Application of slcop1 and slcop1h protein and its encoding gene in regulating tomato overgrowth

Abstract

The application discloses roles of SlCOP1 and SlCOP1H proteins and coding genes thereof in regulating tomato overgrowth. The SlCOP1 or SlCOP1H protein comprises a protein consisting of an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4. The application obtains single knockout mutants cop1 and cop1h and double knockout mutant cch of tomato SlCOP1 and SlCOP1H genes by combining a CRISPR / Cas9 gene editing system with agrobacterium-mediated genetic transformation of tomato. Statistical analysis of growth and development phenotypes of the mutant plants shows that, compared with wild-type tomatoes, the single mutant cop1 has no obvious phenotype change, the cop1h plant is slightly dwarfed, the double mutant cch has a significantly reduced internode length, a dwarfed plant height, a smaller leaf, a darker color, and smaller fruits, indicating that SlCOP1 and SlCOPH jointly regulate tomato growth and development. The application can be used for creating new tomato germplasm resistant to weak light and high temperature overgrowth, and has potential breeding value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the application of SlCOP1 and SlCOP1H (SlCOP1-Homolog) proteins and their encoding genes in regulating tomato elongation, and particularly relates to the application of tomato SlCOP1 and SlCOP1H proteins and their encoding genes in regulating tomato elongation caused by high temperature and low light. Background Art

[0002] Light is one of the key environmental factors affecting plant growth and development. It not only provides plants with the energy they need for photosynthesis, but also acts as an environmental signal to regulate multiple plant growth and development processes. With the rapid development of facility agriculture technology, the area of ​​vegetable cultivation in my country has gradually increased, and weak light levels are one of the main challenges faced in facility cultivation. Weak light levels can cause rapid elongation of seedlings, severe plant growth, thin stems, and susceptibility to lodging. It can also reduce the synthesis and accumulation of nutrients in fruits, affecting commercial properties.

[0003] Tomatoes, a key horticultural crop, are widely cultivated worldwide, enjoying widespread consumer demand for their diverse nutrients. However, tomato production in my country primarily relies on greenhouse cultivation, which also faces the challenge of insufficient light. Inadequate light can increase internode length, weaken plants, and hinder flowering and fruit set. Later, it can also affect fruit coloration and nutritional quality. In addition to low light levels, adverse environmental conditions such as high temperatures can also cause tomato plants to grow excessively, a significant issue impacting tomato production.

[0004] The E3 ubiquitin ligase COP1 is a key regulator of the light signal transduction pathway. Current research in Arabidopsis is relatively clear. COP1, a RING-type E3 ubiquitin ligase, negatively regulates the light signaling pathway by mediating the ubiquitination of positive regulators in the light signal transduction pathway, leading to degradation by the 26S proteasome. Mutations in COP1 in Arabidopsis result in a constitutive photomorphogenic phenotype, with cotyledons opening in darkness, hypocotyl shortening, accumulation of anthocyanins, and dwarf growth. Furthermore, plants exhibit an early flowering phenotype under short-day conditions. However, different plants in nature operate in diverse environments, with distinct evolutionary histories and growth habits. Whether the biological function of the tomato COP1 gene is similar to that reported for Arabidopsis COP1, whether COP1 regulates tomato seedling elongation, and whether COP1 affects tomato growth and development remain to be verified and explored.

[0005] Problems with the existing technology: The existing art has not reported on the biological functions of the tomato homologous genes COP1 and COP1-Homolog in regulating tomato seedling elongation. This invention uses the tomato homologous genes SlCOP1 and SlCOP1-Homolog as a starting point, utilizes the CRISPR / Cas9 gene editing system to generate mutants, and systematically studies the biological functions of SlCOP1 and SlCOP1-Homolog in tomato. This aims to provide a theoretical and technical basis for the exploration and utilization of genetic resources and the study of light signal regulation of tomato growth and development. This has important theoretical and practical significance for the creation of new germplasm for application in tomato breeding. Summary of the Invention

[0006] The present invention aims to overcome the shortcomings and deficiencies of the prior art and to provide insights into the role of SlCOP1 and SlCOP1H proteins and their encoding genes in regulating tomato growth. Using the CRISPR / Cas9 gene editing system combined with Agrobacterium-mediated genetic transformation of tomatoes, the present invention generates single knockout mutants (cop1 and cop1h) and a double knockout mutant (cch) of the SlCOP1 and SlCOP1H genes, respectively. Low light levels and high temperatures can cause tomato growth, but knockout of SlCOP1 and SlCOP1H significantly inhibits these conditions. Analysis of the growth and development phenotypes of the mutant plants revealed that compared to wild-type tomatoes, the single cop1 mutant showed no significant phenotypic changes, while cop1h plants showed slight dwarfing. The double mutant (cch) exhibited significantly reduced internode length, shorter plant height, smaller and darker leaves, and smaller fruit, demonstrating that SlCOP1 and SlCOP1H jointly regulate tomato growth and development. The present invention can be used to create new tomato germplasm that tolerates low light levels and high temperatures and exhibits potential breeding value.

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

[0008] In a first aspect, the present invention provides an application of SlCOP1 and SlCOP1H proteins and their encoding genes in regulating tomato elongation, wherein the application inhibits tomato elongation by knocking out the encoding genes of 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 one embodiment of the present invention, the S1COP1 protein is one or more proteins selected from 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 one or more amino acid residues substituted and / or deleted and / or added and having the same function;

[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 having one or more amino acid residues substituted and / or deleted and / or added 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 biomaterial includes 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 bioengineered 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 a use 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] Alternatively, the product includes a recombinant expression vector for regulating the activity or content of SlCOP1 and SlCOP1H proteins, or a recombinant expression vector for regulating the expression of genes encoding SlCOP1 and SlCOP1H proteins.

[0036] As one embodiment of the present invention, the product further comprises a CRISPR / Cas9 system, and the nucleotide sequences of the gRNA target sites of the SlCOP1 and SlCOP1H protein encoding genes in the CRISPR / Cas9 system are shown as SEQ ID NO.9-SEQ ID NO.14.

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

[0038] Alternatively, the application is to introduce the product to regulate the expression of genes encoding SlCOP1 and / or SlCOP1H proteins;

[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 deficient, thereby affecting the growth and development of plants.

[0041] As one 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 excessive 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 using the CRISPR / Cas9 gene editing system, successfully obtaining single cop1 and cop1h mutants and a cch double mutant. When the mutant seedlings obtained using the present invention were compared with wild-type tomatoes, the single mutants showed no significant changes; the double mutant seedlings exhibited a phenotype of shortened hypocotyls, open cotyledons, and anthocyanin accumulation in darkness. Under low-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 regulate tomato seedling growth and have functional redundancy. During growth and development, the double mutants exhibited shortened internode length, smaller and darker leaves, and reduced plant height. This invention plays an important role in agricultural production and can be used to obtain new germplasm and be applied to tomato breeding. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0054] Figure 1 This is the transcriptional expression of SlCOP1H induced by light in Example 1;

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

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

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

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

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

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

[0061] The present invention discloses tomato SlCOP1 and SlCOP1H proteins, their encoding genes, and their applications. Any methods not mentioned in the examples are conventional experimental methods and will not be described in detail here.

[0062] In the present invention, there are no particular limitations on the plants suitable for this invention, as long as they are suitable for genetic transformation. In a specific embodiment of the present invention, the recombinant expression vectors pTX-SlCOP1 and pTX-SlCOP1H are introduced into tomatoes via Agrobacterium-mediated genetic transformation using the CRISPR / Cas9 gene editing system. Plants with altered target site sequences are screened for tomato cop1 and cop1h mutants. Double mutants are obtained by two methods: one is to design target sites in conserved regions of the amino acid sequence of two proteins and perform co-knockout; the other is to obtain double mutants by genetically crossing single mutants. The mutants are then cultured, and the changes in their traits are observed and 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 a constant temperature of 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 in the figure, SlCOP1 is constitutively expressed 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-edited SlCOP1 and SlCOP1H mutant materials

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

[0074] The CRISPR / Cas9 vector used in this study is a dual-target vector, with two target sites designed on each gene based on the SlCOP1 and SlCOP1H gene sequences. Sequence alignment was performed to select specific sequence segments, and target sequences for knocking out the SlCOP1 and SlCOP1H genes were screened using 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, we designed a target site in the conserved region of the two sequences to knock out both SlCOP1 and SlCOP1H. 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] After adding a homologous recombination arm sequence to the target sequence through the homologous recombination method, it was ligated to the pTX expression vector. The sequence of the pTX vector is shown as 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 entire pTX vector.

[0083] 2.2 Tomato genetic transformation and mutant identification

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

[0085] The T0 generation transgenic tomatoes obtained above were transplanted into a culture medium for cultivation. After survival, young leaves were taken for DNA extraction and identification. PCR was first used to identify the presence of the Cas9 gene in the plants. Plants containing the Cas9 gene were considered transgenic-positive. 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 containing the target sequence were amplified by PCR. Sequencing was used to identify whether the target sequence had changed and confirm whether the gene had been edited, and then mutant plants with altered proteins encoded by the SlCOP1 and SlCOP1H genes were screened out respectively.

[0089] The target identification primers for SlCOP1 and SlCOP1H single mutants are:

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

[0091] SlCOP1-CRdet-Rv: GGAAACAGCCTTCTCACCACAA (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 identifying 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 that did not contain the Cas9 gene, but whose SlCOP1 and SlCOP1H genes were edited and whose encoded proteins were changed. The SlCOP1 single mutant was denoted as cop1, the SlCOP1H single mutant was denoted as cop1h, and the double mutant was denoted 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 low light conditions

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

[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 that germinated uniformly were placed on 1 / 2MS medium and cultured in a constant temperature incubator at 25℃ and 30℃ respectively. The hypocotyl phenotype 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 the 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 (* indicates significant difference, ** indicates extremely significant difference), 10 days after germination, the double mutant cch showed dwarfed seedlings and darker cotyledon color, while the single mutants cop1 and cop1h showed no significant changes compared to the wild type. As the tomato plants grew, the single mutant cop1 showed no significant differences from the wild type; the single mutant cop1h exhibited reduced internode length and shorter stature compared to the wild type; the double mutant cch showed significantly shorter internodes, slower growth, and shorter stature, becoming even shorter than the cop1h single mutant. This suggests that SlCOP1 and SlCOP1H jointly regulate tomato growth and development, but that SlCOP1H is more important.

[0108] Example 6: Identification of mutant fruit traits

[0109] Marking was performed during the flowering period, and the changes in the mutant fruits compared with the wild type at the same growth and development period were observed. Figure 6As shown (dpa: days post anthesis), the fruits of cop1 and cop1h single mutants did not show obvious changes compared with the wild type, but the fruit color of the double mutant cch at the green fruit stage was darker than that of the wild type, and the fruit size of the double mutant was smaller than that of the wild type and 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] TGGATGGAGAGAGAGTTAATGAATGATATGGTCCTTTTGTTCATTCTCAAATTAATT

[0221] ATTTGTTTTTTCTCTTATTTGTTGTGTGTTGAATTTGAAATTATAAGAGATATGCAAA

[0222] CATTTTGTTTTGAGTAAAAATGTGTCAAATCGTGGCCTCTAATGACCGAAGTTAATA

[0223] TGAGGGTAAAACACTTGTAGTTGTACCATTATGCTTATTCACTAGGCAACAAATAT

[0224] ATTTTCAGACCTAGAAAAGCTGCAAATGTTACTGAATACAAGTATGTCCCTCTTGTGT

[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] GTGGTGGCTCTGGTTCCGGTGATTTTGATTATGAAAAGATGGCAAACGCTAATAAG

[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 variations or modifications 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 inhibits tomato elongation by knocking out the genes encoding SlCOP1 and SlCOP1H proteins; Tomato leggy growth is caused by high temperatures and low light levels; The amino acid sequence of the SlCOP1 protein is shown in SEQ ID NO.3, and the amino acid sequence of the SlCOP1H protein is shown in SEQ ID NO.

4.

2. Application of SlCOP1 and SlCOP1H proteins in regulating tomato growth; The amino acid sequence of the SlCOP1 protein is shown in SEQ ID NO.3, and the amino acid sequence of the SlCOP1H protein is shown in SEQ ID NO.4; The tomato growth regulation is to regulate any one or more of the following tomato growth and development by knocking out the coding genes of SlCOP1 and SlCOP1H proteins: (A) Reduce internode length of tomatoes; (B) Reduce the height of tomato plants; (C) Reduce tomato leaf size; (D) Deepen the color of tomato leaves; (E) Deepen the color of tomato fruit; (F) Improve the tolerance of tomato seedlings to weak light and excessive growth.

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