CN110656114B

CN110656114B - Tobacco pigment synthesis related gene and application thereof

Info

Publication number: CN110656114B
Application number: CN201910995642.2A
Authority: CN
Inventors: 李雪梅; 曾婉俐; 向海英; 李晶; 杨光宇; 宋春满; 高茜; 张建铎; 许力; 蒋佳芮; 邓乐乐; ***
Original assignee: China Tobacco Yunnan Industrial Co Ltd
Current assignee: China Tobacco Yunnan Industrial Co Ltd
Priority date: 2019-10-18
Filing date: 2019-10-18
Publication date: 2022-07-01
Anticipated expiration: 2039-10-18
Also published as: CN110656114A

Abstract

The invention relates to a gene related to tobacco pigment synthesis and application thereof, belonging to the technical field of plant genetic engineering. The gene Ntab0036220 related to the synthesis of the tobacco pigments provided by the invention is found to be expressed in leaves through RT-PCR. In the plants in which the gene Ntab0036220 related to tobacco pigment synthesis was edited, the expression level of the gene Ntab0036220 related to tobacco pigment synthesis in leaves was significantly reduced as compared with that of the control (untransformed) plants. According to the invention, a CRISPR/Cas9 editing vector for knocking out the Ntab0036220 gene is constructed through a CRISPR/Cas9 mediated gene editing technology, a safflower macrogol plant with the Ntab0036220 gene edited is obtained after genetic transformation, and the contents of neoxanthin, violaxanthin, lutein, chlorophyll b, chlorophyll a and carotene of the obtained plant are obviously reduced.

Description

Tobacco pigment synthesis related gene and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a gene related to tobacco pigment synthesis and application thereof.

Background

The tobacco contains a great variety of pigments, and fresh tobacco leaves contain chlorophyll, carotenoid, yellow pigment and anthocyan. The pigment is not only an important substance for photosynthesis of tobacco plants, but also a key precursor substance of tobacco fragrance. Therefore, the function research of the pigment synthesis gene can be expected to be used for improving the quality of the tobacco. As an important model plant and economic crop, the tobacco is a raw material source of the tobacco industry in China. With the implementation of the tobacco genome plan in China, the whole genome sequencing of a plurality of varieties is completed, and the research of gene functions provides genetic materials and theoretical bases for the improvement of tobacco leaf quality and the genetic improvement of tobacco varieties.

Flue-cured tobacco is the most widely cultivated common tobacco. The flue-cured tobacco planting area and the total yield of China are in the first place in the world. The flue-cured tobacco is used as an economic crop, and the quality of the tobacco leaves is very important. The quality of tobacco leaves directly influences the quality of cigarettes, and the quality of the tobacco leaves is continuously improved, so that the method is the fundamental purpose of tobacco breeding research. The tobacco pigment comprises a plastid pigment and a cytosol pigment, wherein the plastid pigment exists in plastids (chloroplasts and chromoplasts) of plant cells of tobacco leaves, comprises chlorophyll, carotenoid and the like, and is an important substance for photosynthesis in the growth process of tobacco. The plastid pigment is an important aroma precursor, does not have aroma characteristics per se, but can form aroma components which have important contribution to the quality of tobacco aroma through decomposition and conversion. The chlorophyll degradation product neophytadiene can improve the taste and fragrance of the flue-cured tobacco, and is degraded and converted into a fragrant component. When the flavor quality of the tobacco is improved, the degradation products of the carotenoid are obviously increased, and the content is also obviously increased. Therefore, the research on the gene function which influences the synthesis of the pigment and changes the content of the pigment provides genetic materials and theoretical basis for the improvement of the quality of tobacco leaves and the genetic improvement of tobacco varieties.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides a gene related to tobacco pigment synthesis and application thereof, and provides genetic materials and theoretical basis for improving the quality of tobacco leaves and genetic improvement of tobacco varieties.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a nucleotide sequence of a gene related to tobacco pigment synthesis is shown in SEQ ID NO.1 and comprises 2202bp basic groups, and the gene is derived from tobacco (Nicotiana tabacum) and is named as Ntab 0036220.

The invention also provides a coding protein of the gene related to the synthesis of the tobacco pigment.

Further, preferably, the amino acid sequence of the protein encoded by the gene related to tobacco pigment synthesis is shown in SEQ ID NO.2 and comprises 733 amino acids.

The invention also provides application of the gene related to tobacco pigment synthesis in regulating and controlling the pigment content in tobacco leaves. After the gene is edited, the pigment in the tobacco leaves is reduced, and the research on the gene function provides genetic materials and theoretical basis for the improvement of the tobacco leaf quality and the genetic improvement of tobacco varieties.

The invention also provides the application of the coding protein of the gene related to tobacco pigment synthesis in regulating and controlling the pigment content in tobacco leaves.

The present invention also provides the above tobacco pigment synthesis-related gene editing vector, wherein: the constitutive promoter cauliflower mosaic virus CaMV35S (P35) was selected to drive transcription of the Cas9 gene, and the type III RNA polymerase promoter of the arabidopsis U6-26 gene to drive transcription of the sgRNA.

Further, preferably, the sequence shown in SEQ ID No.8 is linked into a CRISPR/Cas9 vector to obtain an editing vector CRISPR/Cas9-Ntab 0036220; the sequence of the editing vector CRISPR/Cas9-Ntab0036220 is shown in SEQ ID NO. 9.

The invention further provides application of the editing vector of the gene related to tobacco pigment synthesis in regulating and controlling the pigment content in tobacco leaves. The editing vector is used for editing a gene Ntab0036220 related to tobacco pigment synthesis, and the content of the pigment in tobacco leaves is reduced after the gene is edited.

The invention finally provides a method for reducing the pigment content in tobacco leaves, which comprises the following steps: knocking out Ntab0036220 gene in tobacco plant or making Ntab0036220 protein function appear to be deficient.

Further, it is preferable to knock out a gene Ntab0036220 related to tobacco pigment synthesis in a tobacco plant body by using the above-mentioned editing vector.

The gene editing adopts a CRISPR/Cas9 system, takes the specific nucleotide sequence of 20nt of the gene Ntab0036220 related to the pigment synthesis of tobacco as a guide sequence, and links the sequence into a CRISPR/Cas9 vector, thereby constructing an editing vector CRISPR/Cas9-Ntab 0036220.

A novel tobacco variety which can be constructed by using an editing vector CRISPR/Cas9-Ntab0036220 comprises the following specific steps: by utilizing agrobacterium tumefaciens-mediated genetic transformation, an editing vector CRISPR/Cas9-Ntab0036220 is transformed into a safflower large gold ingot, and positive transformation and positive editing plants are obtained by screening and identifying, wherein leaf tobacco is obviously lightened compared with a control (untransformed) plant, as shown in figure 1.

The gene Ntab0036220 related to tobacco pigment synthesis edits plants, the pigment in tobacco leaves is reduced, and the research of the gene function provides genetic materials and theoretical basis for the improvement of the tobacco leaf quality and the genetic improvement of tobacco varieties.

Compared with the prior art, the invention has the beneficial effects that:

the gene Ntab0036220 related to the tobacco pigment synthesis provided by the invention is found to be expressed in leaves by fluorescent quantitative PCR. In the plants in which the gene Ntab0036220 related to tobacco pigment synthesis was edited, the expression level of the gene Ntab0036220 related to tobacco pigment synthesis in leaves was significantly reduced as compared with that of the control (untransformed) plants.

According to the invention, a CRISPR/Cas9 editing vector for knocking out the Ntab0036220 gene is constructed through a CRISPR/Cas9 mediated gene editing technology, a safflower macrogol plant with the Ntab0036220 gene edited is obtained through genetic transformation, the contents of neoxanthin, violaxanthin, lutein, chlorophyll b, chlorophyll a and carotene of the obtained plant are obviously reduced, the contents of the chlorophyll a and the carotenoid are reduced by about 10 times, and the reduction multiple of the violaxanthin is lower by about 5 times, which indicates that the knocking-out of the gene can change the pigment synthesis of the plant leaf and influence the pigment content.

In conclusion, the gene editing plant with reduced pigment content can be obtained by knocking out the Ntab0036220 gene by using the CRISPR/Cas9 mediated gene editing technology, so that genetic materials and theoretical bases are provided for the research of tobacco pigment synthesis paths, the improvement of tobacco leaf quality and the genetic improvement of tobacco varieties.

Drawings

FIG. 1 is a comparison of control (untransformed) and edited plants; wherein (a) is a control (untransformed) plant and (b) is an editing plant;

FIG. 2 is a graph showing the relative expression levels of Ntab0036220 in leaves of control (untransformed) plants and leaves of gene-edited plants;

FIG. 3 is a graph comparing the lutein, chlorophyll b, chlorophyll a content in leaves of control (untransformed) plants and leaves of gene-edited plants;

FIG. 4 is a graph comparing the content of neoxanthin, violaxanthin, and carotene in leaves of control (untransformed) plants and leaves of gene-edited plants.

Detailed Description

The present invention will be described in further detail with reference to examples.

It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.

The percentage numbers are volume percentages and the ratios are volume ratios unless otherwise specified.

Biological material:

tobacco material: safflower Dajinyuan, a commercial tobacco variety;

gene editing vector: CRISPR/Cas9 from Bombyx mori focal laboratory at southwest university; gene sequencing and primer synthesis are completed by Shenzhen Huada gene science and technology service company;

experimental reagent:

BsaI restriction enzyme, T4 ligase, plasmid extraction kit, gel recovery kit and the like were purchased from NEB company;

rifampin, kanamycin, streptomycin, and carbenicillin were purchased from sigma;

MS was purchased from Sigma;

RNA extraction kits were purchased from QIAGEN;

the fluorescent quantitative PCR kit is purchased from TaKaRa company;

peptone, yeast extract, etc. were purchased from Oxoid;

the formula and preparation method of part of the reagents are briefly described as follows:

(1) LB liquid medium (1L): 10g peptone (peptone), 10g sodium chloride (NaCl), 5g yeast extract (yeast extract), autoclaved;

(2) YEB liquid medium (1L): 5g beef extract (beef extract), 5g peptone (peptone), 5g sucrose (sucrose), 1g yeast extract (yeast extract), 0.5g magnesium sulfate (MgSO)₄) Sterilizing at high temperature and high pressure;

(3) MS liquid medium (1L): 4.43g of MS powder, 5g of sucrose (sucrose), 1mol/L NaOH or 1mol/L HCl to adjust the pH value to 5.7, and sterilizing at high temperature and high pressure;

(4) LB (LB) solid medium (1L), YEB (YEB) solid medium (1L) and MS (MS) solid medium (1L) 15g of agar were added to (1), (2), (3), respectively.

(5) NAA (0.5 mg/mL) stock: 0.05g of alpha-naphthylacetic acid (NAA), 10 mL of absolute ethyl alcohol is added, after the alpha-naphthylacetic acid is completely dissolved, sterile water is added to the mixture to reach a constant volume of 100 mL, and the mixture is filtered, sterilized and subpackaged at the temperature of minus 20 ℃ for storage and standby;

(6) 6-BA (2.0 mg/mL) stock: 0.2g of 6-benzylaminopurine (6-BA), adding sterile water to the solution until the volume is 100 mL after the 6-benzylaminopurine (6-BA) is completely dissolved, filtering, sterilizing and subpackaging, and storing in a refrigerator at the temperature of-20 ℃ for later use.

(7) Rifampicin (50 mg/mL) stock: dissolving 1g rifampicin in 20 mL methanol, subpackaging, and storing at-20 deg.C for use.

(8) Stock kanamycin (50 mg/mL): 5g kanamycin in 100 mL ddH₂Filtering with 0.22 μm filter membrane, sterilizing, packaging, and storing at-20 deg.C.

(9) Streptomycin (100 mg/mL) stock: 1g streptomycin dissolved in 10 mL ddH₂Filtering with 0.22 μm filter membrane, sterilizing, packaging, and storing at-20 deg.C.

(10) Stock solution of carbenicillin (250 mg/ml): 10g of carbenicillin dissolved in 40mL of ddH₂Filtering with 0.22 μm filter membrane, sterilizing, packaging, and storing at-20 deg.C.

An experimental instrument:

the real-time fluorescent quantitative PCR system LightCycler 480II is a product of Rogowski company.

Example 1

This example is described briefly below mainly with respect to the procedure for obtaining a gene Ntab0036220 related to tobacco pigment synthesis.

Taking cultivated species tobacco safflower large gold element leaves as samples, extracting total RNA of the tobacco leaves by utilizing an RNA extraction kit, and carrying out reverse transcription to obtain cDNA for later use:

total RNA was extracted using the RNeasy Plant Mini Kit from QIAGEN according to the instructions.

Mu.g of total RNA was extracted from leaves for reverse transcription in the following transcription system:

Total RNA 1μg

Oligo(dT) (10μM) 1.5μL

ddH₂O up to 15μL

mixing the above system, placing in PCR, keeping temperature at 70 deg.C for 5min, removing, immediately placing on ice for 5min, and adding the following reagents into the system:

M-MLV Buffer（5X） 5μL

M-MLV reverse transcriptase 0.5. mu.L

0.5 μ L RNase inhibitor

dNTP Mixture 4μL

ddH₂O up to 25μL

The system is put into a PCR instrument, is kept at 42 ℃ for 65min, 65 ℃ for 10min and 4 ℃ and then is stored in a refrigerator at the temperature of minus 20 ℃ for use.

By a homologous comparison method, referring to the sequence of an arabidopsis gene and a known tobacco partial gene sequence, an amplification primer sequence is designed as follows:

F：5’-attgcagccatgaaattcttga-3’，（SEQ ID No.3）

R：5’-gaaagagaaagcaaatagacat-3’；（SEQ ID No.4）

and (3) performing PCR amplification by using the prepared cDNA as a template and the primers:

amplification system (50 μ L):

cDNA 0.5μL

5×Reaction Buffer 10μL

2.5. mu.L of upstream primer (10 mmol/L)

Downstream primer (10 mmol/L) 2.5. mu.L

dNTP (10 mM) 5μL

Phusion DNA Polymerase 0.5μL

ddH₂O up to 50μL

Mixing, centrifuging and performing PCR amplification, wherein the PCR reaction conditions are as follows: 30 cycles of 95 ℃ for 10sec, 52 ℃ for 30sec, and 72 ℃ for 2.5 min; 10min at 72 ℃; hold at 12 ℃.

And purifying and sequencing the amplified product to obtain a gene Ntab0036220 sequence related to the synthesis of the tobacco pigment, wherein the base sequence is shown as SEQ ID No.1 and comprises 2202bp bases in total. After the gene sequence is translated, the coded protein sequence is shown as SEQ ID No.2 and comprises 733 amino acids in total, and further comparative analysis shows that the protein contains a sequence with high homology and is highly conserved. In order to detect the expression specificity of the Ntab0036220 gene in the leaf, the cultivated tobacco leaf was sampled and the expression level of the gene in the leaf was detected by the fluorescent quantitative PCR method. SYBR Premix Ex Taq TMII from TaKaRa was used as a fluorescent quantitative PCR reagent.

Ntab0036220 qPCR amplification primers were as follows:

qPCR-Ntab0036220-F1：gatagcaatcacacgctcaaat（SEQ ID NO.10）

qPCR-Ntab0036220-R1：gataatcttgctgcaaagcctt（SEQ ID NO.11）

the primers for amplification of the internal reference gene (L25 ribosomal protein) were as follows:

L25-F：cccctcaccacagagtctgc（SEQ ID NO.12）

L25-R：aagggtgttgttgtcctcaatctt（SEQ ID NO.13）

three technical replicates were performed for each sample, and the reaction system was as follows:

SYBR Premix-Ex Taq^TMII （2X）10μL

forward primer (10. mu.M) 0.8. mu.L

Reverse primer (10. mu.M) 0.8. mu.L

ROX reference DyeII 0.4μL

cDNA template 2. mu.L

ddH₂O up to 20μL

Amplification conditions: 30s at 95 ℃; at 95 ℃ for 10s and at 60 ℃ for 10s, for 40 cycles.

Dissolution curve: 95 ℃ for 10s, 60 ℃ for 60s, 95-0.29 ℃/s.

The results showed that the gene Ntab0036220 related to tobacco pigment synthesis was expressed in tobacco leaves (the results are shown in FIG. 2).

Example 2

By utilizing the tobacco pigment synthesis related gene Ntab0036220 obtained in the example 1, the invention further constructs a CRISPR/Cas9 vector, and the related construction process is briefly introduced as follows.

The more specific 20nt nucleotide sequence in this gene [ tcttggattgcagcagcata (SEQ ID No. 8)]An upstream primer and a downstream primer F5 '-gatttcttggattgcagcagcata-3' (SEQ ID No. 5), R5 '-caaatatgctgctgcaatccaaga-3' (SEQ ID No. 6) are synthesized as guide sequences of CRISPR/Cas 9. The synthesized primer was treated with ddH₂After O is diluted to be 100 ng/. mu.L, 5. mu.L of each of the upstream and downstream primers are mixed into a PCR tube, and the annealing procedure of the PCR instrument is as follows: denaturation at 95 ℃ for 2 min, decreasing every 8sCooling to 25 ℃ at 0.1 ℃, annealing to form a double-chain structure with a specific guide sequence in the middle as a target fragment:

gatttcttggattgcagcagcata

agaacctaacgtcgtcgtataaac。

the CRISPR/Cas9 vector was digested with BsaI:

CRISPR/Cas9 vector 10uL

BsaI 5uL

10×Butter 5uL

ddH2O up to 50μL

Incubation at 37 ℃ for 1 hour, electrophoresis on a 1.5% agarose gel, and gel recovery of the cleaved vector fragment.

Connecting the enzyme digestion vector with the annealing product:

10×T4 DNA Ligase Buffer 2μL

5 μ L of the fragment of interest

Enzyme digestion vector 2. mu.L

T4 DNA Ligase 1μL

ddH2O up to 20μL

Ligation was carried out at 25 ℃ for 30 min.

The Trans-T1 competent cells were placed on ice, 10. mu.L of the ligation product was pipetted into the competent cells after thawing, mixed gently, ice-bathed for 10min, heat-shocked at 42 ℃ for 90s, and rapidly placed back on ice for 2 min. 60. mu.L of the transformed product was spread evenly on a solid medium containing 50mg/L Kan LB, and cultured at 37 ℃ for 12 to 18 hours.

A single clone was picked as a template to detect primer [ JC-F: 5'-ttaggtttacccgccaata-3' (SEQ ID No. 7) and a target site downstream primer R5 '-caaatatgctgctgcaatccaaga-3' (SEQ ID No. 6) as an amplification primer for PCR amplification:

1 monoclonal

5×Reaction Buffer 5μL

Upstream primer (10 mmol/L) 1.5. mu.L

Downstream primer (10 mmol/L) 1.5. mu.L

dNTP (10 mM) 2.5μL

Phusion DNA Polymerase 0.3μL

ddH₂O up to 25μL

Mixing, centrifuging and performing PCR amplification, wherein the PCR reaction conditions are as follows: 30 cycles of 95 ℃ for 10sec, 47 ℃ for 30sec, and 72 ℃ for 0.5 min; 10min at 72 ℃; hold at 12 ℃.

The PCR positive clone was sent to sequencing company for sequencing confirmation (sequencing primer: JC-F: 5'-ttaggtttacccgccaata-3' (SEQ ID No. 7)) to obtain a CRISPR/Cas9-Ntab0036220 editing vector.

Example 3

By using the CRISPR/Cas9-Ntab0036220 editing vector constructed in example 2 and taking a safflower large gold element as an example, the inventors further performed a genetic transformation test to knock out a tobacco pigment synthesis-related gene Ntab0036220 in a plant body, and the related experimental process is briefly described as follows.

The tobacco seeds are sowed in a seedling culture dish for seedling culture, when 4 cotyledons grow (about 30 days), the tobacco seeds can be transferred into a culture bottle (containing 80mL of MS liquid culture medium), two plants in each bottle are cultured for 30 days under the conditions of 26 ℃ and 16h light/8 h dark condition for standby.

LBA4404 chemically competent Agrobacterium cells stored at-80 ℃ were removed and frozen and thawed on ice. When the competence is just thawed, 10 mu LCRISPR/Cas9-Ntab0036220 is added to edit the vector, the vector is flicked evenly and placed on ice for 30 min. The cells were snap frozen in liquid nitrogen and then incubated in a 37 th water bath for 5 min. 1mL of YEB broth was added thereto at 28 ℃ and cultured at 200rpm for 3 hours. The cells were centrifuged at 8,000rpm to discard the supernatant medium, and then suspended in 200. mu.L of YEB liquid medium, and plated on YEB solid medium containing 50mg/L rifampicin, 50mg/L streptomycin and 50mg/L kanamycin for 2-3 days in an inverted dark state at 28 ℃.

In a clean bench, the leaves are punched into a circular leaf disc with the diameter of 5mm by a puncher, and agrobacterium colonies containing the CRISPR/Cas9-Ntab0036220 editing vector on a YEB plate are suspended into a bacterial liquid by 40 mM LMS liquid culture medium and then placed in a 50 mL centrifugal tube (OD 600: 0.6-0.8). The leaf discs are transferred into a 50 mL centrifugal tube containing bacterial liquid, and soaked and infected for 10 min. Then the suspension was spread on MS solid medium containing 2.0mg/L NAA +0.5 mg/L6-BA, and cultured at 28 ℃ in the dark for 3 days. Then, the lower surface of the leaf disc is contacted with a culture medium, placed on an MS solid culture medium containing 2.0mg/L NAA, 0.5 mg/L6-BA, 250mg/L Cb and 50mg/L Kan, and cultured under the conditions of (28 ℃, 16h of illumination)/(25 ℃, 8h of darkness); when the differentiated bud grows out (about 2 months later), the differentiated bud (1-2 cm) is cut off and inserted into a solid culture medium (containing 250mg/L Cb +100mg/L Kan) containing MS for rooting culture, and the differentiated bud grows out after about 10 days, as shown in figure 1. The leaves were taken for molecular detection and fluorescent quantitative PCR, and the specific results are shown in FIG. 2. Among them, the control (untransformed) plant was a normal plant and the leaf taking was performed at the same time in the same manner as the above reaction except that the CRISPR/Cas9-Ntab0036220 editing vector was not added.

Example 4

Molecular detection is adopted to show that 10 leaves of a plant with positive editing (light color, as shown in figure 1) and 10 leaves of a control (as shown in figure 1) are respectively subjected to freeze drying and then ground into powder for further color detection, and the specific detection method is as follows:

the obtained edited plants and control (untransformed) plants were tested for pigment content. The detection method comprises the following steps:

accurately weighing 0.2g (accurate to 0.1 mg) of lyophilized sample in 50 mL triangular flask, adding 25 mL of extractant (90% acetone solution), performing ultrasonic extraction for 20 min in ice bath, filtering appropriate amount of supernatant with 0.45 μm organic microfilm, placing the filtrate in brown chromatographic flask, and analyzing with high performance liquid chromatography. The results are shown in FIGS. 3 and 4. The result shows that the content of various pigments in the leaves of the plant edited by the Ntab0036220 gene is reduced by times compared with that of a control (untransformed) plant, and the chlorophyll a and the carotenoid are reduced by about 10 times.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, and such changes and modifications are within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

<110> Limited liability company for tobacco industry in Yunnan province

<120> a gene related to tobacco pigment synthesis and application thereof

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2202

<212> DNA

<213> Artificial sequence ()

<400> 1

atggttagct attttccatg gccaactatt cagagccctt tagatactta tatagttcaa 60

gttgaatcac aagaaagcca aatttccact caatctttat cagatcagga tcttgagagc 120

tggtaccgat cttttttgcc aaatacgata gcaatcacac gctcaaatga cgaagaagga 180

ccacgtttag tgtattcata tcgcaacgtg atgaaaggct ttgcagcaag attatcagca 240

gagcatgtta aagaaatgga gaagaaacca ggctttgtat ctgcatggcc tgagaggata 300

atgtctttac acactaccca tactccaagt tttcttggat tgcagcagca tatgggctta 360

tggaaagatt caaactatgg aaatggtgtg atcattggag tcttggacac tggcatttcg 420

cctgaccatc cttcatttag cgacgaaggg atgcttcctc cgcctgctaa gtggaagggc 480

aagtgtgaat caaatttcac tacaaagtgt aacaacaagc tcattggtgc gaggtcctac 540

atacaagaag atcgctcgcc aatagatgat gatggacatg gcacccacac agccagcact 600

gctgctggag gttttgtgca aggtgccaat gtatatggga atgctaaagg tactgcagtt 660

ggggttgccc ctcttgcaca cttggctatt tataaggttt gtgactcttc tggttgcgct 720

gacagtgaaa ttttagctgc catggatgca gctattgatg atggtgtcga tatcctgtcc 780

ctttcccttg gtggatttag tagtcccttg catagtgatc ccattgcact cggtgcatat 840

agtgcgacag aaagaggcat tcttgtaagt tgttctgccg gcaatgaagg tccatacaat 900

agctctactt caaatgaagc cccatggatt ctgacagtag gtgcaagcac tctcgacagg 960

aaaattaagg ctactgttaa gcttggaaac aaaaaggaat tcgagggcga atcaactttt 1020

catccaaagt ctcccaatgt aacatttttc cctctatttg atcctgcaaa gaatgcaagt 1080

gattttgaca gcccttattg cggaacaggt acactgactg accctgctat tagaggcaaa 1140

atagttttgt gtatggcagg tggtggttat acaaggattg gaaaaggaca agcagtaaag 1200

gatgctggag gtgttggcat gatcatctac aatgggctag aatatggtgt tactacgtta 1260

gccgatgctc atgtccttcc tgccctggat gttacttatg ctgatggaat gaaaattctt 1320

gactatatga attcaacaga gaaaccagta gctagaatta cgtttcaagg aacgataatc 1380

ggagatagaa atgcaccaat ggttgcttca ttttcttctc gaggaccaag catggctagt 1440

cctggaatct tgaagcctga cattattggt cctggtgtta acattcttgc tgcatggcct 1500

gcttctgtag agaacaacac aaactcaaaa tctaccttca atattatttc tggcacctcc 1560

atgtcttgtc ctcacctcag tggagttgca gcattgctaa aaagcgcaca ccctacttgg 1620

tctcctgcag ctattaaatc agcaatcatg acaactgctg atacagtaaa cctcgccaac 1680

aatcccatct tagacgaaag gctccttcct gctaatatct ttgccgttgg ttcaggacat 1740

gtcaatccat caagagcaaa tgatccggga ctaatttatg acaccccatt cgacgactac 1800

ttaccttatt tgtgtggttt gaattacaca aatcgacagg taggaaacct tctacaacgc 1860

aagatcaatt gctcggaagt gaaaagtatt cctgaagcac aactaaatta tccgtcattt 1920

tccatcacac tgggaacaaa tactcacaca tatactagaa cagtaaccaa tgttggcgat 1980

gctaaatcat cttacaatgt agagatagtt tcaccacgag gagtttccgt gatggttaag 2040

ccctctactc taacattctc cgagttgaac cagaagttga cataccaagt gaccttttcc 2100

aaaacagcca atagctcaaa cagtgatgta gttgaaggat tcttgaaatg gactagtaat 2160

aggcactctg tacgaagtcc aattgcagtt gtgctagtct ag 2202

<210> 2

<211> 733

<212> PRT

<213> Artificial sequence ()

<400> 2

Met Val Ser Tyr Phe Pro Trp Pro Thr Ile Gln Ser Pro Leu Asp Thr

1 5 10 15

Tyr Ile Val Gln Val Glu Ser Gln Glu Ser Gln Ile Ser Thr Gln Ser

20 25 30

Leu Ser Asp Gln Asp Leu Glu Ser Trp Tyr Arg Ser Phe Leu Pro Asn

35 40 45

Thr Ile Ala Ile Thr Arg Ser Asn Asp Glu Glu Gly Pro Arg Leu Val

50 55 60

Tyr Ser Tyr Arg Asn Val Met Lys Gly Phe Ala Ala Arg Leu Ser Ala

65 70 75 80

Glu His Val Lys Glu Met Glu Lys Lys Pro Gly Phe Val Ser Ala Trp

85 90 95

Pro Glu Arg Ile Met Ser Leu His Thr Thr His Thr Pro Ser Phe Leu

100 105 110

Gly Leu Gln Gln His Met Gly Leu Trp Lys Asp Ser Asn Tyr Gly Asn

115 120 125

Gly Val Ile Ile Gly Val Leu Asp Thr Gly Ile Ser Pro Asp His Pro

130 135 140

Ser Phe Ser Asp Glu Gly Met Leu Pro Pro Pro Ala Lys Trp Lys Gly

145 150 155 160

Lys Cys Glu Ser Asn Phe Thr Thr Lys Cys Asn Asn Lys Leu Ile Gly

165 170 175

Ala Arg Ser Tyr Ile Gln Glu Asp Arg Ser Pro Ile Asp Asp Asp Gly

180 185 190

His Gly Thr His Thr Ala Ser Thr Ala Ala Gly Gly Phe Val Gln Gly

195 200 205

Ala Asn Val Tyr Gly Asn Ala Lys Gly Thr Ala Val Gly Val Ala Pro

210 215 220

Leu Ala His Leu Ala Ile Tyr Lys Val Cys Asp Ser Ser Gly Cys Ala

225 230 235 240

Asp Ser Glu Ile Leu Ala Ala Met Asp Ala Ala Ile Asp Asp Gly Val

245 250 255

Asp Ile Leu Ser Leu Ser Leu Gly Gly Phe Ser Ser Pro Leu His Ser

260 265 270

Asp Pro Ile Ala Leu Gly Ala Tyr Ser Ala Thr Glu Arg Gly Ile Leu

275 280 285

Val Ser Cys Ser Ala Gly Asn Glu Gly Pro Tyr Asn Ser Ser Thr Ser

290 295 300

Asn Glu Ala Pro Trp Ile Leu Thr Val Gly Ala Ser Thr Leu Asp Arg

305 310 315 320

Lys Ile Lys Ala Thr Val Lys Leu Gly Asn Lys Lys Glu Phe Glu Gly

325 330 335

Glu Ser Thr Phe His Pro Lys Ser Pro Asn Val Thr Phe Phe Pro Leu

340 345 350

Phe Asp Pro Ala Lys Asn Ala Ser Asp Phe Asp Ser Pro Tyr Cys Gly

355 360 365

Thr Gly Thr Leu Thr Asp Pro Ala Ile Arg Gly Lys Ile Val Leu Cys

370 375 380

Met Ala Gly Gly Gly Tyr Thr Arg Ile Gly Lys Gly Gln Ala Val Lys

385 390 395 400

Asp Ala Gly Gly Val Gly Met Ile Ile Tyr Asn Gly Leu Glu Tyr Gly

405 410 415

Val Thr Thr Leu Ala Asp Ala His Val Leu Pro Ala Leu Asp Val Thr

420 425 430

Tyr Ala Asp Gly Met Lys Ile Leu Asp Tyr Met Asn Ser Thr Glu Lys

435 440 445

Pro Val Ala Arg Ile Thr Phe Gln Gly Thr Ile Ile Gly Asp Arg Asn

450 455 460

Ala Pro Met Val Ala Ser Phe Ser Ser Arg Gly Pro Ser Met Ala Ser

465 470 475 480

Pro Gly Ile Leu Lys Pro Asp Ile Ile Gly Pro Gly Val Asn Ile Leu

485 490 495

Ala Ala Trp Pro Ala Ser Val Glu Asn Asn Thr Asn Ser Lys Ser Thr

500 505 510

Phe Asn Ile Ile Ser Gly Thr Ser Met Ser Cys Pro His Leu Ser Gly

515 520 525

Val Ala Ala Leu Leu Lys Ser Ala His Pro Thr Trp Ser Pro Ala Ala

530 535 540

Ile Lys Ser Ala Ile Met Thr Thr Ala Asp Thr Val Asn Leu Ala Asn

545 550 555 560

Asn Pro Ile Leu Asp Glu Arg Leu Leu Pro Ala Asn Ile Phe Ala Val

565 570 575

Gly Ser Gly His Val Asn Pro Ser Arg Ala Asn Asp Pro Gly Leu Ile

580 585 590

Tyr Asp Thr Pro Phe Asp Asp Tyr Leu Pro Tyr Leu Cys Gly Leu Asn

595 600 605

Tyr Thr Asn Arg Gln Val Gly Asn Leu Leu Gln Arg Lys Ile Asn Cys

610 615 620

Ser Glu Val Lys Ser Ile Pro Glu Ala Gln Leu Asn Tyr Pro Ser Phe

625 630 635 640

Ser Ile Thr Leu Gly Thr Asn Thr His Thr Tyr Thr Arg Thr Val Thr

645 650 655

Asn Val Gly Asp Ala Lys Ser Ser Tyr Asn Val Glu Ile Val Ser Pro

660 665 670

Arg Gly Val Ser Val Met Val Lys Pro Ser Thr Leu Thr Phe Ser Glu

675 680 685

Leu Asn Gln Lys Leu Thr Tyr Gln Val Thr Phe Ser Lys Thr Ala Asn

690 695 700

Ser Ser Asn Ser Asp Val Val Glu Gly Phe Leu Lys Trp Thr Ser Asn

705 710 715 720

Arg His Ser Val Arg Ser Pro Ile Ala Val Val Leu Val

725 730

<210> 3

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 3

attgcagcca tgaaattctt ga 22

<210> 4

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 4

gaaagagaaa gcaaatagac at 22

<210> 5

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 5

gatttcttgg attgcagcag cata 24

<210> 6

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 6

caaatatgct gctgcaatcc aaga 24

<210> 7

<211> 19

<212> DNA

<213> Artificial sequence ()

<400> 7

ttaggtttac ccgccaata 19

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 8

tcttggattg cagcagcata 20

<210> 9

<211> 12133

<212> DNA

<213> Artificial sequence ()

<400> 9

ggcgccccgc ggaaagctta gctttcgttt tcttcttttt aactttccat tcggagtttt 60

tgtatcttgt ttcatagttt gtcccaggat tagaatgatt aggcatcgaa ccttcaagaa 120

tttgattgaa taaaacatct tcattcttaa gatatgaaga taatcttcaa aaggcccctg 180

ggaatctgaa agaagagaag caggcccatt tatatgggaa agaacaatag tatttcttat 240

ataggcccat ttaagttgaa aacaatcttc aaaagtccca catcgcttag ataagaaaac 300

gaagctgagt ttatatacag ctagagtcga agtagtgatt gtcttggatt gcagcagcat 360

agttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag 420

tggcaccgag tcggtgcttt ttttgcaaaa ttttccagat cgatttcttc ttcctctgtt 480

cttcggcgtt caatttctgg gtttttctct tcgttttctg gctagcgttt aaacttaagc 540

tgatccacta gtcctgcagg tcaacatggt ggagcacgac acacttgtct actccaaaaa 600

tatcaaagat acagtctcag aagaccaaag ggcaattgag acttttcaac aaagggtaat 660

atccggaaac ctcctcggat tccattgccc agctatctgt cactttattg tgaagatagt 720

ggaaaaggaa ggtggctcct acaaatgcca tcattgcgat aaaggaaagg ccatcgttga 780

agatgcctct gccgacagtg gtcccaaaga tggaccccca cccacgagga gcatcgtgga 840

aaaagaagac gttccaacca cgtcttcaaa gcaagtggat tgatgtgata acatggtgga 900

gcacgacaca cttgtctact ccaaaaatat caaagataca gtctcagaag accaaagggc 960

aattgagact tttcaacaaa gggtaatatc cggaaacctc ctcggattcc attgcccagc 1020

tatctgtcac tttattgtga agatagtgga aaaggaaggt ggctcctaca aatgccatca 1080

ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc gacagtggtc ccaaagatgg 1140

acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca 1200

agtggattga tgtgatatct ccactgacgt aagggatgac gcacaatccc actatccttc 1260

gcaagaccct tcctctatat aaggaagttc atttcatttg gagaggacct cgacctcaac 1320

acaacatata caaaacaaac gaatctcaag caatcaagca ttctacttct attgcagcaa 1380

tttaaatcat ttcttttaaa gcaaaagcaa ttttctgaaa attttcacca tttacgaacg 1440

ataccatggc cccaaagaaa aagagaaagg ttgattacaa agaccacgac ggagactaca 1500

aagaccacga cattgattat aaagatgatg atgataaagg aacgatggac aaaaagtata 1560

gcatcggtct ggatattgga actaactccg tcggctgggc tgtaatcacc gacgaataca 1620

aggtcccgtc aaaaaagttc aaggtattgg gtaacacaga tcgtcactct atcaaaaaga 1680

atctcattgg agctctgttg ttcgacagcg gcgaaacagc tgaggccact agactgaagc 1740

gcaccgccag acgccgttac acgaggagaa agaacagaat ctgctacttg caagaaatat 1800

tctcaaacga gatggccaaa gtggacgatt cgttctttca taggttagaa gagagtttcc 1860

ttgttgaaga ggataaaaag cacgaaagac atccgatatt tggaaacatc gtggacgaag 1920

ttgcttatca cgagaagtac cccacgatct atcatctgcg taaaaagttg gtggactcga 1980

cagataaggc cgacctcagg ttaatatacc ttgcactggc gcacatgatc aaattcagag 2040

gccattttct gattgaaggt gacctgaacc ctgacaatag tgatgtggac aaactcttca 2100

ttcaattagt tcagacctac aatcaactgt ttgaagagaa ccctatcaac gcttcaggag 2160

ttgacgctaa ggccatcctt agtgcgagac tgagcaaatc ccgccgtctc gaaaacttaa 2220

tcgcacagtt gcctggagag aaaaagaacg gtttgttcgg aaatctcatt gcgttgtcac 2280

tcggactcac gccaaacttc aagtctaact tcgatttggc agaagacgcg aaactgcaac 2340

tgagcaaaga cacatatgac gatgacctcg ataacctctt agctcagatc ggcgatcaat 2400

acgccgactt gttcctcgct gccaaaaatc tgtcggacgc tatacttctg agtgatatct 2460

tgcgcgtcaa cacagaaatt actaaggctc ctctgtcggc cagtatgata aaacgctatg 2520

acgaacacca tcaggatttg acattgctca aagccctcgt gcgtcaacag ctcccagaaa 2580

agtacaagga gattttcttt gatcagtcca agaatggcta cgcaggttat atagacggtg 2640

gagcgtcgca agaagagttc tacaagttca tcaagccaat attagaaaag atggacggca 2700

cggaagagtt acttgttaag ctgaatcgtg aggacctgtt gcgtaaacag aggacattcg 2760

ataacggatc aattccgcac caaatacatc ttggcgaact gcacgctatc ctcaggagac 2820

aagaggactt ctaccccttt ttaaaggata accgtgaaaa gatcgagaaa atcctgactt 2880

tcaggattcc ttactatgtc ggcccactgg ctcgtggtaa tagcaggttt gcctggatga 2940

ccaggaagtc cgaagagaca attactccgt ggaacttcga agaggtggtt gataaaggag 3000

catcagcgca gtctttcata gaacgcatga caaattttga caagaactta ccgaatgaga 3060

aggtccttcc caaacactca ctcctctacg aatacttcac agtatacaac gagctcacta 3120

aagtcaagta cgtaaccgag ggtatgcgca aacccgcttt cctgtctgga gagcagaaaa 3180

aggccatcgt ggaccttctg ttcaagacaa accgtaaggt cactgtaaag caactcaagg 3240

aagactactt caaaaagata gagtgtttcg attcagtgga aatctctggc gttgaggaca 3300

gatttaacgc ttccttgggt acttaccacg atttgctcaa gatcattaaa gataaggact 3360

tcctcgacaa cgaagagaac gaagatatct tagaggacat agttctcacc cttacgctgt 3420

ttgaagatag agagatgatt gaagagcgcc tgaagactta tgctcatttg ttcgatgaca 3480

aagtcatgaa gcaactgaaa cgccgtaggt acaccggctg gggtagatta tcgcgcaaac 3540

ttattaatgg tataagggac aagcagtcgg gaaaaacgat attggacttt ctcaagagtg 3600

atggtttcgc caacagaaat tttatgcaac tcatacacga tgacagctta acattcaagg 3660

aagatatcca aaaagcacag gtgtcgggac agggcgacag tttgcacgaa catattgcta 3720

acctcgccgg ctccccggcg ataaaaaagg gtatccttca gactgtgaaa gtcgtagatg 3780

aactggtgaa ggttatgggt cgtcataaac ccgagaacat agttatcgaa atggctaggg 3840

agaatcaaac aactcagaag ggacagaaaa actcaagaga acgcatgaag cgcattgaag 3900

agggtatcaa agagcttggc agtcaaatcc tgaaggaaca ccctgtcgag aacacgcaac 3960

ttcagaacga aaaattgtac ctctactatc tgcagaatgg tagagatatg tacgtagacc 4020

aagaattgga tattaaccgc ctctcagatt acgacgtgga tcatatagtt ccgcagtcat 4080

tcttgaagga tgactctatc gacaacaaag tcctcacaag atcagacaag aaccgcggaa 4140

aatcagataa tgtaccctct gaagaggtgg ttaaaaagat gaaaaactac tggagacagt 4200

tacttaacgc taagttgatc acgcaaagaa agttcgataa cctcacaaag gctgaacgcg 4260

gcggtttaag cgagcttgac aaggccggtt tcataaaacg tcagttagtc gaaaccaggc 4320

aaattacgaa acacgtagcc caaatattgg attcccgcat gaacactaaa tacgatgaaa 4380

atgacaagct catccgtgag gtcaaagtaa ttaccctgaa aagcaagttg gtgtccgact 4440

tcagaaagga tttccagttc tacaaagttc gcgaaatcaa caactaccac catgcacatg 4500

acgcttacct gaacgcagtc gtaggcactg cgttaattaa aaagtaccct aaactggaat 4560

ctgagttcgt gtacggtgac tataaagtgt acgatgttag aaagatgatc gctaaaagcg 4620

aacaggagat tggaaaggct accgccaagt atttctttta ctccaacatc atgaatttct 4680

ttaagaccga aatcacgtta gcaaatggcg agatacgtaa aaggccactt atcgaaacaa 4740

acggagaaac tggcgagata gtgtgggaca agggtagaga ttttgccact gtccgcaaag 4800

tactgtcgat gccgcaagtg aatatcgtta aaaagaccga agttcaaacg ggaggcttca 4860

gcaaagagtc catcctgccc aagcgtaaca gtgataaatt gatagctagg aaaaaggact 4920

gggatcctaa aaagtatggt ggattcgaca gcccaactgt cgcatactcc gtattggtgg 4980

ttgcgaaagt cgaaaaagga aagagcaaaa agctcaagtc cgtaaaagag ctgttgggca 5040

ttaccataat ggaaagatca tctttcgaga agaatcctat cgattttctg gaagccaagg 5100

gatataaaga ggtcaaaaag gacctcataa tcaagttacc aaaatacagt ctgttcgaat 5160

tggagaacgg cagaaaacgc atgcttgcat cagcgggtga actgcaaaag ggaaatgagt 5220

tagcacttcc ttctaaatac gtcaacttcc tgtatttggc gtcacactac gaaaaactga 5280

agggctctcc agaagataac gagcaaaagc agttatttgt ggaacagcac aaacattacc 5340

ttgacgaaat tatagagcaa atctcggagt tcagtaagag agtgattttg gctgacgcca 5400

atcttgataa agttctgtct gcttacaaca agcaccgtga taaaccgatt agggaacagg 5460

ccgagaacat catacatctc ttcacactca ctaaccttgg tgcacccgca gcgttcaaat 5520

attttgacac cacgatagat cgtaagaggt acaccagcac gaaagaagtt ttggacgcga 5580

cactcatcca tcaatcaatc acgggcctgt acgaaaccag aatcgacctg tcccagctcg 5640

gtggcgaccc caagaagaag agaaaggtgt agcggccgca tcgatactgc aggagctcgg 5700

taccttttac tagtgatatc cctgtgtgaa attgttatcc gctacgcgtg atcgttcaaa 5760

catttggcaa taaagtttct taagattgaa tcctgttgcc ggtcttgcga tgattatcat 5820

ataatttctg ttgaattacg ttaagcatgt aataattaac atgtaatgca tgacgttatt 5880

tatgagatgg gtttttatga ttagagtccc gcaattatac atttaatacg cgatagaaaa 5940

caaaatatag cgcgcaaact aggataaatt atcgcgcgcg gtgtcatcta tgttactaga 6000

tcccatggga agttcctatt ccgaagttcc tattctctga aaagtatagg aacttcagcg 6060

atcgcagacg tcgggatctt ctgcaagcat ctctatttcc tgaaggtcta acctcgaaga 6120

tttaagattt aattacgttt ataattacaa aattgattct agtatcttta atttaatgct 6180

tatacattat taattaattt agtactttca atttgttttc agaaattatt ttactatttt 6240

ttataaaata aaagggagaa aatggctatt taaatactag cctattttat ttcaatttta 6300

gcttaaaatc agccccaatt agccccaatt tcaaattcaa atggtccagc ccaattccta 6360

aataacccac ccctaacccg cccggtttcc ccttttgatc catgcagtca acgcccagaa 6420

tttccctata taatttttta attcccaaac acccctaact ctatcccatt tctcaccaac 6480

cgccacatag atctatcctc ttatctctca aactctctcg aaccttcccc taaccctagc 6540

agcctctcat catcctcacc tcaaaaccca ccggggccgg ccatgattga acaagatgga 6600

ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa 6660

cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gaggccggtt 6720

ctttttgtca agaccgacct gtccggtgcc ctgaatgaac ttcaagacga ggcagcgcgg 6780

ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa 6840

gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac 6900

cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt 6960

gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact 7020

cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg 7080

ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg 7140

actcatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc 7200

atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt 7260

gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc 7320

gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgaggc 7380

gcgccgatcg ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc 7440

ttgcgatgat tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt 7500

aatgcatgac gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt 7560

aatacgcgat agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt 7620

catctatgtt actagatccc tagggaagtt cctattccga agttcctatt ctctgaaaag 7680

tataggaact tctttgcgta ttgggcgctc ttggcctttt tggccaccgg tcgtacggtt 7740

aaaaccaccc cagtacatta aaaacgtccg caatgtgtta ttaagttgtc taagcgtcaa 7800

tttgtttaca ccacaatata tcctgccacc agccagccaa cagctccccg accggcagct 7860

cggcacaaaa tcaccactcg atacaggcag cccatcagtc cactagagac gctcaccggg 7920

ctggttgccc tcgccgctgg gctggcggcc gtctatggcc ctgcaaacgc gccagaaacg 7980

ccgtcgaagc cgtgtgcgag acaccgcagc cgccggcgtt gtggatacct cgcggaaaac 8040

ttggccctca ctgacagatg aggggcggac gttgacactt gaggggccga ctcacccggc 8100

gcggcgttga cagatgaggg gcaggctcga tttcggccgg cgacgtggag ctggccagcc 8160

tcgcaaatcg gcgaaaacgc ctgattttac gcgagtttcc cacagatgat gtggacaagc 8220

ctggggataa gtgccctgcg gtattgacac ttgaggggcg cgactactga cagatgaggg 8280

gcgcgatcct tgacacttga ggggcagagt gctgacagat gaggggcgca cctattgaca 8340

tttgaggggc tgtccacagg cagaaaatcc agcatttgca agggtttccg cccgtttttc 8400

ggccaccgct aacctgtctt ttaacctgct tttaaaccaa tatttataaa ccttgttttt 8460

aaccagggct gcgccctgtg cgcgtgaccg cgcacgccga aggggggtgc ccccccttct 8520

cgaaccctcc cggcccgctc tcgcgttggc agcatcaccc ataattgtgg tttcaaaatc 8580

ggctccgtcg atactatgtt atacgccaac tttgaaaaca actttgaaaa agctgttttc 8640

tggtatttaa agttttagaa tgcaaggaac agtgaattgg agttcgtctt gttataatta 8700

gcttcttggg gtatctttaa atactgtaga aaagaggaag gaaataataa atggctaaaa 8760

tgagaatatc accggaattg aaaaaactga tcgaaaaata ccgctgcgta aaagatacgg 8820

aaggaatgtc tcctgctaag gtatataagc tggtgggaga aaatgaaaac ctatatttaa 8880

aaatgacgga cagccggtat aaagggacca cctatgatgt ggaacgggaa aaggacatga 8940

tgctatggct ggaaggaaag ctgcctgttc caaaggtcct gcaccttgaa cggcatgatg 9000

gctggagcaa tctgctcatg agtgaggccg atggcgtcct ttgctcggaa gagtatgaag 9060

atgaacaaag ccctgaaaag attatcgagc tgtatgcgga gtgcatcagg ctctttcact 9120

ccatcgacat atcggattgt ccctatacga atagcttaga cagccgctta gccgaattgg 9180

attacttact gaataacgat ctggccgatg tggattgcga aaactgggaa gaagacaccc 9240

catttaaaga tccgcgcgag ctgtatgatt ttttaaagac ggaaaagccc gaagaggaac 9300

ttgtcttttc ccacggcgac ctgggagaca gcaacatctt tgtgaaagat ggcaaagtaa 9360

gtggctttat tgatcttggg agaagcggca gggcggacaa gtggtatgac attgccttct 9420

gcgtccggtc gatcagggag gatattgggg aagaacagta tgtcgagcta ttttttgact 9480

tactggggat caagcctgat tgggagaaaa taaaatatta tattttactg gatgaattgt 9540

tttagtacct agatgtggcg caacgatgct ggcgacaagc aggagcgcac cgacttcttc 9600

cgcatcaagt gttttggctc tcaggccgag gcccacggca agtatttggg caaggggtcg 9660

ctggtattcg tgcagggcaa gattcggaat accaagtacg agaaggacgg ccagacggtc 9720

tacgggaccg acttcattgc cgataaggtg gattatctgg acaccaaggc accaggcgga 9780

tcaaatcagg aataagggca cattgccccg gcgtgagtcg gggcaatccc gcaaggaggg 9840

tgaatgaatc ggacgtttga ccggaaggca tacaggcaag aactgatcga cgcggggttt 9900

tccgccgagg atgccgaaac catcgcaagc cgcaccgtca tgcgtgcgcc ccgcgaaacc 9960

ttccagtccg tcggctcgat ggcccagcaa gctacggcca agatcgagcg cgacagcgtg 10020

caactggctc cccctgccct gcccgcgcca tcggccgccg tggagcgttc gcgtcgtctc 10080

gaacaggagg cggcaggttt ggcgaagtcg atgaccatcg acacgcgagg aactatgacg 10140

accaagaagc gaaaaaccgc cggcgaggac ctggcaaaac aggtcagcga ggccaagcaa 10200

gccgcgttgc tgaaacacac gaagcagcag atcaaggaaa tgcagctttc cttgttcgat 10260

attgcgccgt ggccggacac gatgcgagcg atgccaaacg acacggcccg ctctgccctg 10320

ttcaccacgc gcaacaagaa aatcccgcgc gaggcgctgc aaaacaaggt cattttccac 10380

gtcaacaagg acgtgaagat cacctacacc ggcgtcgagc tgcgggccga cgatgacgaa 10440

ctggtgtggc agcaggtgtt ggagtacgcg aagcgcaccc ctatcggcga gccgatcacc 10500

ttcacgttct acgagctttg ccaggacctg ggctggtcga tcaatggccg gtattacacg 10560

aaggccgagg aatgcctgtc gcgcctacag gcgacggcga tgggcttcac gtccgaccgc 10620

gttgggcacc tggaatcggt gtcgctgctg caccgcttcc gcgtcctgga ccgtggcaag 10680

aaaacgtccc gttgccaggt cctgatcgac gaggaaatcg tcgtgctgtt tgctggcgac 10740

cactacacga aattcatatg ggagaagtac cgcaagctgt cgccgacggc ccgacggatg 10800

ttcgactatt tcagctcgca ccgggagccg tacccgctca agctggaaac cttccgcctc 10860

atgtgcggat cggattccac ccgcgtgaag aagtggcgcg agcaggtcgg cgaagcctgc 10920

gaagagttgc gaggcagcgg cctggtggaa cacgcctggg tcaatgatga cctggtgcat 10980

tgcaaacgct agggccttgt ggggtcagtt ccggctgggg gttcagcagc cagcgcttta 11040

ctgagatcct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 11100

cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 11160

gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 11220

ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 11280

agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 11340

tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 11400

ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 11460

gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 11520

ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 11580

gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 11640

aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 11700

aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 11760

ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 11820

gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 11880

gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt ggatctcctg 11940

tggttggcat gcacatacaa atggacgaac ggataaacct tttcacgccc ttttaaatat 12000

ccgattattc taataaacgc tcttttctct taggtttacc cgccaatata tcctgtcaaa 12060

cactgatagt ttaaactgaa ggcgggaaac gacaatctgc tagtggatct cccagtcacg 12120

acgttgtaaa acg 12133

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 10

gatagcaatc acacgctcaa at 22

<210> 11

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 11

gataatcttg ctgcaaagcc tt 22

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 12

cccctcacca cagagtctgc 20

<210> 13

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 13

aagggtgttg ttgtcctcaa tctt 24

Claims

1. A gene related to tobacco pigment synthesis is characterized in that a nucleotide sequence is shown as SEQ ID No. 1.

2. A protein encoded by the tobacco pigment synthesis-associated gene according to claim 1.

3. Use of the tobacco pigment synthesis-associated gene of claim 1 for regulating the pigment content in tobacco leaves.

4. Use of a protein encoded by a tobacco pigment synthesis-associated gene according to claim 1 for regulating the pigment content in tobacco leaves.

5. The gene editing vector for a gene involved in tobacco pigment synthesis according to claim 1, characterized in that: the constitutive promoter cauliflower mosaic virus CaMV35S P35 was selected to drive the transcription of Cas9 gene, and the type III RNA polymerase promoter of Arabidopsis U6-26 gene drives the transcription of sgRNA.

6. The method of constructing a gene editing vector for a gene related to tobacco pigment synthesis according to claim 5, wherein: connecting the sequence shown in SEQ ID No.8 into a CRISPR/Cas9 vector to obtain a gene editing vector CRISPR/Cas9-Ntab 0036220; the sequence of the gene editing vector CRISPR/Cas9-Ntab0036220 is shown in SEQ ID NO. 9.

7. Use of a gene-editing vector for a tobacco pigment synthesis-associated gene as defined in claim 5 or 6 for regulating the pigment content in tobacco leaves.

8. A method of reducing the pigment content of tobacco lamina comprising: knocking out the gene of claim 1 or causing a defect in the function of the protein of claim 2 in a tobacco plant.

9. The method of reducing the pigment content in tobacco leaves according to claim 8, wherein the gene of claim 1 is knocked out in a tobacco plant using the gene editing vector of claim 5 or 6.