CN115029378B - Method for creating flower-spot ornamental poplar by PtrDJ1C gene - Google Patents

Abstract

The application discloses a method for creating a flower-spotted ornamental poplar by using PtrDJ1C genes. The present application provides the use of a substance that reduces the activity or content of a protein PtrDJ1C or a substance that inhibits the expression of a nucleic acid molecule encoding said protein PtrDJ1C in any of the following: changing the green leaves of poplar into the flower spot leaves; cultivating poplar with flower spot leaves; inhibiting the development of poplar leaf chloroplasts; inhibit development of chloroplast thylakoid membrane of poplar leaf. The application has important significance in the aspects of directional breeding of the ornamental character of the flower spots of the gardening tree species such as poplar, improving the breeding efficiency and the like, and has extremely high commercial value and scientific research value.

Description

Method for creating flower-spot ornamental poplar by PtrDJ1C gene

Technical Field

The application belongs to the technical field of biology, and particularly relates to a method for creating a flower-spot ornamental poplar by using PtrDJ1C genes.

Background

The ornamental value of the gardening landscape tree is mainly reflected in the aspects of tree shape, color and the like. Poplar is used as a most common ornamental tree species, has the advantages of high and straight property, beautiful tree shape, developed root system, wind prevention, sand fixation and the like, and is widely used in landscaping, street trees and other scenes. However, the method is sometimes limited by greening space, and poplar trees are used as landscape plants and need to be frequently trimmed to adapt to the overall landscape layout and space requirements; in addition, poplar color is also more single. Therefore, how to seek a method for improving the ornamental value of poplar gardening from both tree shape and color becomes a key problem to be solved urgently.

Leaf spot plants have green and non-green (yellow or white) portions on the same leaf, which can lead to slow plant growth due to reduced overall photosynthesis while providing different leaf-color ornamental traits. Plaque is formed because the green tissue contains normal chloroplasts, while tissue in the yellow or white region contains dysplastic chloroplasts. The method solves the problem that poplar tree forms and color expression need to be changed, and is an important research direction for cultivating ornamental poplar trees in the future.

The plaque mutant in the nature is only used for cultivation and planting of colorful flowers and green plants in a small amount at present, and the mechanism of generating the plaque is not clear yet. Therefore, the molecular design breeding and gene editing technology are combined, so that the creation of the plaque mutant is carried out on gardening ornamental trees such as poplar, and the method has extremely important significance for the enrichment of gardening species resources and the yield increase and income creation of under-forest economy.

The genetic transformation technology of woody plants including poplar trees is difficult, so that transgenic plants are difficult to obtain, and meanwhile, the problems of long period, low transformation rate and the like exist, so that the research of obtaining target poplar trees by a transgenic means is relatively slow.

Disclosure of Invention

It is an object of the present application to provide the use of a substance that reduces the activity or content of a protein PtrDJ1C or a substance that inhibits the expression of a nucleic acid molecule encoding said protein PtrDJ 1C.

The application provides the application of a substance for reducing the activity or content of protein PtrDJ1C or a substance for inhibiting the expression of nucleic acid molecules encoding the protein PtrDJ1C in any one of the following A-F;

A. changing the color of poplar leaves;

B. changing the green leaves of poplar into the flower spot leaves;

C. cultivating poplar with flower spot leaves;

D. inhibiting the development of poplar leaf chloroplasts;

E. inhibiting development of a chloroplast thylakoid membrane of the poplar leaf;

F. reducing the expression quantity of proteins related to chloroplast development or photosynthesis in poplar leaves;

the expression amounts of the proteins D1, psaA, cytf, CF1 α, rbcL and ATPase related to chloroplast development or photosynthesis were verified in the examples of the present application as described above.

The protein PtrDJ1C is as follows (1) or (2) or (3):

(1) A protein consisting of an amino acid sequence shown as a sequence 1 in a sequence table;

(2) A protein formed by adding a tag sequence at the tail end of an amino acid sequence shown in a sequence 1 in a sequence table;

(3) And (3) the protein which is derived from the (1) or (2) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table.

In the application, the substance is a CRISPR/Cas9 system;

the CRISPR/Cas9 system comprises 1) or 2) as follows:

1) The target point of the sgRNA is 146 th to 165 th positions of a sequence 4;

2) A CRISPR/Cas9 vector expressing the sgRNA;

the above system further comprises Cas9 protein or mRNA thereof or a nucleic acid molecule encoding Cas9 protein or a plasmid expressing Cas9 protein.

In an embodiment of the present application, the carrier may specifically be: the linearized sgRNA expression cassette and the pYLCRISPR/Cas9Pubi-H plasmid were ligated using the Golden Gate method (restriction enzyme Bsa I was used) to obtain the recombinant plasmid. The linearized sgRNA expression cassette is shown as a sequence 4 in a sequence table. The plasmid contains a target sequence (146 th to 165 th positions of a sequence 4), and the expressed specific sgRNA is shown as a sequence 5 in a sequence table.

The Cas9 protein is a protein coded by Cas9 genes.

The Cas9 gene is specifically shown as 724 th-4926 th nucleotide in sequence 7 of a sequence table.

It is another object of the present application to provide a method of growing poplar with non-green leaves.

The method provided by the application is as follows 1), 2) or 3):

1) The method comprises the following steps: firstly, reducing the activity or content of protein PtrDJ1C in poplar with green leaf phenotype to obtain transgenic plants, and selecting the plants with non-green leaf phenotype, namely the poplar plants with non-green leaf phenotype;

2) The method comprises the following steps: firstly, inhibiting the expression of nucleic acid molecules of coding protein PtrDJ1C in poplar with green leaf phenotype to obtain transgenic plants, and selecting plants with non-green leaf phenotype, namely poplar plants with non-green leaf phenotype;

3) The method comprises the following steps: performing gene editing on nucleic acid molecules for encoding the protein PtrDJ1C in poplar with green leaf phenotype to terminate protein translation in advance to obtain a gene editing plant, and selecting a plant with a non-green leaf phenotype, namely a poplar plant with a non-green leaf;

the protein PtrDJ1C is as follows (1) or (2) or (3):

(1) A protein consisting of an amino acid sequence shown as a sequence 1 in a sequence table;

(2) A protein formed by adding a tag sequence at the tail end of an amino acid sequence shown in a sequence 1 in a sequence table;

(3) And (3) the protein which is derived from the (1) or (2) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table.

Plants with leaf phenotypes selected as non-green as described above: plants with leaf phenotypes other than green can be selected by naked eye identification; sequencing can be further carried out, and a plant edited by the homozygous gene is selected as a plant with a leaf phenotype of non-green.

The homozygous gene-edited plants were all subjected to gene editing for PtrDJ1C genes on 2 homologous chromosomes.

In the above method, the gene editing is achieved by: introducing a CRISPR/Cas9 system into the plant of interest;

the CRISPR/Cas9 system comprises 1) or 2) as follows:

1) The target point of the sgRNA is 146 th to 165 th positions of a sequence 4;

2) CRISPR/Cas9 vectors expressing the sgrnas.

The above CRISPR/Cas9 system further comprises Cas9 protein or mRNA thereof or a nucleic acid molecule encoding Cas9 protein or a plasmid expressing Cas9 protein.

It is a further object of the present application to provide a material that alters the color of poplar leaves.

The present application provides a substance that reduces the activity or content of a protein PtrDJ1C or inhibits the expression of a nucleic acid molecule encoding the protein PtrDJ 1C.

The substance is a CRISPR/Cas9 system;

the CRISPR/Cas9 system comprises 1) or 2) as follows:

1) The target point of the sgRNA is 146 th to 165 th positions of a sequence 4;

2) CRISPR/Cas9 vectors expressing the sgrnas.

In the above, the color of the poplar leaves is changed into a green leaf of the poplar; the non-green (non-pure green) leaves are in particular white leaves or flower-spot leaves.

The application of the PtrDJ1C protein or the coding gene thereof in regulating and controlling the flower spot phenotype of poplar leaves is also the protection scope of the application;

the protein PtrDJ1C is as follows (1) or (2) or (3):

(1) A protein consisting of an amino acid sequence shown as a sequence 1 in a sequence table;

(2) A protein formed by adding a tag sequence at the tail end of an amino acid sequence shown in a sequence 1 in a sequence table;

(3) And (3) the protein which is derived from the (1) or (2) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table.

The application of the PtrDJ1C protein or the coding gene thereof in regulation and control of the development of poplar chloroplasts is also the protection scope of the application;

the protein PtrDJ1C is as follows (1) or (2) or (3):

(1) A protein consisting of an amino acid sequence shown as a sequence 1 in a sequence table;

(2) A protein formed by adding a tag sequence at the tail end of an amino acid sequence shown in a sequence 1 in a sequence table;

(3) And (3) the protein which is derived from the (1) or (2) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table.

Any of the above poplar is dicotyledonous. The dicotyledonous poplar is of the family Salicaceae. The poplar of the salicaceae is poplar of the genus populus. The populus can be populus pie populus, such as 84K populus.

The plant having a mosaic phenotype as described above may be a leaf plant having a mosaic phenotype.

Any of the above poplar with a mosaic phenotype may be a leaf poplar with a mosaic phenotype.

According to the application, the PtrDJ1C gene is subjected to gene editing by a CRISPR/Cas9 gene editing technology, the PtrDJ1C homozygous mutant shows an obvious white-green plaque phenotype, and chloroplast development at a whitened tissue is affected, but the life cycle of the whole plant is not affected. PtrDJ1C protein is positioned in chloroplast, plays an important role in plastid gene transcription and plastid ribosomal RNA (rRNA) shearing processes, and PtrDJ1C deletion causes chloroplast development to be affected, so that chlorophyll-deleted albino spots are generated.

In actual production, the application can obtain the flower-spotted phenotype poplar, and provides theoretical and technical support for landscaping and colorful flower culture. The application has very important theoretical and practical significance on the aspects of directionally, rapidly and efficiently cultivating the plaque ornamental poplar, improving the breeding efficiency and the like, and has extremely high commercial value and scientific research value.

Drawings

FIG. 1 shows the sequencing results of the target region.

FIG. 2 is an exemplary photograph of wild-type plants and plants of the ptrdj1c mutant strain.

FIG. 3 is a photograph showing the result of reverse transcription PCR on wild-type plants and plants of the ptrdj1c mutant line.

FIG. 4 is a photograph of chloroplast ultrastructures of wild type plants and ptrdj1c mutant lines.

FIG. 5 is a photograph of the accumulation of photosynthetic key proteins from wild type plants and mutant strains of ptrdj1 c.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

AtU3d-sgRNA plasmid: wuhan vast, biological technology Co., ltd; atU3d-sgRNA plasmid is shown as sequence 6 in the sequence table. pYLCRISPR/Cas9Pubi-H plasmid: wuhan vast, biological technology Co., ltd; the pYLCRISPR/Cas9Pubi-H plasmid is shown as a sequence 7 of a sequence table.

The inventor discovers a novel protein for regulating and controlling the mosaic phenotype from 84K poplar in the research process, and the protein is named PtrDJ1C protein, and the amino acid sequence of the protein is shown as a sequence 1 of a sequence table. The gene for coding PtrDJ1C protein is named PtrDJ1C gene, and the nucleotide sequence of the cDNA of the gene is shown as the sequence 2 of the sequence table. The genome sequence is shown as a sequence 3 in a sequence table.

Example 1 obtaining Gene-editing lines

1. Construction of CRISPR/Cas9 Gene editing vector

1. Primers for preparing sgRNA are designed and synthesized according to the sequence of PtrDJ1C gene

Synthesizing single-stranded DNA molecules guide-F and single-stranded DNA molecules guide-R respectively, and then mixing and annealing to obtain double-stranded DNA molecules with sticky ends at two ends.

guide-F：5’-gtcaGGAGGAATGCCAGGCTCCGCA-3’；

guide-R：5’-aaacTGCGGAGCCTGGCATTCCTCC-3’。

2. Taking AtU d-sgRNA plasmid, adopting restriction enzyme Bsa I to carry out single enzyme digestion, and recovering enzyme digestion products.

3. And (2) connecting the double-stranded DNA molecule with the sticky end obtained in the step (1) with the enzyme digestion product obtained in the step (2) to obtain a connecting product.

4. And (3) taking the connection product obtained in the step (3), adopting a primer pair consisting of UF and guide-R to carry out PCR amplification, and recovering the amplification product.

5. And (3) taking the connection product obtained in the step (3), adopting a primer pair consisting of gR-R and guide-F to carry out PCR amplification, and recovering the amplification product.

6. And (3) mixing the amplification product in the step (4) and the amplification product in the step (5) in an equimolar manner, and performing PCR (polymerase chain reaction) amplification by using a primer pair consisting of Pps-R and Pgs-L to obtain the amplification product (linearized sgRNA expression cassette).

The sequence is sequenced, the linearized sgRNA expression cassette is shown as a sequence 4 in a sequence table, the expression cassette expresses the sgRNA shown as a sequence 5 in the sequence table, and the target sequence of the sgRNA is 146 th to 165 th positions of the sequence 4.

In practical application, the linearized sgRNA expression cassette can also be directly synthesized artificially.

7. And (3) connecting the linearized sgRNA expression cassette obtained in the step (6) with a pYLCRISPR/Cas9Pubi-H plasmid by utilizing a Golden Gate method (adopting restriction enzyme Bsa I) to obtain the CRISPR/Cas9 gene editing vector. The CRISPR/Cas9 gene editing vector contains a target sequence, wherein the target sequence is 146 th-165 th positions of a sequence 4.

The primers used above were as follows:

UF：5’-CTCCGTTTTACCTGTGGAATCG-3’。

gR-R：5’-CGGAGGAAAATTCCATCCAC-3’。

Pps-R：5’-TTCAGAggtctcTACCGACTAGTATGGAATCGGCAGCAAAGG-3’。

Pgs-L：5’-AGCGTGggtctcGCTCGACGCGTATCCATCCACTCCAAGCTC-3’。

2. obtaining Gene editing plants and identifying

1. And (3) taking the CRISPR/Cas9 gene editing vector constructed in the step one, and introducing the CRISPR/Cas9 gene editing vector into the competence of the agrobacterium EHA105 to obtain the recombinant agrobacterium.

And (3) extracting plasmids of the recombinant bacteria for sequencing verification, and naming the EHA105-CRISPR/Cas9 gene editing vector of the recombinant bacteria with correct sequencing verification.

2. And (3) taking the recombinant agrobacterium EHA105-CRISPR/Cas9 gene editing vector obtained in the step (1), and infecting leaves of 84K poplar by adopting a leaf disk method (the method is seen in MiR156 regulates anthocyanin biosynthesis through SPL targets and other microRNAs in poplar).

https:// doi.org/10.1038/s 41438-020-00341-w) to obtain regenerated poplar plants (leaves with three phenotypes of green, white and mottled; the mottled color is between non-green and green).

3. Screening gene editing positive plants from the obtained regenerated poplar plants, wherein the method comprises the following specific steps:

taking leaves of a regenerated poplar plant, extracting genome DNA, adopting a primer pair consisting of a primer F and a primer R to carry out PCR amplification, then recovering PCR amplification products, connecting a T vector, carrying out monoclonal sequencing, and screening a gene editing plant (the form of the gene editing plant can be a heterozygous gene editing plant, a chimeric gene editing plant or a homozygous gene editing plant) according to a sequencing result, and naming the gene editing plant as a ptrdj1c strain.

Primer F:5'-TATGCCTACTTGAACTTGC-3';

primer R:5'-ACCTCCTTGGCAACAGAC-3'.

As a result, the occurrence of a set peak at the target point is considered to be editing of the plant.

As shown in FIG. 1, both WT-1 and WT-2 were wild 84K poplar seedlings (leaf phenotype green) and dj1c-L1 to dj1c-L5 were gene-edited plants (leaf phenotype mosaic color).

And (3) statistics: 19 regenerated poplar plants in 30 regenerated poplar plants are edited (PtrDJ 1C gene appears to shift the frame and stop protein translation in advance) and named as gene editing plants, the gene editing efficiency reaches 63.3%, and the sgRNA has stronger editing efficiency.

Of the 19 edited plants, 2 were heterozygous gene edited and the leaf phenotype was green; 17 strains are homozygous gene editions, wherein 14 strains of leaf phenotype is a mottled color, and 3 strains of leaf phenotype is white.

The homozygous gene-edited plants were all subjected to gene editing for PtrDJ1C genes on 2 homologous chromosomes.

The heterozygous gene edited plant is that PtrDJ1C genes on 1 homologous chromosome are subjected to gene editing, and PtrDJ1C genes on the other homologous chromosome are not subjected to gene editing.

The homozygous gene-edited plant was designated as gene-edited plant.

3. Transcript level identification step

1) Extraction of total RNA from leaves

(1) About 0.1g of the leaf albino part or the non-green part of the gene editing plant is placed into a 2mL centrifuge tube without RNase, steel balls with proper size are added into the tube, and liquid nitrogen is frozen quickly. The quick frozen material is fully ground into powder.

(2) Subsequent RNA extraction operations were performed using a total plant RNA extraction kit (DP 432) purchased from the root biochemistry technology (beijing) limited.

(3) 1. Mu.L of RNA solution is taken, RNA concentration is measured by a NanoDrop 2000 ultra-micro spectrophotometer, and the labeled RNA is preserved for a long time in a refrigerator at-80 ℃.

2) Synthesis of cDNA

gDNA removal and cDNA Synthesis kit (AT 311) was purchased from Beijing full gold Biotechnology Co.

(1) In 200. Mu.L of RNase-free PCR tube, the preparation of the reverse transcription reaction solution was carried out according to the following system:

Total RNA 50 ng-2μg

OligodT primer 1. Mu.L

ddH ₂ O To 8μL

(2) Incubating the mixed solution in a PCR instrument at 65 ℃ for 5min, taking out, and then carrying out ice bath for 2min.

(3) The reaction solution of the following system was continuously added to the PCR tube to a total volume of 20. Mu.L.

2×TS Reaction Mix 10 μL

RT/RI Enzyme Mix 1 μL

gDNA Remover 1 μL

(4) After thoroughly mixing, incubation was performed in a PCR apparatus at 42℃for 15min.

(5) The RT/RI Enzyme and gDNA reverse were thoroughly inactivated by heating at 85℃for 5s in a PCR apparatus.

3) Reverse transcription PCR

(1) The design of primers is selected for the exon regions near the gene editing and the primers are made to span the intron structure on the genome as much as possible, avoiding experimental inaccuracy.

The primer sequences are as follows:

RT-F:5'TTTGGTACAGAAGAAATGG 3'

RT-R:5'CTCCTTGGCAACAGAC 3'

(3) Amplification was performed in steps using Taq enzyme, with no more than 30 cycles, using an action as a control.

As a result, as shown in FIG. 3, it was found that PtrDJ1C gene was not expressed or the expression amount was low in the gene-edited plant (for example, dj 1C-L1) as compared with the wild type.

The above gene-edited plants were designated ptrdj1c plants.

3. Plant leaf phenotype identification

The above verified gene editing plants and wild 84K plants were cultured under parallel conditions for 60 days under conditions of 80. Mu. Mol m in a greenhouse ^-2 s ^-1 Light intensity, 25 degrees celsius, 16/8 hours light/dark period.

The photograph is shown in FIG. 2, and it can be seen that leaves of wild type 84K plants (WT) are all green and have no whitened areas. The albino area of the leaf of the gene-edited plants (dj 1c-L2 and dj1 c-L3) was 66.5% of the leaf area (average of 10 plant leaves, measurement by Image J software).

The above results indicate that inhibiting or reducing PtrDJ1C gene expression can effect a change in leaf green phenotype, specifically, the appearance of non-green regions in green leaves.

Example 2 chloroplast development assay and accumulation of Critical proteins

1. Chloroplast developmental status detection

Test plants: wild type 84K plants, ptrdj1c strain plants dj1c-L2.

The transmission electron microscope observation is carried out on the chloroplast ultrastructure, and the specific steps are as follows: leaves of the test plants grown for about 4 weeks were fixed with 3% glutaraldehyde solution for about 2 hours, then rinsed with 0.1M PBS buffer, then fixed with 1% osmium solution overnight at 4 ℃, then rinsed with 0.1M PBS buffer, then alcohol dehydrated (30% -95% alcohol each for 2-30min, and finally 100% alcohol three times for 20min each), then infiltrated (1/3 epoxy+2/3 propylene oxide for 2 hours, then 1/2 epoxy+1/2 propylene oxide for 2 hours, then 2/3 epoxy+1/3 propylene oxide for 2 hours, then pure epoxy; placed in a desiccator for 1-2 days), and then embedded (polymerization 37 ℃,24h;45 ℃,24h;55 ℃,24h;60 ℃ for 2 hours). And (5) trimming and marking, positioning the ultrathin slice, and observing by an electron transmission electron microscope.

The photo of the ultrastructure of chloroplast is shown in figure 4, and the leaf of wild type plant (WT) has normal development of chloroplast, and can clearly see structures such as thylakoid membrane; leaves of the ptrdj1c strain plant (dj 1 c): chloroplasts in the albino region appear undeveloped or dysplastic, with only some residual structures being visible.

2. Detection of key protein accumulation affecting chloroplast development

Test plants: wild type 84K plants, ptrdj1c plants dj1c-L2.

Sample supply: test plants were grown for approximately 4 weeks.

1. Extraction of Total protein

A50 mg fresh weight of sample specimen was taken and 200. Mu.L of buffer E (0.1514 g/mL Tris,0.01g/mL SDS,10% glycerol, 0.095g/mL Na) was added ₂ S ₂ O ₅ pH 8.8) was ground to a homogenate, centrifuged at 13000g for 10 min at room temperature, and the supernatant was collected.

2. Quantification of Total protein

mu.L of the supernatant was used for total protein quantification (in mg/mL) using a BioRad DcProtein Assay kit (Bio-Rad, hercules, calif., USA).

3. And (3) leveling the total protein concentration of the supernatant obtained by the two sample samples according to the total protein quantitative result to obtain a protein solution. The protein solutions obtained from the wild plants were diluted to 2-fold and 4-fold volumes, respectively.

4. Protein electrophoresis and Western blot analysis were performed on the protein solution obtained from the plants of the ptrdj1c strain, the protein solution obtained from the wild type plants, the 2-fold dilution of the protein solution obtained from the wild type plants, and the 4-fold dilution of the protein solution obtained from the wild type plants, respectively (Actin protein was used as an internal reference, and the antibodies for detecting the proteins of each optical system were all commercially available antibodies).

As a result, as shown in FIG. 5, the content of each component protein (such as D1, psaA, cytf, CF 1. Alpha., rbcL and ATPase) affecting chloroplast development and photosynthesis was significantly reduced in plants of the ptrdj1c strain (shown as dj1c in the figure) as compared with wild type plants. Reduced levels of the related subunits of the photosynthetic complex can lead to chloroplast thylakoid membrane and chloroplast dysplasia, while reduced levels of chlorophyll associated with the photosystem, ultimately leading to a white (yellowish) phenotype.

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

SEQUENCE LISTING

<110> college of North and Hebei

<120> a method for creating a flower-spotted ornamental poplar using PtrDJ1C gene

<160> 7

<170> PatentIn version 3.5

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ctaaaggata agagagccac tgcacacccc tctgttgtga ccaacctaaa caatgtatca 1260

aatggtgcca aagtagttat tgacggtaaa ttgatcacaa gcaagggact ttctaccgta 1320

acagattttg ctttggctat tgtgagcaag ctttttggcc atgcaaggac aagatgtgta 1380

gctgagggcc ttgtttttga ctatcccagg agttag 1416

<210> 3

<211> 4662

<212> DNA

<213> Artificial sequence

<400> 3

aaacagtcta tagcactgca agctccaatc aatggagtct atgctctgtc ttctctcacc 60

atctcctaca aagctctctc ctttcaagaa actaacttca acttgtgcct taaaaactac 120

attttcctct ttatcatttg cttccatgac ctctcctcca cagccaaaaa caccctctac 180

aaaaaaactc tcttcttcaa aacccaccaa aaccctatct ccaaaaacac ccacaacatc 240

aacgagtgtt caagaaacaa gcactccatt ctctcctcct ctcaaaaagg tgcctttttg 300

cttaaacctt ataccctttt accttaaaat gcttataaaa aaaacatagc ttttcaattg 360

ttgttttgta ttcaagaatt ctagactccc ctttcgacat gatggtagaa agttgatata 420

actatgccta cttgaacttg cagtgaccaa agtgttgatt ttttttatgg gttttgaaat 480

gtttgtggca ttgataggtt ctggtgccaa taggttttgg tacagaagaa atggaagcag 540

ttattatcgt tgatgttttg aggagagctg gtgctgaggt aattgttgct tcagtagaac 600

cacagcttga agttgaagct gcaggtggca ctaggcttgt tgctgatacc tccatttcca 660

aatgtgccaa tgaagttttc gatcttgttg ctttgccggt ttgttgtttt ttactttgaa 720

tcattcatgg tttgcaaatt aattcatgat tgttacattt atttctcatt gataatgtag 780

ccggggcaaa tattatgaca tagctttttt ttttttttaa ttttttcaat aaatggttgg 840

ttagtgactg acactcattg agaaattaat aaaaagtatg gtttagttga tttatgatcg 900

tgccggtaaa gatagttata catacaagcg tgaatgttaa gtataggaaa aagatatact 960

caacgacagg atttaattgg aaaatcgaga agattgtgtt ttctttgggg ttgtaacagg 1020

gagtttggtt gcttgttagt acagtaaatt ttatttggaa aacaactgta gcttttgtag 1080

tttgatctgg ccatgtaatt tgtaacaaag gcttgcgtgt tctgaatggc ttcaagggag 1140

gaatgccagg ctccgcaagg ttgagagatt gtgaagttct caggcaaatt acaagcaaac 1200

aagctgagga taagagatta tatggagcta tatgtgccgc tccggcaatc actcttcttc 1260

catggggcct tctgaggaga aagcaggtgg ctttcatgtg ttgcttttgc ttgaattttg 1320

ttgaatacac atgaatgtat tttggttgac taaatgtgct gtagcatgtg atgtaagatg 1380

acaggtcacc ctgcatttat ggacaagctt cctactttct gggctgttgc atcaaaaatt 1440

caagtttcag gagagcttac aacaagccgc ggtccaggaa cttctttcga gtttgctcta 1500

tccttagtag accagctgtt tggagagtct gttgccaagg aggttggaca attgttggta 1560

agtttaattg ctttgccagt ttcattttta cttttcatgc atgcgtcaac atagcatttt 1620

ggaaatcaag ttcctgtcat taacttaagt gcatcgaaag catgtgcagc ttagtcatgc 1680

cattctttac tgtgaacctc ttattcctaa ttgaatagaa gttaaaatta cctttttcag 1740

tggtgtataa tcatgattta aactctgcct ttttttctgg acatgatctg ctgaagttca 1800

cagggataat tagcgaactg ttttcaactt ttcataccat tgaagattct gtgcagaatt 1860

ctgaaatgaa atgcctgata acaggctcat aaaatttcta ttatcctgtt aattactagg 1920

atcttagcac aacaaattct gctgaaagca atacaaatta ttcaagtttt tttttttttt 1980

ggcatctgtc cattcattta actcaactgg gattcagacc tcgtcatgca catcttaaat 2040

attttttgtt cagtcagatt attgaaggat cgaatggatt tctttctatc ctttaataca 2100

gtatttttat agtggggaaa gaaagtctgg atggtatgaa agaagcagta tgagttaaag 2160

cattaattac cttagtgatc cttcaagttt tctaaaacag aatgcaaaga gtttgtgata 2220

tcttcattta gttcatatct tcaaagcgtc actacttttt acccatgtaa tggtttttta 2280

taggattttt aaaaaatgca cagaaaacca atgacaaaag gtagagaaat ttgataagtg 2340

actgtgtgta tggaatgcat agatcaggtg agctgttttc ttttgttttg taaaagattc 2400

atctgtcaac tgacttgtct tctcttacat gtttaccctt tggaagatgc tttgtggtta 2460

atcatcttga gctaacatct ctttacacct tatattagct aatgcaagct gatgatgaca 2520

cccagagaaa agaagaatat aacaaagttg aatggtcttt tgatcacaat cctcgtgtaa 2580

gtttcttcta ccttcctgtt tctgttaatc acctattggt ttataatctt ctttgtctgg 2640

agaacactcg atgcggggat gtggaacaca ttaaaaacaa gagctcctca tatttgaaac 2700

aattttgcat tttttcagtt gctaggtgga cgcactgata ttttttgaat gcatctttag 2760

tttaatcatt tgcgtaaagt tgcttcatga agtagttccc gcatccctgt gcaatcccaa 2820

tttgtagcat ttgtttgtct tctcatccct agtgcatttg agtttcttcc tatttatcat 2880

caaattaatc ttccacatgt tacacaggtt ctccttccaa ttgcaaatgg ttctgaagag 2940

attgaaatag ttgctattgt agatatttta aggcgggcaa aggtggatgt tgtggttgct 3000

tcaattgaaa aatcagtgca aattttggca tcacgaggca taaaaattgt cgctgacaag 3060

ttaattggtg atgctgctga atcagtatat gatctaatca ttctaccagt aagacaactc 3120

aatttttttt tataatattg aaaattgctt ttcttgcatt ctttcctaat agagttgtga 3180

gggttttata gtctttggaa ttcccactgt gatatcaaca gatgcctttg gaccttgcaa 3240

actagtctaa tatcataaga gggctttgag tacattggta gtcttaactt cagctttatg 3300

ttgatgaaaa aaacttcagc ttcaaggtaa tttgcaacat cttgtggaga tactgtcggt 3360

agcttgggct actgaaatta ctgcattagc tgttatcatt tgctcaggtt aacgccatgc 3420

cacatttctt gggatgaagt gctcaacaag tagcacattc caaaatctag gttttgctaa 3480

atattataga tctttctgaa attgaggctg ctgtctagtg cgttctttga catctacttt 3540

tgtgtttgtg ttgtatcctt gaagagtttt gatttgctgt gtcctttcag ggaggaaatg 3600

ctggggctga gcggctacac aaatctaagg ttctaaagaa gctgctccaa gagcaatata 3660

cagctggcag aatatatgga gcagtttgtt cttcccctgc agttcttcac agacagggtt 3720

tactaaaggt aatttgaact aattgaaatc tcttttctaa acagtaggac gctgaaggat 3780

ctgcttttct tcccaactat aggataagag agccactgca cacccctctg ttgtgaccaa 3840

cctaaacaat gtatcaaatg gtgccaaagt agttattgac ggtaaattga tcacaagcaa 3900

gggactttct accgtaacag attttgcttt ggctattgtg agcaagcttt ttggccatgc 3960

aaggacaaga tgtgtagctg agggccttgt ttttgactat cccaggagtt aggccatcag 4020

gtccttctgg acgaaaggca atttggaaga tccctgttcc atggagaaca gtctattccc 4080

tgaactcata tcagccagtt aagaaagaaa atcctctttc tgaggtgctt gcctaatgga 4140

tatttacgag actcatgttc gtccagatac agaatcctga ttgctttttt ggatagcaga 4200

acgagggaga gttgacattt gcctgtctgt gaagcccaaa gctactcgcc caatggcgat 4260

taacagaaga tgtgttgtga gctgaagcta acatctccaa atgtctgctg aagaacagac 4320

ttgtttgcaa actttatatg ataattcagt gccgaacagt atgaatggac atagaaatgg 4380

ctctgtgtaa caactgggca acgcagctgc tttttattca tcccatcttg ctcatgttgc 4440

attagcaaca ccagggattt cagttcggac ctgtgttatt gtggactttt tctcgttgcc 4500

aacgaatgat aaagattaca agcttaaatc aggactttca tgtatggtta tgatgttgcc 4560

gagattaatc ataaccatat atttcctctt atgcttgcct gttcttgact ggatatttta 4620

gaactggctg ctgttgtgtc tgaaaagcat ccgttcaatt gt 4662

<210> 4

<211> 293

<212> DNA

<213> Artificial sequence

<400> 4

ttcagaggtc tctaccgact agtatggaat cggcagcaaa ggaataagct tatgatttct 60

tttttcttac gaattttgcg tcccacatcg gtaagcgagt gaagaaataa ctgctttata 120

tatggctaca aagcaccatt ggtcagagga atgccaggct ccgcagtttt agagctagaa 180

atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg 240

ctttttttca agagcttgga gtggatggat acgcgtcgag cgagacccac gct 293

<210> 5

<211> 104

<212> DNA

<213> Artificial sequence

<400> 5

agaggaaugc caggcuccgc aguuuuagag cuagaaauag caaguuaaaa uaaggcuagu 60

ccguuaucaa cuugaaaaag uggcaccgag ucggugcuuu uuuu 104

<210> 6

<211> 2944

<212> DNA

<213> Artificial sequence

<400> 6

ggatccagcg tgggtctcgg ttttagagct agaaatagca agttaaaata aggctagtcc 60

gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttcaagagct tggagtggat 120

ggaattttcc tccgttttac ctgtggaatc ggcagcaaag gaataagctt atgatttctt 180

ttttcttacg aattttgcgt cccacatcgg taagcgagtg aagaaataac tgctttatat 240

atggctacaa agcaccattg gtcaagagac ctctgaagat aacatactaa gcttggcact 300

ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct 360

tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc 420

ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac 480

gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc 540

cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg 600

tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca 660

gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt 720

tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg 780

aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 840

catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 900

tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 960

tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 1020

ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 1080

ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 1140

cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 1200

ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 1260

tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 1320

gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 1380

ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 1440

aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 1500

acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 1560

tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 1620

cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 1680

gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 1740

taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 1800

tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 1860

cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 1920

ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 1980

accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 2040

cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 2100

cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 2160

tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 2220

taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 2280

gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 2340

agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 2400

ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 2460

acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 2520

caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 2580

tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 2640

tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc 2700

aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag 2760

gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca 2820

ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag 2880

cggataacaa tttcacacag gaaacagcta tgaccatgat tacgaattcg agctcggtac 2940

ccgg 2944

<210> 7

<211> 17619

<212> DNA

<213> Artificial sequence

<400> 7

ttgccctttt cctttatttc aatatatgcc gtgcacttgt ttgtcgggtc atcttttcat 60

gctttttttt gtcttggttg tgatgatgtg gtctggttgg gcggtcgttc tagatcggag 120

tagaattctg tttcaaacta cctggtggat ttattaattt tggatctgta tgtgtgtgcc 180

atacatattc atagttacga attgaagatg atggatggaa atatcgatct aggataggta 240

tacatgttga tgcgggtttt actgatgcat atacagagat gctttttgtt cgcttggttg 300

tgatgatgtg gtgtggttgg gcggtcgttc attcgttcta gatcggagta gaatactgtt 360

tcaaactacc tggtgtattt attaattttg gaactgtatg tgtgtgtcat acatcttcat 420

agttacgagt ttaagatgga tggaaatatc gatctaggat aggtatacat gttgatgtgg 480

gttttactga tgcatataca tgatggcata tgcagcatct attcatatgc tctaaccttg 540

agtacctatc tattataata aacaagtatg ttttataatt attttgatct tgatatactt 600

ggatgatggc atatgcagca gctatatgtg gattttttta gccctgcctt catacgctat 660

ttatttgctt ggtactgttt cttttgtcga tgctcaccct gttgtttggt gttacttctg 720

cagatggctc ctaagaagaa gcggaaggtt ggtattcacg gggtgcctgc ggctgacaag 780

aagtactcca tcggcctcga catcggcacc aacagcgtcg gctgggcggt gatcaccgac 840

gagtacaagg tcccgtccaa gaagttcaag gtcctgggca acaccgaccg ccactccatc 900

aagaagaacc tcatcggcgc cctcctcttc gactccggcg agacggcgga ggcgacccgc 960

ctcaagcgca ccgcccgccg ccgctacacc cgccgcaaga accgcatctg ctacctccag 1020

gagatcttct ccaacgagat ggcgaaggtc gacgactcct tcttccaccg cctcgaggag 1080

tccttcctcg tggaggagga caagaagcac gagcgccacc ccatcttcgg caacatcgtc 1140

gacgaggtcg cctaccacga gaagtacccc actatctacc accttcgtaa gaagcttgtt 1200

gactctactg ataaggctga tcttcgtctc atctaccttg ctctcgctca catgatcaag 1260

ttccgtggtc acttccttat cgagggtgac cttaaccctg ataactccga cgtggacaag 1320

ctcttcatcc agctcgtcca gacctacaac cagctcttcg aggagaaccc tatcaacgct 1380

tccggtgtcg acgctaaggc gatcctttcc gctaggctct ccaagtccag gcgtctcgag 1440

aacctcatcg cccagctccc tggtgagaag aagaacggtc ttttcggtaa cctcatcgct 1500

ctctccctcg gtctgacccc taacttcaag tccaacttcg acctcgctga ggacgctaag 1560

cttcagctct ccaaggatac ctacgacgat gatctcgaca acctcctcgc tcagattgga 1620

gatcagtacg ctgatctctt ccttgctgct aagaacctct ccgatgctat cctcctttcg 1680

gatatcctta gggttaacac tgagatcact aaggctcctc tttctgcttc catgatcaag 1740

cgctacgacg agcaccacca ggacctcacc ctcctcaagg ctcttgttcg tcagcagctc 1800

cccgagaagt acaaggagat cttcttcgac cagtccaaga acggctacgc cggttacatt 1860

gacggtggag ctagccagga ggagttctac aagttcatca agccaatcct tgagaagatg 1920

gatggtactg aggagcttct cgttaagctt aaccgtgagg acctccttag gaagcagagg 1980

actttcgata acggctctat ccctcaccag atccaccttg gtgagcttca cgccatcctt 2040

cgtaggcagg aggacttcta ccctttcctc aaggacaacc gtgagaagat cgagaagatc 2100

cttactttcc gtattcctta ctacgttggt cctcttgctc gtggtaactc ccgtttcgct 2160

tggatgacta ggaagtccga ggagactatc accccttgga acttcgagga ggttgttgac 2220

aagggtgctt ccgcccagtc cttcatcgag cgcatgacca acttcgacaa gaacctcccc 2280

aacgagaagg tcctccccaa gcactccctc ctctacgagt acttcacggt ctacaacgag 2340

ctcaccaagg tcaagtacgt caccgagggt atgcgcaagc ctgccttcct ctccggcgag 2400

cagaagaagg ctatcgttga cctcctcttc aagaccaacc gcaaggtcac cgtcaagcag 2460

ctcaaggagg actacttcaa gaagatcgag tgcttcgact ccgtcgagat cagcggcgtt 2520

gaggaccgtt tcaacgcttc tctcggtacc taccacgatc tcctcaagat catcaaggac 2580

aaggacttcc tcgacaacga ggagaacgag gacatcctcg aggacatcgt cctcactctt 2640

actctcttcg aggataggga gatgatcgag gagaggctca agacttacgc tcatctcttc 2700

gatgacaagg ttatgaagca gctcaagcgt cgccgttaca ccggttgggg taggctctcc 2760

cgcaagctca tcaacggtat cagggataag cagagcggca agactatcct cgacttcctc 2820

aagtctgatg gtttcgctaa caggaacttc atgcagctca tccacgatga ctctcttacc 2880

ttcaaggagg atattcagaa ggctcaggtg tccggtcagg gcgactctct ccacgagcac 2940

attgctaacc ttgctggttc ccctgctatc aagaagggca tccttcagac tgttaaggtt 3000

gtcgatgagc ttgtcaaggt tatgggtcgt cacaagcctg agaacatcgt catcgagatg 3060

gctcgtgaga accagactac ccagaagggt cagaagaact cgagggagcg catgaagagg 3120

attgaggagg gtatcaagga gcttggttct cagatcctta aggagcaccc tgtcgagaac 3180

acccagctcc agaacgagaa gctctacctc tactacctcc agaacggtag ggatatgtac 3240

gttgaccagg agctcgacat caacaggctt tctgactacg acgtcgacca cattgttcct 3300

cagtctttcc ttaaggatga ctccatcgac aacaaggtcc tcacgaggtc cgacaagaac 3360

aggggtaagt cggacaacgt cccttccgag gaggttgtca agaagatgaa gaactactgg 3420

aggcagcttc tcaacgctaa gctcattacc cagaggaagt tcgacaacct cacgaaggct 3480

gagaggggtg gcctttccga gcttgacaag gctggtttca tcaagaggca gcttgttgag 3540

acgaggcaga ttaccaagca cgttgctcag atcctcgatt ctaggatgaa caccaagtac 3600

gacgagaacg acaagctcat ccgcgaggtc aaggtgatca ccctcaagtc caagctcgtc 3660

tccgacttcc gcaaggactt ccagttctac aaggtccgcg agatcaacaa ctaccaccac 3720

gctcacgatg cttaccttaa cgctgtcgtt ggtaccgctc ttatcaagaa gtaccctaag 3780

cttgagtccg agttcgtcta cggtgactac aaggtctacg acgttcgtaa gatgatcgcc 3840

aagtccgagc aggagatcgg caaggccacc gccaagtact tcttctactc caacatcatg 3900

aacttcttca agaccgagat caccctcgcc aacggcgaga tccgcaagcg ccctcttatc 3960

gagacgaacg gtgagactgg tgagatcgtt tgggacaagg gtcgcgactt cgctactgtt 4020

cgcaaggtcc tttctatgcc tcaggttaac atcgtcaaga agaccgaggt ccagaccggt 4080

ggcttctcca aggagtctat ccttccaaag agaaactcgg acaagctcat cgctaggaag 4140

aaggattggg accctaagaa gtacggtggt ttcgactccc ctactgtcgc ctactccgtc 4200

ctcgtggtcg ccaaggtgga gaagggtaag tcgaagaagc tcaagtccgt caaggagctc 4260

ctcggcatca ccatcatgga gcgctcctcc ttcgagaaga acccgatcga cttcctcgag 4320

gccaagggct acaaggaggt caagaaggac ctcatcatca agctccccaa gtactctctt 4380

ttcgagctcg agaacggtcg taagaggatg ctggcttccg ctggtgagct ccagaagggt 4440

aacgagcttg ctcttccttc caagtacgtg aacttcctct acctcgcctc ccactacgag 4500

aagctcaagg gttcccctga ggataacgag cagaagcagc tcttcgtgga gcagcacaag 4560

cactacctcg acgagatcat cgagcagatc tccgagttct ccaagcgcgt catcctcgct 4620

gacgctaacc tcgacaaggt cctctccgcc tacaacaagc accgcgacaa gcccatccgc 4680

gagcaggccg agaacatcat ccacctcttc acgctcacga acctcggcgc ccctgctgct 4740

ttcaagtact tcgacaccac catcgacagg aagcgttaca cgtccaccaa ggaggttctc 4800

gacgctactc tcatccacca gtccatcacc ggtctttacg agactcgtat cgacctttcc 4860

cagcttggtg gtgataagcg tcctgctgcc accaaaaagg ccggacaggc taagaaaaag 4920

aagtaggatc ctcccgatcg ttcaaacatt tggcaataaa gtttcttaag attgaatcct 4980

gttgccggtc ttgcgatgat tatcatataa tttctgttga attacgttaa gcatgtaata 5040

attaacatgt aatgcatgac gttatttatg aggtgggttt ttatgattag agtcccgcaa 5100

ttatacattt aatacgcgat agaaaacaaa atatagcgcg caaactagga taaattatcg 5160

cgcgcggtgt catctatgtt actagatcgg gagcaccggt aaggcgcgcc gtagtgctcg 5220

agagacctct gaagtggccg attcattaat gcagctggca cgacaggttt cccgactgga 5280

aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg 5340

ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc 5400

acacaagaaa cagctatgac catgattacg ccaagctatt taggtgacac tatagaatac 5460

tcaagctatg catcaagctc aatgggtcta gtctgtagat acccatcaca ctggcgaccg 5520

ctcgaacatc agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg 5580

gatgtacaga gtgatattat tgacacgccg gggcgacgga tggtgatccc cctggccagt 5640

gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat 5700

gaaagctggc gcatgatgac caccgatatg gccagtgtgc ctgtctccgt tatcggggaa 5760

gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc 5820

tggggaatat aaatgtcagg cctgaatggc gaatggacgc gccctgtagc ggcgcattaa 5880

gcgcggcggg tgagcgtggg tctcgcggta tcattggcgc gcctctcgag ctagcggccg 5940

catgcatcga tctcctacat cgtataaatt agcctatacg aagttattgc atctatgtcg 6000

ggtgcggaga aagaggtaat gaaatggcag tattagatct gataacttcg tataatgtat 6060

gctatacgaa gttatgactg caggtcgaca cccataatag ctgtttgcca agcttggcac 6120

tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc 6180

ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc 6240

cttcccaaca gttgcgcagc ctgaatggcg aatgctagag cagcttgagc ttggatcaga 6300

ttgtcgtttc ccgccttcag tttaaactat cagtgtttga caggatatat tggcgggtaa 6360

acctaagaga aaagagcgtt tattagaata atcggatatt taaaagggcg tgaaaaggtt 6420

tatccgttcg tccatttgta tgtgcatgcc aaccacaggg ttcccctcgg gatcaaagta 6480

ctttgatcca acccctccgc tgctatagtg cagtcggctt ctgacgttca gtgcagccgt 6540

cttctgaaaa cgacatgtcg cacaagtcct aagttacgcg acaggctgcc gccctgccct 6600

tttcctggcg ttttcttgtc gcgtgtttta gtcgcataaa gtagaatact tgcgactaga 6660

accggagaca ttacgccatg aacaagagcg ccgccgctgg cctgctgggc tatgcccgcg 6720

tcagcaccga cgaccaggac ttgaccaacc aacgggccga actgcacgcg gccggctgca 6780

ccaagctgtt ttccgagaag atcaccggca ccaggcgcga ccgcccggag ctggccagga 6840

tgcttgacca cctacgccct ggcgacgttg tgacagtgac caggctagac cgcctggccc 6900

gcagcacccg cgacctactg gacattgccg agcgcatcca ggaggccggc gcgggcctgc 6960

gtagcctggc agagccgtgg gccgacacca ccacgccggc cggccgcatg gtgttgaccg 7020

tgttcgccgg cattgccgag ttcgagcgtt ccctaatcat cgaccgcacc cggagcgggc 7080

gcgaggccgc caaggcccga ggcgtgaagt ttggcccccg ccctaccctc accccggcac 7140

agatcgcgca cgcccgcgag ctgatcgacc aggaaggccg caccgtgaaa gaggcggctg 7200

cactgcttgg cgtgcatcgc tcgaccctgt accgcgcact tgagcgcagc gaggaagtga 7260

cgcccaccga ggccaggcgg cgcggtgcct tccgtgagga cgcattgacc gaggccgacg 7320

ccctggcggc cgccgagaat gaacgccaag aggaacaagc atgaaaccgc accaggacgg 7380

ccaggacgaa ccgtttttca ttaccgaaga gatcgaggcg gagatgatcg cggccgggta 7440

cgtgttcgag ccgcccgcgc acgtctcaac cgtgcggctg catgaaatcc tggccggttt 7500

gtctgatgcc aagctggcgg cctggccggc cagcttggcc gctgaagaaa ccgagcgccg 7560

ccgtctaaaa aggtgatgtg tatttgagta aaacagcttg cgtcatgcgg tcgctgcgta 7620

tatgatgcga tgagtaaata aacaaatacg caaggggaac gcatgaaggt tatcgctgta 7680

cttaaccaga aaggcgggtc aggcaagacg accatcgcaa cccatctagc ccgcgccctg 7740

caactcgccg gggccgatgt tctgttagtc gattccgatc cccagggcag tgcccgcgat 7800

tgggcggccg tgcgggaaga tcaaccgcta accgttgtcg gcatcgaccg cccgacgatt 7860

gaccgcgacg tgaaggccat cggccggcgc gacttcgtag tgatcgacgg agcgccccag 7920

gcggcggact tggctgtgtc cgcgatcaag gcagccgact tcgtgctgat tccggtgcag 7980

ccaagccctt acgacatatg ggccaccgcc gacctggtgg agctggttaa gcagcgcatt 8040

gaggtcacgg atggaaggct acaagcggcc tttgtcgtgt cgcgggcgat caaaggcacg 8100

cgcatcggcg gtgaggttgc cgaggcgctg gccgggtacg agctgcccat tcttgagtcc 8160

cgtatcacgc agcgcgtgag ctacccaggc actgccgccg ccggcacaac cgttcttgaa 8220

tcagaacccg agggcgacgc tgcccgcgag gtccaggcgc tggccgctga aattaaatca 8280

aaactcattt gagttaatga ggtaaagaga aaatgagcaa aagcacaaac acgctaagtg 8340

ccggccgtcc gagcgcacgc agcagcaagg ctgcaacgtt ggccagcctg gcagacacgc 8400

cagccatgaa gcgggtcaac tttcagttgc cggcggagga tcacaccaag ctgaagatgt 8460

acgcggtacg ccaaggcaag accattaccg agctgctatc tgaatacatc gcgcagctac 8520

cagagtaaat gagcaaatga ataaatgagt agatgaattt tagcggctaa aggaggcggc 8580

atggaaaatc aagaacaacc aggcaccgac gccgtggaat gccccatgtg tggaggaacg 8640

ggcggttggc caggcgtaag cggctgggtt gcctgccggc cctgcaatgg cactggaacc 8700

cccaagcccg aggaatcggc gtgagcggtc gcaaaccatc cggcccggta caaatcggcg 8760

cggcgctggg tgatgacctg gtggagaagt tgaaggccgc gcaggccgcc cagcggcaac 8820

gcatcgaggc agaagcacgc cccggtgaat cgtggcaagc ggccgctgat cgaatccgca 8880

aagaatcccg gcaaccgccg gcagccggtg cgccgtcgat taggaagccg cccaagggcg 8940

acgagcaacc agattttttc gttccgatgc tctatgacgt gggcacccgc gatagtcgca 9000

gcatcatgga cgtggccgtt ttccgtctgt cgaagcgtga ccgacgagct ggcgaggtga 9060

tccgctacga gcttccagac gggcacgtag aggtttccgc agggccggcc ggcatggcca 9120

gtgtgtggga ttacgacctg gtactgatgg cggtttccca tctaaccgaa tccatgaacc 9180

gataccggga agggaaggga gacaagcccg gccgcgtgtt ccgtccacac gttgcggacg 9240

tactcaagtt ctgccggcga gccgatggcg gaaagcagaa agacgacctg gtagaaacct 9300

gcattcggtt aaacaccacg cacgttgcca tgcagcgtac gaagaaggcc aagaacggcc 9360

gcctggtgac ggtatccgag ggtgaagcct tgattagccg ctacaagatc gtaaagagcg 9420

aaaccgggcg gccggagtac atcgagatcg agctagctga ttggatgtac cgcgagatca 9480

cagaaggcaa gaacccggac gtgctgacgg ttcaccccga ttactttttg atcgatcccg 9540

gcatcggccg ttttctctac cgcctggcac gccgcgccgc aggcaaggca gaagccagat 9600

ggttgttcaa gacgatctac gaacgcagtg gcagcgccgg agagttcaag aagttctgtt 9660

tcaccgtgcg caagctgatc gggtcaaatg acctgccgga gtacgatttg aaggaggagg 9720

cggggcaggc tggcccgatc ctagtcatgc gctaccgcaa cctgatcgag ggcgaagcat 9780

ccgccggttc ctaatgtacg gagcagatgc tagggcaaat tgccctagca ggggaaaaag 9840

gtcgaaaagt tctctttcct gtggatagca cgtacattgg gaacccaaag ccgtacattg 9900

ggaaccggaa cccgtacatt gggaacccaa agccgtacat tgggaaccgg tcacacatgt 9960

aagtgactga tataaaagag aaaaaaggcg atttttccgc ctaaaactct ttaaaactta 10020

ttaaaactct taaaacccgc ctggcctgtg cataactgtc tggccagcgc acagccgaag 10080

agctgcaaaa agcgcctacc cttcggtcgc tgcgctccct acgccccgcc gcttcgcgtc 10140

ggcctatcgc ggccgctggc cgctcaaaaa tggctggcct acggccaggc aatctaccag 10200

ggcgcggaca agccgcgccg tcgccactcg accgccggcg cccacatcaa ggcaccctgc 10260

ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc 10320

acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt 10380

gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact 10440

ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa 10500

taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca 10560

ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 10620

taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 10680

agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 10740

cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 10800

tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 10860

tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 10920

gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 10980

acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 11040

acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 11100

cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 11160

gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 11220

gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 11280

agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 11340

ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgcat tctaggtact 11400

aaaacaattc atccagtaaa atataatatt ttattttctc ccaatcaggc ttgatcccca 11460

gtaagtcaaa aaatagctcg acatactgtt cttccccgat atcctccctg atcgaccgga 11520

cgcagaaggc aatgtcatac cacttgtccg ccctgccgct tctcccaaga tcaataaagc 11580

cacttacttt gccatctttc acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga 11640

caagttcctc ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc ggatctttaa 11700

atggagtgtc ttcttcccag ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt 11760

aatccaattc ggctaagcgg ctgtctaagc tattcgtata gggacaatcc gatatgtcga 11820

tggagtgaaa gagcctgatg cactccgcat acagctcgat aatcttttca gggctttgtt 11880

catcttcata ctcttccgag caaaggacgc catcggcctc actcatgagc agattgctcc 11940

agccatcatg ccgttcaaag tgcaggacct ttggaacagg cagctttcct tccagccata 12000

gcatcatgtc cttttcccgt tccacatcat aggtggtccc tttataccgg ctgtccgtca 12060

tttttaaata taggttttca ttttctccca ccagcttata taccttagca ggagacattc 12120

cttccgtatc ttttacgcag cggtattttt cgatcagttt tttcaattcc ggtgatattc 12180

tcattttagc catttattat ttccttcctc ttttctacag tatttaaaga taccccaaga 12240

agctaaggaa ggtgcgaaca agtccctgat atgagatcat gtttgtcatc tggagccata 12300

gaacagggtt catcatgagt catcaactta ccttcgccga cagtgaattc agcagtaagc 12360

gccgtcagac cagaaaagag attttcttgt cccgcatgga gcagattctg ccatggcaaa 12420

acatggtgga agtcatcgag ccgttttacc ccaaggctgg taatggccgg cgaccttatc 12480

cgctggaaac catgctacgc attcactgca tgcagcattg gtacaacctg agcgatggcg 12540

cgatggaaga tgctctgtac gaaatcgcct ccatgcgtct gtttgcccgg ttatccctgg 12600

atagcgcctt gccggaccgc accaccatca tgaatttccg ccacctgctg gagcagcatc 12660

aactggcccg ccaattgttc aagaccatca atcgctggct ggccgaagca ggcgtcatga 12720

tgactcaagg caccttggtc gatgccacca tcattgaggc acccagctcg accaagaaca 12780

aagagcagca acgcgatccg gagatgcatc agaccaagaa aggcaatcag tggcactttg 12840

gcatgaaggc ccacattggt gtcgatgcca agagtggcct gacccacagc ctggtcacca 12900

ccgcggccaa cgagcatgac ctcaatcagc tgggtaatct gctgcatgga gaggagcaat 12960

ttgtctcagc cgatgccggc taccaagggg cgccacagcg cgaggagctg gccgaggtgg 13020

atgtggactg gctgatcgcc gagcgccccg gcaaggtaag aaccttgaaa cagcatccac 13080

gcaagaacaa aacggccatc aacatcgaat acatgaaagc cagcatccgg gccagggtgg 13140

agcacccatt tcgcatcatc aagcgacagt tcggcttcgt gaaagccaga tacaaggggt 13200

tgctgaaaaa cgataaccaa ctggcgatgt tattcacgct ggccaacctg tttcgggcgg 13260

accaaatgat acgtcagtgg gagagatctc actaaaaact ggggataacg ccttaaatgg 13320

cgaagaaacg gtctaaatag gctgattcaa ggcatttacg ggagaaaaaa tcggctcaaa 13380

catgaagaaa tgaaatgact gagtcagccg agaagaattt ccccgcttat tcgcaccttc 13440

cctaattata acaagacgaa ctccaattca ctgttccttg cattctaaaa ccttaaatac 13500

cagaaaacag ctttttcaaa gttgttttca aagttggcgt ataacatagt atcgacggag 13560

ccgattttga aaccgcggtg atcacaggca gcaacgctct gtcatcgtta caatcaacat 13620

gctaccctcc gcgagatcat ccgtgtttca aacccggcag cttagttgcc gttcttccga 13680

atagcatcgg taacatgagc aaagtctgcc gccttacaac ggctctcccg ctgacgccgt 13740

cccggactga tgggctgcct gtatcgagtg gtgattttgt gccgagctgc cggtcgggga 13800

gctgttggct ggctggtggc aggatatatt gtggtgtaaa caaattgacg cttagacaac 13860

ttaataacac attgcggacg tttttaatgt actgaattaa cgccgaatta attcggggga 13920

tctggatttt agtactggat tttggtttta ggaattagaa attttattga tagaagtatt 13980

ttacaaatac aaatacatac taagggtttc ttatatgctc aacacatgag cgaaacccta 14040

taggaaccct aattccctta tctgggaact actcacacat tattatggag aaactcgagc 14100

ttgtcgatcg acagatcccg gtcggcatct actctatttc tttgccctcg gacgagtgct 14160

ggggcgtcgg tttccactat cggcgagtac ttctacacag ccatcggtcc agacggccgc 14220

gcttctgcgg gcgatttgtg tacgcccgac agtcccggct ccggatcgga cgattgcgtc 14280

gcatcgaccc tgcgcccaag ctgcatcatc gaaattgccg tcaaccaagc tctgatagag 14340

ttggtcaaga ccaatgcgga gcatatacgc ccggagtcgt ggcgatcctg caagctccgg 14400

atgcctccgc tcgaagtagc gcgtctgctg ctccatacaa gccaaccacg gcctccagaa 14460

gaagatgttg gcgacctcgt attgggaatc cccgaacatc gcctcgctcc agtcaatgac 14520

cgctgttatg cggccattgt ccgtcaggac attgttggag ccgaaatccg cgtgcacgag 14580

gtgccggact tcggggcagt cctcggccca aagcatcagc tcatcgagag cctgcgcgac 14640

ggacgcactg acggtgtcgt ccatcacagt ttgccagtga tacacatggg gatcagcaat 14700

cgcgcatatg aaatcacgcc atgtagtgta ttgaccgatt ccttgcggtc cgaatgggcc 14760

gaacccgctc gtctggctaa gatcggccgc agcgatcgca tccatagcct ccgcgaccgg 14820

ttgtagaaca gcgggcagtt cggtttcagg caggtcttgc aacgtgacac cctgtgaacg 14880

gcgggagatg caataggtca ggctctcgct aaactcccca atgtcaagca cttccggaat 14940

cgggagcgcg gccgatgcaa agtgccgata aacataacga tctttgtaga aaccatcggc 15000

gcagctattt acccgcagga catatccacg ccctcctaca tcgaagctga aagcacgaga 15060

ttcttcgccc tccgagagct gcatcaggtc ggagacgctg tcgaactttt cgatcagaaa 15120

cttctcgaca gacgtcgcgg tgagttcagg ctttttcata tctcattgcc ccccggatct 15180

gcgaaagctc gagagagata gatttgtaga gagagactgg tgatttcagc gtgtcctctc 15240

caaatgaaat gaacttcctt atatagagga agggtcttgc gaaggatagt gggattgtgc 15300

gtcatccctt acgtcagtgg agatatcaca tcaatccact tgctttgaag acgtggttgg 15360

aacgtcttct ttttccacga tgctcttcgt gggtgggggt ccatctttgg gaccactgtc 15420

ggcagaggca tcttgaacga tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc 15480

accttccttt tctactgtcc ttttgatgaa gtgacagata gctgggcaat ggaatccgag 15540

gaggtttccc gatattaccc tttgttgaaa agtctcaata gccctttggt cttctgagac 15600

tgtatctttg atattcttgg agtagacgag agtgtcgtgc tccaccatgt tcacatcaat 15660

ccacttgctt tgaagacgtg gttggaacgt cttctttttc cacgatgctc ctcgtgggtg 15720

ggggtccatc tttgggacca ctgtcggcag aggcatcttg aacgatagcc tttcctttat 15780

cgcaatgatg gcatttgtag gtgccacctt ccttttctac tgtccttttg atgaagtgac 15840

agatagctgg gcaatggaat ccgaggaggt ttcccgatat taccctttgt tgaaaagtct 15900

caatagccct ttggtcttct gagactgtat ctttgatatt cttggagtag acgagagtgt 15960

cgtgctccac catgttggca agctgctcta gccaatacgc aaaccgcctc tccccgcgcg 16020

ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga 16080

gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat 16140

gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag 16200

ctatgacatg attacgaatt cgagctcggt accgtaacta taacggtcct aaggtagcga 16260

aggatccgct cgctacctta agagaggata tccctccatc ctataatgta ggctatagga 16320

actagggcaa ggccggccat gcggccgcaa gctgggtgca gcgtgacccg gtcgtgcccc 16380

tctctagaga taatgagcat tgcatgtcta agttataaaa aattaccaca tatttttttt 16440

gtcacacttg tttgaagtgc agtttatcta tctttataca tatatttaaa ctttactcta 16500

cgaataatat aatctatagt actacaataa tatcagtgtt ttagagaatc atataaatga 16560

acagttagac atggtctaaa ggacaattga gtattttgac aacaggactc tacagtttta 16620

tctttttagt gtgcatgtgt tctccttttt ttttgcaaat agcttcacct atataatact 16680

tcatccattt tattagtaca tccatttagg gtttagggtt aatggttttt atagactaat 16740

ttttttagta catctatttt attctatttt agcctctaaa ttaagaaaac taaaactcta 16800

ttttagtttt tttatttaat aatttagata taaaatagaa taaaataaag tgactaaaaa 16860

ttaaacaaat accctttaag aaattaaaaa aactaaggaa acatttttct tgtttcgagt 16920

agataatgcc agcctgttaa acgccgtcga cgagtctaac ggacaccaac cagcgaacca 16980

gcagcgtcgc gtcgggccaa gcgaagcaga cggcacggca tctctgtcgc tgcctctgga 17040

cccctctcga gagttccgct ccaccgttgg acttgctccg ctgtcggcat ccagaaattg 17100

cgtggcggag cggcagacgt gagccggcac ggcaggcggc ctcctcctcc tctcacggca 17160

ccggcagcta cgggggattc ctttcccacc gctccttcgc tttcccttcc tcgcccgccg 17220

taataaatag acaccccctc cacaccctct ttccccaacc tcgtgttgtt cggagcgcac 17280

acacacacaa ccagatctcc cccaaatcca cccgtcggca cctccgcttc aaggtacgcc 17340

gctcgtcctc cccccccccc ctctctacct tctctagatc ggcgttccgg tccatggtta 17400

gggcccggta gttctacttc tgttcatgtt tgtgttagat ccgtgtttgt gttagatccg 17460

tgctgctagc gttcgtacac ggatgcgacc tgtacgtcag acacgttctg attgctaact 17520

tgccagtgtt tctctttggg gaatcctggg atggctctag ccgttccgca gacgggatcg 17580

atttcatgat tttttttgtt tcgttgcata gggtttggt 17619

Claims (7)

1. Use of a substance that reduces the activity or content of a protein PtrDJ1C or a substance that inhibits the expression of a nucleic acid molecule encoding said protein PtrDJ1C in any one of the following a-F;

A. changing the color of poplar leaves;

B. changing the green leaves of poplar into the flower spot leaves;

C. cultivating poplar with flower spot leaves;

D. inhibiting the development of poplar leaf chloroplasts;

E. inhibiting development of a chloroplast thylakoid membrane of the poplar leaf;

F. reducing the expression quantity of proteins related to chloroplast development or photosynthesis in poplar leaves;

the protein PtrDJ1C is as follows (1) or (2):

(1) A protein consisting of an amino acid sequence shown as a sequence 1 in a sequence table;

(2) And a protein formed by adding a tag sequence at the tail end of an amino acid sequence shown in a sequence 1 in a sequence table.

2. The use according to claim 1, characterized in that:

the substance is a CRISPR/Cas9 system;

the CRISPR/Cas9 system comprises 1) or 2) as follows:

1) The target point of the sgRNA is 146 th to 165 th positions of a sequence 4;

2) CRISPR/Cas9 vectors expressing the sgrnas.

3. A method of growing a non-green leaf poplar, 1) or 2) or 3) as follows:

1) The method comprises the following steps: firstly, reducing the activity or content of protein PtrDJ1C in poplar with green leaf phenotype to obtain transgenic plants, and selecting the plants with non-green leaf phenotype, namely the poplar plants with non-green leaf phenotype;

2) The method comprises the following steps: firstly, inhibiting the expression of nucleic acid molecules of coding protein PtrDJ1C in poplar with green leaf phenotype to obtain transgenic plants, and selecting plants with non-green leaf phenotype, namely poplar plants with non-green leaf phenotype;

3) The method comprises the following steps: performing gene editing on nucleic acid molecules for encoding the protein PtrDJ1C in poplar with green leaf phenotype to terminate protein translation in advance to obtain a gene editing plant, and selecting a plant with a non-green leaf phenotype, namely a poplar plant with a non-green leaf;

the protein PtrDJ1C is as follows (1) or (2):

(1) A protein consisting of an amino acid sequence shown as a sequence 1 in a sequence table;

(2) And a protein formed by adding a tag sequence at the tail end of an amino acid sequence shown in a sequence 1 in a sequence table.

4. A method according to claim 3, characterized in that:

the gene editing is achieved by: introducing a CRISPR/Cas9 system into the plant of interest;

the CRISPR/Cas9 system comprises 1) or 2) as follows:

1) The target point of the sgRNA is 146 th to 165 th positions of a sequence 4;

2) CRISPR/Cas9 vectors expressing the sgrnas.

5. Use according to claim 1 or 2, characterized in that: the changing of the color of the poplar leaves is to change the green leaves of the poplar into non-green leaves.

Application of PtrDJ1C protein or coding gene thereof in regulation and control of the leaf spot phenotype of poplar;

the protein PtrDJ1C is as follows (1) or (2):

(1) A protein consisting of an amino acid sequence shown as a sequence 1 in a sequence table;

(2) And a protein formed by adding a tag sequence at the tail end of an amino acid sequence shown in a sequence 1 in a sequence table.

Application of PtrDJ1C protein or coding gene thereof in regulation and control of poplar chloroplast development;

the protein PtrDJ1C is as follows (1) or (2):

(1) A protein consisting of an amino acid sequence shown as a sequence 1 in a sequence table;

(2) And a protein formed by adding a tag sequence at the tail end of an amino acid sequence shown in a sequence 1 in a sequence table.