacggtg acctccactt tcagcgacaa acgagcactt atacatacgc 3360

gactattctg ccgctataca taaccactca gctagcttaa gatcccatca agcttgcatg 3420

ccgggcgcgc cagaaggagc gcagccaaac caggatgatg tttgatgggg tatttgagca 3480

cttgcaaccc ttatccggaa gccccctggc ccacaaaggc taggcgccaa tgcaagcagt 3540

tcgcatgcag cccctggagc ggtgccctcc tgataaaccg gccagggggc ctatgttctt 3600

tactttttta caagagaagt cactcaacat cttaaaatgg ccaggtgagt cgacgagcaa 3660

gcccggcgga tcaggcagcg tgcttgcaga tttgacttgc aacgcccgca ttgtgtcgac 3720

gaaggctttt ggctcctctg tcgctgtctc aagcagcatc taaccctgcg tcgccgtttc 3780

catttgcagg atggccatgc atatggccaa gctgaccagc gccgttccgg tgctcaccgc 3840

gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgggactt 3900

cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt 3960

ccaggaccag gtgagtcgac gagcaagccc ggcggatcag gcagcgtgct tgcagatttg 4020

acttgcaacg cccgcattgt gtcgacgaag gcttttggct cctctgtcgc tgtctcaagc 4080

agcatctaac cctgcgtcgc cgtttccatt tgcaggacca ggtggtgccg gacaacaccc 4140

tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt 4200

ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg 4260

ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc 4320

aggacgcccc ggtgaagcag accctgaact tcgacctgct gaagctggcg ggcgacgtgg 4380

agagcaaccc gggccccctc gagatggtga gcaagggcga ggagctgttc accggggtgg 4440

tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgcgcggcg 4500

agggcgaggg cgatgccacc aacggcaagc tgaccctgaa gttcatctgc accaccggca 4560

agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca 4620

gccgctaccc cgaccacatg aagcgccacg acttcttcaa gtccgccatg cccgaaggct 4680

acgtccagga gcgcaccatc agcttcaagg acgacggcac ctacaagacc cgcgccgagg 4740

tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg 4800

aggacggcaa catcctgggg cacaagctgg agtacaactt caacagccac aacgtctata 4860

tcaccgccga caagcagaag aacggcatca aggccaactt caagatccgc cacaacgtgg 4920

aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc 4980

ccgtgctgct gcccgacaac cactacctga gcacccagtc cgtgctgagc aaagacccca 5040

acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactcacg 5100

gcatggacga gctgtacaag ggatccggca tgcgtgaggt catctccatc cacatcggcc 5160

aggccggtat ccaggtgggc aatgcctgct gggagctcta ctgcctggag cacggcatcc 5220

agcccgatgg ccagatgccc tccgacaaga ccattggcgg tggcgacgat gccttcaaca 5280

ccttcttctc ggagaccggt gccggcaagc acgtgccccg ctgcatcttc ctggacctgg 5340

agcccaccgt ggtggatgag gtccgcaccg gcacctaccg ccagctgttc caccccgagc 5400

agctgatctc cggcaaggag gatgccgcca acaacttcgc ccgcggccac tacaccatcg 5460

gcaaggagat tgtcgacctg gccctggacc gcatccgcaa gctggccgac aactgcaccg 5520

gtctgcaggg cttcctggtc ttcaacgccg tcggcggtgg caccggttcc ggcctgggct 5580

ctctgctact ggagcgcctg tcggtcgact acggcaagaa gtccaagctg ggcttcaccg 5640

tctacccctc gccccaggtg tcgaccgccg tcgtcgagcc ctacaactcg gtgctgtcca 5700

cccactccct gctggagcac accgatgtcg ccgtgatgct cgacaacgag gccatctacg 5760

acatttgccg ccgctccctg gacattgagc gccccaccta caccaacctg aaccgcctga 5820

tcgcccaggt catctcgtcg ctgaccgcct cgcttcgctt tgacggtgcc ctgaacgtgg 5880

atatcactga gttccagacc aacctggtgc cctacccccg catccacttc atgctcagct 5940

cgtacgcgcc catcatctcg gccgagaagg cgtaccacga gcagctgtcg gtggccgaga 6000

tcaccaacgc cgccttcgag cccgcctcga tgatggtcaa gtgcgacccc cgccacggca 6060

agtacatggc ctgctgcctg atgtaccgcg gtgacgtcgt gcccaaggac gttaacgcgt 6120

ccgtggccac catcaagacc aagcgcacca tccagttcgt cgactggtgc cccaccggct 6180

tcaagtgcgg tatcaactac cagcccccca ccgtcgtgcc cggcggtgac ctggccaagg 6240

tgcagcgcgc cgtgtgcatg atctccaaca gcactgctat cggcgagatc ttcagccgcc 6300

tggaccacaa gttcgacctg atgtacgcca agcgtgcctt cgtgcactgg tacgtcggtg 6360

agggtatgga ggagggtgag ttctccgagg cccgcgagga cctggctgcc ctggagaagg 6420

acttcgagga ggtcggcgcc gagtccgccg agggcgctgg cgagggcgag ggtgaggagt 6480

actag 6485

<210> 6

<211> 11318

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cacacacctg cccgtctgcc tgacaggaag tgaacgcatg tcgagggagg cctcaccaat 60

cgtcacacga gccctcgtca gaaacacgtc tccgccacgc tctccctctc acggccgacc 120

ccgcagccct tttgcccttt cctaggccac cgacaggacc caggcgctct cagcatgcct 180

caacaacccg tactcgtgcc agcggtgccc ttgtgctggt gatcgcttgg aagcgcatgc 240

gaagacgaag gggcggagca ggcggcctgg ctgttcgaag ggctcgccgc cagttcgggt 300

gcctttctcc acgcgcgcct ccacacctac cgatgcgtga aggcaggcaa atgctcatgt 360

ttgcccgaac tcggagtcct taaaaagccg cttcttgtcg tcgttccgag acatgttagc 420

agatcgcagt gccacctttc ctgacgcgct cggccccata ttcggacgca attgtcattt 480

gtagcacaat tggagcaaat ctggcgaggc agtaggcttt taagttgcaa ggcgagagag 540

caaagtggga cgcggcgtga ttattggtat ttacgcgacg gcccggcgcg ttagcggccc 600

ttcccccagg ccagggacga ttatgtatca atattgttgc gttcgggcac tcgtgcgagg 660

gctcctgcgg gctggggagg gggatctggg aattggaggt acgaccgaga tggcttgctc 720

ggggggaggt ttcctcgccg agcaagccag ggttaggtgt tgcgctcttg actcgttgtg 780

cattctagga ccccactgct actcacaaca agcccatatg atgtgcgata actggcaggc 840

aaccattgac accctgcagg gtgcaagccc tgtttttgac aagcctaagc tgtctcagaa 900

gctgttggag aagcctcctt ttcgcttcct tcacgatgtg gtcactgcgg tgagaccgcg 960

tagggtttgt ggtggagctt aggacctagt ctcctgtcaa acatcaaagg cattcagcag 1020

cctgtgctgg gcagaagcga gcaagtcctg gaactcgggc atgccgggtg gtccttccac 1080

gacctcgcct gagtgtcatg gtttgctgta caggtgcaac aagcgactgg ctttgcgcca 1140

gggctgtatc aaggagacga gctggacggc aaggccattc aggtcagtgc acaacttgcg 1200

agcacgccac aaccggcgcc tgcccccggc caacctgcca actgggggtg tcatgcttcc 1260

cataacacag gagaaggatg ccaaggtagc gtacctgaag aaaatcatcg aggtggtcag 1320

catggtgctg ggggagcaat gccccgctcg gcccaacaag gtgcggcgtg ccagggcctg 1380

gtcatggggc cttgcaaggg tgatttccta tgcatgtgtg tcatagtctc aagctctttg 1440

atattgacac gcgcagatcg tggctggcct ggagccggag aacacaaaca tctttctgca 1500

gatgctgggg cgggcgtgcc agaagggtaa cggcgcgaaa gcagtccagg tgggtgtcaa 1560

aggcccttgc gcttcgcaga caacttcata tcacatgcat gcactgagcc catgctttgg 1620

caggggacat gctacacggg ttccgaagta ccgggtagtc tctgggtgac tgggcaccag 1680

cgcgcaacgc gcaacggata acggatagca tggcctactg cacgctgtca ccgaactcgc 1740

atgcaatcac ttgatgcgtg cagaaagtac taggcggtgg gggtgctgag cccgcgccgg 1800

ccaaggagga ggccccgccg ccggagaaga agcccgagaa gaaggagaag aaggaggaga 1860

agccggcgga aaagagccgg gcggaggcgt cgccagcgcg gaaaaaggcc gcggagccgg 1920

aggcagaaaa gaaggcatcg agcaagagct cgtctaggac gaaggaggag ccgccggcta 1980

aggcgccggc gaagaagaag gaggagccgg cgccggagaa gccgtccaag tcaaaggccg 2040

cgcccgcagg tgcgggcgag tggcattgca tggcacatgg cgttaaggca ggggcgtgtg 2100

ggagtaaaca tggtgggaat gctgggatgt cttatggtca tgcgccgtca tgccgctacc 2160

cgcaagcact gggttgcttc actgcatcaa cactgccctg cccatctgtc cttgcagctg 2220

aggaagcgcc gccgccgccg ccgcctgccg cggagccgcc cgctcgctcc gcgtcgcctg 2280

gaggcgagga cccgctcaac aagagcggca gtgccgcgcc caagttccag cggcccacgt 2340

ccgcgaggaa ggcgccgcct cgcgtgcccc agccgcagca gtgagtttac cggatggacg 2400

cggcttagca gacacagggc gaatcgagct ccctacggca atgcttgggt gggcaaggga 2460

tgggtaatag tgtagcgcgt atcatgtttt aacgactttg cacctcatgt tgacgggcac 2520

tcactcacca caggccaaca atgctggcgg gcacgggcat tcgccccggc accgcgaccc 2580

ggcgacccaa tgagcccaag cccacggaca gcaaggcgag tacagttgca tgtgggcgtg 2640

ttgacgaggt gcacgccgtt gccgcacggc gtgccaatgc tctgcccctc cctttacggc 2700

ttgtggtaac ccctagcaag acctactgcg aaggcgctaa tagcaacacg ggcgccattt 2760

ccttaatcct cgctccacag gttaccaagc ccgtggcggt gttcacggac aacgccaagg 2820

acaacagcga cgacgaagtg gaggtggtgc acgagcagac gccggtgctg tcgggcggcg 2880

ccaacatgac gggcgagcag ggtgtcctgg tcaaggacat cctggcggcg gagaagggcc 2940

tcaaggtgcg gcgttgaggg tggcgggcgg gcggtcatgt gcgacgcggg cttgcctttg 3000

cgcacggggg gagggaggcc gttgatgggg aacggagtga ttgggggacg tggggaacgt 3060

ctctgcacac gtgaaggaca cggcggcgct ctagtactga agcggggcgg agcgtgtgca 3120

acggagtgca acactgcagc agaacaagtt cactcgacat cctatccctt gcgcaacctg 3180

caacacctcc atgcggccac agaaagcggg cgtggacgcc accgcggata acgcggacac 3240

gtctgaccag ggcagcaccg gtatcatcct caagcgcctg ggcggcaagg cggccggtgc 3300

gggtgcggcc gctgccgggc ctcgcgcaca cgacccctcc agcgtgcgcg agctggtgca 3360

gaagctgtgc cacagcagca cgccgctggc caagtcgatg gactacttgc aggaggacat 3420

tgagaacatg cgcaaggagt ataagtaagc tggcgcagaa gcgtttggtc ctcctccaca 3480

tgtatgccgc cgtctggttg catgcagtgc acccctttgc ctgcctgtca tccgcgtgtc 3540

cgccatcctc tcgcatgctt actcctgctt actcaactta ctccaggttc tggctgacgg 3600

agaagcgcat gtaccaggac gagctggcgc gtgagctgcg gctgcagggc gaggccgcca 3660

acgtggacgc gcagctggcg gacctggacg gccagattaa gcaggtggga tgatggaaga 3720

ggtgtggaca gctgcgccat gcgttctgag tgccccttcc cggcatactt ggcatcgcct 3780

ctccccgcgc cgttcattcg gggcttagct tacttccccg caagcggaaa cgcgtttatg 3840

caatacgatg gggtcgatgg gggcatgcgc gtatcgttgt gttgtgacct gcggggaggc 3900

ggcggttgcc tgcctgcctt ggcaacacac aatgtgggac ccgccgagac atgccatgca 3960

tgctacagcg cggacagcct tctgtcagct acacgtgagg aatgcgccac tgacgcacga 4020

tgctccttgc ccgtcattcc tactgcgtaa ttgacaggcg cgcgaccgca tcattggcat 4080

gaagggccag atcctgcgga acgacgagac gctggggaag ctgctggcaa tggcaaccgc 4140

tgggcggaga tctatggcgt cgctagcgga tccgtcgcga tacccctacg acgtgcccga 4200

ctacgcctac ccctacgacg tgcccgacta cgccgatcga tccggaccgt acccctacga 4260

cgtgcccgac tacgcctaac ctaggagatc cgcggccgcg gagcttcggg cgcgcgggag 4320

ttggactcag gcggtagcgc ggggggcgct agggacatag cggggagggg tgcaaatgta 4380

atcatgaatc tgtacagtgt atgtgtgctc ttcataagac ggctggggcc ttaggtagaa 4440

ggaggggcgg agcgcggtct ctggggcccc tcgatcgggc cgcgcctcct tggtggactg 4500

atagaaaact gacggacatg actgatgaca acatgacgtt tatggtgctt ggaggactcg 4560

catttgacgc ggcatgactt caagagccct gcaaatgcag cggcattctc gcggggggct 4620

tctgtaacgg cggacgtatc gcagttgcct cgctggtatc atgctatcgc tgatcgtgat 4680

ggtggggctt cagagcggca gcagcagcgc cggtcaaggc ccggtcgtcg gtccgaaagt 4740

ttccatgttc cagaagttca gcggaagcgg gcacaagggg cagtctaatg gtaaaggggc 4800

aatggatatc ggcgcctccg gccagggcgc ctccggcgcg atggccaagg gcgaggagct 4860

gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt 4920

cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat 4980

ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcggctacgg 5040

cctgatgtgc ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc 5100

catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa 5160

gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg 5220

catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag 5280

ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat 5340

ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc 5400

catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagctacc agtccgccct 5460

gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc 5520

cgggatcact ctcggcatgg acgagctgta caagtaagaa ttctggcagc agctggaccg 5580

cctgtaccat ggagaagagc tttacttgcc gggatggccg atttcgctga ttgatacggg 5640

atcggagctc ggaggctttc gcgctagggg ctaggcgaag ggcagtggtg accagggtcg 5700

gtgtggggtc ggcccacggt caattagcca caggaggatc agggggaggt aggcacgtcg 5760

acttggtttg cgaccccgca gttttggcgg acgtgctgtt gtagatgtta gcgtgtgcgt 5820

gagccagtgg ccaacgtgcc acacccattg agaagaccaa ccaacttact ggcaatatct 5880

gccaatgcca tactgcatgt aatggccagg ccatgtgaga gtttgccgtg cctgcgcgcg 5940

ccccgggggc gcagtttagc tgaccagccg tgggatgatg cacgcatttg caaggacagg 6000

gtaatcacag cagcaacatg gtgggcttag gacagctgtg ggtcagtgga cggacggcag 6060

gggagggacg gcgcagctcg ggagacaggg ggagacagcg tgactgtgca atcaagctaa 6120

ttcgatctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa 6180

tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat 6240

gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta 6300

cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctcc accgcggtgg 6360

cggccgctct agaactagtg gatccactat agggcgaatt ggagctccac cgcggtggcg 6420

gccgctctag agacggcggg gagctcgctg aggcttgaca tgattggtgc gtatgtttgt 6480

atgaagctac aggactgatt tggcgggcta tgagggcggg ggaagctctg gaagggccgc 6540

gatggggcgc gcggcgtcca gaaggcgcca tacggcccgc tggcggcacc catccggtat 6600

aaaagcccgc gaccccgaac ggtgacctcc actttcagcg acaaacgagc acttatacat 6660

acgcgactat tctgccgcta tacataacca ctcagctagc ttaagatccc atcaagcttc 6720

tttcttgcgc tatgacactt ccagcaaaag gtagggcggg ctgcgagacg gcttcccggc 6780

gctgcatgca acaccgatga tgcttcgacc ccccgaagct ccttcggggc tgcatgggcg 6840

ctccgatgcc gctccagggc gagcgctgtt taaatagcca ggcccccgat tgcaaagaca 6900

ttatagcgag ctaccaaagc catattcaaa cacctagatc actaccactt ctacacaggc 6960

cactcgagct tgtgatcgca ctccgctaag ggggcgcctc ttcctcttcg tttcagtcac 7020

aacccgcaaa catgacacaa gaatccctgt tacttctcga ccgtattgat tcggatgatt 7080

cctacgcgag cctgcggaac gaccaggaat tctgggaggt gagtcgacga gcaagcccgg 7140

cggatcaggc agcgtgcttg cagatttgac ttgcaacgcc cgcattgtgt cgacgaaggc 7200

ttttggctcc tctgtcgctg tctcaagcag catctaaccc tgcgtcgccg tttccatttg 7260

cagccgctgg cccgccgagc cctggaggag ctcgggctgc cggtgccgcc ggtgctgcgg 7320

gtgcccggcg agagcaccaa ccccgtactg gtcggcgagc ccggcccggt gatcaagctg 7380

ttcggcgagc actggtgcgg tccggagagc ctcgcgtcgg agtcggaggc gtacgcggtc 7440

ctggcggacg ccccggtgcc ggtgccccgc ctcctcggcc gcggcgagct gcggcccggc 7500

accggagcct ggccgtggcc ctacctggtg atgagccgga tgaccggcac cacctggcgg 7560

tccgcgatgg acggcacgac cgaccggaac gcgctgctcg ccctggcccg cgaactcggc 7620

cgggtgctcg gccggctgca cagggtgccg ctgaccggga acaccgtgct caccccccat 7680

tccgaggtct tcccggaact gctgcgggaa cgccgcgcgg cgaccgtcga ggaccaccgc 7740

gggtggggct acctctcgcc ccggctgctg gaccgcctgg aggactggct gccggacgtg 7800

gacacgctgc tggccggccg cgaaccccgg ttcgtccacg gcgacctgca cgggaccaac 7860

atcttcgtgg acctggccgc gaccgaggtc accgggatcg tcgacttcac cgacgtctat 7920

gcgggagact cccgctacag cctggtgcaa ctgcatctca acgccttccg gggcgaccgc 7980

gagatcctgg ccgcgctgct cgacggggcg cagtggaagc ggaccgagga cttcgcccgc 8040

gaactgctcg ccttcacctt cctgcacgac ttcgaggtgt tcgaggagac cccgctggat 8100

ctctccggct tcaccgatcc ggaggaactg gcgcagttcc tctgggggcc gccggacacc 8160

gcccccggcg cctgataagg atccccgctc cgtgtaaatg gaggcgctcg ttgatctgag 8220

ccttgccccc tgacgaacgg cggtggatgg aagatactgc tctcaagtgc tgaagcggta 8280

gcttagctcc ccgtttcgtg ctgatcagtc tttttcaaca cgtaaaaagc ggaggagttt 8340

tgcaattttg ttggttgtaa cgatcctccg ttgattttgg cctctttctc catgggcggg 8400

ctgggcgtat ttgaagcggg taccagcttt tgttcccttt agtgagggtt aatttcgagc 8460

ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 8520

cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 8580

ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 8640

ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 8700

gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 8760

cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 8820

tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 8880

cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 8940

aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 9000

cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 9060

gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 9120

ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 9180

cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 9240

aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 9300

tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 9360

ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 9420

tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 9480

ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 9540

agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 9600

atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 9660

cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 9720

ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 9780

ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 9840

agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 9900

agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 9960

gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 10020

cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 10080

gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 10140

tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 10200

tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 10260

aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 10320

cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 10380

cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 10440

aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 10500

ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 10560

tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 10620

ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 10680

gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 10740

ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 10800

cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 10860

agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 10920

aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 10980

gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 11040

aaatttaacg cgaattttaa caaaatatta acgcttacaa tttccattcg ccattcaggc 11100

tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga 11160

aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac 11220

gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattggagc 11280

tccaccgcgg tggcggccgc tctagaacta gtggatcc 11318

<210> 7

<211> 66

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gtgaagcaga ccctgaactt cgacctgctg aagctggcgg gcgacgtgga gagcaacccg 60

ggcccc 66

<210> 8

<211> 262

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Ala Lys Ala Val Gln Lys Val Leu Gly Gly Gly Gly Ala Glu Pro Ala

1 5 10 15

Pro Ala Lys Glu Glu Ala Pro Pro Pro Glu Lys Lys Pro Glu Lys Lys

20 25 30

Glu Lys Lys Glu Glu Lys Pro Ala Glu Lys Ser Arg Ala Glu Ala Ser

35 40 45

Pro Ala Arg Lys Lys Ala Ala Glu Pro Glu Ala Glu Lys Lys Ala Ser

50 55 60

Ser Lys Ser Ser Ser Arg Thr Lys Glu Glu Pro Pro Ala Lys Ala Pro

65 70 75 80

Ala Lys Lys Lys Glu Glu Pro Ala Pro Glu Lys Pro Ser Lys Ser Lys

85 90 95

Ala Ala Pro Ala Ala Glu Glu Ala Pro Pro Pro Pro Pro Pro Ala Ala

100 105 110

Glu Pro Pro Ala Arg Ser Ala Ser Pro Gly Gly Glu Asp Pro Leu Asn

115 120 125

Lys Ser Gly Ser Ala Ala Pro Lys Phe Gln Arg Pro Thr Ser Ala Arg

130 135 140

Lys Ala Pro Pro Arg Val Pro Gln Pro Gln Gln Pro Thr Met Leu Ala

145 150 155 160

Gly Thr Gly Ile Arg Pro Gly Thr Ala Thr Arg Arg Pro Asn Glu Pro

165 170 175

Lys Pro Thr Asp Ser Lys Val Thr Lys Pro Val Ala Val Phe Thr Asp

180 185 190

Asn Ala Lys Asp Asn Ser Asp Asp Glu Val Glu Val Val His Glu Gln

195 200 205

Thr Pro Val Leu Ser Gly Gly Ala Asn Met Thr Gly Glu Gln Gly Val

210 215 220

Leu Val Lys Asp Ile Leu Ala Ala Glu Lys Gly Leu Lys Lys Ala Gly

225 230 235 240

Val Asp Ala Thr Ala Asp Asn Ala Asp Thr Ser Asp Gln Gly Ser Thr

245 250 255

Gly Ile Ile Leu Lys Arg

260

<210> 9

<211> 202

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Gly Cys Ser Ser Ser Lys Asp Glu Val Glu Leu Ala Gln Gln Val

1 5 10 15

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

20 25 30

Ala Leu Gly Leu Glu Asp Gly Glu Val Trp Ile Thr Arg Gln Thr Thr

35 40 45

Asp Ile Arg Lys Val Phe Lys Phe Gly Gln Gln Ile Asn Lys Gly Gln

50 55 60

Phe Gly Val Ile His Val Val Thr Asp Ala Ala Gly Asn Lys Tyr Ala

65 70 75 80

Cys Lys Gln Ile Ser Lys Arg Lys Leu Thr Gly Pro Asn Ser Ile Arg

85 90 95

Asp Ile Arg Arg Glu Ile Glu Ile Met His His Leu Arg Gly His Pro

100 105 110

Ser Val Ile Thr Phe His Gly Val Tyr Glu Asp Val Ser Asp Val Tyr

115 120 125

Met Val Met Glu Leu Cys Thr Gly Gly Asp Leu Phe Glu Lys Ile Glu

130 135 140

Lys Met Ser Thr Val Thr Glu Arg Ala Ala Ala Ala Leu Phe Arg Glu

145 150 155 160

Leu Val Glu Cys Val Ala Tyr Cys His Thr Leu Gly Val Met His Arg

165 170 175

Asp Leu Lys Pro Glu Asn Phe Leu Val Thr Asp Arg Thr Gly Asp Ala

180 185 190

Arg Ile Lys Leu Ser Asp Phe Gly Leu Ser

195 200

Claims

1. Application of a Nolserine-N-acetyltransferase gene as a screening marker in genetic transformation of Chlamydomonas.

2. Application of the Nolsemiin-N-acetyltransferase gene as a screening marker in preparation of a chlamydomonas target gene expression strain and/or a transgenic chlamydomonas strain.

3. Use according to claim 1 or 2, characterized in that:

the nourseothricin-N-acetyltransferase gene is a DNA molecule of any one of the following 1) to 5):

1) a DNA molecule shown as a sequence 1 in a sequence table;

3) a DNA molecule shown as a sequence 2in a sequence table;

4. Use of a biomaterial related to the nourseothricin-N-acetyltransferase gene as claimed in claim 3 in the preparation of a chlamydomonas target gene expression strain or a transgenic chlamydomonas strain, wherein the biomaterial is any of the following:

C1) an expression cassette comprising the nourseothricin-N-acetyltransferase gene of claim 3;

C2) a recombinant vector comprising the nourseothricin-N-acetyltransferase gene of claim 3, or a recombinant vector comprising the expression cassette of C1);

C3) a recombinant microorganism comprising the nourseothricin-N-acetyltransferase gene of claim 3, or a recombinant microorganism comprising C1) said expression cassette, or a recombinant microorganism comprising C2) said recombinant vector;

C4) a transgenic chlamydomonas cell line comprising the nourseothricin-N-acetyltransferase gene of claim 1 or 2, or a transgenic chlamydomonas cell line comprising the expression cassette of C1), or a transgenic chlamydomonas cell line comprising the recombinant vector of C2).

5. Use according to claim 4, characterized in that: C1) the expression cassette is HR-NAT-HA or HR-NAT, the sequence of the HR-NAT-HA is shown as a sequence 4 in a sequence table, wherein the 1 st to 509 th sites of the sequence 4 are sequences of a promoter HSP70a/RBCS2, the 518 th to 662 th sites of the sequence 4 are sequences of an enhancer RBCS2intron1, the 679 th to 1248 th sites of the sequence 4 are sequences of a nourseothricin-N-acetyltransferase gene, the 1255 th to 1380 th sites of the sequence 4 are sequences of HA, and the 1400 th to 1632 th sites of the sequence 4 are sequences of a terminator RBCS 2; the HR-NAT sequence is obtained by removing 1255 th to 1380 th bits of the sequence 4.

6. Use according to claim 5, characterized in that: C1) the expression cassette further comprises a 2A sequence and a gene of interest, the 2A sequence, the gene of interest sharing a promoter and a terminator with the nourseothricin-N-acetyltransferase gene of any one of claims 1 to 5, and the 2A sequence being located between the nourseothricin-N-acetyltransferase gene and the gene of interest.

7. A DNA molecule characterized by:

the DNA molecule is any one of the following 1) to 5):

1) a DNA molecule shown as a sequence 1 in a sequence table;

3) a DNA molecule shown as a sequence 2in a sequence table;

8. The biomaterial associated with the DNA molecule of claim 7, wherein the biomaterial is any one of:

C1) an expression cassette comprising the DNA molecule of claim 7;

C2) a recombinant vector comprising the DNA molecule of claim 7, or a recombinant vector comprising the expression cassette of C1);

C3) a recombinant microorganism comprising the DNA molecule of claim 7, or a recombinant microorganism comprising C1) the expression cassette, or a recombinant microorganism comprising C2) the recombinant vector;

C4) a transgenic chlamydomonas cell line comprising the DNA molecule of claim 7, or a transgenic chlamydomonas cell line comprising the expression cassette of C1), or a transgenic chlamydomonas cell line comprising the recombinant vector of C2).

9. The biomaterial of claim 8, wherein: C1) the expression cassette is HR-NAT-HA or HR-NAT, the sequence of the HR-NAT-HA is shown as a sequence 4 in a sequence table, wherein the 1 st to 509 th sites of the sequence 4 are sequences of a promoter HSP70a/RBCS2, the 518 th to 662 th sites of the sequence 4 are sequences of an enhancer RBCS2intron1, the 679 th to 1248 th sites of the sequence 4 are sequences of the DNA molecule of any one of claims 7 to 8, the 1255 th to 1380 th sites of the sequence 4 are sequences of HA, and the 1400 th to 1632 th sites of the sequence 4 are sequences of a terminator RBCS 2; the HR-NAT sequence is obtained by removing 1255 th to 1380 th bits of the sequence 4.

10. The biomaterial of claim 9, wherein: C1) the expression cassette further comprises a 2A sequence and a gene of interest, the 2A sequence, the gene of interest sharing a promoter and a terminator with the DNA molecule of any one of claims 7-9, and the 2A sequence being located between the DNA molecule of any one of claims 7-9 and the gene of interest.