Application of SlCOP1 and SlCOP1H proteins and coding genes thereof in regulating and controlling spindling of tomatoes

Abstract

The invention discloses an effect of SlCOP1 and SlCOP1H proteins and coding genes of the SlCOP1 and SlCOP1H proteins in regulation and control of spindling of tomatoes. The SlCOP1 protein or the SlCOP1H protein comprises a protein composed of an amino acid sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 4. The invention further discloses a preparation method of the SlCOP1 protein or the SlCOP1H protein. According to the invention, single knockout mutants cop1 and cop1h and double knockout mutants cch of tomato SlCOP1 and SlCOP1H genes are respectively obtained through a CRISPR / Cas9 gene editing system in combination with agrobacterium tumefaciens-mediated tomato genetic transformation. Statistics of plant growth and development phenotypes of the mutant discovers that compared with wild tomatoes, a single mutant cop1 has no obvious phenotype change, a cop1h plant is slightly shortened, the internode length of a double mutant cch is remarkably reduced, the plant height is shortened, leaves are reduced, the color is darkened, and fruits are reduced, which indicates that the SlCOP1 and the SlCOPH jointly regulate and control the growth and development of the tomatoes. The method can be used for creating new tomato germplasm resistant to weak light and high-temperature spindling, and has potential breeding value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and relates to application of SlCOP1 and SlCOP1H (SlCOP1-Homolog) proteins and encoding genes thereof in regulating tomato leggy growth, and specifically relates to application of tomato SlCOP1 and SlCOP1H proteins and encoding genes thereof in regulating tomato leggy growth caused by high temperature and weak light. Background Art

[0002] Light is one of the important environmental factors that affect plant growth and development. It not only provides plants with the energy needed for photosynthesis, but also acts as an environmental signal to participate in regulating multiple growth and development processes of plants. With the rapid development of facility agriculture technology, the area of ​​vegetable cultivation in my country has gradually increased, and weak light is one of the main problems faced in facility cultivation. Weak light can cause plant seedlings to elongate rapidly, plants to grow too long, stems to be thin and weak, and easy to fall over. It can also reduce the synthesis and accumulation of nutrients in fruits, affecting commercial traits.

[0003] As an important horticultural crop, tomatoes are loved by consumers because they contain a variety of nutrients and are widely planted around the world. my country's tomato production is mainly based on facility cultivation, which also faces the problem of weak light. Weak light will increase the length of the internodes of tomato plants, make the plants thin and weak, affect flowering and fruiting, and later affect the formation of fruit coloring and nutritional quality. In addition to weak light, adverse environmental conditions such as high temperature can also cause tomato to grow too long, which is also an important problem affecting tomato production.

[0004] E3 ubiquitin ligase COP1 is one of the important regulatory factors in the light signal transduction pathway. At present, the research in Arabidopsis is relatively clear. As a RING-type E3 ubiquitin ligase, COP1 mediates the ubiquitination of positive regulatory factors in the light signal transduction pathway and is degraded by the 26S proteasome, thereby negatively regulating the light signal pathway. After the COP1 mutation in Arabidopsis, the plant exhibits a constitutive photomorphogenic phenotype, with cotyledons opening, hypocotyls shortening, anthocyanin accumulation in the dark, short plant growth, and early flowering phenotype under short days. However, different plants in nature are in different environments, and their evolutionary history and growth habits have their own characteristics. Whether the biological function of the tomato COP1 gene is similar to the reported function of Arabidopsis COP1, whether COP1 regulates the leggy growth of tomato seedlings, and whether COP1 affects the growth and development of tomatoes, etc., need to be verified and explored.

[0005] Problems in the prior art: The prior art does not report the biological functions of homologous genes COP1 and COP1-Homolog in tomatoes in regulating the leggy growth of tomato seedlings. The present invention uses homologous genes SlCOP1 and SlCOP1-Homolog in tomatoes as the starting point, uses the CRISPR / Cas9 gene editing system to obtain mutants, and systematically studies the biological functions of SlCOP1 and SlCOP1-Homolog in tomatoes, aiming to provide a theoretical and technical basis for the mining and utilization of gene resources and the exploration of light signal regulation of tomato growth and development, which has important theoretical and practical significance for the creation of new germplasm for tomato breeding. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide the role of SlCOP1 and SlCOP1H proteins and their encoding genes in regulating tomato leggy growth. The present invention uses the CRISPR / Cas9 gene editing system and combines Agrobacterium-mediated tomato genetic transformation to obtain single knockout mutants cop1 and cop1h and double knockout mutant cch of tomato SlCOP1 and SlCOP1H genes respectively. Weak light and high temperature conditions can cause tomato leggy growth, and knocking out SlCOP1 and SlCOP1H can significantly inhibit the leggy growth caused by weak light and high temperature. The growth and development phenotypes of mutant plants were statistically analyzed, and it was found that compared with wild-type tomatoes, the single mutant cop1 had no obvious phenotypic changes, the cop1h plant was slightly dwarfed, and the double mutant cch had significantly reduced internode length, shorter plant height, smaller leaves, darker color, and smaller fruits, indicating that SlCOP1 and SlCOPH jointly regulate tomato growth and development. The present invention can be used to create new tomato germplasm that is resistant to weak light and high temperature leggy growth, and has potential breeding value.

[0007] The purpose of the present invention can be achieved by the following scheme:

[0008] In a first aspect, the present invention provides an application of SlCOP1 and SlCOP1H proteins and genes encoding the same in regulating tomato leggy growth, wherein the application inhibits tomato leggy growth by knocking out genes encoding the SlCOP1 and SlCOP1H proteins.

[0009] As an embodiment of the present invention, the tomato elongation is caused by high temperature and weak light.

[0010] As an embodiment of the present invention, the S1COP1 protein is one or more proteins of A1 to A3:

[0011] A1, a protein consisting of the amino acid sequence shown in SEQ ID NO.3;

[0012] A2, a protein derived from A1 with the amino acid sequence shown in SEQ ID NO.3 having the same function as that of A1 after one or more amino acid residues are replaced and / or deleted and / or added;

[0013] A3, a fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of A1 or A2;

[0014] The S1COP1H protein is one or more proteins from B1 to B3:

[0015] B1, a protein consisting of the amino acid sequence shown in SEQ ID NO.4;

[0016] B2, a protein derived from B1 with the amino acid sequence shown in SEQ ID NO.4 being substituted and / or deleted and / or added with one or more amino acid residues and having the same function;

[0017] B3: A fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of B1 or B2.

[0018] In a second aspect, the present invention provides a biomaterial related to SlCOP1 and SlCOP1H proteins, wherein the related biomaterial comprises any one of the following:

[0019] C1, a polynucleotide encoding the SlCOP1 protein as shown in SEQ ID NO.1 and a polynucleotide encoding the SlCOP1H protein as shown in SEQ ID NO.2;

[0020] Or, a degenerate variant comprising the sequences of SEQ ID NO.1 and SEQ ID NO.2;

[0021] Or, a polynucleotide encoding the SlCOP1 protein as shown in SEQ ID NO.1, a polynucleotide encoding the SlCOP1H protein as shown in SEQ ID NO.2, or a degenerate variant comprising the sequences of SEQ ID NO.1 and SEQ ID NO.2;

[0022] C2, a recombinant expression vector containing the polynucleotide described in C1;

[0023] C3, a bioengineered bacterium containing the polynucleotide described in C1;

[0024] C4, a bioengineering bacterium containing the recombinant expression vector described in C2;

[0025] C5, a transgenic plant cell containing the polynucleotide described in C1;

[0026] C6, a transgenic plant containing the recombinant expression vector described in C2;

[0027] The plant is tomato.

[0028] As an embodiment of the present invention, the CDS sequences of the protein encoding genes SlCOP1 (Solyc12g005950) and SlCOP1H (Solyc11g011980) are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0029] In a third aspect, the present invention provides an application of SlCOP1 and SlCOP1H proteins and related biological materials in regulating tomato growth and / or tomato organ development and / or tomato breeding.

[0030] In the present invention, plant growth and development are inhibited after the SlCOP1 and SlCOP1H genes are knocked out.

[0031] In a fourth aspect, the present invention provides a product, wherein the product is prepared by using SlCOP1 and SlCOP1H proteins, or polynucleotides as targets;

[0032] Alternatively, the product is obtained by screening SlCOP1 and SlCOP1H proteins, or polynucleotides as targets.

[0033] As one embodiment of the present invention, the product includes a recombinant expression vector for regulating the activity or content of SlCOP1 and SlCOP1H proteins;

[0034] Or, the product includes a recombinant expression vector that regulates the expression of genes encoding SlCOP1 and SlCOP1H proteins;

[0035] Or, the product includes a recombinant expression vector for regulating the activity or content of SlCOP1 and SlCOP1H proteins, and a recombinant expression vector for regulating the expression of genes encoding SlCOP1 and SlCOP1H proteins.

[0036] As an embodiment of the present invention, the product also includes a CRISPR / Cas9 system, and the gRNA target site nucleotide sequences of the SlCOP1 and SlCOP1H protein encoding genes in the CRISPR / Cas9 system are shown in SEQ ID NO.9-SEQID NO.14.

[0037] In a fifth aspect, the present invention provides an application of the product in regulating tomato growth and / or organ growth and development and / or tomato breeding, wherein the application is to introduce the product to regulate the content or activity of SlCOP1 and / or SlCOP1H proteins;

[0038] Or, the application is to introduce the product to regulate the expression of SlCOP1 and / or SlCOP1H protein encoding gene;

[0039] Alternatively, the application is to introduce the product to regulate the content or activity of SlCOP1 and / or SlCOP1H proteins, and to regulate the expression of genes encoding SlCOP1 and / or SlCOP1H proteins.

[0040] In the present invention, the SlCOP1 and SlCOP1H genes are knocked out to cause the SlCOP1 and SlCOP1H proteins to be missing, thereby affecting the growth and development of plants.

[0041] As an embodiment of the present invention, the application is to regulate any one or more of the following plant growth and development:

[0042] (A) Regulates plant internode length;

[0043] (B) regulating plant height;

[0044] (C) Regulate plant leaf size;

[0045] (D) Regulate plant leaf color;

[0046] (E) regulating plant fruit color;

[0047] (F) Regulate plant fruit size;

[0048] (G) regulating the weak light tolerance and leggy growth of plant seedlings;

[0049] The plant is tomato.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] 1. The tomato SlCOP1 and SlCOP1H gene knockout vectors constructed in the present invention are reported for the first time and can be directly used for Agrobacterium-mediated tomato genetic transformation to obtain new germplasms of SlCOP1 and SlCOP1H mutants.

[0052] 2. In the present invention, the SlCOP1 and SlCOP1H genes were knocked out by the CRISPR / Cas9 gene editing system, and cop1, cop1h single mutants and cch double mutants were successfully obtained. The mutant seedlings obtained by the present invention were compared with wild-type tomatoes, and the single mutants did not change significantly; the double mutant seedlings showed a phenotype of shortened hypocotyls, open cotyledons, and anthocyanin accumulation in the dark. Under weak light conditions, the hypocotyls of the double mutant seedlings were significantly shorter than those of the wild type and single mutants, and the double mutant seedlings accumulated more anthocyanins, indicating that SlCOP1 and SlCOP1H jointly regulated the growth of tomato seedlings, and there was functional redundancy between the two; during the growth and development process, the internode length of the double mutant shortened, the leaves became smaller, the color became darker, and the plant height decreased. The invention plays an important role in agricultural production, can be used to obtain new germplasm, and is applied to tomato breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0054] Figure 1 The transcriptional expression of SlCOP1H induced by light in Example 1;

[0055] Figure 2 It is the mutant form of Example 2;

[0056] Figure 3 This is the growth phenotype of mutant seedlings in Example 3 under weak light;

[0057] Figure 4 This is the growth phenotype of mutant seedlings under high temperature in Example 4;

[0058] Figure 5 The mutant plant phenotypes of Example 5; Figures AD are the mutant plant phenotypes at 10d, 20d, 35d, and 80d after germination, respectively, and Figure E is the statistical result of the plant height at 35 days after germination;

[0059] Figure 6 This is the mutant fruit phenotype of Example 6. DETAILED DESCRIPTION

[0060] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples are implemented on the premise of the technical solution of the present invention, provide detailed implementation methods and specific operation processes, and will help those skilled in the art to further understand the present invention. It should be pointed out that the protection scope of the present invention is not limited to the following embodiments, and several adjustments and improvements made under the premise of the concept of the present invention all belong to the protection scope of the present invention.

[0061] The embodiments of the present invention disclose tomato SlCOP1 and SlCOP1H proteins and their encoding genes and applications. The methods not mentioned in the embodiments are conventional test methods and will not be described in detail here.

[0062] In the present invention, there is no particular limitation on the plants applicable to the present invention, as long as they are suitable for gene transformation operations. In a specific embodiment of the present invention, the recombinant expression vectors pTX-SlCOP1 and pTX-SlCOP1H are introduced into tomatoes by Agrobacterium-mediated genetic transformation using the CRISPR / Cas9 gene editing system, and the materials with changed target site sequences are screened as tomato cop1 and cop1h mutants. Double mutants are obtained in two ways, one is to design target sites in the conserved regions of the amino acid sequences of two proteins for co-knockout; the other is to obtain single mutants respectively and then perform genetic hybridization to obtain double mutants. After the mutants are obtained, they are cultured and the changes in the mutant traits are observed and statistically analyzed.

[0063] The amino acid sequence of the SlCOP1 protein of the present invention is shown in SEQ ID NO.3, and the amino acid sequence of the SlCOP1H protein is shown in SEQ ID NO.4. The nucleotide sequence of the gene encoding the SlCOP1 protein is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the SlCOP1H protein is shown in SEQ ID NO.2.

[0064] Example 1: Detection of transcriptional expression levels of SlCOP1 and SlCOP1H after light treatment

[0065] Cultivated tomato (Ailsa Craig) seeds were sterilized and sown on 1 / 2MS medium, grown in the dark at 25°C for 4 days, and then transferred to white light (100 μmol m -2 s -1 ) for 3 h to detect the transcriptional expression of SlCOP1 and SlCOP1H (the expression level of the wild type in the dark was used as reference 1 to calculate the relative expression level in the sample), with three biological replicates.

[0066] Tomato seedlings were sampled after light treatment, RNA was extracted, reverse transcribed into cDNA, and qRT-PCR was performed using the following primers:

[0067] SlCOP1-Q-Fw: GGACAAAGAGGAGGGGGGTAG (SEQ ID NO.5)

[0068] SlCOP1-Q-Rv:CCCACAAGCTGTTAAAAATGCAT(SEQ ID NO.6)

[0069] SlCOP1H-Q-Fw: CTGCAATGACAGTCAGGCTCC (SEQ ID NO.7)

[0070] SlCOP1H-Q-Rv: GTGGTTGTCATCCTTCCGTTGC (SEQ ID NO.8)

[0071] The results are as follows Figure 1 As shown, SlCOP1 is constitutively transcribed and its transcription level is not affected by light, while the transcriptional expression of SlCOP1H is significantly induced by light.

[0072] Example 2: Acquisition of CRISPR / Cas9 gene editing SlCOP1 and SlCOP1H mutant materials

[0073] 2.1 Construction of expression vectors pTX-SlCOP1 and pTX-SlCOP1H

[0074] The CRISPR / Cas9 vector used in the present invention is a dual-target vector, and two target sites are designed on each gene according to the SlCOP1 and SlCOP1H gene sequences. By sequence alignment, sequence-specific segments are selected, and the target sequences for knocking out the SlCOP1 and SlCOP1H genes are screened through the CRISPR / Cas9 target analysis website (http: / / crispr.dbcls.jp / ). The target sequences are as follows:

[0075] SlCOP1-CR-1: CAAGCAGCTCAGCTTAGATC (SEQ ID NO.9)

[0076] SlCOP1-CR-2:ATATGAAGAGCATGAGAAAC (SEQ ID NO.10)

[0077] SlCOP1H-CR-1:GTTTCTCATGCTCTTCATAT (SEQ ID NO.11)

[0078] SlCOP1H-CR-2: GGCAAGACAGTCGCAGAAAC (SEQ ID NO. 12)

[0079] Through sequence alignment, the target was designed in the conserved region of the two sequences to knock out SlCOP1 and SlCOP1H together. The target sequence is as follows:

[0080] cch-CR-1: GATGAGTTGTTTGCTACTGC (SEQ ID NO.13)

[0081] cch-CR-2: CGGGTAGACATTTCAACAAC (SEQ ID NO.14)

[0082] By homologous recombination, after adding the homologous recombination arm sequence to the target sequence, it was connected to the pTX expression vector. The sequence of the pTX vector is shown in SEQ ID NO.25-SEQ ID NO.26; wherein SEQ ID NO.25 is the upper half sequence of the pTX vector, SEQ ID NO.26 is the lower half sequence of the pTX vector, and the downstream sequence CCACGCCCG in the upper half sequence is connected to the upstream sequence CAGTTCCGC in the lower half sequence to constitute the pTX vector as a whole.

[0083] 2.2 Tomato genetic transformation and mutant identification

[0084] The recombinant expression vector constructed above was transferred into Agrobacterium competent GV3101, single colonies were picked for identification and activation, and the cotyledons of sterile cultivated tomato (Ailsa Craig) seedlings were infected, and resistant T0 generation transgenic tomatoes were obtained after screening.

[0085] The T0 transgenic tomatoes obtained above were transplanted into the substrate for cultivation. After survival, young leaves were taken to extract DNA for identification. First, PCR was used to identify whether the Cas9 gene existed in the plants. Plants containing the Cas9 gene were transgenic positive plants. The Cas9 identification primers were:

[0086] Cas9-det-Fw:CGGCCTCGATATTGGGACTAACTCT(SEQ ID NO.15)

[0087] Cas9-det-Rv:CTTATCTGTGGAGTCCACGAGCTTC(SEQ ID NO.16)

[0088] Using the DNA of the obtained transgenic positive plants as a template, the gDNA fragments of SlCOP1 and SlCOP1H including the target sequence were amplified by PCR, and sequencing was used to identify whether the target sequence had changed and whether the gene had been edited, and then mutant plants in which the proteins encoded by the SlCOP1 and SlCOP1H genes had been changed were screened out respectively.

[0089] The primers for identifying the target sites of SlCOP1 and SlCOP1H single mutants are:

[0090] SlCOP1-CRdet-Fw:TCCCCATAATGGCATATAGTCG (SEQ ID NO.17)

[0091] SlCOP1-CRdet-Rv: GGAAACAGCCTCTCTACCACAA (SEQ ID NO. 18)

[0092] SlCOP1H-CRdet-Fw:GTACATTAAAGAAGACATAAATGCTGTGGAGA(SEQ IDNO.19)

[0093] SlCOP1H-CRdet-Rv: AAAAATGTCAGAATGCCTGAGTTCAGCAA (SEQ ID NO. 20)

[0094] The single mutants obtained by screening were genetically crossed and double mutants were obtained by self-segregation.

[0095] Using the transgenic positive plants obtained by co-knockout, PCR amplification and sequencing were used to detect whether SlCOP1 and SlCOP1H were edited.

[0096] The primers for identification of SlCOP1 and SlCOP1H co-knockout double mutants are:

[0097] cch-COP1-Fw:AAATGGCTTTGCTGGAGTGCT(SEQ ID NO.21)

[0098] cch-COP1-Rv: GCTTTCAAGCCGATTCGGAAT (SEQ ID NO.22)

[0099] cch-COP1H-Fw:GCTGAACTCAGGCATTCTGACAT(SEQ ID NO.23)

[0100] cch-COP1H-Rv:CATGCTCCTCGTACTCCATCACA(SEQ ID NO.24)

[0101] All mutants obtained above were self-pollinated to screen for mutants without Cas9 gene, but SlCOP1 and SlCOP1H genes were edited and the encoded proteins were changed. The SlCOP1 single mutant was recorded as cop1, the SlCOP1H single mutant was recorded as cop1h, and the double mutant was recorded as cch for subsequent phenotypic statistics. Figure 2 As shown, the cop1 mutant was obtained by deleting 213 bp in the SlCOP1 gene, and the cop1h mutant was obtained by deleting 4 bp in the SlCOP1H gene; in the double mutant cch, the SlCOP1 gene deleted 12 bp and the SlCOP1H gene deleted 2 bp.

[0102] Example 3: Phenotypic identification of mutant seedlings growing under weak light

[0103] After the seeds were sterilized, they were germinated on 1 / 2MS medium. The seeds with consistent germination were placed on 1 / 2MS medium and placed in a constant temperature incubator for dark (Dark) or weak white light (WL) culture. The phenotype of the hypocotyl of the seedlings was observed after 4 days. Figure 3 As shown in the figure, the single mutants cop1 and cop1h had no obvious phenotypic changes compared with the wild type in darkness and weak light, while the double mutant cch seedlings had significantly shorter hypocotyls and open cotyledons compared with the wild type in darkness, indicating that SlCOP1 and SlCOP1H have functional redundancy in regulating the growth of tomato seedlings.

[0104] Example 4: Phenotypic identification of mutant seedlings growing under high temperature

[0105] After the seeds were sterilized, they were germinated on 1 / 2MS medium. The seeds with consistent germination were placed on 1 / 2MS medium and cultured in constant temperature incubators at 25℃ and 30℃, respectively. The phenotype of the hypocotyls of the seedlings was observed after 4 days. Figure 4 As shown, the single mutant cop1 had no obvious changes compared with the wild type, and its hypocotyl elongated rapidly at 30℃; the single mutant cop1h had partially inhibited hypocotyl elongation at 30℃; while the double mutant cch had completely inhibited hypocotyl elongation at 30℃, which was consistent with the hypocotyl length of seedlings grown at 25℃.

[0106] Example 5: Identification of mutant plant traits

[0107] Seeds with uniform germination were sown in soil and placed in a 100 μmol / m -2 s -1 Grow under light conditions. Figure 5 As shown in the figure (* indicates significant difference, ** indicates extremely significant difference), 10 days after germination, the double mutant cch seedlings were short and the cotyledon color became darker, while the single mutants cop1 and cop1h showed no significant changes compared with the wild type. As the tomato grew, the single mutant cop1 plant had no significant difference compared with the wild type; the single mutant cop1h showed a decrease in internode length and a shorter plant compared with the wild type; while the double mutant cch had a significantly reduced internode length, slowed growth, and a shorter plant, which was even shorter than the cop1h single mutant. This indicates that SlCOP1 and SlCOP1H jointly regulate tomato growth and development, but the function of SlCOP1H is more important.

[0108] Example 6: Identification of mutant fruit traits

[0109] Marking was performed during the flowering period to observe the changes in mutant fruits compared with wild-type fruits at the same growth and development period. Figure 6As shown (dpa: days post anthesis), there is no obvious change in the fruits of cop1 and cop1h single mutants compared with the wild type, but the fruit color of the double mutant cch during the green fruit stage is darker than that of the wild type, and the fruit size of the double mutant is smaller than that of the wild type and the single mutant.

[0110] The sequence of the pTX vector (SEQ ID NO.25-SEQ ID NO.26) is as follows:

[0111] CAAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATCGATGGTTTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGTGATTTTGCTGGCTCTAATTCCCAAATGGCTCAAGTCGGTGACGGTGATAATTCACCTTTAATGAATAATTTCCGTCAATATTTACCTTCCCTCCCTCAATCGGTTGAATGTCGCCCTTTTGTCTTTGGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTATAAATCTTTTTAATTTATAGTATATTTATGTAAGTTTTCACGTTGAGTAAATAGCGAAGAAGTTGGGCCCAACCAAGTAAAATAAGAAGGCCGGGCCATTACAATTAAGTCGTCACACAACTGGGCTTCATTGAAAAAAGCGCAAAACCGATTCCAGGCCCGTGTTAGCATGAAGACTCAACTCAACCAGAGATTTCTCCCTCATCGCTTACAGAAAAAAGCTATATGCTGTTTATATTGCGAATCTAACAGTGTAGTTTGNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTTGCAAAATTTTCCAGATCGATTTCTTCTTCCTCTGTTCTTCGGCGTTCAATTTCTGGGGTTTTCTCTTCGTTTTCTGTAACTGAAACCTAAAATTTGACCTAAAAAAAATCTCAAATAATATGATTCAGTGGTTTTG

[0112] TACTTTTCAGTTAGTTGAGTTTTGCAGTTCCGATGAGATAAACCAATAATAAATCTT

[0113] TTTAATTTATAGTATATTTATGTAAGTTTTCACGTTGAGTAAATAGCGAAGAAGTTGG

[0114] GCCCAACCAAGTAAAATAAGAAGGCCGGGCCATTACAATTAAGTCGTCACACAAC

[0115] TGGGCTTCATTGAAAAAAGCGCAAAACCGATTCCAGGCCCGTGTTAGCATGAAGA

[0116] CTCAACTCAACCAGAGATTTCTCCCTCATCGCTTACAGAAAAAAGCTATATGCTGT

[0117] TTATATTGCGAATCTAACAGTGTAGTTTGNNNNNNNNNNNNNNNNNNNGTTTTAGA

[0118] GCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCA

[0119] CCGAGTCGGTGCTTTTTTTTGCAAAATTTTCCAGATCGATTTCTTCTTCCTCTGTTCT

[0120] TCGGCGTTCAATTTCTGGGGTTTTCTCTTCGTTTTCTGTAACTGAAACCTAAAATTT

[0121] GACCTAAAAAAAATCTCAAATAATATGATTCAGTGGTTTTGTACTTTTCAGTTAGTT

[0122] GAGTTTTGCAGTTCCGATGAGATAAACCAATAAGCTTGCATGCCTGCAGGTCAACA

[0123] TGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGA

[0124] AGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTC

[0125] GGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGG

[0126] TGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCT

[0127] CTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAA

[0128] AAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGT

[0129] GGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGAC

[0130] CAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATT

[0131] CCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCT

[0132] CCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCC

[0133] GACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAA

[0134] GACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGT

[0135] AAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAA

[0136] GTTCATTTCATTTGGAGAGGACCTCGACCTCAACACAACATATACAAAACAAACGA

[0137] ATCTCAAGCAATCAAGCATTCTACTTCTATTGCAGCAATTTAAATCATTTCTTTTAAA

[0138] GCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATACTCGAGTAATCTAG

[0139] ATGGATTACAAGGACCACGACGGGGATTACAAGGACCACGACATTGATTACAAGG

[0140] ATGATGATGACAAGATGGCTCCGAAGAAGAAGAGGAAGGTTGGCATCCACGGGGT

[0141] GCCAGCTGCTGACAAGAAGTACTCGATCGGCCTCGATATTGGGACTAACTCTGTTG

[0142] GCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCTCAAAGAAGTTCAAGGTCCT

[0143] GGGCAACACCGATCGGCATTCCATCAAGAAGAATCTCATTGGCGCTCTCCTGTTCG

[0144] ACAGCGGCGAGACGGCTGAGGCTACGCGGCTCAAGCGCACCGCCCGCAGGCGGT

[0145] ACACGCGCAGGAAGAATCGCATCTGCTACCTGCAGGAGATTTTCTCCAACGAGAT

[0146] GGCGAAGGTTGACGATTCTTTCTTCCACAGGCTGGAGGAGTCATTCCTCGTGGAG

[0147] GAGGATAAGAAGCACGAGCGGCATCCAATCTTCGGCAACATTGTCGACGAGGTTG

[0148] CCTACCACGAGAAGTACCCTACGATCTACCATCTGCGGAAGAAGCTCGTGGACTCC

[0149] ACAGATAAGGCGGACCTCCGCCTGATCTACCTCGCTCTGGCCCACATGATTAAGTT

[0150] CAGGGGCCATTTCCTGATCGAGGGGGATCTCAACCCGGACAATAGCGATGTTGAC

[0151] AAGCTGTTCATCCAGCTCGTGCAGACGTACAACCAGCTCTTCGAGGAGAACCCCA

[0152] TTAATGCGTCAGGCGTCGACGCGAAGGCTATCCTGTCCGCTAGGCTCTCGAAGTCT

[0153] CGGCGCCTCGAGAACCTGATCGCCCAGCTGCCGGGCGAGAAGAAGAACGGCCTG

[0154] TTCGGGAATCTCATTGCGCTCAGCCTGGGGCTCACGCCCAACTTCAAGTCGAATTT

[0155] CGATCTCGCTGAGGACGCCAAGCTGCAGCTCTCCAAGGACACATACGACGATGAC

[0156] CTGGATAACCTCCTGGCCCAGATCGGCGATCAGTACGCGGACCTGTTCCTCGCTGC

[0157] CAAGAATCTGTCGGACGCCATCCTCCTGTCTGATATTCTCAGGGTGAACACCGAGA

[0158] TTACGAAGGCTCCGCTCTCAGCCTCCATGATCAAGCGCTACGACGAGCACCATCAG

[0159] GATCTGACCCTCCTGAAGGCGCTGGTCAGGCAGCAGCTCCCCGAGAAGTACAAGG

[0160] AGATCTTCTTCGATCAGTCGAAGAACGGCTACGCTGGGTACATTGACGGCGGGGC

[0161] CTCTCAGGAGGAGTTCTACAAGTTCATCAAGCCGATTCTGGAGAAGATGGACGGC

[0162] ACGGAGGAGCTGCTGGTGAAGCTCAATCGCGAGGACCTCCTGAGGAAGCAGCGG

[0163] ACATTCGATAACGGCAGCATCCCACACCAGATTCATCTCGGGGAGCTGCACGCTAT

[0164] CCTGAGGAGGCAGGAGGACTTCTACCCTTTCCTCAAGGATAACCGCGAGAAGATC

[0165] GAGAAGATTCTGACTTTCAGGATCCCGTACTACGTCGGCCCACTCGCTAGGGGCAA

[0166] CTCCCGCTTCGCTTGGATGACCCGCAAGTCAGAGGAGACGATCACGCCGTGGAAC

[0167] TTCGAGGAGGTGGTCGACAAGGGCGCTAGCGCTCAGTCGTTCATCGAGAGGATGA

[0168] CGAATTTCGACAAGAACCTGCCAAATGAGAAGGTGCTCCCTAAGCACTCGCTCCT

[0169] GTACGAGTACTTCACAGTCTACAACGAGCTGACTAAGGTGAAGTATGTGACCGAG

[0170] GGCATGAGGAAGCCGGCTTTCCTGTCTGGGGAGCAGAAGAAGGCCATCGTGGACC

[0171] TCCTGTTCAAGACCAACCGGAAGGTCACGGTTAAGCAGCTCAAGGAGGACTACTT

[0172] CAAGAAGATTGAGTGCTTCGATTCGGTCGAGATCTCTGGCGTTGAGGACCGCTTCA

[0173] ACGCCTCCCTGGGGACCTACCACGATCTCCTGAAGATCATTAAGGATAAGGACTTC

[0174] CTGGACAACGAGGAGAATGAGGATATCCTCGAGGACATTGTGCTGACACTCACTC

[0175] TGTTCGAGGACCGGGAGATGATCGAGGAGCGCCTGAAGACTTACGCCCATCTCTT

[0176] CGATGACAAGGTCATGAAGCAGCTCAAGAGGAGGAGGTACACCGGCTGGGGGAG

[0177] GCTGAGCAGGAAGCTCATCAACGGCATTCGGGACAAGCAGTCCGGGAAGACGAT

[0178] CCTCGACTTCCTGAAGAGCGATGGCTTCGCGAACCGCAATTTCATGCAGCTGATTC

[0179] ACGATGACAGCCTCACATTCAAGGAGGATATCCAGAAGGCTCAGGTGAGCGGCCA

[0180] GGGGGACTCGCTGCACGAGCATATCGCGAACCTCGCTGGCTCGCCAGCTATCAAG

[0181] AAGGGGATTCTGCAGACCGTGAAGGTTGTGGACGAGCTGGTGAAGGTCATGGGC

[0182] AGGCACAAGCCTGAGAACATCGTCATTGAGATGGCCCGGGAGAATCAGACCACGC

[0183] AGAAGGGCCAGAAGAACTCACGCGAGAGGATGAAGAGGATCGAGGAGGGCATTA

[0184] AGGAGCTGGGGTCCAGATCCTCAAGGAGCACCCGGTGGAGAACACGCAGCTGC

[0185] AGAATGAGAAGCTCTACCTGTACTACCTCCAGAATGGCCGCGATATGTATGTGGAC

[0186] CAGGAGCTGGATATTAACAGGCTCAGCGATTACGACGTCGATCATATCGTTCCACA

[0187] GTCATTCCTGAAGGATGACTCCATTGACAACAAGGTCCTCACCAGGTCGGACAAG

[0188] AACCGGGGCAAGTCTGATAATGTTCCTTCAGAGGAGGTCGTTAAGAGATGAAGA

[0189] ACTACTGGCGCCAGCTCCTGAATGCCAAGCTGATCACGCAGCGGAAGTTCGATAA

[0190] CCTCACAAAGGCTGAGAGGGGCGGGCTCCTGAGCTGGACAAGCGGGCTTCATC

[0191] AAGAGGCAGCTGGTCGAGACACGGCAGATCACTAAGCACGTTGCGCAGATTCTCG

[0192] ACTCACGGATGAACACTAAGTACGATGAGAATGACAAGCTGATCCGCGAGGTGAA

[0193] GGTCATCACCCTGAAGTCCAAAGCTCGTCTCCGACTTCAGGAAGGATTTCCAGTTCT

[0194] ACAAGGTTCGGGAGATCAACAATTACCACCATGCCCATGACGCGTACCTGAACGC

[0195] GGTGGTCGGCACAGCTCTGATCAAGAAGTACCCAAAGCTCGAGAGCGAGTTCGTG

[0196] TACGGGGACTACAAGGTTTACGATGTGAGGAAGATGATCGCCAAGTCGGAGCAGG

[0197] AGATTGGCAAGGCTACCGCCAAGTACTTCTTCTACTCTAACATTATGAATTTCTTCA

[0198] AGACAGAGATCACTCTGGCCAATGGCGAGATCCGGAAGCGCCCCCTCATCGAGAC

[0199] GAACGGCGAGACGGGGGAGATCGTGTGGGACAAGGGCAGGGATTTCGCGACCGT

[0200] CAGGAAGGTTCTCTCCATGCCACAAGTGAATATCGTCAAGAAGACAGAGGTCCAG

[0201] ACTGGCGGGTTCTCTAAGGAGTCAATTCTGCCTAAGCGGAACAGCGACAAGCTCA

[0202] TCGCCCGCAAGAAGGACTGGGATCCGAAGAAGTACGGCGGGTTCGACAGCCCCA

[0203] CTGTGGCCTACTCGGTCCTGGTTGTGGCGAAGGTTGAGAAGGGCAAGTCCAAGAA

[0204] GCTCAAGAGCGTGAAGGAGCTGCTGGGGATCACGATTATGGAGCGCTCCAGCTTC

[0205] GAGAAGAACCCGATCGATTTCCTGGAGGCGAAGGGCTACAAGGAGGTGAAGAAG

[0206] GACCTGATCATTAAGCTCCCCAAGTACTCACTCTTCGAGCTGGAGAACGGCAGGA

[0207] AGCGGATGCTGGCTTCCGCTGGCGAGCTGCAGAAGGGGAACGAGCTGGCTCTGCC

[0208] GTCCAAGTATGTGAACTTCCTCTACCTGGCCTCCCACTACGAGAAGCTCAAGGGCA

[0209] GCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTCGAGCAGCACAAGCATTACC

[0210] TCGACGAGATCATTGAGCAGATTTCCGAGTTCTCCAAGCGCGTGATCCTGGCCGAC

[0211] GCGAATCTGGATAAGGTCCTCTCCGCGTACAACAAGCACCGCGACAAGCCAATCA

[0212] GGGAGCAGGCTGAGAATATCATTCATCTCTTCACCCTGACGAACCTCGGCGCCCCT

[0213] GCTGCTTTCAAGTACTTCGACACAACTATCGATCGCAAGAGGTACACAAGCACTAA

[0214] GGAGGTCCTGGACGCGACCCTCATCCACCAGTCGATTACCGGCCTCTACGAGACG

[0215] CGCATCGACCTGTCTCAGCTCGGGGGCGACAAGCGGCCAGCGGCGACGAAGAAG

[0216] GCGGGGCAGGCGAAGAAGAAGAAGTGAGCTCAGAGCTTTCGTTCGTATCATCGGT

[0217] TTCGACAACGTTCGTCAAGTTCAATGCATCAGTTTCATTGCGCACACACCAGAATC

[0218] CTACTGAGTTTGAGTATTATGGCATTGGGAAAACTGTTTTTCTTGTACCATTTGTTG

[0219] TGCTTGTAATTTACTGTGTTTTTTATTCGGTTTTCGCTATCGAACTGTGAAATGGAAA

[0220] TGGATGGAGAGAGAGTTAATGAATGATATGGTCCTTTTGTTCATTCTCAAATTAATATT

[0221] ATTTGTTTTTTCTCTTATTTGTTGTGTGTTGAATTTGAAATTATAAGAGATATGCAAA

[0222] CATTTTGTTTTGAGTAAAAATGTGTCAAATCGTGGCCTCTAATGACCGAAGTTAATA

[0223] TGAGGGTAAAACACTTGTAGTTGTACCATTATGCTTATTCACTAGGCAACAAATAT

[0224] ATTTTCAGACCTAGAAAAGCTGCAAATGTTACTGAATACAAGTATGTCCTCTTGTGT

[0225] TTTAGACATTTATGAACTTTCCTTTATGTAATTTTCCAGAATCCTTGTCAGATTCTAA

[0226] TCATTGCTTTATAATTATAGTTATACTCATGGATTTGTAGTTGAGTATGAAAATATTTT

[0227] TTAATGCATTTTATGACTTGCCAATTGATTGACAACGAATTCACTGGCCGTCGTTTT

[0228] ACAACGTCGTGACTGGGAAAACCCTGGGCGTTACCCAACTTAATCGCCTTGCAGCA

[0229] CATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTC

[0230] CCAACAGTTGCGCAGCCTGAATGGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTC

[0231] TCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGG

[0232] TTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGG

[0233] TTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGT

[0234] CCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATC

[0235] TCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGAACCACCATCAAAC

[0236] AGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA

[0237] GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAA

[0238] AACCACCCCAGTACATTAAAAACGTCCGCAATGTGTTATTAAGTTGTCTAAGCGTC

[0239] AATTTGTTTACACCACAATATATCCTGCCACCAGCCAGCCAACAGCTCCCCGACCG

[0240] GCAGCTCGGCACAAAATCACCACTCGATACAGGCAGCCCATCAGTCCGGGACGGC

[0241] GTCAGCGGGAGAGCCGTTGTAAGGCGGCAGACTTTGCTCATGTTACCGATGCTATT

[0242] CGGAAGAACGGCAACTAAGCTGCCGGGTTTGAAACACGGATGATCTCGCGGAGG

[0243] GTAGCATGTTGATTGTAACGATGACAGAGCGTTGCTGCCTGTGATCAAATATCATCT

[0244] CCCTCGCAGAGATCCGAATTATCAGCCTTCTTATTCATTTCTCGCTTAACCGTGACA

[0245] GGCTGTCGATCTTGAGAACTATGCCGACATAATAGGAAATCGCTGGATAAAGCCGC

[0246] TGAGGAAGCTGAGTGGCGCTATTTCTTTAGAAGTGAACGTTGACGATATCAACTCC

[0247] CCTATCCATTGCTCACCGAATGGTACAGGTCGGGGACCCGAAGTTCCGACTGTCGG

[0248] CCTGATGCATCCCCGGCTGATCGACCCCAGATCTGGGGCTGAGAAAGCCCAGTAA

[0249] GGAAACAACTGTAGGTTCGAGTCGCGAGATCCCCCGGAACCAAAGGAAGTAGGTT

[0250] AAACCCGCTCCGATCAGGCCGAGCCACGCCAGGCCGAGAACATTGGTTCCTGTAG

[0251] GCATCGGGATTGGCGGATCAAACACTAAAGCTACTGGAACGAGCAGAAGTCCTCC

[0252] GGCCGCCAGTTGCCAGGCGGTAAAGGTGAGCAGAGGCACGGGAGGTTGCCACTT

[0253] GCGGGTCAGCACGGTTCCGAACGCCATGGAAACCGCCCCCGCCAGGCCCGCTGCG

[0254] ACGCCGACAGGATCTAGCGCTGCGTTTGGTGTCAACACCAACAGCGCCACGCCCG

[0255] CAGTTCCGCAAATAGCCCCCAGGACCGCCATCAATCGTATCGGGCTACCTAGCAGA

[0256] GCGGCAGAGATGAACACGACCATCAGCGGCTGCACAGCGCCTACCGTCGCCGCGA

[0257] CCCCGCCCGGCAGGCGGTAGACCGAAATAAACAACAAGCTCCAGAATAGCGAAAT

[0258] ATTAAGTGCGCCGAGGATGAAGATGCGCATCCACCAGATTCCCGTTGGAATCTGTC

[0259] GGACGATCATCACGAGCAATAAACCCGCCGGCAACGCCCGCAGCAGCATACCGGC

[0260] GACCCCTCGGCCTCGCTGTTCGGGCTCCACGAAAACGCCGGACAGATGCGCCTTG

[0261] TGAGCGTCCTTGGGGCCGTCCTCCTGTTTGAAGACCGACAGCCCAATGATCTCGCC

[0262] GTCGATGTAGGCGCCGAATGCCACGGCATCTCGCAACCGTTCAGCGAACGCCTCC

[0263] ATGGGCTTTTTCTCCTCGTGCTCGTAAACGGACCCGAACATCTCTGGAGCTTTCTT

[0264] CAGGGCCGACAATCGGATCTCGCGGAAATCCTGCACGTCGGCCGCTCCAAGCCGT

[0265] CGAATCTGAGCCTTAATCACAATTGTCAATTTTAATCCTCTGTTTATCGGCAGTTCGT

[0266] AGAGCGCGCCGTGCGTCCCGAGCGATACTGAGCGAAGCAAGTGCGTCGAGCAGT

[0267] GCCCGCTTGTTCCTGAAATGCCAGTAAAGCGCTGGCTGCTGAACCCCCAGCCGGA

[0268] ACTGACCCCACAAGGCCCTAGCGTTTGCAATGCACCAGGTCATCATTGACCCAGG

[0269] CGTGTTCCACCAGGCCGCTGCCTCGCCAACTCTTCGCAGGCTTCGCCGACCTGCTCG

[0270] CGCCACTTCTTCACGCGGGTGGAATCCGATCCGCACATGAGGCGGAAGGTTTCCA

[0271] GCTTGAGCGGGTACGGCTCCCGGTGCGAGCTGAAATAGTCGAACATCCGTCGGGC

[0272] CGTCGGCGACAGCTTGCGGTACTTCTCCCATATGAATTTCGTGTAGTGGTCGCCAG

[0273] CAAACAGCACGACGATTTCCTCGTCGATCAGGACCTGGCAACGGGACGTTTTCTT

[0274] GCCACGGTCCAGGACGCGGAAGCGGTGCAGCAGCGACACCGATTCCAGGTGCCC

[0275] AACGCGGTCGGACGTGAAGCCCATCGCCGTCGCCTGTAGGCGCGACAGGCATTCC

[0276] TCGGCCTTCGTGTAATACCGGCCATTGATCGACCAGCCCAGGTCCTGGCAAAGCTC

[0277] GTAGAACGTGAAGGTGATCGGCTCGCCGATAGGGTGCGCTTCGCGTACTCCAAC

[0278] ACCTGCTGCCACACCAGTTCGTCATCGTCGGCCCGCAGCTCGACGCCGGTGTAGG

[0279] TGATCTTCACGTCCTTGTTGACGTGGAAAATGACCTTGTTTTGCAGCGCCTCCGGC

[0280] GGGATTTTCTTGTTGCGCGTGGTGAACAGGGCAGAGCGGGCCGTGTCGTTTGGCA

[0281] TCGCTCGCATCGTGTCCGGCCACGGCGCAATATCGAACAAGGAAAGCTGCATTTCC

[0282] TTGATCTGCTGCTTCGTGTGTTTCAGCAACGCGGCCTGCTTGGCCTCGCTGACCTG

[0283] TTTTGCCAGGTCCTCGCCGGCGGTTTTTCGCTTCTTGGTCGTCATAGTTCCTCGCGT

[0284] GTCGATGGTCATCGACTTCGCCAAACCTGCCGCCTCCTGTTCGAGACGACGCGAA

[0285] CGCTCCACGGCGGCCGATGGCGCGGGCAGGGCAGGGGGAGCCAGTTGCACGCTG

[0286] TCGCGCTCGATCTTGGCCGTAGCTTGCTGGACCATCGAGCCGACGGACTGGAAGG

[0287] TTTCGCGGGGCGCACGCATGACGGTGCGGCTTGCGATGGTTTCGGCATCCTCGGCG

[0288] GAAAACCCCGCGTCGATCAGTTCTTGCCTGTATGCCTTCCGGTCAAACGTCCGATT

[0289] CATTCACCCTCCTTGCGGGATTGCCCCGACTCACGCCGGGGCAATGTGCCCTTATT

[0290] CCTGATTTGACCCGCCTGGTGCCTTGGTGTCCAGATAATCCACCTTATCGGCAATGA

[0291] AGTCGGTCCCGTAGACCGTCTGGCCGTCCTTCTCGTACTTGGTATTCCGAATCTTGC

[0292] CCTGCACGAATACCAGCGACCCCTTGCCCAAATACTTGCCGTGGGCCTCGGCCTGA

[0293] GAGCCAAAACACTTGATGCGGAAGAAGTCGGTGCGCTCCTGCTTGTCGCCGGCAT

[0294] CGTTGCGCCACATCTAGGTACTAAAACAATTCATCCAGTAAAATATAATATTTTATTT

[0295] TCTCCCAATCAGGCTTGATCCCCAGTAAGTCAAAAAATAGCTCGACATACTGTTCT

[0296] TCCCCGATATCCTCCCTGATCGACCGGACGCAGAAGGCAATGTCATACCACTTGTC

[0297] CGCCCTGCCGCTTCTCCCAAGATCAATAAAGCCACTTACTTTGCCATCTTTCACAA

[0298] AGATGTTGCTGTCTCCCAGGTCGCCGTGGGAAAAGACAAGTTCCTCTTCGGGCTTT

[0299] TCCGTCTTTAAAAAATCATACAGCTCGCGCGGATCTTTAAATGGAGTGTCTTCTTCC

[0300] CAGTTTTCGCAATCCACATCGGCCAGATCGTTATTCAGTAAGTAATCCAATTCGGCT

[0301] AAGCGGCTGTCTAAGCTATTCGTATAGGGACAATCCGATATGTCGATGGAGTGAAA

[0302] GAGCCTGATGCACTCCGCATACAGCTCGATAATCTTTTCAGGGCTTTGTTCATCTTC

[0303] ATACTCTTCCGAGCAAAGGACGCCATCGGCCTCACTCATGAGCAGATTGCTCCAGC

[0304] CATCATGCCGTTCAAAGTGCAGGACCTTTGGAACAGGCAGCTTTCCTTCCAGCCAT

[0305] AGCATCATGTCCTTTTCCCGTTCCACATCATAGGTGGTCCCTTTATACCGGCTGTCC

[0306] GTCATTTTTAAATATAGGTTTTCATTTTCTCCCACCAGCTTATATACCTTAGCAGGAG

[0307] ACATTCCTTCCGTATCTTTTACGCAGCGGTATTTTTCGATCAGTTTTTTCAATTCCGG

[0308] TGATATTCTCATTTTAGCCATTTATTATTTCCTTCCTCTTTTCTACAGTATTTAAAGAT

[0309] ACCCCAAGAAGCTAATTATAACAAGACGAACTCCAATTCACTGTTCCTTGCATTCT

[0310] AAAACCTTAAATACCAGAAAACAGCTTTTTCAAAGTTGTTTTCAAAGTTGGCGTAT

[0311] AACATAGTATCGACGGAGCCGATTTTGAAACCACAATTATGGGTGATGCTGCCAAC

[0312] TTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTGTCTATC

[0313] AGCTGTCCCTCCTGTTCAGCTACTGACGGGGTGGTGCGTAACGGCAAAAGCACCG

[0314] CCGGACATCAGCGCTATCTCTGCTCTCACTGCCGTAAAACATGGCAACTGCAGTTC

[0315] ACTTACACCGCTTCTCAACCCGGTACGCACCAGAAAATCATTGATATGGCCATGAA

[0316] TGGCGTTGGATGCCGGGCAACAGCCCGCATTATGGGCGTTGGCCTCAACACGATTT

[0317] TACGTCACTTAAAAAACTCAGGCCGCAGTCGGTAACCTCGCGCATACAGCCGGGC

[0318] AGTGACGTCATCGTCTGCGCGGAAATGGACGAACAGTGGGGCTATGTCGGGGCTA

[0319] AATCGCGCCAGCGCTGGCTGTTTTACGCGTATGACAGTCTCCGGAAGACGGTTGTT

[0320] GCGCACGTATTCGGTGAACGCACTATGGCGACGCTGGGGCGTCTTATGAGCCTGCT

[0321] GTCACCCTTTGACGTGGTGATATGGATGACGGATGGCTGGCCGCTGTATGAATCCC

[0322] GCCTGAAGGGAAAGCTGCACGTAATCAGCAAGCGATATACGCAGCGAATTGAGCG

[0323] GCATAACCTGAATCTGAGGCAGCACCTGGCACGGCTGGGACGGAAGTCGCTGTCG

[0324] TTCTCAAAATCGGTGGAGCTGCATGACAAAGTCATCGGGCATTATCTGAACATAAA

[0325] ACACTATCAATAAGTTGGAGTCATTACCCAATTATGATAGAATTTACAAGCTATAAG

[0326] GTTATTGTCCTGGGTTTCAAGCATTAGTCCATGCAAGTTTTTATGCTTTGCCCATTCT

[0327] ATAGATATATTGATAAGCGCGCTGCCTATGCCTTGCCCCCTGAAATCCTTACATACGG

[0328] CGATATCTTCTATATAAAAGATATATTATCTTATCAGTATTGTCAATATATTCAAGGCA

[0329] ATCTGCCTCCTCATCCTCTTCATCCTCTTCGTCTTGGTAGCTTTTTAAATATGGCGCT

[0330] TCATAGAGTAATTCTGTAAAGGTCCAATTCTCGTTTTCATACCTCGGTATAATCTTAC

[0331] CTATCACCTCAAATGGTTCGCTGGGTTTATCGCACCCCCGAACACGAGCACGGCAC

[0332] CCGCGACCACTATGCCAAGAATGCCCAAGGTAAAAATTGCCGGCCCCGCCATGAA

[0333] GTCCGTGAATGCCCCGACGGCCGAAGTGAAGGGCAGGCCGCCACCCAGGCCGCC

[0334] GCCCTCACTGCCCGGCACCTGGTCGCTGAATGTCGATGCCAGCACCTGCGGCACG

[0335] TCAATGCTTCCGGGCGTCGCGCTCGGGCTGATCGCCCATCCCGTTACTGCCCCGAT

[0336] CCCGGCAATGGCAAGGACTGCCAGCGCTGCCATTTTTGGGGTGAGGCCGTTCGCG

[0337] GCCGAGGGGCGCAGCCCCTGGGGGGATGGGAGGCCCGCGTTAGCGGGCCGGGAG

[0338] GGTTCGAGAAGGGGGGGCACCCCCCTTCGGCGTGCGCGGTCACGCGCACAGGGC

[0339] GCAGCCCTGGTTAAAAACAAGGTTTATAAATATTGGTTTAAAAGCAGGTTAAAAGA

[0340] CAGGTTAGCGGTGGCCGAAAAACGGGCGGAAACCCTTGCAAATGCTGGATTTTCT

[0341] GCCTGTGGACAGCCCCTCAAATGTCAATAGGTGCGCCCCTCATCTGTCAGCACTCT

[0342] GCCCCTCAAGTGTCAAGGATCGCGCCCCTCATCTGTCAGTAGTCGCGCCCCTCAAG

[0343] TGTCAATACCGCAGGGCACTTATCCCCAGGCTTGTCCACATCATCTGTGGGAAACT

[0344] CGCGTAAAATCAGGCGTTTTCGCCGATTTGCGAGGCTGGCCAGCTCCACGTCGCC

[0345] GGCCGAAATCGAGCCTGCCCCTCATCTGTCAACGCCGCGCCGGGTGAGTCGGCCC

[0346] CTCAAGTGTCAACGTCCGCCCCTCATCTGTCAGTGAGGGCCAAGTTTTCCGCGAG

[0347] GTATCCACAACGCCGGCGGCCGCGGTGTCTCGCACACGGCTTCGACGGCGTTTCT

[0348] GGCGCGTTTGCAGGGCCATAGACGGCCGCCAGCCCAGCGGCGAGGGCAACCAGC

[0349] CCGGTGAGCGTCGCAAAGGCGCTCGGTCTTGCCTTGCTCGTCGGTGATGTACTTCA

[0350] CCAGCTCCGCGAAGTCGCTCTTCTTGATGGAGCGCATGGGGACGTGCTTGGCAATC

[0351] ACGCGCACCCCCCGGCCGTTTTAGCGGCTAAAAAAGTCATGGCTCTGCCCTCGGG

[0352] CGGACCACGCCCATCATGACCTTGCCAAGCTCGTCCTGCTTCTCTTCGATCTTCGC

[0353] CAGCAGGGCGAGGATCGTGGCATCACCGAACCGCGCCGTGCGCGGGTCGTCGGTG

[0354] AGCCAGAGTTTCAGCAGGCCGCCCAGGCGGCCCAGGTCGCCATTGATGCGGGCCA

[0355] GCTCGCGGACGTGCTCATAGTCCACGACGCCCGTGATTTTGTAGCCCTGGCCGACG

[0356] GCCAGCAGGTAGGCCGACAGGCTCATGCCGGCCGCCGCCGCCTTTTCCTCAATCG

[0357] CTCTTCGTTCGTCTGGAAGGCAGTACACCTTGATAGGTGGGCTGCCCTTCCTGGTT

[0358] GGCTTGGTTTCATCAGCCATCCGCTTGCCCTCATCTGTTACGCCGGCGGTAGCCGG

[0359] CCAGCCTCGCAGAGCAGGATTCCCGTTGAGCACCGCCAGGTGCGAATAAGGGACA

[0360] GTGAAGAAGGAACACCCGCTCGCGGGTGGGCCTACTTCACCTATCCTGCCCGGCT

[0361] GACGCCGTTGGATACACCAAGGAAAGTCTACACGAACCCTTTGGCAAAATCCTGT

[0362] ATATCGTGCGAAAAAGGATGGATATACCGAAAAAATCGCTATAATGACCCCGAAGC

[0363] AGGGTTATGCAGCGGAAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGG

[0364] TATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGG

[0365] GGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCG

[0366] TCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAAC

[0367] GCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCT

[0368] GCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACC

[0369] GCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAA

[0370] GAGCGCCAGAAGGCCGCCAGAGAGGCCGAGCGCGGCCGTGAGGCTTGGACGCTA

[0371] GGGCAGGGCATGAAAAAGCCCGTAGCGGGCTGCTACGGGCGTCTGACGCGGTGG

[0372] AAAGGGGGAGGGGATGTTGTCTACATGGCTCTGCTGTAGTGAGTGGGTTGCGCTC

[0373] CGGCAGCGGTCCTGATCAATCGTCACCCTTTCTCGGTCCTTCAACGTTCCTGACAA

[0374] CGAGCCTCCTTTTCGCCAATCCATCGACAATCACCGCGAGTCCCTGCTCGAACGCT

[0375] GCGTCCGGACCGGCTTCGTCGAAGGCGTCTATCGCGGCCCGCAACAGCGGCGAGA

[0376] GCGGAGCCTGTTCAACGGTGCCGCCGCGCTCGCCGGCATCGCTGTCGCCGGCCTG

[0377] CTCCTCAAGCACGGCCCCAACAGTGAAGTAGCTGATTGTCATCAGCGCATTGACG

[0378] GCGTCCCCGGCCGAAAAACCCGCCTCGCAGAGGAAGCGAAGCTGCGCGTCGGCC

[0379] GTTTCCATCTGCGGTGCGCCCGGTCGCGTGCCGGCATGGATGCGCGCGCCATCGCG

[0380] GTAGGCGAGCAGCGCCTGCCTGAAGCTGCGGGCATTCCCGATCAGAAATGAGCGC

[0381] CAGTCGTCGTCGGCTCTCGGCACCGAATGCGTATGATTCTCCGCCAGCATGGCTTC

[0382] GGCCAGTGCGTCGAGCAGCGCCCGCTTGTTCCTGAAGTGCCAGTAAAGCGCCGGC

[0383] TGCTGAACCCCCAACCGTTCCGCCAGTTTGCGTGTCGTCAGACCGTCTACGCCGA

[0384] CCTCGTTCAACAGGTCCAGGGCGGCACGGATCACTGTATTCGGCTGCAACTTTGTC

[0385] ATGCTTGACACTTTATCACTGATAAACATAATATGTCCACCAACTTATCAGTGATAA

[0386] AGAATCCGCGCGTTCAATCGGACCAGCGGAGGCTGGTCCGGAGGCCAGACGTGA

[0387] AACCCAACATACCCCTGATCGTAATTCTGAGCACTGTCGCGCTCGACGCTGTCGGC

[0388] ATCGGCCTGATTATGCCGGTGCTGCCGGGCCTCCTGCGCGATCTGGTTCACTCGAA

[0389] CGACGTCACCGCCCACTATGGCATTCTGCTGGCGCTGTATGCGTTGGTGCAATTTG

[0390] CCTGCGCACCTGTGCTGGGCGCGCTGTCGGATCGTTTCGGGCGGCGGCCAATCTTG

[0391] CTCGTCTCGCTGGCCGGCGCCAGATCTGGGGAACCCTGTGGTTGGCATGCACATAC

[0392] AAATGGACGAACGGATAAACCTTTTCACGCCCTTTTAAATATCCGATTATTCTAATA

[0393] AACGCTCTTTTCTCTTAGGTTTACCCGCCAATATATCCTGTCAAACACTGATAGTTT

[0394] AAACTGAAGGCGGGAAACGACAATCTGATCATGAGCGGAGAATTAAGGGAGTCA

[0395] CGTTATGACCCCCGCCGATGACGCGGGACAAGCCGTTTTACGTTTGGAACTGACA

[0396] GAACCGCAACGTTGAAGGAGCCACTCAGCCGCGGGTTTCTGGAGTTTAATGAGCT

[0397] AAGCACATACGTCAGAAACCATTATTGCGCGTTCAAAAGTCGCCTAAGGTCACTAT

[0398] CAGCTAGCAAATATTTCTTGTCAAAAATGCTCCACTGACGTTCCATAAATTCCCCTC

[0399] GGTATCCAATTAGAGTCTCATATTCACTCTCAATCCAAATAATCTGCACCGGATCTG

[0400] GATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGG

[0401] GTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGC

[0402] CGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGAC

[0403] CTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGG

[0404] CCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAG

[0405] GGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTG

[0406] CTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTT

[0407] GATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCAC

[0408] GTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCA

[0409] GGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGC

[0410] GATGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAA

[0411] TGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATC

[0412] AGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCT

[0413] GACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTC

[0414] TATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGAC

[0415] CAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGA

[0416] AAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGC

[0417] GGGGATCTCATGCTGGAGTTCTTCGCCCACGGGATCTCTGCGGAACAGGCGGTCG

[0418] AAGGTGCCGATATCATTACGACAGCAACGGCCGACAAGCACAACGCCACGATCCT

[0419] GAGCGACAATATGATCGGGCCCGGCGTCCACATCAACGGCGTCGGCGGCGACTGC

[0420] CCAGGCAAGACCGAGATGCACCGCGATATCTTGCTGCGTTCGGATATTTTCGTGGA

[0421] GTTCCCGCCACAGACCCGGATGATCCCCGATCGTTCAAACATTTGGCAATAAAGTT

[0422] TCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAA

[0423] TTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTT

[0424] TTTATGATTAGAGGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGC

[0425] GCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGCCT

[0426] CCTGTCAATGCTGGCGGCGGCTCTGGTGGTGGTTCTGGTGGCGGCTCTGAGGGTG

[0427] GTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGCTCTGAGGGAGGCGGTTCCG

[0428] GTGGTGGCTCTGGTTCCGGTGATTTTGATTATGAAAAGATGGCAACGCTAATAAG

[0429] GGGGCTATGACCGAAAATGCCGATGAAAACGCGCTACAGTCTGACGCTAAAGG

[0430] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. Application of SlCOP1 and SlCOP1H proteins in regulating tomato elongation, characterized in that: The application is used to inhibit tomato elongation by knocking out the coding genes of SlCOP1 and SlCOP1H proteins.

2. The use according to claim 1, characterized in that: The tomato leggy growth is caused by high temperature and low light.

3. The use according to claim 1, characterized in that: The S1COP1 protein is one or more proteins from A1 to A3: A1, a protein consisting of the amino acid sequence shown in SEQ ID NO.3; A2, a protein derived from A1 with the amino acid sequence shown in SEQ ID NO.3 having the same function as that of A1 after one or more amino acid residues are replaced and / or deleted and / or added; A3, a fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of A1 or A2; The S1COP1H protein is one or more proteins from B1 to B3: B1, a protein consisting of the amino acid sequence shown in SEQ ID NO.4; B2, a protein derived from B1 with the amino acid sequence shown in SEQ ID NO.4 having the same function as that of B1 after one or more amino acid residues are replaced and / or deleted and / or added; B3: A fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of B1 or B2.

4. Biomaterials related to SlCOP1 and SlCOP1H proteins, characterized in that The relevant biological materials include any one of the following: C1, a polynucleotide encoding the SlCOP1 protein as shown in SEQ ID NO.1 and a polynucleotide encoding the SlCOP1H protein as shown in SEQ ID NO.2; Or, a degenerate variant comprising SEQ ID NO.1 and SEQ ID NO.2; Or, a polynucleotide encoding the SlCOP1 protein as shown in SEQ ID NO.1, a polynucleotide encoding the SlCOP1H protein as shown in SEQ ID NO.2, or a degenerate variant comprising the sequences of SEQ ID NO.1 and SEQ ID NO.2; C2, a recombinant expression vector containing the polynucleotide described in C1; C3, a bioengineered bacterium containing the polynucleotide described in C1; C4, a bioengineering bacterium containing the recombinant expression vector described in C2; C5, a transgenic plant cell containing the polynucleotide described in C1; C6, a transgenic plant containing the recombinant expression vector described in C2; The plant is tomato.

5. Application of SlCOP1 and SlCOP1H proteins and related biological materials in regulating tomato growth and / or tomato organ development and / or tomato breeding.

6. A product, characterized in that The product is prepared by using SlCOP1 and SlCOP1H proteins, or polynucleotides as targets; Alternatively, the product is obtained by screening SlCOP1 and SlCOP1H proteins, or polynucleotides as targets.

7. The product according to claim 6, characterized in that The product includes a recombinant expression vector for regulating the activity or content of SlCOP1 and SlCOP1H proteins; Or, the product includes a recombinant expression vector that regulates the expression of genes encoding SlCOP1 and SlCOP1H proteins; Or, the product includes a recombinant expression vector for regulating the activity or content of SlCOP1 and SlCOP1H proteins, and a recombinant expression vector for regulating the expression of genes encoding SlCOP1 and SlCOP1H proteins.

8. The product according to claim 6, characterized in that The product also includes a CRISPR / Cas9 system, and the gRNA target site nucleotide sequences of the SlCOP1 and SlCOP1H protein encoding genes in the CRISPR / Cas9 system are shown in SEQ ID NO.9-SEQ ID NO.

14.

9. Use of the product according to any one of claims 6 to 8 in regulating tomato growth and / or organ growth and development and / or tomato breeding, characterized in that: The application is to introduce the product to regulate the content or activity of SlCOP1 and / or SlCOP1H protein; Or, the application is to introduce the product to regulate the expression of SlCOP1 and / or SlCOP1H protein encoding gene; Alternatively, the application is to introduce the product to regulate the content or activity of SlCOP1 and / or SlCOP1H proteins, and to regulate the expression of genes encoding SlCOP1 and / or SlCOP1H proteins.

10. The use according to claim 5 or 9, characterized in that: The application is to regulate any one or more of the following plant growth and development: (A) Regulates plant internode length; (B) regulating plant height; (C) Regulate plant leaf size; (D) Regulate plant leaf color; (E) regulating plant fruit color; (F) Regulate plant fruit size; (G) regulating the weak light tolerance and leggy growth of plant seedlings; The plant is tomato.

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