CN108424917B - Maize C-type cytoplasmic male sterile nuclear restoring gene and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a maize C-type cytoplasmic male sterile nuclear restoring gene, which comprises the following components: a nucleotide sequence shown in any one of SEQ ID No. 1-4, or a nucleotide sequence complementary or identical with the nucleotide sequence, an active fragment and the like. The invention also relates to a promoter, an expression polypeptide, a recombinant construct, a host cell of the restoring gene and application of the restoring gene in crop improvement. The maize C-type cytoplasmic male sterile nuclear restoring gene can not only solve the limitation of artificial factors, but also effectively make up for the defects of low accuracy and poor timeliness of the traditional maize breeding selection technology and accelerate the breeding process.

Description

Maize C-type cytoplasmic male sterile nuclear restoring gene and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a maize C-type cytoplasmic male sterile nuclear restoring gene and application thereof.

Background

Corn is one of the most successful crops in utilizing heterosis worldwide, and the production of high-purity and high-quality hybrid corn seeds is an essential prerequisite for ensuring the utilization of heterosis. Because the corn has the biological characteristics of isofloria of hermaphrodite isofloria, the hybrid seed production can be carried out by adopting modes of manual emasculation or mechanical emasculation and the like, but with the continuous increase of labor cost, the manual emasculation cost becomes a key factor of the hybrid seed cost, and the international competitiveness of seed enterprises in China is restricted to a certain extent. In addition, due to untimely and incomplete artificial emasculation, the purity of hybrid seeds is often reduced, and the yield of the corn is influenced to a certain extent. Mechanical detasseling can save labor cost, but because the plants grow in the field inconsistently, the manual detasseling is also required to be checked manually whether the detasseling is complete, so that a part of labor cost is still required to be increased in production. Corn is the first large grain crop in China, the annual sowing area is about 5 hundred million mu, the annual seed demand is about 10 hundred million kilograms, and the annual seed production area is maintained at about 250 thousand mu. Because the local population is less, the labor shortage in the castration season becomes a limiting factor for further development of corn seed production.

The male sterility has the hereditary characteristics of pollen abortion and normal female gamete, and can be divided into two types of cytoplasmic male sterility and nuclear male sterility according to the hereditary characteristics. The abortion of the nuclear male sterility occurs in the meiosis period, and the method has the characteristics of complete abortion, stable sterility and the like, and needs to utilize an endosperm color gene or a yellow-green seedling gene linked with a nuclear sterile gene for early identification in the seed production by utilizing a sterile line, so that the method has higher application risk due to the linked tightness degree and the deviation of artificial identification. In order to utilize the nuclear sterile gene, pioneer in the United states constructs a vector containing gamete lethal, color display and other genes through ingenious design, and introduces the vector into a female parent through genetic transformation to form an SPT (seed Production technology) inbred line. The SPT inbred line is inbred to generate two filial generations of a non-transgenic sterile line and an SPT fertile line in a ratio of 1:1, and the propagation problem of the sterile line is solved and the diffusion of transgenic pollen is effectively avoided through fluorescent screening. Subsequently, the rice develops a corresponding SPT technology, and the technology has wide application prospect in future crossbreeding. However, the expensive large-scale sorting equipment still represents a small investment for many small and medium-sized seed enterprises. Related art development attempts have also been made in corn, and the application thereof has been limited due to relative difficulty in transformation and insufficient related patent reserves.

Corn is a crop which firstly utilizes cytoplasmic male sterile line to produce hybrid seeds, the T-type cytoplasmic male sterile line is utilized for producing the hybrid seeds in a large area in the American corn production in the 50 th century, the hybrid seeds utilized in the American corn production in the 70 th century almost all adopt sterility to produce the seeds, but the specific infection of T microspecium of corn causes the prevalence of microspecium, and the utilization of T-type cytoplasmic male sterility is forced to stop. Turning to research on the utilization of maize for type C and S sterile cytoplasm, maize geneticbreeders reported that 33.9% of sterile cytoplasm, 22.1% of type C and 11.5% of type S, were present in hybrid seeds sold in the last 90S of the united states. The corn C-type cytoplasm sterility has the outstanding characteristics of stable sterility, easy recovery and the like, and can be divided into three sterile cytoplasm types of CI, CII and CIII according to the difference of enzyme digestion polymorphism of mitochondria, wherein the CII and CIII sterile cytoplasm types do not have specific infection on the small blotch C, and become an important type for the production of corn hybrid seeds. Restoration of maize C-type cytoplasmic male sterility is controlled by two pairs of overlapping restorer genes, Rf4 and Rf5, located on chromosome 8 and chromosome 2, respectively, of maize, wherein the restorer gene Rf5 also presents a dominant suppressor gene, Rf-I, which is designated on chromosome 7.

The transformation of the traditional sterile line and the restorer line is mainly identified according to the field phenotype of the corn plant in the pollen scattering period, the transformation can be completed only by 6-8 generations, and the selected timeliness is limited to a certain extent.

Therefore, a new technology is urgently needed to be developed, which can not only solve the limitation of artificial factors, but also make up the defects of low accuracy and poor timeliness of the traditional selection technology, and accelerate the breeding process.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a gene capable of recovering the C-type cytoplasm fertility of corn, which can not only solve the limitation of artificial factors, but also effectively make up the defects of low accuracy and poor timeliness of the traditional corn breeding selection technology and accelerate the breeding process.

In order to solve the technical problems, the invention is realized by the following technical scheme:

the invention provides a maize C-type cytoplasmic male sterile nuclear restoring gene, which comprises a nucleotide sequence of any one of the following:

(1) a nucleotide sequence shown as any one of SEQ ID Nos. 1-4;

(2) a nucleotide sequence that hybridizes under at least moderately stringent conditions to the complement of the nucleotide sequence of (1);

(3) a nucleotide sequence having at least 80% identity to the nucleotide sequence of (1);

(4) a nucleotide sequence encoding the same amino acid sequence as the nucleotide sequence of (1);

(5) a nucleotide sequence encoding one of the following amino acid sequences: an amino acid sequence as set forth in any one of SEQ ID Nos. 7 to 8, or an amino acid sequence which differs from SEQ ID Nos. 7 to 8 due to substitution, deletion and/or insertion of one or more amino acid residues, or an amino acid sequence which has at least 70% identity to an amino acid sequence as set forth in any one of SEQ ID Nos. 7 to 8;

(6) an active fragment of the nucleotide sequence of any one of (1) to (5);

(7) a nucleotide sequence complementary to any one of the nucleotide sequences (1) to (5);

(8) and other nucleotide sequences different from SEQ ID Nos. 3 to 4 are generated by selectively cutting and selecting different transcription start sites or transcription termination sites from SEQ ID Nos. 3 to 4.

Wherein, one cDNA sequence of Rf4 and Rf4 is shown as SEQ ID No. 1-2, the nucleotide sequence of the related promoter is shown as SEQ ID No. 5-6, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 7-8. See table 1 below for details.

Table 1 SEQ ID No: 1-8 sequence names and sources thereof

Preferably, the maize C-type cytoplasmic male sterile nuclear restorer gene of the invention is shown in SEQ ID No: 1 is shown.

The invention also provides a promoter of the maize C-type cytoplasmic male sterile nuclear restorer gene, which comprises a nucleotide sequence of any one of the following;

(1) SEQ ID No: 5-6 of any one of the nucleotide sequences shown in the specification;

(2) a nucleotide sequence that hybridizes under at least moderately stringent conditions to the complement of the nucleotide sequence of (1);

(3) a nucleotide sequence having at least 80% identity to the nucleotide sequence of (1);

(4) an active fragment of the nucleotide sequence of any one of (1) to (3);

(5) a nucleotide sequence complementary to the nucleotide sequence of any one of (1) to (4);

(6) consisting of SEQ ID No: 3. 4 different transcription initiation results in the amino acid sequence of SEQ ID No: 5. 6 correspondingly varied nucleotide sequences.

The invention also provides a construct comprising the promoter sequence and a cell comprising the construct, wherein the cell is a plant cell, preferably a maize cell.

The invention also provides a polypeptide encoded by the restorer gene of the invention, comprising an amino acid sequence of any one of:

(1) SEQ ID No: 7. 8, or a pharmaceutically acceptable salt thereof;

(2) a substitution, deletion and/or insertion with one or more amino acid disabilities as compared to SEQ ID No: 7. 8 different amino acid sequences;

(3) and SEQ ID No: 7. 8, an amino acid sequence having at least 70% identity to the amino acid sequence set forth in any one of seq id nos;

(4) an active fragment of the amino acid sequence of (1) or (2) or (3);

(5) the amino acid sequence encoded by the restorer gene of the present invention.

Preferably, the protein has two conserved domains of SBP and ANK; or SBP conserved domain alone.

The invention also relates to a recombinant construct comprising the restorer gene of the invention. Wherein the vector used for the construct is a cloning vector or an expression vector for expressing the restorer gene.

The invention also relates to a recombinant host cell comprising a recombinant construct according to the invention or having integrated in its genome a restorer gene according to the invention. The host cell may be selected from plant cells or microbial cells, such as e.coli cells or agrobacterium cells, preferably plant cells, most preferably corn cells. The cell may be isolated, ex vivo, cultured or part of a plant.

The invention also relates to methods of improving a trait (e.g., generating or knocking out restoration of cytoplasmic C male sterility in a plant) in a plant, the method comprising preparing a plant comprising a restorer gene of the invention, e.g., the method may comprise: transgenic crop plants are obtained by regenerating a transgenic plant from a recombinant plant cell containing a restorer gene of the present invention or crossing a plant containing a restorer gene of the present invention with another plant, or transfecting a crop plant with a recombinant agrobacterium cell containing a restorer gene of the present invention. The trait includes the production or knockout of crop plants for restoration of cytoplasmic male sterility type C. Wherein the plant is preferably a plant which is not negatively affected by other traits than restored function, wherein the crop is preferably a crop plant, such as maize.

The present invention also relates to a method of improving a crop, the method comprising: transfecting a crop plant with a recombinant agrobacterium cell containing a restoring gene to obtain a transgenic crop plant, or adjusting the expression level of the restoring gene in the crop plant in a proper amount, or changing the biological activity of the restoring protein in a proper amount, or crossing a plant containing the restoring gene of the invention with another plant; wherein the plant is preferably a plant which is not negatively affected by other traits than restored function, wherein the crop plant is preferably a crop plant, such as maize.

The method for breeding the maize inbred line having restored maize C-type cytoplasmic male sterility fertility can be performed by: crossing a maize plant containing the restorer gene and allelic variation thereof with another maize plant, and carrying out continuous backcross to obtain a maize inbred line plant containing the restorer gene; or transfecting a maize plant with a recombinant host cell (e.g., an agrobacterium cell) comprising the restorer gene to obtain a transgenic maize plant containing the restorer gene.

The invention has the following positive and beneficial effects:

through intensive research, the invention determines the nucleotide sequence of the maize C-type cytoplasmic male sterile nuclear restorer gene and the expressed amino acid sequence thereof. The sequence of the invention can facilitate the creation of three-line matching materials of corn hybrid, shorten the breeding period, reduce the breeding cost and improve the seed production efficiency.

Drawings

FIG. 1 shows the results of screening of primer X-26 in M1, M2, M3, M4, and M5 super pools, 87-1: and (4) positive control.

FIG. 2 results of primer X-26 screening in the BAC subpool.

FIG. 3 Single clones screened for primer X-26.

FIG. 4 shows that the selected single clones were verified by linkage markers.

FIG. 5 Gene prediction results.

FIG. 6 Gene prediction results (continuation).

FIG. 7 real-time analysis of the differences in expression of gene No. 9 (A, B, R denotes sterile, maintainer and restorer lines respectively; the same numbers following the letters denote the corresponding developmental stages).

FIG. 8 schematic representation of the pCUB-Rf4 vector.

FIG. 9 test cross result of positive plant transformed by pCUB-Rf4 vector and sterile line.

FIG. 10 schematic diagram of the pBLUE411-Rf4 vector.

FIG. 11 shows the result of editing site detection of CRISPR/Cas9 transformed test cross sterile plants.

FIG. 12 CRISPR/Cas9 vector transformation for the test cross sterile phenotype.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It is to be understood that the following examples are intended only to further illustrate the present invention and are not intended to limit the spirit and scope of the present invention.

It should be noted that, unless otherwise specified, the reagents, enzymes and the like used in the following examples are those commercially available from reagent companies as analytical grade reagents or enzymes.

The present inventors have determined, through intensive studies, a fertility restorer gene Rf4 for maize C-type cytoplasm, which is located at the end of the short arm of chromosome eight. In addition to differences in coding regions, the promoter regions of the genes differ by virtue of different transcription initiation. Embodiments of the invention are developed based on their restored function to the C-type cytoplasm of maize.

Plant transformation

In a particularly preferred embodiment, at least one protein of the invention that restores fertility to maize C-type cytoplasmic male sterility is expressed in a higher organism, such as a plant. The nucleotide sequence of the restorer gene Rf4 of the present invention may be inserted into an expression cassette, which is then preferably stably integrated into the plant genome. In another preferred embodiment, the nucleotide sequence of the restorer gene Rf4 is comprised in a non-pathogenic self-replicating virus. Plants transformed according to the present invention may be monocotyledonous or dicotyledonous plants including, but not limited to, maize, wheat, barley, rye, sweet potato, beans, peas, chicory, lettuce, cabbage, cauliflower, onion, garlic, squash, apple, pear, strawberry, pineapple, tomato, sorghum, sunflower, rapeseed rape, carrot, rice, eggplant, cucumber, arabidopsis. Particularly preferred are corn, rice, wheat, barley.

Once the desired nucleotide sequence has been transformed into a particular plant species, it may be propagated in that species or transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques.

Preferably, the nucleotide sequences according to the invention are expressed in transgenic plants, which result in the biosynthesis of the corresponding restored functional proteins in the transgenic plants. In this way, transgenic plants with restored function can be produced. In order to express the nucleotide sequence of the present invention in transgenic plants, the nucleotide sequence of the present invention may need to be modified and optimized. All organisms have a particular codon usage preference, which is known in the art, and the codons can be changed to conform to plant preferences while maintaining the amino acids encoded by the nucleotide sequences of the present invention. Moreover, high levels of expression in plants can best be achieved from coding sequences having at least about 35%, preferably more than about 45%, more preferably more than 50%, and most preferably more than about 60% GC content.

Example 1: acquisition of maize C-type cytoplasmic male sterility restorer gene Rf4 sequence

The restorer gene Rf4 derived from A618 was selfed after 8 generations of continuous backcrossing, and finally the Rf4 gene was introduced into the background of selfed line Yu 87-1. And (3) constructing a BAC library by taking Cms-es87-1Rf4Rf4 as a material. The clone of the recovery region of Rf4 was selected from the maize inbred line 87-1BAC mixed pool by PCR (see Table 1 for reaction system and Table 2 for reaction program). The composition of the maize inbred line 87-1BAC pool is 1296 subpools and 16 super pools. Screening, obtaining a subpool where a positive target clone is located through two PCR reactions, taking 1 mu L of bacterial liquid in the subpool, diluting, coating a plate, selecting a monoclonal to be cultured in a 96-hole cell culture plate, then respectively taking part of the bacterial liquid of 96 monoclonal of each plate and merging the part of the bacterial liquid into a centrifugal tube, finding the plate where the positive clone is located through the PCR reaction, then respectively taking part of the bacterial liquid of each plate transversely crossing 12 holes and longitudinally crossing 8 holes and merging the part of the bacterial liquid into a centrifugal tube to obtain 20 in total, and then obtaining the target positive clone through one-time PCR reaction. Positive clones continued to be verified by closely linked markers (fig. 1-4). The final positive clone entrusted the company to test through the sequence. Sequencing result analysis shows that the physical distance between the two markers X-21-1 and X-33 which are closest to the target gene is about 45000Nt, 9 possible gene regions are shared between the two markers through gene function prediction, and 2, 5 and 8 have no functional annotation; 1. 3 is related to retrotransposon; 4 a protein kinase; 6 is a related protein that regulates the cytoskeleton; 7 is a histone like protein; 9 is a promoter binding protein (FIGS. 5, 6).

The 9 gene transcript was subjected to expression analysis by using real-time, and the 9 gene had significant difference between fertile sterile lines (fig. 7), and was determined to be a candidate gene of the restored gene Rf 4.

TABLE 2 PCR reaction System

TABLE 3 PCR reaction procedure

Labeling the primer sequence:

x-26F：5'-GGGATTTGAACGACTGGAGGT-3'

x-26R：5'-TCGTCTCCTCAGTGTTCCGC-3'

x-33F：5'-CACACACGAGGGGAGGAGGT-3'

x-33R：5'-AAGGTCTTTGTTGTCGGGCA-3'

x-21-1F：5'-TTTCTTCTCAGGGTGGTCTTGC-3'

x-21-1R：5'-CACCTTTCCACCTCGCCTATT-3'。

example 2: rf4 corn transgenic experiment and transgenic material fertility analysis

In this example, the restorer Cms-es87-1Rf4Rf4 binary anther was used as a material, and the full-length cDNA of Rf4 gene was obtained by reverse transcription and amplification.

(1) Construction and transformation of Rf4 overexpression transgenic vector

Primers related to the cDNA sequence of Rf4 Gene:

CUB-BamH1-F: 5’-CGCGGATCCATGGAAGCCGGTTTCCTGTG-3’

CUB-Sma1-R:5’-TCCCCCGGGTCACTTATCGTCGTCATCCTTGTAATCATTGGGGCCCCAACG-3’

the full-length cDNA of Rf4 gene was used as a template to amplify a full-length Rf4 sequence, and the PCR product was digested with SalI and Kpn to recover a target fragment, which was ligated to a pCUB vector recovered by digestion with the same two enzymes, to construct an overexpression vector pCUB-Rf4 (FIG. 8). The vector is introduced into a maize transformation receptor inbred line C01 by an agrobacterium-mediated transformation method. The transgenic positive plants are subjected to test crossing with a Cms-es87-1rf4rf4 sterile line, and the result shows that the transgenic positive plants containing the construct can restore the fertility of the maize C-type cytoplasmic male sterile line (figure 9).

(2) Construction and transformation of Rf4 editing vectors

Based on the amino acid sequence corresponding to the CDS sequence of Rf4 gene, SBP, ANK and TM are predicted to be present in the functional domain of Rf4 gene. Therefore, aiming at the nucleotide sequences corresponding to the two functional domains of SBP and ANK, the following target sequences are involved according to the requirements of constructing the CRISPR vector:

SBP1:5‘-CAGAGAGGGCGGAACACTGAGG-3’

SBP2:5‘-CACGTCTGGCACAACATAATGG-3’

ANK1: 5‘-AAATCTTGCAACAGGGAAGCGG-3’

ANK2: 5‘-TTGACAGTTCAGTCATCGCAGG-3’

synthesizing corresponding oligonucleotide primer sequence and the criprpr vector according to the target sequence for PCR amplification, purifying and recovering the amplification product, and establishing a BsaI-T4Ligase enzyme digestion link system.

TABLE 4 enzyme digestion linking System

5 mu L of the ligation product was used to transform E.coli and screened on Kan plates. Positive clones were identified by colony PCR and confirmed by sequencing (fig. 10). The vector is introduced into a maize transformation receptor inbred line C01 by an agrobacterium-mediated transformation method. The transgenic positive plant and the Cms-es87-1Rf4Rf4 sterile line are subjected to test cross, and a positive sterile individual is screened for PCR sequencing detection, so that the SBP region of the target gene Rf4 is edited (figure 11), and the Rf4 recovery function is lost (figure 12).

The above examples show that the restorer gene of the present invention has a sequence which is the authentic sequence of the Rf4 gene and has fertility for restoring maize C-type cytoplasmic male sterility.

Sequence listing

<110> Henan university of agriculture

<120> maize C-type cytoplasmic male sterile nuclear restoring gene and application thereof

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ctagccgccg gggccgaacg aagtcgggga tgtaattgaa tccagcgatc tttggatgct 180

cgtcatacgt gccaacagca gggtcaattt ccctcgaaag agaagaccga ggaaatggaa 240

gccggtttcc tgtggaaaga gatcggtgac caagccggtg cggcgagaat gtccggtggg 300

aacaagaaca ggagcctcga ctgggatttg aacgactgga ggtgggacgc caacctgttc 360

ctggccacac cagcggccgc cgcgccatcc gagtccatca gcagggagct gagccgaggt 420

caggggagat cgatttcggc gtcgtcgttg acaagaggcg gcggctctca ccagaggagg 480

acggcagcgg cgggtgcagc aattctgcgg tagcagatgg agacaacaat cacgtggtgt 540

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ccactccttc ttgccaggtt gacggctgcc aagctgatct tagtggtgcc agggactacc 660

ataagaggca caaggtgtgt gaagcgcata ccaggacaac agtggtctgc atcaataatg 720

tagagcatcg gttctgtcag cagtgcagca ggtttcacct tcttcacgaa tttgatgatg 780

gtaagaagag ctgccgatca cgtctggcac aacataatgg aaggagaagg aaagttcaac 840

cgcaacctgc tgtgaacggg aattccatga atgaagatca gtctctaagt agcaccttat 900

tccttctgtt aaaacaactt tccgggctcg agtctggtag ctcttctgag caaatcaacc 960

atcctaattc tttggttaac cttttgaaga accttgctgc tattgctagc acacatgcat 1020

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atggctctat aatgcatgag caaaccatac ggtcaattcc tgtcaggaga gaatcattag 1140

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aagaagctga gagccgaaca gataaaattg tgttcaaact ctttggaaaa gagccaaaag 1260

attttcctgt agatctacgg gaacagatcc taaactggtt gtcgcattat ccaactgata 1320

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cagggggaga tcgatttcgg cgtcgtcgtt gacaagaggc ggcggctctc accagaggag 480

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gtagagcatc ggttctgtca gcagtgcagc aggtttcacc ttcttcacga atttgatgat 780

ggtaagaaga gctgccgatc acgtctggca caacataatg gaaggagaag gaaagttcaa 840

ccgcaacctg ctgtgaacgg gaattccatg aatgaagatc agtctctaag tagcacctta 900

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gattttcctg tagatctacg ggaacagatc ctaaactggt tgtcgcatta tccaactgat 1320

atggaaagtt atattagacc tggttgtgtt attctaacta tttaccttca cctccctaat 1380

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tatttaattc aagaagagac acaaatgcta cttgaagatt caactatgca gcaaggccct 1740

caatgcctga ccttctcttg ttcctttcct tgtacaagtg gaagaggatt catagcagat 1800

tgaagactat gatcaaagca gcctttctgt tccctttgtt gtcacggaca aagatgtatg 1860

ttctgagatt cggatgttgg agcatggatt ggatttagtt tcatttgatc aaacctccaa 1920

aagaatagat gatctgatga tttatcgcag tcgagcatta cattttttgc atgaaatcgg 1980

atggcttctt caaaggagcc atgtgcgagc tacgtctgag cagcgacaat attgtcctga 2040

ccgcttccct gttgcaagat ttagatggct gctatccttt gcagttgatc aggaatggtg 2100

tgctgttcta aggaagcttc tgaacaccat gttccagggt gatattgatg tattgtcacc 2160

aattgaattt gccttgggag aaaatctatt gcatactgcg gtcaaaaaac gctcaaagcc 2220

tttagttgaa tttctattaa gatacaccac aacaaatatt gctccagtgg gcggtggaga 2280

tggtgctcca gttcagttct tgttcactcc tgcgatgact gaactgtcaa atatcacacc 2340

tcttcatatt gcagccacaa tcagtgatgc tattggtgtt ttagatgctt taactgatga 2400

tcctcagcag ttgggaatca aagcgtggaa gaatgctcgt gacgccactg gcttcactcc 2460

tgaggaatat gctgcaaaga gaggcaacat atcctatatt caaatggtac aggacaaaat 2520

tgacagaagg gtgaccagag ctcatgtctc ggttaccatc cccagcacaa ttgatactgt 2580

cggaaagcat ggtagccgaa tgaagcctgc cgatcaaatc acatttggtg ttgagaaaaa 2640

acaactaagc atcaaccaaa cattgagctg cagacagtgt gtccagcagg cccagcagct 2700

tgcattccat ccccggacaa ataggtttct gtctaatagg actgcgatgc tttccttggt 2760

ctccattgct gccgtctgcg tctgtgtggg attgatcatg aagagcctgc cacaagttgg 2820

ttgtatgaag cctttcctct gggacaatat acgttggggc cccaattgat agactgcaga 2880

agagccagcc cgattgtatc agtgtatgat gaagatgttt gcgccgagcg aagctagttt 2940

cttgtaatgt acatgtttct tatactaaat gtctaactta tttctgttta tagtaaaacc 3000

tgtgaacttg cctagcttgt aactccattg catttgcatc ctgactgaag taaagctttt 3060

acttgttttg gttgagag 3078

<210> 3

<211> 6273

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aacccacggt acccaatact tgactcggct ctctccatga gctactgttc catctccatt 60

tcttcaatca tttctcgcct ttcatgttca agggcgttgc ttgccaacca cggtatcgtt 120

ctagccgccg gggccgaacg aaggtacttc ttggttcttg tactcttcat tagtttcagg 180

atttgtccca agtcccaacc cagggttggc aatccctttg tgttcgtgtt ggtgttcgta 240

tctaattccc tgtgtttgtt tcggctgtag tcggggatgt aattgaatcc agcgatcttt 300

ggatgctcgt catacgtgcc aacagcaggg tcaatttccc tcgaaagaga agaccgagga 360

aatggaagcc ggtttcctgt ggaaagagat cggtgaccaa gccggtgcgg cgagaatgtc 420

cggtgggaac aagaacagga gcctcgactg ggatttgaac gactggaggt gggacgccaa 480

cctgttcctg gccacaccag cggccgccgc gccatccgag tccatcagca gggagctgag 540

ccgaggtcag ggggagatcg atttcggcgt cgtcgttgac aagaggcggc ggctctcacc 600

agaggaggac ggcagcggcg ggtgcagcaa ttctgcggta gcagatggag acaacaatca 660

cgtggtgtgt gttcagagag ggcggaacac tgaggagacg agacccagga aaggtgccaa 720

ttcgagcacc actccttctt gccaggttga cggctgccaa gctgatctta gtggtgccag 780

ggactaccat aagaggcaca aggtgtgtga agcgcatacc aggacaacag tggtctgcat 840

caataatgta gagcatcggt tctgtcagca gtgcagcagg ttggtcacca atttcgttgt 900

gagattacag ctgctgaatt ttttttctcc agcaagagtc ctgtgcccta cctgtattgg 960

agaaagaaaa agaaacactt ctccggtaca aaacccaaag ggggaaataa aagagataaa 1020

aaggctcatt accttggtgt cacaggcaca caacacaaac gaattactga tcgttaacct 1080

tgagaaatgc tgaacacatt agtttttttt gctccatgat aggatccgtt cataaataag 1140

ttggtgtagg tcagagactc agagttgaaa cccaaccgaa agcacaagtc taaggttcaa 1200

ccacttggat agttgttttt catgcactct tatttaagac taaagcaaac tttatatatt 1260

ccatcctttc aagtcttttt tactgtttct ttttatgtca actttggtct tctcttgtct 1320

ctgttcccat tgctatcccc ttaggatccc actaggcact agtgccttta gaggtcttcg 1380

ttctacgtgt ctgaaccatc tcaaccggtg ttggacaagt ttttctttaa ttggtgccac 1440

ccctagccta tgacatcata gttataggct caatctcttc ttgttcgacc acaaattcat 1500

tatcgcatac atatttccgc aatagttatc tgttggacat gtcatatttt tgtaggccaa 1560

cattttgcat cgtacaacat agcagaagaa aaaaaaccaa acattgctct ttcttctaca 1620

tacatcttaa ctttagcagc ctatcatgat attgagttta gcataaaact tgaaattcat 1680

attttaatac accgagcatt ttttatgttt cctttcattt gctcatcagc tgaagaaagt 1740

aaataatttg tatgccattt taggtttcac cttcttcacg aatttgatga tggtaagaag 1800

agctgccgat cacgtctggc acaacataat ggaaggagaa ggaaagttca accgcaacct 1860

gctgtgaacg ggaattccat gaatgaagat cagtctctaa gtagcacctt attccttctg 1920

ttaaaacaac tttccgggct cgagtgtaag ttctatacaa aatttgatta ccaatttaaa 1980

atacaaatat ttcaaacgac atgcaatcca gacagcaatt ctaactgtgc gcttgtgtgg 2040

atgacagctg gtagctcttc tgagcaaatc aaccatccta attctttggt taaccttttg 2100

aagaaccttg ctgctattgc tagcacacat gcatatcaag atgttctaaa gaatgcaact 2160

tcaatatcat caaatgatgg taataatgct gcaaatggct ctataatgca tgagcaaacc 2220

atacggtcaa ttcctgtcag gagagaatca ttagcaggta aagcacttta tcgtttactt 2280

gattctattt agcatgtcaa atatcaaagg tataaataaa atgatttgaa taacttgtgt 2340

tctcagaaga gcctgcggtg aaaagacgag tacaggactt tgatttgaac gattcatgca 2400

ttgaagaagc tgaggtaaat gtttagtaaa tttcataaac tacaccaatc ttggacaaac 2460

tatcgcaaaa atacactttt tccaaaccaa tctaaaaaac tgtctcagct atattttgag 2520

aagcaacttc atggaattgc atttctattg tccagacaaa tcacagaaaa tacacaatta 2580

ttgccattca atatcacaaa actacacatt ttagctctta aaaactgttg tttttcgata 2640

caaattaacc acaaatgtgt agtctaatga tactatttga caatagaagt ataattttat 2700

ggaaatggtt tttaaaagtg tagctttgtg aaagtagttt gaagtgtagt ttttttttct 2760

cgaacaacac aggagatttg cgtgtcattt catttaagga gagaaaaaac aagaggtctg 2820

gcaaggccag accgattaca aaaggaacac aacaaaaccc acaactcaga cgagagtcct 2880

caaccacgac caacaaaaag aacaccctca agaaaactca aactaaacca tctagcgacc 2940

caaccaggaa ctcctgtagc tttgaagctc tagccatgca ccacattttg cattcttcta 3000

caatcttcct tagaagatca aatatacacg gtcttccatt atcgaaaaca cacagtgctt 3060

ccaaatttcc caggcagcaa gaatgatcag agagttcagc cccctatgcc ttccctttgg 3120

cgtcaaacga accaccttgc tccaccactt agggaaagaa taatcatcca gagcgggaga 3180

tagacgtgga atgtggagtg cctagaacat atgaaaccaa aaacacagtt atttcggtgc 3240

ttccaaattt cccaggcagc aaggatgatc agagagttca gccccctacg ccttcccttt 3300

ggcgtcaaac gaaccacctc gctccaccac ttagggaaag aataatcatc caaagtggga 3360

gatagacctg gaatgtggat gtgaagtgta gttactcagg ttttccaatc ttagtgtaat 3420

tttgtggaag ttactctaga atttgtagat agattctccc agatgtattt ctgatgtttg 3480

attccgactt aatatctctt atgcacaaca tttgcagagc cgaacagata aaattgtgtt 3540

caaactcttt ggaaaagagc caaaagattt tcctgtagat ctacgggaac aggtcagtta 3600

tcatgttaca aactggaatc atatttttct attaactttg tcaatgtgga tattttaata 3660

cgctaatgct aattctttta ttaaatggtt tgaatttaca gatcctaaac tggttgtcgc 3720

attatccaac tgatatggaa agttatatta gacctggttg tgttattcta actatttacc 3780

ttcacctccc taattggatg tgggatgagg tagtattgca ctacattaac agtgaattta 3840

gatctgtata tgttttcttt atagtatatg atgcagaagc aaagtgctta ctgttatttt 3900

tcttttctat tatagtttaa tgatgaccca gcttcatgga tagaaaatct tattagctta 3960

tccaatgatg gattctggag aacaggatgg ttgtatgcta gggtacagga ctgcctaaca 4020

ctgagttgca atggtccgtt catcttattt ggagtagtta ttcatatatt cgaggatcag 4080

cttcatttgt atttctttcc ttatttttgt taggtagtct tatgtttgca tctccctggc 4140

aaccggtaat tggtgacaag catcagagac tgtgtgtaac tccaattgca gttgattgtt 4200

cttcgtcagt aaaattctct gtgaaaggtt tcaatatagt tcagccaacc acaaagtatg 4260

tatctttttt cacatttaaa tgtctcctct aaacttactt ctttagttct tttaactgac 4320

acattctgtt tttgttatag attactttgt gtgtttgatg aaaaatattt aattcaagaa 4380

gagacacaaa tgctacttga agattcaact atgcagcaag gccctcaatg cctgaccttc 4440

tcttgttcct ttccttgtac aagtggaaga ggattcatag aggtacaatg tttgtatatt 4500

ttttatatcc tcattttttg tgtatattac agacttgact ccgttttcag attgaagact 4560

atgatcaaag cagcctttct gttccctttg ttgtcacgga caaagatgta tgttctgaga 4620

ttcggatgtt ggagcatgga ttggatttag tttcatttga tcaaacctcc aaaagaatag 4680

atgatctgat gatttatcgc agtcgagcat tacatttttt gcatgaaatc ggatggcttc 4740

ttcaaaggag ccatgtgcga gctacgtctg agcagcgaca atattgtcct gaccgcttcc 4800

ctgttgcaag atttagatgg ctgctatcct ttgcagttga tcaggaatgg tgtgctgttc 4860

taaggaagct tctgaacacc atgttccagg gtgatattga tgtattgtca ccaattgaat 4920

ttgccttggg agaaaatcta ttgcatactg cggtcaaaaa acgctcaaag cctttagttg 4980

aatttctatt aagatacacc acaacaaata ttgctccagt gggcggtgga gatggtgctc 5040

cagttcagtt cttgttcact cctgcgatga ctgaactgtc aaatatcaca cctcttcata 5100

ttgcagccac aatcagtgat gctattggtg ttttagatgc tttaactgat gatcctcagc 5160

aggtaactac tctaaattgc catgttctta cctgttgtag cctgctttaa ctaaaaaaaa 5220

gcacatctac attcaaattt caaagtagca gatttccgat tcttagtgta aaacagttaa 5280

aataaaaact aagaccacca tttattgtta gaacctaagt ctgcacctga aatattttca 5340

tctgaaatgc attatgtaca tatcaagaaa aagaaaggac ttccatcaaa acattggttt 5400

gtgtattaca ttgattgttg aatagatcgt gtagggtgta taatatcaca tcagagttcc 5460

gcatgaggtg tagtatgata aaaattcatt taccttaaaa acattttctt ttacttttga 5520

gaatttgtgc aagcactgaa acatttgctg tctggcttct ttctgcctaa atgtctcagt 5580

tgggaatcaa agcgtggaag aaagctcgtg acgccactgg ctttactcct gaggaatatg 5640

ctgcaaagag aggcaacata tcctatattc aaatggtaca ggacaaaatt gacagaaggg 5700

tgaccagagc tcatgtctcg gttaccatgc ccagcacaat tgatactgtc ggaaagcatg 5760

ctagccgaat gaagcctgcc gatcaaatca catttggtgt tgagaaaaaa caactaagca 5820

tcaaccaaac attgagctgc agacagtgtg tccagcaggc ccagcagctt gcattccatc 5880

cccgaacaaa taggtttctg tctaatagga ctgcgatgct ttccttggtc tccattgctg 5940

ccgtctgcgt ctgtgtggga ttgatcatga agagcctgcc acaagttggt tgtatgaagc 6000

ctttcctctg ggacaatata cgttggggcc ccaattgata gactgcagaa gagccagccc 6060

gattgtatca gtgtatgatg aagatgtttg cgccgagcga agctagtttc ttgtaatgta 6120

catgtttctt atactaaatg tctaacttat ttctgtttat agtaaaacct gtgaacttgc 6180

ctagcttgta actccattgc atttgcatcc tgactgaagt aaagctttta cttgttttgg 6240

ttgagaggaa gaaaaccttt tacttctaaa ttc 6273

<210> 4

<211> 6257

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aacccacggt acccaatact tgactcggct ctctccatga gctactgttc catctccatt 60

tcttcaatca tttctcgcct ttcatgttca agggcgttgc ttgccaacca cggtatcgtt 120

ctagccgccg gggccgaacg aaggtacttc ttggttcttg tactcttcag tagtttcagg 180

attttgtccc aagtcccaac ccagggttgg cagtcccttt gtgttcgtgt tggtgttcgt 240

atctaattcc ctgtgtttgt ttcggctgta gtcggggatg taattgaatc cagcgatctt 300

tggatgctcg tcatacgtgc caacagcagg gtcaatttcc ctcgaaagag aaggccgagg 360

aaatggaagc cggtttcctg tggaaagaga tcggtgacca agccggtgcg gcgagaatgt 420

ccggtgggaa caagaacagg agcctcgact gggatttgaa cgactggagg tgggacgcca 480

acctgttcct ggccacacca gcggccgccg cgccatccga gtccatcagc agggagctga 540

gccgaggtca gggggagatc gatttcggcg tcgtcgttga caagaggcgg cggctctcac 600

cagaggagga cggcagcggt gggtgcagca attctgcggt agcagatgga gacaacagtc 660

acgtggtgtg tgttcagaga gggcggaaca ctgaggagac gagacccagg aaaggtgcca 720

attcgagcac cactccttct tgccaggttg acggctgcca agctgatctt agtggtgcca 780

gggactacca taagaggcac aaggtgtgtg aagcgcatac caggacaaca gtggtctgca 840

tcaataatgt agagcatcgg ttctgtcagc agtgcagcag gttggtcacc aatttcgttg 900

tgagattaca gctgctgaat tttttttctc cagcaagagt cctgtgccct acctgtattg 960

gagaaagaaa aagaaacact tctccggtac aaaacccaaa gggggaaata aaagagataa 1020

aaaggctcat taccttggtg tcacaggcac acaacacaaa cgaattactg atcgttaacc 1080

ttgagaaatg ctgaacacat tagttttttt tgctccatga taggatccgt tcataaataa 1140

gttggtgtag gtcagagact cagagttgaa acccaaccga aagcacaagt ctaaggttca 1200

accacttgga tagttgtttt tcatgcactc ttatttaaga ctaaagcaaa ctttatatat 1260

tccatccttt caagtctttt ttactgtttc tttttatgtc aactttggtc ttctcttgtc 1320

tctgttccca ttgctatccc cttaggatcc cactaggcac tagtgccttt agaggtcttc 1380

gttctacgtg tctgaaccat ctcaaccggt gttggacaag tttttcttta attggtgcta 1440

cccctagcct atgacatcat agttataggc tcaatctctt cttgttcgac cacaaattca 1500

ttatcgcata catatttccg caatagttat ctgttggaca tgtcatattt ttgtaggcca 1560

acattttgca tcgtacaaca tagcagaaga aaaaaaacca aacattgctc tttcttctac 1620

atacatctta actttagcag cctatcatga tattgagttt agcataaaac ttgaaattca 1680

tattttaata caccgagcat tttttatgtt tcctttcatt tgctcatcag ctgaagaaag 1740

taaataattt gtatgccatt ttaggtttca ccttcttcac gaatttgatg atggtaagaa 1800

gagctgccga tcacgtctgg cacaacataa tggaaggaga aggaaagttc aaccgcaacc 1860

tgctgtgaac gggaattcca tgaatgaaga tcagtctcta agtagcacct tattccttct 1920

gttaaaacaa ctttccgggc tcgagtgtaa gttctataca aaatttgatt accaatttaa 1980

aatacaaata tttcaaacga catgcaatcc agacagcaat tctaactgtg cgcttgtgtg 2040

gatgacagct ggtagctctt ctgagcaaat caaccatcct aattctttgg ttaacctttt 2100

gaagaacctt gctgctattg ctagcacaca tgcatatcaa gatgttctaa agaatgcaac 2160

ttcaatatca tcaaatgatg gtaataatgc tgcaaatggc tctataatgc atgagcaaac 2220

catacggtca attcctgtca ggagagaatc attagcaggt aaagcacttt atcgtttact 2280

tgattctatt tagcatgtca aatatcaaag gtataaataa aatgatttga ataacttgtg 2340

ttctcagaag agcctgcggt gaaaagacga gtacaggact ttgatttgaa cgattcatgc 2400

attgaagaag ccgaggtaaa tgtttagtaa atttcataaa ctacaccaat cttggacaaa 2460

ctatcgcaaa aatacacttt ttccaaacca atctaaaaaa ctgtctcagc tatattttga 2520

gaagcaactt catggaattg catttctatt gtccagacaa atcacagaaa atacacaatt 2580

attgccattc aatatcacaa aactacacat tttagctctt aaaaactgtt gtttttcgat 2640

acaaattaac cacaaatgtg tagtctaatg atactatttg acaatagaag tataatatta 2700

tggaaatggt ttttaaaagt gtagctttgt gaaagtagtt tgaagtgtag tttttttttt 2760

ctcgaacaac acaggagatt tgcgtgtcat ttcatttaag gagagaaaaa acaagaggtc 2820

tggcaaggcc agaccgatta caaaaggaac acaacaaaac ccacaactca gacgagagtc 2880

ctcaaccacg accaacaaaa agaacaccct caagaaaact caaactaaac catctagcga 2940

cccaaccagg aactcctgta gctttgaagc tctagccatg caccacattt tgcattcttc 3000

tacaatcttc cttagaagat caaatataca cggtcttcca ttatcgaaaa cacacagtgc 3060

ttccaaattt cccaggcagc aagaatgatc agagagttca gccccctatg ccttcccttt 3120

ggcgtcaaac gaaccacctt gctccaccac ttagggaaag aataatcatc cagagcggga 3180

gatagacgtg gaatgtggag tgcctagaac atatgaaacc aaaaacacag ttatttcggt 3240

gcttccaaat ttcccaggca gcaaggatga tcagagagtt cagcccccta cgccttccct 3300

ttggcgtcaa acgaaccacc tcgctccacc acttagggaa agaataatca tccaaagtgg 3360

gagatagacc tggaatgtgg atgtgaagtg tagttactca ggttttccaa tcttagtgta 3420

attttgtgga agttactcta gaatttgtag atagattctc ccagatgtat ttctgatgtt 3480

tgattccgac ttaatatctc ttatgcacaa catttgcaga gccgaacaga taaaattgtg 3540

ttcaaactct ttggaaaaga gccaaaagat tttcctgtag atctacggga acaggtcagt 3600

tatcatgtta caaactggaa tcatattttt ctattaactt tgtcaatgtg gatattttaa 3660

tacgctaatg ctaattcttt tattaaatgg tttgaattta cagatcctaa actggttgtc 3720

gcattatcca actgatatgg aaagttatat tagacctggt tgtgttattc taactattta 3780

ccttcacctc cctaattgga tgtgggatga ggtagtattg cactacatta acagtgaatt 3840

tagatctgta tatgttttct ttatagtata tgatgcagaa gcaaagtgct tactgttatt 3900

tttcttttct attatagttt aatgatgacc cagcttcatg gatagaaaat cttattagct 3960

tatccaatga tggattctgg agaacaggat ggttgtatgc tagggtacag gactgcctaa 4020

cactgagttg caatggtccg ttcatcttat ttggagtagt tattcatata ttcgaggatc 4080

agcttcattt gtatttcttt ccttattttt gttaggtagt cttatgtttg catctccctg 4140

gcaaccggta attggtgaca agcatcagag actgtgtgta actccaattg cagttgattg 4200

ttcttcgtca gtaaaattct ctgtgaaagg tttcaatata gttcagccaa ccacaaagta 4260

tgtatctttt ttcacattta aatgtctcct ctaaacttac ttctttagtt cttttaactg 4320

acacattctg attttgttat agattacttt gtgtgtttga tgaaaaatat ttaattcaag 4380

aagagacaca aatgctactt gaagattcaa ctatgcagca aggccctcaa tgcctgacct 4440

tctcttgttc ctttccttgt acaagtggaa gaggattcat agaggtacaa tgtttgtata 4500

ttttttatat cctcattttt tgtgtatatt acagacttga ctccgttttc agattgaaga 4560

ctatgatcaa agcagccttt ctgttccctt tgttgtcacg gacaaagatg tatgttctga 4620

gattcggatg ttggagcatg gattggattt agtttcattt gatcaaacct ccaaaagaat 4680

agatgatctg atgatttatc gcagtcgagc attacatttt ttgcatgaaa tcggatggct 4740

tcttcaaagg agccatgtgc gagctacgtc tgagcagcga caatattgtc ctgaccgctt 4800

ccctgttgca agatttagat ggctgctatc ctttgcagtt gatcaggaat ggtgtgctgt 4860

tctaaggaag cttctgaaca ccatgttcca gggtgatatt gatgtattgt caccaattga 4920

atttgccttg ggagaaaatc tattgcatac tgcggtcaaa aaacgctcaa agcctttagt 4980

tgaatttcta ttaagataca ccacaacaaa tattgctcca gtgggcggtg gagatggtgc 5040

tccagttcag ttcttgttca ctcctgcgat gactgaactg tcaaatatca cacctcttca 5100

tattgcagcc acaatcagtg atgctattgg tgttttagat gctttaactg atgatcctca 5160

gcaggtaact actctaaatt gccatgttct tacctgttgt agcctgcttt aactaaaaaa 5220

aagcacatct acattcaaat ttcaaagtag cagatttccg attcttagtg taaaacagtt 5280

aaaataaaaa ctaagaccac catttattgt tagaacctaa gtctgcacct gaaatatttt 5340

catctgaaat gcattatgta catatcaaga aaaagaaagg acttccatca aaacattggt 5400

ttgtgtatta cattgattgt tgaatagatc gtgtagggtg tataatatca catcagagtt 5460

ccgcatgagg tgtagtatga taaaaattca tttaccttaa aaacattttc ttttactttt 5520

gagaatttgt gcaagcactg aaacatttgc tgtctggctt ctttctgcct aaatgtctca 5580

gttgggaatc aaagcgtgga agaatgctcg tgacgccact ggcttcactc ctgaggaata 5640

tgctgcaaag agaggcaaca tatcctatat tcaaatggta caggacaaaa ttgacagaag 5700

ggtgaccaga gctcatgtct cggttaccat ccccagcaca attgatactg tcggaaagca 5760

tggtagccga atgaagcctg ccgatcaaat cacatttggt gttgagaaaa aacaactaag 5820

catcaaccaa acattgagct gcagacagtg tgtccagcag gcccagcagc ttgcattcca 5880

tccccggaca aataggtttc tgtctaatag gactgcgatg ctttccttgg tctccattgc 5940

tgccgtctgc gtctgtgtgg gattgatcat gaagagcctg ccacaagttg gttgtatgaa 6000

gcctttcctc tgggacaata tacgttgggg ccccaattga tagactgcag aagagccagc 6060

ccgattgtat cagtgtatga tgaagatgtt tgcgccgagc gaagctagtt tcttgtaatg 6120

tacatgtttc ttatactaaa tgtctaactt atttctgttt atagtaaaac ctgtgaactt 6180

gcctagcttg taactccatt gcatttgcat cctgactgaa gtaaagcttt tacttgtttt 6240

ggttgagagg aagaaaa 6257

<210> 5

<211> 2000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atttttctgt tcaccaaacc agccctaaag gcgccgcggt cctgtttttt ttgtcctcag 60

gtgagtttgg gttgctggcg ctgcttcctt gctcatgcgg aatctgctct atttcttgtt 120

agttttagtt cgtaatatat ttcagactat tgactgatgg attcagttaa aatttggaga 180

tgttgtctct taacctttcc tagctagacc ctgcagcgtt tatgagacca tggctgattg 240

gttctccttg gatctggttt tgctatcctg gtgaaacatc cagttttctc tataaaacat 300

ccagttttct acgttttccc aaaaaaaggg gatatgttgg taatacgagc agaaatcatc 360

ctggaaagac ggacacatgt gaccggtttg cttttgcttc acatgtttaa cgagtccatt 420

gttcggatga actgaataac tgatctcatg tatcattgct acatgtacct ttgtctgaat 480

tgaaaatcat tatgttatat tattattaat ccaattaaca agactagcta ccttgcaagg 540

attatacact aatctctcca ttctgaacac cttgaatagc atgctgctgg ctttaatctc 600

gcgctgcacg ttctcaattt tcagatagtt ccttcaatct gatactactt ctagtttccc 660

tcacagattt gtgaactatg aaaccacctt tggagaggaa ccctactaag aagcgccatt 720

atttggtggg acagtcacat tagccctaag aactccgaaa aacatcaaag gttagcgaaa 780

ctatagccaa aatatttttt tacgtgctat ttttagatct aaatttgcta cacgtgttat 840

tcctattata attaaagtaa atttaagaat tttatagagc atacaaaaag ataattgtac 900

cctaaataaa acaccaattc aaattaatct agatccatcc aacaaataac ttctcaatgt 960

gttttctttg cgtacttttt ccatgtttct tctttctcat gcttcctctt actcactcac 1020

atcttctaac caaggacgtc cagatcattc cactcctcac gcacgtcccc accagcgccc 1080

tcacatgcaa acccccttcg tcgccgctcc tagtctctcc cccgttctct ttctcttgct 1140

atggtctctg ccaccgatcc tgatggacga gcaagacgac gactgaggtg ccgttcggtt 1200

ctttaggatt ggtgggtcgg aacgattcct aactggattg tttctctaat ttatataaac 1260

tttgattaac tggaatgatt ccggatgcaa tccgatacaa acgaacaagg ctgatgagga 1320

cgatggcacg acgagcacct cctcgctcgt ctgcttccgc caagcgacac caaggtcgat 1380

gaggcggctc agacagatgt aggtgagggg atgtgtcctg gacttagatg accattttac 1440

tagttttcta cattgagcat aaaaatcagg ggtttttggc ggtaaaccgg cgattttgac 1500

cagtttttcg ttgataaacc gacacaggaa gagcactgat aaatttgaat ttgccctgat 1560

cggttttacc ggaccgaaac cgatgaggga cggttttcgg ttgtaaaacg gatcggtgaa 1620

ccctgagtcc agcccccgga gcaagaggaa acggcctgcg gcgccgcgtc ggccgtgggg 1680

ccgaccgccc agcgagcgag tcgtcatgga aacgaggaat cgtcgcgccg ccctgcgtca 1740

tcaagtcacc tgctcttcca aaggtccaac cggcccttgg tcgtggcgtc gctgtccgag 1800

cgccagacaa gagacagggg agatgaaaaa gaggcggtgg tggttcgagt gtgaatatgc 1860

cggtggggag ggctctgcct ccccaggata cttcgccccc cgttccaccg cgagcccttc 1920

cggcttccgc ctctgttccg ccgctccagc ctccaggatc cgtgagaaga aaagtagtag 1980

ccccatcccc aaccggtcct 2000

<210> 6

<211> 1982

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tcatggtagc cagggcctac aacaagaagg tcaaagcaaa attatttcaa ttaggggacc 60

tggtgtggaa gactattctg cctctaagga ataaagaccg taagttcgga aaatggtcgc 120

caaactggga gggtccttat aaagtaaaac aggtgatgtc tggcaacgcc tatttactac 180

aaacattaca aggcaaggat ttacctaagg ctttgaatgt gcgtttcctc aaatagtacc 240

atcctagtat gtggcaagat gcctaagaaa accgatgtaa tcacatcgag ttagttgctt 300

ttggtttgct cagctccacc gaaaggcagg gggcatatgt tgagcaccgt tttgagcctg 360

gcggacggtc cggccctgag gccggacgat ccgcggtccg gacagtccgc gcctgtgggc 420

cggacggtcc gcgcatgcgc agagcagttt agggttccga gttttgtgcc atgtttgttg 480

gttagatttg cgaaattagc tcggaatcca gtcgtgtaaa gggtctagcc cccctcctct 540

atataaagag aggtctacga ccgatttgta atcatcaatc gaatcaatac aacttctatt 600

cacatttatt tccagtacaa ttaggagtag ttctagtcta gttctagttt agcctctcaa 660

tccccaaatt cttcgcctct cttcgactct acgccgatta gaggagtcta ggtcggccgg 720

cccgagccta gaaaacccct aggatctctc ctccccgacg gggtccctcc cgggagcgag 780

atccaggcgc cgccggcaat cttccgccgc gcggaccgtc cggccgtcag gcaggaaacc 840

ctagcccctg cgccaagtcg cggaccgtcc gaccctaggc cgcggaccgt ccgcgcctga 900

ccagagagca ccgccgccgg tttttcttga gtatttggcg ctccgaaaaa gcgtcaacaa 960

tgggactgtc acattagccc taaaaactcc gaaaaacatc aaaggtgagc gaaactatag 1020

ccaaaatatt ttttacgtgc tatttttaga tctaaatttg ctacacgtgt tattcctatt 1080

ataattaaag taaatttaag aattttatag agcttacaaa aagataattg taccctaaat 1140

aaaacaccaa ttcaaattaa tctagatcca tccaacaaat aacgtctcaa tgcaaccccc 1200

ttcgtcgctg ctcccggtct ctcccccgtt ctctttctct tgctatggtc tctgccaccg 1260

atcctgatgg acgggcaaga cgacgactga gccttgctct ccaccgggga tgcacgagga 1320

cgatggcacg gcgagcacct cctccgacgg tgcgagcggt gctcgtcgct cgtctgcttc 1380

cgccaagcgg caccaaggtc gatgaggcgg ctcagacaac gatgtaggtg aggggatgtg 1440

tcctggactt aggactgttt gtttcagctt atagattata taatttaaat tatataatcc 1500

agattataat ctagatatat tataatctag atatattata atcggttgta ccggaccgaa 1560

accgatgagg gacggttttc ggttgtaaaa cggatcggtg aaccctgagt ccagcccccg 1620

gagcaagagg aaacggcctg cggcgccgcg tcggccgtgg ggccgaccgc ccagcgagcg 1680

agtcgtcatg gaaacgagga atcgtcgcgc cgccctgcgt catcaagtca cctgctcttc 1740

caaaggtcca accggccctt ggtcgtggcg tcgctgtccg agcgccagac aagagacagg 1800

ggagatgaaa aagaggcggt ggtggttcga gtgtgaatat gccggtgggg agggctctgc 1860

ctccccagga tacttcgccc cccgttccac cgcgagccct tccggcttcc gcctctgttc 1920

cgccgctcca gcctccagga tccgtgagag gaaaagtagt agccccatcc ccaaccggtc 1980

ct 1982

<210> 7

<211> 877

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Met Glu Ala Gly Phe Leu Trp Lys Glu Ile Gly Asp Gln Ala Gly Ala

1 5 10 15

Ala Arg Met Ser Gly Gly Asn Lys Asn Arg Ser Leu Asp Trp Asp Leu

20 25 30

Asn Asp Trp Arg Trp Asp Ala Asn Leu Phe Leu Ala Thr Pro Ala Ala

35 40 45

Ala Ala Pro Ser Glu Ser Ile Ser Arg Glu Leu Ser Arg Gly Gln Gly

50 55 60

Glu Ile Asp Phe Gly Val Val Val Asp Lys Arg Arg Arg Leu Ser Pro

65 70 75 80

Glu Glu Asp Gly Ser Gly Gly Cys Ser Asn Ser Ala Val Ala Asp Gly

85 90 95

Asp Asn Asn His Val Val Cys Val Gln Arg Gly Arg Asn Thr Glu Glu

100 105 110

Thr Arg Pro Arg Lys Gly Ala Asn Ser Ser Thr Thr Pro Ser Cys Gln

115 120 125

Val Asp Gly Cys Gln Ala Asp Leu Ser Gly Ala Arg Asp Tyr His Lys

130 135 140

Arg His Lys Val Cys Glu Ala His Thr Arg Thr Thr Val Val Cys Ile

145 150 155 160

Asn Asn Val Glu His Arg Phe Cys Gln Gln Cys Ser Arg Phe His Leu

165 170 175

Leu His Glu Phe Asp Asp Gly Lys Lys Ser Cys Arg Ser Arg Leu Ala

180 185 190

Gln His Asn Gly Arg Arg Arg Lys Val Gln Pro Gln Pro Ala Val Asn

195 200 205

Gly Asn Ser Met Asn Glu Asp Gln Ser Leu Ser Ser Thr Leu Phe Leu

210 215 220

Leu Leu Lys Gln Leu Ser Gly Leu Glu Ser Gly Ser Ser Ser Glu Gln

225 230 235 240

Ile Asn His Pro Asn Ser Leu Val Asn Leu Leu Lys Asn Leu Ala Ala

245 250 255

Ile Ala Ser Thr His Ala Tyr Gln Asp Val Leu Lys Asn Ala Thr Ser

260 265 270

Ile Ser Ser Asn Asp Gly Asn Asn Ala Ala Asn Gly Ser Ile Met His

275 280 285

Glu Gln Thr Ile Arg Ser Ile Pro Val Arg Arg Glu Ser Leu Ala Glu

290 295 300

Glu Pro Ala Val Lys Arg Arg Val Gln Asp Phe Asp Leu Asn Asp Ser

305 310 315 320

Cys Ile Glu Glu Ala Glu Ser Arg Thr Asp Lys Ile Val Phe Lys Leu

325 330 335

Phe Gly Lys Glu Pro Lys Asp Phe Pro Val Asp Leu Arg Glu Gln Ile

340 345 350

Leu Asn Trp Leu Ser His Tyr Pro Thr Asp Met Glu Ser Tyr Ile Arg

355 360 365

Pro Gly Cys Val Ile Leu Thr Ile Tyr Leu His Leu Pro Asn Trp Met

370 375 380

Trp Asp Glu Phe Asn Asp Asp Pro Ala Ser Trp Ile Glu Asn Leu Ile

385 390 395 400

Ser Leu Ser Asn Asp Gly Phe Trp Arg Thr Gly Trp Leu Tyr Ala Arg

405 410 415

Val Gln Asp Cys Leu Thr Leu Ser Cys Asn Gly Ser Leu Met Phe Ala

420 425 430

Ser Pro Trp Gln Pro Val Ile Gly Asp Lys His Gln Arg Leu Cys Val

435 440 445

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

450 455 460

Gly Phe Asn Ile Val Gln Pro Thr Thr Lys Leu Leu Cys Val Phe Asp

465 470 475 480

Glu Lys Tyr Leu Ile Gln Glu Glu Thr Gln Met Leu Leu Glu Asp Ser

485 490 495

Thr Met Gln Gln Gly Pro Gln Cys Leu Thr Phe Ser Cys Ser Phe Pro

500 505 510

Cys Thr Ser Gly Arg Gly Phe Ile Glu Ile Glu Asp Tyr Asp Gln Ser

515 520 525

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

530 535 540

Ile Arg Met Leu Glu His Gly Leu Asp Leu Val Ser Phe Asp Gln Thr

545 550 555 560

Ser Lys Arg Ile Asp Asp Leu Met Ile Tyr Arg Ser Arg Ala Leu His

565 570 575

Phe Leu His Glu Ile Gly Trp Leu Leu Gln Arg Ser His Val Arg Ala

580 585 590

Thr Ser Glu Gln Arg Gln Tyr Cys Pro Asp Arg Phe Pro Val Ala Arg

595 600 605

Phe Arg Trp Leu Leu Ser Phe Ala Val Asp Gln Glu Trp Cys Ala Val

610 615 620

Leu Arg Lys Leu Leu Asn Thr Met Phe Gln Gly Asp Ile Asp Val Leu

625 630 635 640

Ser Pro Ile Glu Phe Ala Leu Gly Glu Asn Leu Leu His Thr Ala Val

645 650 655

Lys Lys Arg Ser Lys Pro Leu Val Glu Phe Leu Leu Arg Tyr Thr Thr

660 665 670

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

675 680 685

Leu Phe Thr Pro Ala Met Thr Glu Leu Ser Asn Ile Thr Pro Leu His

690 695 700

Ile Ala Ala Thr Ile Ser Asp Ala Ile Gly Val Leu Asp Ala Leu Thr

705 710 715 720

Asp Asp Pro Gln Gln Leu Gly Ile Lys Ala Trp Lys Lys Ala Arg Asp

725 730 735

Ala Thr Gly Phe Thr Pro Glu Glu Tyr Ala Ala Lys Arg Gly Asn Ile

740 745 750

Ser Tyr Ile Gln Met Val Gln Asp Lys Ile Asp Arg Arg Val Thr Arg

755 760 765

Ala His Val Ser Val Thr Met Pro Ser Thr Ile Asp Thr Val Gly Lys

770 775 780

His Ala Ser Arg Met Lys Pro Ala Asp Gln Ile Thr Phe Gly Val Glu

785 790 795 800

Lys Lys Gln Leu Ser Ile Asn Gln Thr Leu Ser Cys Arg Gln Cys Val

805 810 815

Gln Gln Ala Gln Gln Leu Ala Phe His Pro Arg Thr Asn Arg Phe Leu

820 825 830

Ser Asn Arg Thr Ala Met Leu Ser Leu Val Ser Ile Ala Ala Val Cys

835 840 845

Val Cys Val Gly Leu Ile Met Lys Ser Leu Pro Gln Val Gly Cys Met

850 855 860

Lys Pro Phe Leu Trp Asp Asn Ile Arg Trp Gly Pro Asn

865 870 875

<210> 8

<211> 877

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Glu Ala Gly Phe Leu Trp Lys Glu Ile Gly Asp Gln Ala Gly Ala

1 5 10 15

Ala Arg Met Ser Gly Gly Asn Lys Asn Arg Ser Leu Asp Trp Asp Leu

20 25 30

Asn Asp Trp Arg Trp Asp Ala Asn Leu Phe Leu Ala Thr Pro Ala Ala

35 40 45

Ala Ala Pro Ser Glu Ser Ile Ser Arg Glu Leu Ser Arg Gly Gln Gly

50 55 60

Glu Ile Asp Phe Gly Val Val Val Asp Lys Arg Arg Arg Leu Ser Pro

65 70 75 80

Glu Glu Asp Gly Ser Gly Gly Cys Ser Asn Ser Ala Val Ala Asp Gly

85 90 95

Asp Asn Ser His Val Val Cys Val Gln Arg Gly Arg Asn Thr Glu Glu

100 105 110

Thr Arg Pro Arg Lys Gly Ala Asn Ser Ser Thr Thr Pro Ser Cys Gln

115 120 125

Val Asp Gly Cys Gln Ala Asp Leu Ser Gly Ala Arg Asp Tyr His Lys

130 135 140

Arg His Lys Val Cys Glu Ala His Thr Arg Thr Thr Val Val Cys Ile

145 150 155 160

Asn Asn Val Glu His Arg Phe Cys Gln Gln Cys Ser Arg Phe His Leu

165 170 175

Leu His Glu Phe Asp Asp Gly Lys Lys Ser Cys Arg Ser Arg Leu Ala

180 185 190

Gln His Asn Gly Arg Arg Arg Lys Val Gln Pro Gln Pro Ala Val Asn

195 200 205

Gly Asn Ser Met Asn Glu Asp Gln Ser Leu Ser Ser Thr Leu Phe Leu

210 215 220

Leu Leu Lys Gln Leu Ser Gly Leu Glu Ser Gly Ser Ser Ser Glu Gln

225 230 235 240

Ile Asn His Pro Asn Ser Leu Val Asn Leu Leu Lys Asn Leu Ala Ala

245 250 255

Ile Ala Ser Thr His Ala Tyr Gln Asp Val Leu Lys Asn Ala Thr Ser

260 265 270

Ile Ser Ser Asn Asp Gly Asn Asn Ala Ala Asn Gly Ser Ile Met His

275 280 285

Glu Gln Thr Ile Arg Ser Ile Pro Val Arg Arg Glu Ser Leu Ala Glu

290 295 300

Glu Pro Ala Val Lys Arg Arg Val Gln Asp Phe Asp Leu Asn Asp Ser

305 310 315 320

Cys Ile Glu Glu Ala Glu Ser Arg Thr Asp Lys Ile Val Phe Lys Leu

325 330 335

Phe Gly Lys Glu Pro Lys Asp Phe Pro Val Asp Leu Arg Glu Gln Ile

340 345 350

Leu Asn Trp Leu Ser His Tyr Pro Thr Asp Met Glu Ser Tyr Ile Arg

355 360 365

Pro Gly Cys Val Ile Leu Thr Ile Tyr Leu His Leu Pro Asn Trp Met

370 375 380

Trp Asp Glu Phe Asn Asp Asp Pro Ala Ser Trp Ile Glu Asn Leu Ile

385 390 395 400

Ser Leu Ser Asn Asp Gly Phe Trp Arg Thr Gly Trp Leu Tyr Ala Arg

405 410 415

Val Gln Asp Cys Leu Thr Leu Ser Cys Asn Gly Ser Leu Met Phe Ala

420 425 430

Ser Pro Trp Gln Pro Val Ile Gly Asp Lys His Gln Arg Leu Cys Val

435 440 445

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

450 455 460

Gly Phe Asn Ile Val Gln Pro Thr Thr Lys Leu Leu Cys Val Phe Asp

465 470 475 480

Glu Lys Tyr Leu Ile Gln Glu Glu Thr Gln Met Leu Leu Glu Asp Ser

485 490 495

Thr Met Gln Gln Gly Pro Gln Cys Leu Thr Phe Ser Cys Ser Phe Pro

500 505 510

Cys Thr Ser Gly Arg Gly Phe Ile Glu Ile Glu Asp Tyr Asp Gln Ser

515 520 525

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

530 535 540

Ile Arg Met Leu Glu His Gly Leu Asp Leu Val Ser Phe Asp Gln Thr

545 550 555 560

Ser Lys Arg Ile Asp Asp Leu Met Ile Tyr Arg Ser Arg Ala Leu His

565 570 575

Phe Leu His Glu Ile Gly Trp Leu Leu Gln Arg Ser His Val Arg Ala

580 585 590

Thr Ser Glu Gln Arg Gln Tyr Cys Pro Asp Arg Phe Pro Val Ala Arg

595 600 605

Phe Arg Trp Leu Leu Ser Phe Ala Val Asp Gln Glu Trp Cys Ala Val

610 615 620

Leu Arg Lys Leu Leu Asn Thr Met Phe Gln Gly Asp Ile Asp Val Leu

625 630 635 640

Ser Pro Ile Glu Phe Ala Leu Gly Glu Asn Leu Leu His Thr Ala Val

645 650 655

Lys Lys Arg Ser Lys Pro Leu Val Glu Phe Leu Leu Arg Tyr Thr Thr

660 665 670

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

675 680 685

Leu Phe Thr Pro Ala Met Thr Glu Leu Ser Asn Ile Thr Pro Leu His

690 695 700

Ile Ala Ala Thr Ile Ser Asp Ala Ile Gly Val Leu Asp Ala Leu Thr

705 710 715 720

Asp Asp Pro Gln Gln Leu Gly Ile Lys Ala Trp Lys Asn Ala Arg Asp

725 730 735

Ala Thr Gly Phe Thr Pro Glu Glu Tyr Ala Ala Lys Arg Gly Asn Ile

740 745 750

Ser Tyr Ile Gln Met Val Gln Asp Lys Ile Asp Arg Arg Val Thr Arg

755 760 765

Ala His Val Ser Val Thr Ile Pro Ser Thr Ile Asp Thr Val Gly Lys

770 775 780

His Gly Ser Arg Met Lys Pro Ala Asp Gln Ile Thr Phe Gly Val Glu

785 790 795 800

Lys Lys Gln Leu Ser Ile Asn Gln Thr Leu Ser Cys Arg Gln Cys Val

805 810 815

Gln Gln Ala Gln Gln Leu Ala Phe His Pro Arg Thr Asn Arg Phe Leu

820 825 830

Ser Asn Arg Thr Ala Met Leu Ser Leu Val Ser Ile Ala Ala Val Cys

835 840 845

Val Cys Val Gly Leu Ile Met Lys Ser Leu Pro Gln Val Gly Cys Met

850 855 860

Lys Pro Phe Leu Trp Asp Asn Ile Arg Trp Gly Pro Asn

865 870 875

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gggatttgaa cgactggagg t 21

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tcgtctcctc agtgttccgc 20

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cacacacgag gggaggaggt 20

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

aaggtctttg ttgtcgggca 20

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tttcttctca gggtggtctt gc 22

<210> 14

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cacctttcca cctcgcctat t 21

<210> 15

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cgcggatcca tggaagccgg tttcctgtg 29

<210> 16

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tcccccgggt cacttatcgt cgtcatcctt gtaatcattg gggccccaac g 51

<210> 17

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

cagagagggc ggaacactga gg 22

<210> 18

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

cacgtctggc acaacataat gg 22

<210> 19

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

aaatcttgca acagggaagc gg 22

<210> 20

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ttgacagttc agtcatcgca gg 22

Claims (4)

1. A maize C-type cytoplasmic male sterile nuclear restorer gene which is characterized in that: the nucleotide sequence of the gene is a nucleotide sequence coded by SEQ ID No: 7.

2. A polypeptide, characterized by: the amino acid sequence is SEQ ID No: 7.

3. a recombinant construct, characterized in that: the vector containing the restorer gene of claim 1, which is a cloning vector or an expression vector for expressing the restorer gene.

4. A method of improving a corn crop comprising: a transgenic maize plant obtained by transfecting a maize plant with a recombinant Agrobacterium cell comprising the restorer gene of claim 1, or a maize plant containing the restorer gene of claim 1 is crossed with another maize plant.

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