Method for preparing rice photosensitive male sterile material and related gene
阅读说明：本技术 一种制备水稻光敏型雄性不育材料的方法及相关基因 (Method for preparing rice photosensitive male sterile material and related gene ) 是由 李莉 邱牡丹 李懿星 张大兵 宋书锋 王天抗 于 2020-04-27 设计创作，主要内容包括：本发明公开了一种制备水稻光敏型雄性不育材料的方法及相关基因。本发明制备光敏型雄性不育水稻的方法,包括：降低目的水稻中蛋白质RMS1的丰度、降低目的水稻中蛋白质RMS1的活性或降低目的水稻中蛋白质RMS1的含量,得到光敏型雄性不育水稻。所述蛋白质RMS1为如下A1)或A2)：A1)其氨基酸序列如序列表中SEQ ID No.1所示；A2)与A1)具有98％以上同一性且来源于水稻的同源蛋白质。本发明通过控制水稻的RMS1基因及其编码蛋白获得了水稻的光敏型雄性不育材料,实现了水稻育性在不同光照条件下的转换。(The invention discloses a method for preparing a rice photosensitive male sterile material and a related gene. The method for preparing the photosensitive male sterile rice comprises the following steps: reducing the abundance of the protein RMS1 in the target rice, reducing the activity of the protein RMS1 in the target rice or reducing the content of the protein RMS1 in the target rice to obtain the photosensitive male sterile rice. The protein RMS1 was either a1) or a2) as follows: A1) the amino acid sequence is shown as SEQ ID No.1 in the sequence table; A2) homologous protein having 98% or more identity to A1) and derived from rice. The invention obtains the photosensitive male sterile material of the rice by controlling the RMS1 gene of the rice and the coding protein thereof, and realizes the conversion of the rice fertility under different illumination conditions.)
1. A method for producing a light-sensitive male-sterile rice, comprising: reducing the abundance of the protein RMS1 in the target rice, reducing the activity of the protein RMS1 in the target rice or reducing the content of the protein RMS1 in the target rice to obtain the photosensitive male sterile rice;
the protein RMS1 was either a1) or a2) as follows:
A1) the amino acid sequence is shown as SEQ ID No.1 in the sequence table;
A2) homologous protein having 98% or more identity to A1) and derived from rice.
2. The method of claim 1, wherein: the reduction of the abundance of the protein RMS1 in the target rice, the reduction of the activity of the protein RMS1 in the target rice or the reduction of the content of the protein RMS1 in the target rice is realized by inhibiting the expression of the coding gene of the protein RMS1 in the target rice or knocking out the coding gene of the protein RMS1 in the target rice.
3. The method of claim 2, wherein: the inhibition of the expression of the coding gene of the protein RMS1 in the target rice or the knockout of the coding gene of the protein RMS1 in the target plant is realized by using a CRISPR/Cas9 system.
4. The method of claim 3, wherein: the CRISPR/Cas9 system includes a vector that expresses sgrnas whose target sequences are as follows: CCAAGGCCGGTAAGCGCCGC are provided.
5. The method according to any one of claims 2-4, wherein: the coding gene of the protein RMS1 is any one of the following b1) -b 4):
b1) a DNA molecule shown as SEQ ID No.2 in the sequence table;
b2) a DNA molecule shown as SEQ ID No.3 in the sequence table;
b3) a DNA molecule having 75% or more 75% identity to the nucleotide sequence defined in b1) or b2) and encoding the protein RMS1 of claim 1;
b4) a DNA molecule which hybridizes with the nucleotide sequence defined by b1) or b2) under strict conditions and codes for the protein RMS1 of claim 1.
sgRNA or a recombinant plasmid expressing the sgRNA;
the target sequence of the sgRNA is as follows: CCAAGGCCGGTAAGCGCCGC are provided.
7. Use of the sgRNA or the recombinant plasmid of claim 6 in rice breeding.
8. A method for preparing transgenic rice, comprising the steps of: the sgRNA coding gene of claim 6 and the Cas9 protein coding gene are introduced into recipient rice to obtain light-sensitive male sterile rice.
9. Any one of the following products:
p1, protein RMS1, said protein RMS1 being a11) or a12 as follows):
A11) the amino acid sequence is shown as SEQ ID No.1 in the sequence table;
A12) homologous protein having 98% or more identity to a11) and derived from rice;
p2, a nucleic acid molecule encoding the protein of P1;
p3, the nucleic acid molecule encoding the protein P1 is any one of the following b1) -b 4):
b11) a DNA molecule shown as SEQ ID No.2 in the sequence table;
b12) a DNA molecule shown as SEQ ID No.3 in the sequence table;
b13) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by b11) or b12) and codes for the protein RMS1 of P1;
b14) a DNA molecule which is hybridized with the nucleotide sequence defined by b11) or b12) under strict conditions and codes for the protein RMS1 of P1;
p4, protein RMS1-4, said protein RMS1-4 being A21) or A22 as follows:
A21) the amino acid sequence is shown as SEQ ID No.6 in the sequence table;
A22) homologous protein having 98% or more identity to a21) and derived from rice;
p5, a nucleic acid molecule encoding the protein of P4;
p6, the nucleic acid molecule encoding the protein P4 is any one of the following b1) -b 4):
b21) a DNA molecule shown as SEQ ID No.7 in the sequence table;
b22) a DNA molecule shown as SEQ ID No.8 in the sequence table;
b23) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by b21) or b22) and codes the protein RMS1-4 of P4;
b24) a DNA molecule which is hybridized with the nucleotide sequence defined by b21) or b22) under strict conditions and codes for the protein RMS1-4 of P4;
p7, protein RMS1-5, said protein RMS1-5 being A31) or A32) as follows:
A31) the amino acid sequence is shown as SEQ ID No.9 in the sequence table;
A32) homologous protein having 98% or more identity to a31) and derived from rice;
p8, a nucleic acid molecule encoding the protein of P7;
p9, the nucleic acid molecule encoding the protein P7 is any one of the following b31) -b 34):
b31) a DNA molecule shown as SEQ ID No.10 in the sequence table;
b32) a DNA molecule shown as SEQ ID No.11 in the sequence table;
b33) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by b31) or b32) and codes for the protein RMS1-5 of P7;
b34) a DNA molecule which is hybridized with the nucleotide sequence defined by b31) or b32) under strict conditions and codes for the protein RMS1-5 of P7;
p10, protein RMS1-11, said protein RMS1-11 being A41) or A42) as follows:
A41) the amino acid sequence is shown as SEQ ID No.12 in the sequence table;
A42) homologous protein having 98% or more identity to a41) and derived from rice;
p11, a nucleic acid molecule encoding the protein of P10;
p12, the nucleic acid molecule encoding the protein P10 is any one of the following b41) -b 44):
b41) a DNA molecule shown as SEQ ID No.13 in the sequence table;
b42) a DNA molecule shown as SEQ ID No.14 in the sequence table;
b43) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by b41) or b42) and codes the protein RMS1-11 of P10;
b44) a DNA molecule which is hybridized with the nucleotide sequence defined by b41) or b42) under strict conditions and codes for the protein RMS1-11 described by P10.
10. The use of the product of claim 9 for regulating photoperiod sensitive fertility of rice;
or, the use of the product of claim 9 for breeding photosensitive male sterile rice;
or, culturing photosensitive male sterile rice by using the product as the target in claim 9.
Technical Field
The invention relates to the field of biotechnology breeding, in particular to a method for preparing a rice photosensitive male sterile material and a related gene thereof.
Background
The crossbreeding technology plays a very important role in improving the rice yield in China and the world, and is an important guarantee for food safety and agricultural sustainable development in China. The two-line sterile line has free matching and simplified seed production process, but the fertility is greatly influenced by the external environment, the seed production has risks, the breeding of the photo-thermo-sensitive male sterile line is the core of the research and development and application of a two-line hybrid rice breeding technical system, the photo-thermo-sensitive male sterile line is widely used in the current production, is mainly controlled by temperature, has relatively changeable temperature environment, the illumination length is more constant in a specific area, and the difference between the years is smaller, so the method has great application prospect in the production of screening the photo-sensitive male sterile line with stable fertility conversion. In recent years, with the development of functional genomics, the research on genetic systems of rice male sterility and fertility restoration has been advanced. However, besides the application of the genes, a plurality of genes which are not discovered temporarily and control photo-thermo-sensitive male sterility exist in the current production, if the genes can be deeply mined and subjected to functional research, the understanding and understanding of the photo-thermo-sensitive male sterility mechanism can be more comprehensively expanded and deepened, and the method has important guiding significance and application value for breeding and creating a novel excellent stable two-line sterile line.
Disclosure of Invention
The invention aims to solve the technical problem of how to prepare photosensitive male sterile rice.
In order to solve the above technical problems, the present invention firstly provides a method for preparing photosensitive male sterile rice.
The method for preparing the photosensitive male sterile rice provided by the invention comprises the following steps: reducing the abundance of the protein RMS1 in the target rice, reducing the activity of the protein RMS1 in the target rice or reducing the content of the protein RMS1 in the target rice to obtain the photosensitive male sterile rice;
the protein RMS1 was either a1) or a2) as follows:
A1) the amino acid sequence is shown as SEQ ID No.1 in the sequence table;
A2) homologous protein having 98% or more than 99% identity to A1) and derived from rice.
In the above proteins, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.
The reduction of the abundance of the protein RMS1 in the target rice, the reduction of the activity of the protein RMS1 in the target rice or the reduction of the content of the protein RMS1 in the target rice is realized by inhibiting the expression of the coding gene of the protein RMS1 in the target rice or knocking out the coding gene of the protein RMS1 in the target rice. The knockout includes the knockout of the entire gene, as well as the knockout of a partial segment of the gene.
The reduction of the abundance of the protein RMS1 in the target rice, the reduction of the activity of the protein RMS1 in the target rice or the reduction of the content of the protein RMS1 in the target rice can also be realized by silencing a coding gene of the protein RMS 1.
The "reduction of the abundance of the protein RMS1 in the target rice, the reduction of the activity of the protein RMS1 in the target rice or the reduction of the content of the protein RMS1 in the target rice" can be specifically realized by gene editing of a coding gene of the protein RMS 1.
The coding gene of the protein RMS1 is any one of the following b1) -b 4):
b1) the nucleotide sequence of the DNA molecule is shown as SEQ ID No.2 in the sequence table;
b2) the nucleotide sequence of the DNA molecule is shown as SEQ ID No.3 in the sequence table;
b3) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by b1) or b2) and codes the protein RMS 1;
b4) a DNA molecule which is hybridized with the nucleotide sequence defined by b1) or b2) under strict conditions and codes the protein RMS 1.
In the above genes, "identity" refers to sequence similarity to the native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence of coding sequence 2 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
The stringent conditions are hybridization and washing of the membrane 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.
The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.
In the above method, any method in the prior art may be adopted to make the RMS1 gene suppression or RMS1 gene knockout, so as to make the gene generate deletion mutation, insertion mutation or base change mutation, and further to suppress RMS1 gene expression or knockout RMS1 gene.
In the above-mentioned method, the method of suppressing the expression of the gene encoding the protein RMS1 in the target rice or the method of deleting the gene encoding the protein RMS1 in the target rice may be a chemical mutagenesis, physical mutagenesis, RNAi, gene site-directed editing, homologous recombination, or the like.
Whichever method is taken, the RMS1 gene may be targeted, and the individual elements that regulate the expression of the RMS1 gene may be targeted, so long as inhibition of RMS1 gene expression or knock-out of the RMS1 gene is achieved. For example, exon 1, exon 2, exon 3 and/or exon 4 of the RMS1 gene may be targeted.
In the above-mentioned genome site-directed editing, Zinc Finger Nuclease (ZFN) technology, Transcription activator effector-like nuclease (TALEN) technology, clustered regularly spaced short palindromic repeats (clustered regularly interspaced short palindromic repeats/CRISPR associated, CRISPR/Cas9 system) technology, and other technologies capable of realizing genome site-directed editing can be adopted.
In the specific embodiment of the invention, the coding gene of the protein RMS1 in rice is knocked out by means of a CRISPR/Cas9 system, wherein the related target sequence is CCAAGGCCGGTAAGCGCCGC, and the coding gene of the used sgRNA (guide RNA) is shown as SEQ ID No.4 in a sequence table.
More specifically, the invention uses a recombinant vector pYLCRISPR/Cas9-MT-RMS1 capable of expressing a guide RNA and Cas 9. The recombinant vector pYLCRISPR/Cas9-MT-RMS1 is a recombinant vector obtained by replacing a fragment between two Bsa I enzyme cutting sites of the vector pYLCRISPR/Cas9-MTmono with a DNA fragment containing a specific sgRNA coding gene and a U3 promoter and keeping other nucleotides of the pYLCRISPR/Cas9-MTmono unchanged, and particularly is obtained by replacing a fragment between two Bsa I enzyme cutting sites of the vector pYLCRISPR/Cas9-MTmono with a DNA molecule shown in SEQ ID No.5 in a sequence table. The above method is applicable to any rice, such as: japonica rice (Oryza sativa subsp. japonica) or indica rice (Oryza sativa subsp. indica) as long as it contains the above target sequence. An example of the present invention is rice variety wuyujing No.7 (Oryza sativa subsp.
The invention also protects specific sgrnas. The target sequences of the specific sgrnas are as follows: CCAAGGCCGGTAAGCGCCGC are provided.
The invention also protects the specific recombinant plasmid. The specific recombinant plasmid is pYLCRISPR/Cas9-MT-RMS 1.
pYLCRISPR/Cas9-MT-RMS1 contains the gene encoding the Cas9 protein and the gene encoding the sgRNA.
The invention also protects the application of the specific sgRNA or the specific recombinant plasmid in rice breeding; the purpose of rice breeding is to cultivate photosensitive male sterile rice.
The invention also provides a method for preparing a transgenic plant, which comprises the following steps: and (3) introducing the coding gene of the specific sgRNA and the coding gene of the Cas9 protein into receptor rice to obtain the photosensitive male sterile rice. The encoding gene of the specific sgRNA and the encoding gene of the Cas9 protein are specifically introduced into receptor rice through the recombinant plasmid.
In order to solve the technical problem, the invention also provides protein RMS 1.
The protein RMS1 was either a11) or a12) as follows:
A11) the amino acid sequence is shown as SEQ ID No.1 in the sequence table;
A12) homologous protein having 98% or more than 99% identity to A11) and derived from rice.
Wherein, the protein shown in SEQ ID No.1 consists of 345 amino acid residues.
In order to solve the technical problem, the invention also provides a gene for coding the protein RMS 1.
The gene coding the protein RMS1 provided by the invention is any one of the following b11) -b 14):
b11) The nucleotide sequence of the DNA molecule is shown as SEQ ID No.2 in the sequence table;
b12) The nucleotide sequence of the DNA molecule is shown as SEQ ID No.3 in the sequence table;
b13) A DNA molecule having 75% or more 75% identity to the nucleotide sequence defined in b11) or b12) and encoding the protein RMS 1;
b14) a DNA molecule which is hybridized with the nucleotide sequence defined by b11) or b22) under strict conditions and codes for protein RMS 1.
Wherein, SEQ ID No.2 in the sequence table consists of 1038 nucleotides and encodes the protein shown by SEQ ID No.1 in the sequence table.
In order to solve the technical problem, the invention also provides protein RMS 1-4.
The protein RMS1-4 is A21) or A22) as follows:
A21) the amino acid sequence is shown as SEQ ID No.6 in the sequence table;
A22) homologous protein having 98% or more than 99% identity to A21) and derived from rice.
Wherein, the protein shown in SEQ ID No.6 consists of 359 amino acid residues.
In order to solve the technical problem, the invention also provides a gene for coding the protein RMS 1-4.
The gene coding protein RMS1-4 provided by the invention is any one of the following b21) -b 24):
b21) the nucleotide sequence of the DNA molecule is shown as SEQ ID No.7 in the sequence table;
b22) the nucleotide sequence of the DNA molecule is shown as SEQ ID No.8 in the sequence table;
b23) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by b21) or b22) and codes the protein RMS 1-4;
b24) a DNA molecule which is hybridized with the nucleotide sequence defined by b21) or b22) under strict conditions and codes for protein RMS 1-4.
Wherein, SEQ ID No.7 in the sequence table is composed of 1080 nucleotides and encodes the protein shown by SEQ ID No.6 in the sequence table.
In order to solve the technical problem, the invention also provides protein RMS 1-5.
The protein RMS1-5 is A31) or A32) as follows:
A31) the amino acid sequence is shown as SEQ ID No.9 in the sequence table;
A32) homologous protein having 98% or more than 99% identity to A31) and derived from rice.
Wherein, the protein shown in SEQ ID No.9 consists of 111 amino acid residues.
In order to solve the technical problem, the invention also provides a gene for coding the protein RMS 1-5.
The gene coding the protein RMS1-5 provided by the invention is any one of the following b31) -b 34):
b31) the nucleotide sequence of the DNA molecule is shown as SEQ ID No.10 in the sequence table;
b32) the nucleotide sequence of the DNA molecule is shown as SEQ ID No.11 in the sequence table;
b33) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by b31) or b32) and codes the protein RMS 1-5;
b34) a DNA molecule which is hybridized with the nucleotide sequence defined by b31) or b32) under strict conditions and codes for protein RMS 1-5.
Wherein, SEQ ID No.10 in the sequence table is composed of 336 nucleotides and encodes the protein shown by SEQ ID No.9 in the sequence table.
In order to solve the technical problem, the invention also provides a protein RMS 1-11.
The protein RMS1-11 is A41) or A42) as follows:
A41) the amino acid sequence is shown as SEQ ID No.12 in the sequence table;
A42) homologous protein having 98% or more than 99% identity to A41) and derived from rice.
Wherein, the protein shown in SEQ ID No.12 consists of 360 amino acid residues.
In order to solve the technical problem, the invention also provides a gene for coding the protein RMS 1-11.
The gene coding the protein RMS1-11 provided by the invention is any one of the following b1) -b 4):
b41) the nucleotide sequence of the DNA molecule is shown as SEQ ID No.13 in the sequence table;
b42) the nucleotide sequence of the DNA molecule is shown as SEQ ID No.14 in the sequence table;
b43) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by b41) or b42) and codes the protein RMS 1-11;
b44) a DNA molecule which is hybridized with the nucleotide sequence defined by b41) or b42) under strict conditions and codes for protein RMS 1-11.
Wherein, SEQ ID No.13 in the sequence table is composed of 1083 nucleotides and encodes the protein shown by SEQ ID No.12 in the sequence table.
In order to solve the technical problems, the invention also provides application of the RMS1 protein or the coding gene of the RMS1 protein in regulating and controlling the photoperiod sensitivity fertility of rice.
In order to solve the technical problems, the invention also provides application of the RMS1 protein or the coding gene of the RMS1 protein in cultivating photosensitive male sterile rice.
In order to solve the technical problems, the invention also provides a method for breeding photosensitive male sterile rice by using the RMS1 protein or the coding gene of the RMS1 protein as a target.
In the present invention, the male sterility is represented by reduced pollen fertility or pollen abortion.
In the invention, the photosensitive male sterility is photosensitive and temperature-sensitive male sterility with the dominant photosensitive effect; the photo-thermo-sensitive male sterility is photo-thermo-sensitive nuclear male sterility.
In the invention, the photosensitive male sterile rice is rice with pollen fertility changed along with the change of illumination time.
The invention utilizes CRISPR/Cas9 technology to edit the rice RMS1 gene at fixed points, and knocks out the rice RMS1 gene through frameshift mutation, so that the protein RSM1 is inactivated, and a new generation of photosensitive male sterile rice germplasm (RMS1 gene knockouts rice) is obtained. Compared with wild rice, the obtained RMS1 gene knockout rice has no obvious difference in the vegetative growth stage, but the pollen fertility changes along with the change of illumination time length, under the condition of short illumination (the illumination time length is 10.5 hours; the illumination time period temperature is 30 ℃, the illumination intensity is 30000 Lx; the dark time period temperature is 24 ℃), the anther of the RMS1 gene knockout rice is whitened and shrunken, the number of pollen grains is obviously reduced, compared with the wild rice, the pollen iodine staining shows that the rice contains a large amount of sterile pollen grains, and the fertility is obviously reduced; under the long-light condition (the illumination time is 13.5 hours; the temperature in the illumination time period is 30 ℃, the illumination intensity is 30000 Lx; and the temperature in the dark time period is 24 ℃), the anther of the RMS1 gene knockout rice is bright yellow, the shape is full, the pollen grain number is normal, the fertility is consistent with that of wild rice, and the fertility is recovered compared with that of a mutant under the short-light condition. In order to further explore the relationship between the pollen fertility and the temperature of the RMS1 mutant material, different temperatures are set for treatment on the basis of short-light conditions. The result shows that the fertility of the RMS1 mutant pollen is obviously lower than that of wild rice in both pollen quantity and dyeing rate under the condition of short light regardless of the temperature, and further proves that the short light environment has a decisive effect on the pollen fertility of the RMS1 mutant. Meanwhile, under the condition of short illumination, the low temperature also has the promotion effect on the reduction of the pollen fertility of the RMS1 mutant, and the pollen sterility characteristic of the mutant RMS1 is enhanced. Thus, the RMS1 gene knock-out rice is photosensitive male sterile rice (photosensitive male nuclear sterile rice). The photosensitive male sterile rice can be used as a female parent to produce hybrid seeds by matching with a dominant variety. The photosensitive male sterile rice not only provides a new sterile line material for two-line hybrid breeding of rice, but also lays a theoretical foundation for the hybrid breeding of other gramineous crops.
Drawings
FIG. 1 is a map of the intermediate vector pYLgRNA-U3.
FIG. 2 is an amplification electrophoretic detection map of the expression cassette of the intermediate vector pYLgRNA-U3-RMS 1.
FIG. 3 is a map of the genome editing vector pYLCRISPR/Cas9-MTmono vector.
FIG. 4 is an electrophoresis diagram showing the result of PCR detection of a single colony of E.coli transformed with the recombinant vector pYLCRISPR/Cas9-MT-RMS 1.
FIG. 5 shows a sequencing alignment of the recombinant vector pYLCRISPR/Cas9-MT-RMS 1.
FIG. 6 shows the mutation type of RMS1 gene and the amino acid type encoded after the mutation; therein, 952238740-targetIs wild type rice 9522; the black part of the encoded protein is the RMS1 core domain, and the gray part is the newly encoded protein region after RMS1 mutation.
FIG. 7 shows a wild type rice variety 9522 and an RMS1 homozygous mutant 952238740-5Hybridization F2Group of generationsAnd (4) performing phenotype analysis.
FIG. 8 shows a RMS1 homozygous mutant 952238740-5Phenotype comparison with wild type rice variety 9522 at different illumination durations; wherein, A is long light irradiation treatment, and B is short light irradiation treatment.
FIG. 9 shows a RMS1 homozygous mutant 952238740-5Pollen grain I treated at different temperatures under short illumination compared with wild type rice 95222-comparison of KI staining effects; wherein A is a pollen grain staining microscopic picture of a wild type rice variety 9522 after a short-day high-temperature treatment, and B is a homozygous mutant 952238740-5Microscopic staining of pollen grains after short-day high-temperature treatment; c is a pollen grain staining microscopic picture of a wild type rice variety 9522 after short-day low-temperature treatment, D is a homozygous mutant 952238740-5Staining microscopic picture of pollen grains after short day low temperature treatment.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The expression vector pYLgRNA-U3 is used for editing a RICE ear development Osal gene at a fixed point in a document of Shijiang Wei, Li yi star, Song Shufeng, Qiu peony, Deng Yao, Li. CRISPR/Cas 9. HYBRID RICE (HYBRID RICE), 2017 and 32 (3): 74-78, the biological material is only used for repeating the experiments related to the present invention and is not used for other purposes.
Expression vector pYLCRISPR/Cas9-MTmono in the literature "Shijiang Wei, Li-yi star, Song front, Qiu peony, Du Yao, Li. CRISPR/Cas9 fixed-point editing RICE tasal gene HYBRID RICE (HYBRID RICE), 2017, 32 (3): 74-78, publicly available from the research center for hybrid rice in Hunan, the biomaterial was used only for repeating the experiments related to the present invention, and was not used for other purposes.
RICE cultivar wuyunjing No.7 (primary No. 9522) RICE panicle development Osal gene was edited at the CRISPR/Cas9 site in the literature HYBRID RICE (HYBRID RICE), 2017, 32 (3): 74-78, the biological material is only used for repeating the experiments related to the present invention and is not used for other purposes.
Example 1 selection of Rice RMS1 Gene target site and construction of knockout vector
The inventor of the invention discovers a gene related to photosensitive Male Sterility, namely RMS1(Reverse Male Sterility) gene from the rice Wuyujing 7. Wherein, the coding sequence of the RMS1 gene is shown as SEQ ID No.2 in the sequence table, and codes a protein RMS1 consisting of 345 amino acid residues, and the amino acid sequence is shown as SEQ ID No.1 in the sequence table. The gDNA total length of the RMS1 gene is 2623bp, contains 3 exons and 4 introns, and the nucleotide sequence thereof is shown as SEQ ID No.3 in the sequence table.
In the embodiment, the rice RMS1 gene is knocked out by a CRISPR/Cas9 gene editing technology to obtain a mutant 9522 with a photosensitive male sterility phenotype38740-4、952238740-5And 952238740-11Mutant 952238740-4、952238740-5And 952238740-11Both are RMS1 knock-out rice. The specific operation method comprises the following steps:
1. design of target sequences
The target sequence used was 5'-CCAAGGCCGGTAAGCGCCGC-3', which is located where the 1 st exon joins the 2 nd intron sequence, i.e.positions 384 to 403 of sequence 3.
2. Construction of intermediate vector pYLgRNA-U3-RMS1
(1) RMS1 target site adapter primer design and synthesis
After the target site sequence is determined, GGCA is added before the 5 'of the sense strand of the target sequence, and AAAC is added before the 5' of the antisense strand to obtain the target site joint primer. The target site linker primer sequences are as follows:
RMS1-Cas9-F：5’-GGCACCAAGGCCGGTAAGCGCCGC-3’
RMS1-Cas9-R：5’-AAACGCGGCGCTTACCGGCCTTGG-3’
(2) preparation of RMS1 target site linker
RMS1 target site linker primers RMS1-Cas9-F and RMS1-Cas9-R are diluted into mother liquor with the concentration of 10 mu M by ddH2O, 10 mu L to 80 mu L of deionized water are respectively taken to reach the final volume of 100 mu L, after fully mixing, the mother liquor is thermally shocked for 30s at 90 ℃, and then the mother liquor is moved to the room temperature to complete annealing, so that RMS1 target site linker is obtained and is marked as RMS1-Cas 9.
(3) Construction of RMS1 intermediate vector
mu.L of pYLgRNA-U3 vector plasmid (shown in FIG. 1), 1. mu.L of 10 XT 4 DNA Ligase Buffer, 1. mu.L of target site linker RMS1-Cas9, 1. mu.L of Bsa I restriction enzyme and 0.5. mu.L of 10 XT 4 DNA Ligase were mixed uniformly and reacted with a PCR instrument under the following reaction conditions: 5min at 37 ℃ and 5min at 20 ℃ for 5 cycles to obtain an intermediate vector containing a rice RMS1 gene target sequence, and the intermediate vector is named as pYLgRNA-U3-RMS 1.
3. Construction and transformation of RMS1 site-directed editing final vector
(1) Amplification of the RMS1 intermediate vector expression cassette
PCR amplification is carried out by taking an intermediate vector pYLgRNA-U3-RMS1 as a template and Uctcg-B1(TTCAGAGGTCTCTCTCGCACTGGAATCGGCAGCAAAGG) and gRCggt-BL (AGCGTGGGTCTCGACCGGGTCCATCCATCCACCAAGCTC) as primers to obtain an amplification product. The amplification product was detected by gel electrophoresis and determined to be a DNA molecule of about 550bp in size (as shown in FIG. 2), and the amplification result was consistent with the expectation. The amplification product was recovered and purified and designated as RMS1 intermediate vector expression cassette. The expression cassette comprises a sgRNA coding gene and a U3 promoter, wherein the sgRNA target sequence is 5'-CCAAGGCCGGTAAGCGCCGC-3', sgRNA coding gene and is shown as SEQ ID No.4 in a sequence table.
(2) Construction and transformation of RMS1 site-directed editing final vector
The gene editing vector pYLCRISPR/Cas9-MTmono (shown in figure 3) and the RMS1 intermediate vector expression cassette were digested and ligated using Bsa I restriction endonuclease and T4 DNA Ligase to obtain RMS1 gene site-directed editing final vector pYLCRISPR/Cas9-MT-RMS 1. Coli was transformed, plated on a plate containing kanamycin, and cultured overnight at 37 ℃.
(3) Detection of recombinant vector pYLCRISPR/Cas9-MT-RMS1
Randomly picking 3 single colonies cultured overnight in the step (2) and respectively named as RMS1-Cas9-1, RMS1-Cas9-2 and RMS1-Cas9-3, and performing PCR detection on the 3 single colonies by using pYLCRISPR/Cas9-MTmono vector detection primers SP1(CCCGACATAGATGCAATAACTTC) and SP2 (GCGCGGTGTCATCTATGTTACT). The PCR amplification products were subjected to gel electrophoresis, and the results of gel electrophoresis (shown in FIG. 4) showed that the RMS1-cas9-2 monoclonal colony amplified a band of about 550bp, which was consistent with the expectation.
Plasmid DNA from a single clone of RMS1-cas9-2 was extracted for sequencing. The sequencing results (as shown in figure 5) show that: the DNA fragment shown as SEQ ID No.5 in the sequence table successfully replaces the DNA fragment between two Bsa I enzyme cutting sites on a gene editing vector pYLCRISPR/Cas 9-MTmono. This shows that the expression cassette containing the U3 promoter and sgRNA encoding gene is successfully constructed on the gene editing vector pYLCISPR/Cas 9-MTmono, i.e. the genome site-directed editing vector of RMS1 is successfully constructed to obtain the recombinant vector pYLCISPR/Cas 9-MT-RMS 1.
Example 2 acquisition of RMS1 mutant Rice Material and phenotypic analysis thereof
First, obtaining of RMS1 mutant Rice Material
A method for transforming rice callus by agrobacterium-mediated transformation is used, a vector pYLCRISPR/Cas9-MT-RMS1 is edited by an RMS1 gene site-specific editing gene to transform rice variety Wuyujing No.7 (original code is 9522, hereinafter referred to as 9522) callus, and a positive mutant is obtained by screening and identifying.
Second, detection of fixed point editing
Homozygous mutants of 3 mutation types with the RMS1 gene knocked out are obtained through PCR detection and are respectively named as homozygous mutants 952238740-4Homozygous mutant 952238740-5Homozygous mutant 952238740-11. The sequencing results showed (as shown in FIG. 6):
homozygous mutant 9522 for the RMS1 gene (wild-type)38740-4A mutation of 2 bases deletion at position 128-129 of the CDS of the RMS1 gene, which results in a displacement of the ORF of the RMS1 gene after position 126 and a new stop codon in the RMS 13' UTR sequence; the gene after frameshift mutation is named as RMS1-4 gene, the nucleotide sequence of the RMS1-4 gene is shown as SEQ ID No.8 in a sequence table, and RMThe coding sequence of the S1-4 gene is shown as SEQ ID No.7 in the sequence table, codes a protein RMS1-4 consisting of 359 amino acid residues, and the amino acid sequence is shown as SEQ ID No.6 in the sequence table.
Homozygous mutant 9522 for the RMS1 gene (wild-type)38740-5The mutation, 1 base is deleted at the 127 th position of the CDS of the RMS1 gene, the mutation causes the displacement of the ORF of the RMS1 gene after the 126 th position, a new stop codon is formed in advance at the 334-336bp position of the CDS of the RMS1 gene, and the translation is stopped; the frame shift mutated gene is named as RMS1-5 gene; the nucleotide sequence of the RMS1-5 gene is shown as SEQ ID No.11 in the sequence table, the coding sequence of the RMS1-5 gene is shown as SEQ ID No.10 in the sequence table, and the coding sequence of the RMS1-5 protein consisting of 111 amino acid residues has the amino acid sequence shown as SEQ ID No.9 in the sequence table.
Homozygous mutant 9522 for the RMS1 gene (wild-type)38740-11A mutation of 1 base inserted at position 127 of CDS of RMS1 gene, which results in displacement of ORF of RMS1 gene after position 126 and formation of new stop codon in RMS 13' UTR sequence; the frame shift mutated gene is named as RMS1-11 gene; the nucleotide sequence of the RMS1-11 gene is shown as SEQ ID No.14 in the sequence table, the coding sequence of the RMS1-11 gene is shown as SEQ ID No.13 in the sequence table, and the coding sequence encodes a protein RMS1-11 consisting of 360 amino acid residues, and the amino acid sequence is shown as SEQ ID No.12 in the sequence table.
2 core domains of SANT of wild-type RMS1 protein are respectively located at 14-61 th and 67-112 th positions of amino acid sequence, therefore, the mutation of the positions all result in deletion of the core domain SANT, and further influence the function of RMS1 gene.
Tris, RMS1 mutant F2Co-segregating population construction and phenotypic analysis
Homozygous mutant 952238740-4、952238740-5、952238740-11Have the same mutant phenotype and agronomic traits. Thus, homozygous mutant 9522 is shown in the subsequent examples of the invention38740-5For example, detailed phenotypic analysis was performed.
1、F2Population construction
Homozygous mutant 9522 with wild 9522 as mother parent38740-5T obtained by selfing1The generation individual plant is taken as a male parent to be hybridized (planting time: 201806-1Seed, F1The generation group is planted in the Hainan Ling water (planting time: 201812 and 201904); f1Inbreeding of the generations to obtain F2Generation group, F2The generation group is planted in the Hunan Changsha (planting time: 201906-.
2. Phenotypic analysis
F of step 12A total of 42 individuals of the generation segregating population, for which F2All the individuals of the segregation population are subjected to genotype identification and analysis, 3 genotypes are separated in total, namely wild genotypes (the corresponding positions of two stainers are both RMS1 genes from wild rice) and RMS1 mutant material homozygous mutant 9522 are respectively obtained38740-5Homozygous genotype (corresponding positions on both chromosomes are from homozygous mutant 952238740-5The RMS1-5 gene of (1), hereinafter referred to as 952238740-5Genotype), as well as homozygous mutants 9522 of wild type and RMS1 mutant material38740-5Heterozygous genotype (i.e., the corresponding position on one chromosome is the RMS1 gene from wild-type rice and the corresponding position on the other chromosome is from homozygous mutant 952238740-5The RMS1-5 gene of (1), hereinafter referred to as heterozygous genotype). Wherein 7 strains of the wild type genotype group, 26 strains of the heterozygous genotype group, 952238740-5Genotype group 9, wild-type genotype: heterozygous genotype: 952238740-5The segregation ratio of genotypes was 7: 26: 9, substantially in accordance with 1: 2: 1 separation ratio.
Found by planting, F2The leaves of each individual plant in the generation segregation population are consistent in shape. The anthers with different genotypes are found to have different shapes by microscopic observation. F2In the generation separation population, the anther of the wild type population is bright yellow, the anther shape is full, the number of pollen grains is normal, and the anther can be cultivated by microscopic examination; the heterozygous genotype population is identical to the wild-type population; and 952238740-5Abnormal whitening of anthers and anthers in genotypic groupThe morphology was shriveled, the number of pollen grains decreased suddenly, and the anther microscopic examination showed that a large number of sterile pollen grains were contained (as shown in FIG. 7). Corresponds to F2Population segregation ratio, which indicates that the RMS1 gene mutation causes anther dysplasia and further influences the fertility of the pollen of the receptor plant.
Example 3 analysis of photosensitivity of RMS1 mutant Rice Material
1. Photosensitive characteristic analysis of RMS1 mutant rice material
Homozygous mutant 9522 of RMS1 mutant material grown in natural conditions in Hainan Ling water (18 ° 51 '23 "N, 110 ° 5' 6" E)38740-4T2Generation and homozygous mutant 952238740-5T2The generation and wild type rice 9522, and the result shows that the homozygous mutant 952238740-4T2Generation and homozygous mutant 952238740-5T2The fructification rate of the generation plants is 4.56 percent and 3.13 percent respectively, while the fructification rate of the wild type rice 9522 is 95.6 percent (201812-; homozygous mutant 9522 was grown in Hunan Changsha (28 ℃ 13 '07 "N, 113 ℃ 15' 10" E) under natural conditions38740-4T3Generation and homozygous mutant 952238740-5T3The generation and wild type rice 9522, and the result shows that the homozygous mutant 952238740-4T3Generation and homozygous mutant 952238740-5T3The maturing rates of the generation mutants were 35.29% and 16.02%, respectively, while that of the wild type rice 9522 was 96.75% (201906-201910). The same RMS1 mutant rice line has obvious difference in planting maturing rate in different regions, and the illumination length in different regions is considered to influence the fertility of pollen, thereby causing the maturing rate to change.
To further explore the effect of illumination on pollen fertility of RMS1 mutant plants, 7 homozygous mutants 9522 were each added during full-time growth38740-5T3The plants were tested by exploratory short light treatment (light duration 10.5 hours, light intensity 30000 Lx; light time period temperature 30 ℃ C., dark time period temperature 24 ℃ C.) and long light treatment (light duration 13.5 hours, light intensity 30000 Lx; light time period temperature 30 ℃ C., dark time period temperature 24 ℃ C.), all of which were temperature and light controllableIn a greenhouse. Wild type rice 9522 was used as a control.
Collection of wild type Rice 9522 and mutant 952238740-5T3The mature anthers of the plants were subjected to microscopic examination and iodine staining. During iodine staining, anthers of 3 florets of a single rice plant are taken and put on a glass slide, the anthers are mashed by tweezers to release pollen grains, and 1-2 drops of I are added2KI solution, coverslipped and observed under microscope. The blue-dyed pollen grains are the pollen grains with strong vitality, and the yellow-brown pollen grains are the dysplasia pollen grains. Taking 3 visual fields for each piece of the pollen, counting the staining rate of the pollen, and expressing the fertility of the pollen by using the staining rate.
The results show that: under the condition of short illumination, the anther of the wild rice 9522 is bright yellow, the shape is full, and the number of pollen grains is normal; the pollen is stained by iodine, and the staining rate is 95.64 percent, which shows that the pollen fertility of the wild rice 9522 is normal; homozygous mutant 9522 under equivalent conditions38740-5T3The anther of the plant generation is whitened and shrunken, and the number of pollen grains is obviously reduced; the iodine staining rate of the pollen is only 28.17%, which shows that the mutant 952238740-5T3The pollen fertility of the generations of plants was significantly reduced (as shown in table 1 and fig. 8B).
Under the long-time illumination condition, the anther of the wild rice 9522 is bright yellow, full in shape and normal in pollen grain number; the pollen is stained by iodine, and the staining rate is 96.18 percent, which shows that the pollen fertility of the wild rice 9522 is normal; homozygous mutant 9522 under equivalent conditions38740-5T3The anther of the plant substitute is bright yellow, has full shape and normal pollen grain number; pollen was iodine stained with 86.40% (as shown in table 1 and fig. 8A).
Homozygous mutant 9522 under long light conditions compared to short light conditions38740-5T3The number of pollen grains and the dyeing rate of the pollen grains of the generation plants are obviously recovered, which shows that the homozygous mutant 952238740-5T3The pollen fertility of the generation plants is restored.
TABLE 1 homozygous mutant 9522 RMS1 under different lighting conditions38740-5Pollen grain I of T3 plant and wild rice 9522 variety2-KI staining results statistics
Note: p < 0.01.
Therefore, under different illumination conditions, the color, the shape and the pollen fertility microscopic examination of the anther of the control material 9522 are consistent, which indicates that the illumination time does not influence the pollen fertility of the receptor material 9522. However, the RMS1 homozygous mutant 952238740-5T3The difference of anther color, shape, pollen grain number and the like of the pollen of the generation plants is obvious under the treatment of different illumination time lengths. Homozygous mutant 9522 under short light conditions38740-5T3The anther of the plant substitute is whitish in color and shriveled in shape; iodine staining results show that the number of pollen grains is greatly reduced, and the pollen grains contain a large amount of sterile pollen grains, and have obvious difference compared with a control under the same condition; whereas under long-term light conditions, homozygous mutant 952238740-5T3The number and fertility of pollen grains of generation plants are obviously restored, and the homozygous mutant 952238740-5T3The anther color, shape, pollen grain number, etc. of the generation plants are consistent with those of the control material under the same conditions. The result shows that the fertility of the RMS1 mutant material pollen is sensitive to the illumination duration, the fertility of the RMS1 mutant pollen is suddenly reduced under the short illumination environment, and the fertility of the RMS1 mutant pollen can be restored under the long illumination environment.
2. Effect of temperature on fertility of RMS1 mutant Rice pollen
(1) Effect of temperature on fertility of RMS1 mutant Rice under greenhouse conditions
In order to further explore the relationship between pollen fertility and temperature of the RMS1 mutant material, different temperatures were set for treatment on the basis of 12-hour short-light conditions.
6 plants to be planted in the field are 952238740-5T3The generation plants and 6 wild type 9522 plants were transferred into a culture pot during the jointing period so that the plants grew mildly. And when the plant is in the early stage of the booting ear, transferring the plant into an incubator to perform short-illumination high-low temperature treatment. Wherein, the short illumination low temperature (hereinafter referred to as short day low temperature) treatment condition is that the illumination time is 12 hours,the illumination intensity is 30000Lx, and the temperature is 23 ℃; the conditions of short-time irradiation high-temperature (hereinafter referred to as short-time high-temperature) treatment were an irradiation time of 12 hours, an irradiation intensity of 30000Lx, and a temperature of 33 ℃. And after the plants are subjected to ear sprouting, performing pollen microscopic examination on the plants treated at different temperatures. 3 florets of each individual plant were subjected to mixed microscopic examination. The pollen fertility microscopic examination method is the same as the above, each slide takes 3 visual fields, the dyeing rate of the pollen is counted, and the pollen fertility is expressed by the dyeing rate.
The results show that: under the condition of short day and high temperature, the iodine staining rate of pollen of the wild rice 9522 is 94.41 percent, and under the same condition, the iodine staining rate of pollen of the wild rice 9522 is 952238740-5T3The iodine staining rate of pollen of the generation plants is 23.86 percent; under the condition of short day and low temperature, the iodine staining rate of pollen of the wild rice 9522 is 89.75%, and the homozygous mutant 9522 under the same condition38740-5T3The iodine staining rate of pollen of the generation plants was 0 (as shown in table 2 and fig. 9). This indicates that the RMS1 mutant material homozygous mutant 952238740-5Under the condition of short illumination, the number of pollen and the dyeing rate are obviously lower than those of wild rice no matter the temperature is high or low, and the fact that the short illumination environment has a decisive effect on the pollen fertility of the RMS1 mutant is further verified. Meanwhile, under the condition of short illumination, the low temperature also has the promotion effect on the reduction of the pollen fertility of the RMS1 mutant.
TABLE 2 homozygous mutant 9522 RMS1 under different temperature conditions38740-5T3Pollen grain I of plant and wild rice 9522 variety2-KI staining results statistics
Note: p < 0.01.
(2) Influence of temperature in natural environment on fertility of rice pollen of RMS1 mutant
The homozygous mutant 9522 of RMS1 mutant material was planted in two batches under the natural conditions of Hainan Ling water (18 degrees 51 '23' N, 110 degrees 5 '6' E, 201912-reservoir 202004)38740-5T4The first batch of the materials are sown for 12 months and 3 days in 2019, and the booting period of the first batch of the materials is aboutThe average temperature of the time is 22.2 ℃ in 2 months and 10 months in 2020 to 5 months in 2020 and the seeding time of the second batch of materials is 12 and 13 days in 2019, the booting period is about 2 and 20 days in 2020 to 3 and 15 days in 2020, the average temperature of the time is 23.78 ℃ and the average temperature of the booting period of the two batches of materials is different by 1.58 ℃. After the plant shoots, homozygous mutant 9522 sown in different batches is collected38740-5T4Mature anthers of the generation plants and wild type rice 9522 plants are subjected to pollen iodine staining, 3 single plants are randomly selected from each group, and 1 visual field is taken from each single plant for statistics.
As a result, it was found that: first homozygous mutant 952238740-5T4The microscopic pollen staining rate of the generation plants is 0 percent, and the microscopic pollen staining rate of the wild type 9522 pollen of the same batch is 94.87 percent; while the second batch of homozygous mutant 952238740-5T4The microscopic staining rate of the pollen of the generation plants is 10.97%, and the microscopic staining rate of the pollen of the wild type 9522 of the same batch is 92.19% (shown in Table 3). This indicates that: the change of the iodine staining rate of the pollen occurs when the mutant material of the same strain is sowed in batch in the same place, which shows that the temperature difference between different sowing periods can cause the change of the pollen fertility.
Table 3RMS1 homozygous mutant 9522 sown in different sowing batches38740-5T4Pollen grain I of plant and wild rice 9522 variety2-KI staining results statistics
In conclusion, the pollen of the RMS1 mutant material is sensitive to long-short response to light, and is specifically shown in that the pollen of the RMS1 mutant material can be cultivated under the long-day (long-light) condition; the fertility of the pollen of the short-day (short-day) RMS1 mutant material is obviously reduced, and the low temperature can promote the complete abortion of the mutant RMS1 pollen and enhance the sterility characteristic of the mutant RMS1 pollen. Therefore, RMS1 is considered to have photosensitivity characteristic and is a photosensitivity fertility related gene, and photosensitivity type male sterile rice can be obtained by knocking out the gene.
<110> research center for hybrid rice in Hunan province
<120> method for preparing rice photosensitive male sterile material and related gene
<130> GNCRJ200346
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 345
<212> PRT
<213> Rice (Oryza sativa)
<400> 1
Met Gly Arg Ser Pro Cys Cys Glu Lys Glu Gly Leu Lys Lys Gly Pro
1 5 10 15
Trp Thr Pro Glu Glu Asp Gln Lys Leu Leu Ala Tyr Ile Glu Gln His
20 25 30
Gly His Gly Cys Trp Arg Ser Leu Pro Ser Lys Ala Gly Leu Gln Arg
35 40 45
Cys Gly Lys Ser Cys Arg Leu Arg Trp Thr Asn Tyr Leu Arg Pro Asp
50 55 60
Ile Lys Arg Gly Lys Phe Ser Leu Gln Glu Glu Gln Thr Ile Ile Gln
65 70 75 80
Leu His Ala Leu Leu Gly Asn Arg Trp Ser Ala Ile Ala Thr His Leu
85 90 95
Pro Lys Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr His Leu
100 105 110
Lys Lys Arg Leu Ala Lys Met Gly Ile Asp Pro Val Thr His Lys Pro
115 120 125
Arg Ser Asp Val Ala Gly Ala Gly Gly Gly Gly Gly Gly Ala Ala Gly
130 135 140
Gly Ala Ala Gly Ala Gln His Ala Lys Ala Ala Ala His Leu Ser His
145 150 155 160
Thr Ala Gln Trp Glu Ser Ala Arg Leu Glu Ala Glu Ala Arg Leu Ala
165 170 175
Arg Glu Ala Lys Leu Arg Ala Leu Ala Ala Ser Ala Thr Pro Gly Ala
180 185 190
Pro His Leu Pro Ala Pro Pro Ala Ser Ala Ala Ala Ala Ala Ala Ala
195 200 205
His Gly Leu Asp Ser Pro Thr Ser Thr Leu Ser Phe Ser Glu Ser Ala
210 215 220
Val Leu Ala Thr Val Leu Glu Ala His Gly Ala Ala Ala Ala Ala Ala
225 230 235 240
Ala Arg Ala Ala Met Gln Pro Met Gln Ala Tyr Asp Glu Ala Cys Lys
245 250 255
Asp Gln His Trp Gly Asp Val Asp Ala Ala Asp Val Gly Phe Pro Gly
260 265 270
Ala Gly Ala Gly Phe Thr Gly Leu Leu Leu Glu Gly Ser Leu Asn Gln
275 280 285
Ile Pro Arg Pro Ala Gly Arg Asp Ala Glu Ala Asp Gly Glu Phe Gln
290 295 300
Glu Thr Glu Glu Glu Lys Asn Tyr Trp Asn Ser Ile Leu Asn Leu Val
305 310 315 320
Asn Ser Ser Ser Ala Pro Met Ser Thr Ala Val Val Val Pro Ala Ser
325 330 335
His Ala Tyr Ser Pro Ala Pro Asp Phe
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<212> DNA
<213> Rice (Oryza sativa)
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atggggcgat cgccgtgctg cgagaaggag gggctcaaga aggggccatg gacgccggag 60
gaggaccaga agctgctggc ctacatcgag cagcacggcc acggctgctg gcgctcgcta 120
ccctccaagg ccgggctgca gcggtgcggc aagagctgcc gactccggtg gacgaactac 180
ctccggccgg acatcaagag gggcaagttc agcctgcagg aggagcagac catcatccag 240
ctccacgccc ttctcggcaa caggtggtcg gcgatcgcga cgcacctgcc gaagcgcacg 300
gacaacgaga tcaagaacta ctggaacacg cacctaaaga agcggctggc caagatgggg 360
atcgacccgg tcacgcacaa gccgcgctcc gacgtggccg gcgcgggcgg cggcggcgga 420
ggtgcggccg gcggcgcggc gggcgcgcag cacgccaagg ccgcggcgca cctcagccac 480
acggcgcagt gggagagcgc gcggctcgag gcggaggcgc gcttggctcg ggaggccaag 540
ctgcgcgcgc tcgcggcctc cgcgaccccg ggcgcgccgc acctcccggc accccccgcg 600
tcggcggcgg cggcggcggc ggctcacggc ctcgactcgc cgacgtccac gctgagcttc 660
tcggagagcg cggtgctcgc caccgtgctg gaggcgcacg gcgccgccgc cgcggcggcc 720
gcgcgcgccg ccatgcagcc catgcaggcg tacgacgagg cgtgcaagga ccagcactgg 780
ggcgacgtcg acgccgccga cgtgggcttc cccggcgccg gagcggggtt cacgggccta 840
ctcctcgaag gctccttgaa ccagatcccg aggccggcgg ggagagacgc ggaagccgac 900
ggcgagttcc aggagaccga ggaggagaag aactactgga acagcatact gaacctggtg 960
aactcctcgt cggcacctat gtcgacggcc gtggttgtgc ccgcctccca cgcgtactcg 1020
ccggcgccgg acttctga 1038
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<213> Rice (Oryza sativa)
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gcctcgatca cgtactacta cacatagacc tactacttga gcccgagaga agaggagagg 180
aggaagaacc agagggcgtc gaagatcatc ggggaggagt tttcctagag ctcgctgctg 240
ctgttgcttt tctccggcga tggggcgatc gccgtgctgc gagaaggagg ggctcaagaa 300
ggggccatgg acgccggagg aggaccagaa gctgctggcc tacatcgagc agcacggcca 360
cggctgctgg cgctcgctac cctccaaggc cggtaagcgc cgcttatcta gcttaaattt 420
cttctcaacc tctgcaatcc tagctgcaat gttcggtcga ggcgatcgat cctcgaggct 480
ggctactctc tgaactctga tctgaggtgc atgcaaaccg tgacaatcgt gtgcagggct 540
gcagcggtgc ggcaagagct gccgactccg gtggacgaac tacctccggc cggacatcaa 600
gaggggcaag ttcagcctgc aggaggagca gaccatcatc cagctccacg cccttctcgg 660
caacaggtga tcgattactt cgttttcgca tggatgcatc atgcatacaa gatacgtagt 720
gcacaactct ccctcctgct agctgctcgc tcgttcttca cctcgcaccc ggagcacatt 780
taattccgta atcgcgatgg aacccttgat tctcctgcac gaattttgac tgctagtact 840
tgttgctgac cggcaggtca agaacacact agccagtagc caccattctg caccgtagtc 900
ttggcagaca tttatgaaag ggttatgcaa tgcaagggtt ggaacacgga gcttagccag 960
gggatgtgta aatttcgcag gccgtgctac ttacttgctg tccccgtaca cacctgcttc 1020
agcattttgt ccgtaacaaa ccgtactgtc catagattaa cacacaagct aggctaaaaa 1080
ttcttacgtt agaacagaat catcacttgt tttcgttttg ttcacacgta atgctgcatt 1140
gctcatcttt tgcccgtcga acaaccacgc attagctgtg agcacagacc aatcaatgca 1200
tgcatcaaca agggaaaaag tgtgaaaagg ttgggcagtg agaggctcgg cccagaattt 1260
tcctttcttt tctcccatat gattcggcat tcaagctcgt catttaagga ggcgagcccc 1320
cccatcattg tggaccaaaa ctggggtttg gtccactgtt gccacctgcc cctcttccca 1380
ttttgactca cagcttccga tcatctctgc cctctgtctg tactacgcca cgcacgcctt 1440
aaatcacacc gccgattatt tacgttttca agagtgctgt ttgtttaatt ttgtcactgc 1500
gaatggaggg cttttgacgt gcgattttcc tgatcttttt tcttggccgg cgttggcgtt 1560
gttgttgttt tgcaggtggt cggcgatcgc gacgcacctg ccgaagcgca cggacaacga 1620
gatcaagaac tactggaaca cgcacctaaa gaagcggctg gccaagatgg ggatcgaccc 1680
ggtcacgcac aagccgcgct ccgacgtggc cggcgcgggc ggcggcggcg gaggtgcggc 1740
cggcggcgcg gcgggcgcgc agcacgccaa ggccgcggcg cacctcagcc acacggcgca 1800
gtgggagagc gcgcggctcg aggcggaggc gcgcttggct cgggaggcca agctgcgcgc 1860
gctcgcggcc tccgcgaccc cgggcgcgcc gcacctcccg gcaccccccg cgtcggcggc 1920
ggcggcggcg gcggctcacg gcctcgactc gccgacgtcc acgctgagct tctcggagag 1980
cgcggtgctc gccaccgtgc tggaggcgca cggcgccgcc gccgcggcgg ccgcgcgcgc 2040
cgccatgcag cccatgcagg cgtacgacga ggcgtgcaag gaccagcact ggggcgacgt 2100
cgacgccgcc gacgtgggct tccccggcgc cggagcgggg ttcacgggcc tactcctcga 2160
aggctccttg aaccagatcc cgaggccggc ggggagagac gcggaagccg acggcgagtt 2220
ccaggagacc gaggaggaga agaactactg gaacagcata ctgaacctgg tgaactcctc 2280
gtcggcacct atgtcgacgg ccgtggttgt gcccgcctcc cacgcgtact cgccggcgcc 2340
ggacttctga gcggagaaaa cctcgccggc atcaaacttg attcgatctc atgacacagt 2400
aaatagttta gcaatgattg tcgaataaga tgggactaat attaatgtta gtaattatta 2460
atcacgttct tgggttaacc tgacaatgct ttcgattaaa cttgtgggca agaacttcaa 2520
ctgttcaagg ctgtatcgac atttgaaatt cgatgcttgt tttgttcggt gttcttatag 2580
aatgtgtaaa acactgaaac tactatcaga gaatgtagca tcc 2623
<210> 4
<211> 72
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca 60
ccgagtcggt gc 72
<210> 5
<211> 533
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ctcgcactgg aatcggcagc aaaggaagga atctttaaac atacgaacag atcacttaaa 60
gttcttctga agcaacttaa agttatcagg catgcatgga tcttggagga atcagatgtg 120
cagtcaggga ccatagcaca agacaggcgt cttctactgg tgctaccagc aaatgctgga 180
agccgggaac actgggtacg ttggaaacca cgtgtgatgt gaaggagtaa gataaactta 240
ggagaaaagc atttcgtagt gggccatgaa gcctttcagg acatgtattg cagtatgggc 300
cggcccatta cgcaattgga cgacaacaaa gactagtatt agtaccacct cggctatcca 360
catagatcaa agctggttta aaagagttgt gcagatgatc cgtggcacca aggccggtaa 420
gcgccgcgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg 480
aaaaagtggc accgagtcgg tgcttttttt caagagcttg gagtggatgg acc 533
<210> 6
<211> 359
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Met Gly Arg Ser Pro Cys Cys Glu Lys Glu Gly Leu Lys Lys Gly Pro
1 5 10 15
Trp Thr Pro Glu Glu Asp Gln Lys Leu Leu Ala Tyr Ile Glu Gln His
20 25 30
Gly His Gly Cys Trp Arg Ser Leu Pro Ser Ser Arg Ala Ala Ala Val
35 40 45
Arg Gln Glu Leu Pro Thr Pro Val Asp Glu Leu Pro Pro Ala Gly His
50 55 60
Gln Glu Gly Gln Val Gln Pro Ala Gly Gly Ala Asp His His Pro Ala
65 70 75 80
Pro Arg Pro Ser Arg Gln Gln Val Val Gly Asp Arg Asp Ala Pro Ala
85 90 95
Glu Ala His Gly Gln Arg Asp Gln Glu Leu Leu Glu His Ala Pro Lys
100 105 110
Glu Ala Ala Gly Gln Asp Gly Asp Arg Pro Gly His Ala Gln Ala Ala
115 120 125
Leu Arg Arg Gly Arg Arg Gly Arg Arg Arg Arg Arg Cys Gly Arg Arg
130 135 140
Arg Gly Gly Arg Ala Ala Arg Gln Gly Arg Gly Ala Pro Gln Pro His
145 150 155 160
Gly Ala Val Gly Glu Arg Ala Ala Arg Gly Gly Gly Ala Leu Gly Ser
165 170 175
Gly Gly Gln Ala Ala Arg Ala Arg Gly Leu Arg Asp Pro Gly Arg Ala
180 185 190
Ala Pro Pro Gly Thr Pro Arg Val Gly Gly Gly Gly Gly Gly Gly Ser
195 200 205
Arg Pro Arg Leu Ala Asp Val His Ala Glu Leu Leu Gly Glu Arg Gly
210 215 220
Ala Arg His Arg Ala Gly Gly Ala Arg Arg Arg Arg Arg Gly Gly Arg
225 230 235 240
Ala Arg Arg His Ala Ala His Ala Gly Val Arg Arg Gly Val Gln Gly
245 250 255
Pro Ala Leu Gly Arg Arg Arg Arg Arg Arg Arg Gly Leu Pro Arg Arg
260 265 270
Arg Ser Gly Val His Gly Pro Thr Pro Arg Arg Leu Leu Glu Pro Asp
275 280 285
Pro Glu Ala Gly Gly Glu Arg Arg Gly Ser Arg Arg Arg Val Pro Gly
290 295 300
Asp Arg Gly Gly Glu Glu Leu Leu Glu Gln His Thr Glu Pro Gly Glu
305 310 315 320
Leu Leu Val Gly Thr Tyr Val Asp Gly Arg Gly Cys Ala Arg Leu Pro
325 330 335
Arg Val Leu Ala Gly Ala Gly Leu Leu Ser Gly Glu Asn Leu Ala Gly
340 345 350
Ile Lys Leu Asp Ser Ile Ser
355
<210> 7
<211> 1080
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atggggcgat cgccgtgctg cgagaaggag gggctcaaga aggggccatg gacgccggag 60
gaggaccaga agctgctggc ctacatcgag cagcacggcc acggctgctg gcgctcgcta 120
ccctccagcc gggctgcagc ggtgcggcaa gagctgccga ctccggtgga cgaactacct 180
ccggccggac atcaagaggg gcaagttcag cctgcaggag gagcagacca tcatccagct 240
ccacgccctt ctcggcaaca ggtggtcggc gatcgcgacg cacctgccga agcgcacgga 300
caacgagatc aagaactact ggaacacgca cctaaagaag cggctggcca agatggggat 360
cgacccggtc acgcacaagc cgcgctccga cgtggccggc gcgggcggcg gcggcggagg 420
tgcggccggc ggcgcggcgg gcgcgcagca cgccaaggcc gcggcgcacc tcagccacac 480
ggcgcagtgg gagagcgcgc ggctcgaggc ggaggcgcgc ttggctcggg aggccaagct 540
gcgcgcgctc gcggcctccg cgaccccggg cgcgccgcac ctcccggcac cccccgcgtc 600
ggcggcggcg gcggcggcgg ctcacggcct cgactcgccg acgtccacgc tgagcttctc 660
ggagagcgcg gtgctcgcca ccgtgctgga ggcgcacggc gccgccgccg cggcggccgc 720
gcgcgccgcc atgcagccca tgcaggcgta cgacgaggcg tgcaaggacc agcactgggg 780
cgacgtcgac gccgccgacg tgggcttccc cggcgccgga gcggggttca cgggcctact 840
cctcgaaggc tccttgaacc agatcccgag gccggcgggg agagacgcgg aagccgacgg 900
cgagttccag gagaccgagg aggagaagaa ctactggaac agcatactga acctggtgaa 960
ctcctcgtcg gcacctatgt cgacggccgt ggttgtgccc gcctcccacg cgtactcgcc 1020
ggcgccggac ttctgagcgg agaaaacctc gccggcatca aacttgattc gatctcatga 1080
<210> 8
<211> 2621
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ggcctctctc tctctctctc tctctctcac acacacacac actctcactg actctgctgc 60
tgcattagtc actcgcagag agccacagct ccctgcaaag aagatctctc gtagtgaatt 120
gcctcgatca cgtactacta cacatagacc tactacttga gcccgagaga agaggagagg 180
aggaagaacc agagggcgtc gaagatcatc ggggaggagt tttcctagag ctcgctgctg 240
ctgttgcttt tctccggcga tggggcgatc gccgtgctgc gagaaggagg ggctcaagaa 300
ggggccatgg acgccggagg aggaccagaa gctgctggcc tacatcgagc agcacggcca 360
cggctgctgg cgctcgctac cctccagccg gtaagcgccg cttatctagc ttaaatttct 420
tctcaacctc tgcaatccta gctgcaatgt tcggtcgagg cgatcgatcc tcgaggctgg 480
ctactctctg aactctgatc tgaggtgcat gcaaaccgtg acaatcgtgt gcagggctgc 540
agcggtgcgg caagagctgc cgactccggt ggacgaacta cctccggccg gacatcaaga 600
ggggcaagtt cagcctgcag gaggagcaga ccatcatcca gctccacgcc cttctcggca 660
acaggtgatc gattacttcg ttttcgcatg gatgcatcat gcatacaaga tacgtagtgc 720
acaactctcc ctcctgctag ctgctcgctc gttcttcacc tcgcacccgg agcacattta 780
attccgtaat cgcgatggaa cccttgattc tcctgcacga attttgactg ctagtacttg 840
ttgctgaccg gcaggtcaag aacacactag ccagtagcca ccattctgca ccgtagtctt 900
ggcagacatt tatgaaaggg ttatgcaatg caagggttgg aacacggagc ttagccaggg 960
gatgtgtaaa tttcgcaggc cgtgctactt acttgctgtc cccgtacaca cctgcttcag 1020
cattttgtcc gtaacaaacc gtactgtcca tagattaaca cacaagctag gctaaaaatt 1080
cttacgttag aacagaatca tcacttgttt tcgttttgtt cacacgtaat gctgcattgc 1140
tcatcttttg cccgtcgaac aaccacgcat tagctgtgag cacagaccaa tcaatgcatg 1200
catcaacaag ggaaaaagtg tgaaaaggtt gggcagtgag aggctcggcc cagaattttc 1260
ctttcttttc tcccatatga ttcggcattc aagctcgtca tttaaggagg cgagcccccc 1320
catcattgtg gaccaaaact ggggtttggt ccactgttgc cacctgcccc tcttcccatt 1380
ttgactcaca gcttccgatc atctctgccc tctgtctgta ctacgccacg cacgccttaa 1440
atcacaccgc cgattattta cgttttcaag agtgctgttt gtttaatttt gtcactgcga 1500
atggagggct tttgacgtgc gattttcctg atcttttttc ttggccggcg ttggcgttgt 1560
tgttgttttg caggtggtcg gcgatcgcga cgcacctgcc gaagcgcacg gacaacgaga 1620
tcaagaacta ctggaacacg cacctaaaga agcggctggc caagatgggg atcgacccgg 1680
tcacgcacaa gccgcgctcc gacgtggccg gcgcgggcgg cggcggcgga ggtgcggccg 1740
gcggcgcggc gggcgcgcag cacgccaagg ccgcggcgca cctcagccac acggcgcagt 1800
gggagagcgc gcggctcgag gcggaggcgc gcttggctcg ggaggccaag ctgcgcgcgc 1860
tcgcggcctc cgcgaccccg ggcgcgccgc acctcccggc accccccgcg tcggcggcgg 1920
cggcggcggc ggctcacggc ctcgactcgc cgacgtccac gctgagcttc tcggagagcg 1980
cggtgctcgc caccgtgctg gaggcgcacg gcgccgccgc cgcggcggcc gcgcgcgccg 2040
ccatgcagcc catgcaggcg tacgacgagg cgtgcaagga ccagcactgg ggcgacgtcg 2100
acgccgccga cgtgggcttc cccggcgccg gagcggggtt cacgggccta ctcctcgaag 2160
gctccttgaa ccagatcccg aggccggcgg ggagagacgc ggaagccgac ggcgagttcc 2220
aggagaccga ggaggagaag aactactgga acagcatact gaacctggtg aactcctcgt 2280
cggcacctat gtcgacggcc gtggttgtgc ccgcctccca cgcgtactcg ccggcgccgg 2340
acttctgagc ggagaaaacc tcgccggcat caaacttgat tcgatctcat gacacagtaa 2400
atagtttagc aatgattgtc gaataagatg ggactaatat taatgttagt aattattaat 2460
cacgttcttg ggttaacctg acaatgcttt cgattaaact tgtgggcaag aacttcaact 2520
gttcaaggct gtatcgacat ttgaaattcg atgcttgttt tgttcggtgt tcttatagaa 2580
tgtgtaaaac actgaaacta ctatcagaga atgtagcatc c 2621
<210> 9
<211> 111
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Met Gly Arg Ser Pro Cys Cys Glu Lys Glu Gly Leu Lys Lys Gly Pro
1 5 10 15
Trp Thr Pro Glu Glu Asp Gln Lys Leu Leu Ala Tyr Ile Glu Gln His
20 25 30
Gly His Gly Cys Trp Arg Ser Leu Pro Ser Arg Pro Gly Cys Ser Gly
35 40 45
Ala Ala Arg Ala Ala Asp Ser Gly Gly Arg Thr Thr Ser Gly Arg Thr
50 55 60
Ser Arg Gly Ala Ser Ser Ala Cys Arg Arg Ser Arg Pro Ser Ser Ser
65 70 75 80
Ser Thr Pro Phe Ser Ala Thr Gly Gly Arg Arg Ser Arg Arg Thr Cys
85 90 95
Arg Ser Ala Arg Thr Thr Arg Ser Arg Thr Thr Gly Thr Arg Thr
100 105 110
<210> 10
<211> 336
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atggggcgat cgccgtgctg cgagaaggag gggctcaaga aggggccatg gacgccggag 60
gaggaccaga agctgctggc ctacatcgag cagcacggcc acggctgctg gcgctcgcta 120
ccctccaggc cgggctgcag cggtgcggca agagctgccg actccggtgg acgaactacc 180
tccggccgga catcaagagg ggcaagttca gcctgcagga ggagcagacc atcatccagc 240
tccacgccct tctcggcaac aggtggtcgg cgatcgcgac gcacctgccg aagcgcacgg 300
acaacgagat caagaactac tggaacacgc acctaa 336
<210> 11
<211> 2622
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ggcctctctc tctctctctc tctctctcac acacacacac actctcactg actctgctgc 60
tgcattagtc actcgcagag agccacagct ccctgcaaag aagatctctc gtagtgaatt 120
gcctcgatca cgtactacta cacatagacc tactacttga gcccgagaga agaggagagg 180
aggaagaacc agagggcgtc gaagatcatc ggggaggagt tttcctagag ctcgctgctg 240
ctgttgcttt tctccggcga tggggcgatc gccgtgctgc gagaaggagg ggctcaagaa 300
ggggccatgg acgccggagg aggaccagaa gctgctggcc tacatcgagc agcacggcca 360
cggctgctgg cgctcgctac cctccaggcc ggtaagcgcc gcttatctag cttaaatttc 420
ttctcaacct ctgcaatcct agctgcaatg ttcggtcgag gcgatcgatc ctcgaggctg 480
gctactctct gaactctgat ctgaggtgca tgcaaaccgt gacaatcgtg tgcagggctg 540
cagcggtgcg gcaagagctg ccgactccgg tggacgaact acctccggcc ggacatcaag 600
aggggcaagt tcagcctgca ggaggagcag accatcatcc agctccacgc ccttctcggc 660
aacaggtgat cgattacttc gttttcgcat ggatgcatca tgcatacaag atacgtagtg 720
cacaactctc cctcctgcta gctgctcgct cgttcttcac ctcgcacccg gagcacattt 780
aattccgtaa tcgcgatgga acccttgatt ctcctgcacg aattttgact gctagtactt 840
gttgctgacc ggcaggtcaa gaacacacta gccagtagcc accattctgc accgtagtct 900
tggcagacat ttatgaaagg gttatgcaat gcaagggttg gaacacggag cttagccagg 960
ggatgtgtaa atttcgcagg ccgtgctact tacttgctgt ccccgtacac acctgcttca 1020
gcattttgtc cgtaacaaac cgtactgtcc atagattaac acacaagcta ggctaaaaat 1080
tcttacgtta gaacagaatc atcacttgtt ttcgttttgt tcacacgtaa tgctgcattg 1140
ctcatctttt gcccgtcgaa caaccacgca ttagctgtga gcacagacca atcaatgcat 1200
gcatcaacaa gggaaaaagt gtgaaaaggt tgggcagtga gaggctcggc ccagaatttt 1260
cctttctttt ctcccatatg attcggcatt caagctcgtc atttaaggag gcgagccccc 1320
ccatcattgt ggaccaaaac tggggtttgg tccactgttg ccacctgccc ctcttcccat 1380
tttgactcac agcttccgat catctctgcc ctctgtctgt actacgccac gcacgcctta 1440
aatcacaccg ccgattattt acgttttcaa gagtgctgtt tgtttaattt tgtcactgcg 1500
aatggagggc ttttgacgtg cgattttcct gatctttttt cttggccggc gttggcgttg 1560
ttgttgtttt gcaggtggtc ggcgatcgcg acgcacctgc cgaagcgcac ggacaacgag 1620
atcaagaact actggaacac gcacctaaag aagcggctgg ccaagatggg gatcgacccg 1680
gtcacgcaca agccgcgctc cgacgtggcc ggcgcgggcg gcggcggcgg aggtgcggcc 1740
ggcggcgcgg cgggcgcgca gcacgccaag gccgcggcgc acctcagcca cacggcgcag 1800
tgggagagcg cgcggctcga ggcggaggcg cgcttggctc gggaggccaa gctgcgcgcg 1860
ctcgcggcct ccgcgacccc gggcgcgccg cacctcccgg caccccccgc gtcggcggcg 1920
gcggcggcgg cggctcacgg cctcgactcg ccgacgtcca cgctgagctt ctcggagagc 1980
gcggtgctcg ccaccgtgct ggaggcgcac ggcgccgccg ccgcggcggc cgcgcgcgcc 2040
gccatgcagc ccatgcaggc gtacgacgag gcgtgcaagg accagcactg gggcgacgtc 2100
gacgccgccg acgtgggctt ccccggcgcc ggagcggggt tcacgggcct actcctcgaa 2160
ggctccttga accagatccc gaggccggcg gggagagacg cggaagccga cggcgagttc 2220
caggagaccg aggaggagaa gaactactgg aacagcatac tgaacctggt gaactcctcg 2280
tcggcaccta tgtcgacggc cgtggttgtg cccgcctccc acgcgtactc gccggcgccg 2340
gacttctgag cggagaaaac ctcgccggca tcaaacttga ttcgatctca tgacacagta 2400
aatagtttag caatgattgt cgaataagat gggactaata ttaatgttag taattattaa 2460
tcacgttctt gggttaacct gacaatgctt tcgattaaac ttgtgggcaa gaacttcaac 2520
tgttcaaggc tgtatcgaca tttgaaattc gatgcttgtt ttgttcggtg ttcttataga 2580
atgtgtaaaa cactgaaact actatcagag aatgtagcat cc 2622
<210> 12
<211> 360
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Met Gly Arg Ser Pro Cys Cys Glu Lys Glu Gly Leu Lys Lys Gly Pro
1 5 10 15
Trp Thr Pro Glu Glu Asp Gln Lys Leu Leu Ala Tyr Ile Glu Gln His
20 25 30
Gly His Gly Cys Trp Arg Ser Leu Pro Ser Lys Gly Arg Ala Ala Ala
35 40 45
Val Arg Gln Glu Leu Pro Thr Pro Val Asp Glu Leu Pro Pro Ala Gly
50 55 60
His Gln Glu Gly Gln Val Gln Pro Ala Gly Gly Ala Asp His His Pro
65 70 75 80
Ala Pro Arg Pro Ser Arg Gln Gln Val Val Gly Asp Arg Asp Ala Pro
85 90 95
Ala Glu Ala His Gly Gln Arg Asp Gln Glu Leu Leu Glu His Ala Pro
100 105 110
Lys Glu Ala Ala Gly Gln Asp Gly Asp Arg Pro Gly His Ala Gln Ala
115 120 125
Ala Leu Arg Arg Gly Arg Arg Gly Arg Arg Arg Arg Arg Cys Gly Arg
130 135 140
Arg Arg Gly Gly Arg Ala Ala Arg Gln Gly Arg Gly Ala Pro Gln Pro
145 150 155 160
His Gly Ala Val Gly Glu Arg Ala Ala Arg Gly Gly Gly Ala Leu Gly
165 170 175
Ser Gly Gly Gln Ala Ala Arg Ala Arg Gly Leu Arg Asp Pro Gly Arg
180 185 190
Ala Ala Pro Pro Gly Thr Pro Arg Val Gly Gly Gly Gly Gly Gly Gly
195 200 205
Ser Arg Pro Arg Leu Ala Asp Val His Ala Glu Leu Leu Gly Glu Arg
210 215 220
Gly Ala Arg His Arg Ala Gly Gly Ala Arg Arg Arg Arg Arg Gly Gly
225 230 235 240
Arg Ala Arg Arg His Ala Ala His Ala Gly Val Arg Arg Gly Val Gln
245 250 255
Gly Pro Ala Leu Gly Arg Arg Arg Arg Arg Arg Arg Gly Leu Pro Arg
260 265 270
Arg Arg Ser Gly Val His Gly Pro Thr Pro Arg Arg Leu Leu Glu Pro
275 280 285
Asp Pro Glu Ala Gly Gly Glu Arg Arg Gly Ser Arg Arg Arg Val Pro
290 295 300
Gly Asp Arg Gly Gly Glu Glu Leu Leu Glu Gln His Thr Glu Pro Gly
305 310 315 320
Glu Leu Leu Val Gly Thr Tyr Val Asp Gly Arg Gly Cys Ala Arg Leu
325 330 335
Pro Arg Val Leu Ala Gly Ala Gly Leu Leu Ser Gly Glu Asn Leu Ala
340 345 350
Gly Ile Lys Leu Asp Ser Ile Ser
355 360
<210> 13
<211> 1083
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
atggggcgat cgccgtgctg cgagaaggag gggctcaaga aggggccatg gacgccggag 60
gaggaccaga agctgctggc ctacatcgag cagcacggcc acggctgctg gcgctcgcta 120
ccctccaaag gccgggctgc agcggtgcgg caagagctgc cgactccggt ggacgaacta 180
cctccggccg gacatcaaga ggggcaagtt cagcctgcag gaggagcaga ccatcatcca 240
gctccacgcc cttctcggca acaggtggtc ggcgatcgcg acgcacctgc cgaagcgcac 300
ggacaacgag atcaagaact actggaacac gcacctaaag aagcggctgg ccaagatggg 360
gatcgacccg gtcacgcaca agccgcgctc cgacgtggcc ggcgcgggcg gcggcggcgg 420
aggtgcggcc ggcggcgcgg cgggcgcgca gcacgccaag gccgcggcgc acctcagcca 480
cacggcgcag tgggagagcg cgcggctcga ggcggaggcg cgcttggctc gggaggccaa 540
gctgcgcgcg ctcgcggcct ccgcgacccc gggcgcgccg cacctcccgg caccccccgc 600
gtcggcggcg gcggcggcgg cggctcacgg cctcgactcg ccgacgtcca cgctgagctt 660
ctcggagagc gcggtgctcg ccaccgtgct ggaggcgcac ggcgccgccg ccgcggcggc 720
cgcgcgcgcc gccatgcagc ccatgcaggc gtacgacgag gcgtgcaagg accagcactg 780
gggcgacgtc gacgccgccg acgtgggctt ccccggcgcc ggagcggggt tcacgggcct 840
actcctcgaa ggctccttga accagatccc gaggccggcg gggagagacg cggaagccga 900
cggcgagttc caggagaccg aggaggagaa gaactactgg aacagcatac tgaacctggt 960
gaactcctcg tcggcaccta tgtcgacggc cgtggttgtg cccgcctccc acgcgtactc 1020
gccggcgccg gacttctgag cggagaaaac ctcgccggca tcaaacttga ttcgatctca 1080
tga 1083
<210> 14
<211> 2624
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ggcctctctc tctctctctc tctctctcac acacacacac actctcactg actctgctgc 60
tgcattagtc actcgcagag agccacagct ccctgcaaag aagatctctc gtagtgaatt 120
gcctcgatca cgtactacta cacatagacc tactacttga gcccgagaga agaggagagg 180
aggaagaacc agagggcgtc gaagatcatc ggggaggagt tttcctagag ctcgctgctg 240
ctgttgcttt tctccggcga tggggcgatc gccgtgctgc gagaaggagg ggctcaagaa 300
ggggccatgg acgccggagg aggaccagaa gctgctggcc tacatcgagc agcacggcca 360
cggctgctgg cgctcgctac cctccaaagg ccggtaagcg ccgcttatct agcttaaatt 420
tcttctcaac ctctgcaatc ctagctgcaa tgttcggtcg aggcgatcga tcctcgaggc 480
tggctactct ctgaactctg atctgaggtg catgcaaacc gtgacaatcg tgtgcagggc 540
tgcagcggtg cggcaagagc tgccgactcc ggtggacgaa ctacctccgg ccggacatca 600
agaggggcaa gttcagcctg caggaggagc agaccatcat ccagctccac gcccttctcg 660
gcaacaggtg atcgattact tcgttttcgc atggatgcat catgcataca agatacgtag 720
tgcacaactc tccctcctgc tagctgctcg ctcgttcttc acctcgcacc cggagcacat 780
ttaattccgt aatcgcgatg gaacccttga ttctcctgca cgaattttga ctgctagtac 840
ttgttgctga ccggcaggtc aagaacacac tagccagtag ccaccattct gcaccgtagt 900
cttggcagac atttatgaaa gggttatgca atgcaagggt tggaacacgg agcttagcca 960
ggggatgtgt aaatttcgca ggccgtgcta cttacttgct gtccccgtac acacctgctt 1020
cagcattttg tccgtaacaa accgtactgt ccatagatta acacacaagc taggctaaaa 1080
attcttacgt tagaacagaa tcatcacttg ttttcgtttt gttcacacgt aatgctgcat 1140
tgctcatctt ttgcccgtcg aacaaccacg cattagctgt gagcacagac caatcaatgc 1200
atgcatcaac aagggaaaaa gtgtgaaaag gttgggcagt gagaggctcg gcccagaatt 1260
ttcctttctt ttctcccata tgattcggca ttcaagctcg tcatttaagg aggcgagccc 1320
ccccatcatt gtggaccaaa actggggttt ggtccactgt tgccacctgc ccctcttccc 1380
attttgactc acagcttccg atcatctctg ccctctgtct gtactacgcc acgcacgcct 1440
taaatcacac cgccgattat ttacgttttc aagagtgctg tttgtttaat tttgtcactg 1500
cgaatggagg gcttttgacg tgcgattttc ctgatctttt ttcttggccg gcgttggcgt 1560
tgttgttgtt ttgcaggtgg tcggcgatcg cgacgcacct gccgaagcgc acggacaacg 1620
agatcaagaa ctactggaac acgcacctaa agaagcggct ggccaagatg gggatcgacc 1680
cggtcacgca caagccgcgc tccgacgtgg ccggcgcggg cggcggcggc ggaggtgcgg 1740
ccggcggcgc ggcgggcgcg cagcacgcca aggccgcggc gcacctcagc cacacggcgc 1800
agtgggagag cgcgcggctc gaggcggagg cgcgcttggc tcgggaggcc aagctgcgcg 1860
cgctcgcggc ctccgcgacc ccgggcgcgc cgcacctccc ggcacccccc gcgtcggcgg 1920
cggcggcggc ggcggctcac ggcctcgact cgccgacgtc cacgctgagc ttctcggaga 1980
gcgcggtgct cgccaccgtg ctggaggcgc acggcgccgc cgccgcggcg gccgcgcgcg 2040
ccgccatgca gcccatgcag gcgtacgacg aggcgtgcaa ggaccagcac tggggcgacg 2100
tcgacgccgc cgacgtgggc ttccccggcg ccggagcggg gttcacgggc ctactcctcg 2160
aaggctcctt gaaccagatc ccgaggccgg cggggagaga cgcggaagcc gacggcgagt 2220
tccaggagac cgaggaggag aagaactact ggaacagcat actgaacctg gtgaactcct 2280
cgtcggcacc tatgtcgacg gccgtggttg tgcccgcctc ccacgcgtac tcgccggcgc 2340
cggacttctg agcggagaaa acctcgccgg catcaaactt gattcgatct catgacacag 2400
taaatagttt agcaatgatt gtcgaataag atgggactaa tattaatgtt agtaattatt 2460
aatcacgttc ttgggttaac ctgacaatgc tttcgattaa acttgtgggc aagaacttca 2520
actgttcaag gctgtatcga catttgaaat tcgatgcttg ttttgttcgg tgttcttata 2580
gaatgtgtaa aacactgaaa ctactatcag agaatgtagc atcc 2624
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