A method for synergistic regulation of cotton fiber elongation and Verticillium wilt resistance based on the GhMP gene in upland cotton and transgenic cotton plants

Abstract

This invention provides a method based on upland cotton GhMP This research relates to the field of genetic engineering technology and describes a method for the coordinated regulation of cotton fiber elongation and Verticillium wilt resistance using genes, as well as transgenic cotton plants. The gene encodes a membrane protein, and experiments using overexpression and RNA interference transgenic lines have demonstrated its effectiveness. GhMP The expression level of this compound is positively correlated with fiber development and disease resistance: increased expression significantly increases fiber length and improves resistance to Verticillium wilt, while decreased expression leads to fiber shortening and weakened resistance. Further studies have shown that... GhMP Proteins are located on the cell membrane and can interact with... GhPIP2.7 and GhTIP1.1 Aquaporins interact and jointly participate in the transmembrane transport of hydrogen peroxide, forming a molecular complex that regulates fiber elongation and disease resistance. This invention provides a key target gene that combines quality improvement and disease resistance enhancement, offering a new strategy and gene resource for multi-trait molecular breeding of cotton, and has significant application value and promising prospects.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a method based on upland cotton. GhMP Methods for gene-coordinated regulation of cotton fiber elongation and Verticillium wilt resistance, and transgenic cotton plants. Background Technology

[0002] Cotton is the world's most important natural fiber crop, with my country accounting for over 20% of global cotton production. Fiber length and resistance to Verticillium wilt are core traits determining cotton yield and quality. Cotton fiber quality indicators include more than ten factors such as length, strength, fineness, maturity, and uniformity, among which fiber length is one of the key traits determining cotton quality and economic benefits. For every 1 mm increase in fiber length, yarn strength can increase by 2%-3%, and long-fiber cotton (≥28 mm) is more efficient in spinning than short-fiber cotton (<25 mm), thus reducing costs. In recent years, with rising living standards, the demand for high-end textiles has increased significantly. However, some high-quality cotton varieties in China still lag behind international advanced levels, making it difficult to fully meet the demands of the high-end textile market. High-quality raw cotton suitable for spinning yarns of 80 count and above still relies to some extent on imports. Therefore, utilizing modern biotechnology through molecular breeding to improve cotton fiber quality, especially fiber length, is not only an urgent industry need but also a national policy direction.

[0003] Verticillium wilt of cotton ( Verticillium dahliae Verticillium wilt (Variegata wilt) is a semi-living, soil-borne fungal pathogen that can infect a variety of plants. It infects plants through the roots, multiplies in the vascular system, and causes wilting symptoms in the host. Variegata wilt causes significant yield losses in cotton-growing areas every year. To cope with Variegata wilt stress, plants initiate complex physiological and biochemical responses, including tissue structure modification, accumulation of antimicrobial substances, reactive oxygen species (ROS) bursts and homeostasis, calcium signaling, mitogen-activated protein kinase (MAPK) cascades, hormone signaling, and PAMPs / effective agent-triggered immune responses (PTI / ETI). Furthermore, Variegata wilt stress leads to excessive accumulation of ROS in plants; if not cleared in time, it can damage plant biofilms, thereby affecting normal plant physiological functions. Therefore, to address the serious threat of Variegata wilt to cotton production, breeding superior disease-resistant varieties is the fundamental measure to solve this problem.

[0004] In recent years, global climate change has led to frequent outbreaks of Verticillium wilt, while the need for improved fiber quality is urgent, posing a dual challenge to cotton breeding. Fiber development involves cell elongation and secondary cell wall synthesis, while resistance to Verticillium wilt depends on the accumulation of secondary metabolites (such as lignin and flavonoids) and the activation of immune signaling pathways. However, current research mostly focuses on genes regulating single traits, making it difficult to overcome the bottleneck of "high yield but low disease resistance, and disease resistance but low yield." Therefore, identifying key genes that simultaneously regulate fiber development and disease resistance, and elucidating their synergistic mechanisms, is of great significance for achieving synergistic improvement of multiple traits. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide upland cotton GhMP The application of gene synergistic regulation of cotton fiber elongation and Verticillium wilt resistance, through regulation of upland cotton GhMP The gene expression level was positively regulated to improve cotton fiber length and resistance to Verticillium wilt, providing key gene resources and technical means for molecular breeding of high-quality disease-resistant cotton varieties.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] A type of upland cotton GhMP Methods for gene-mediated synergistic regulation of cotton fiber elongation and Verticillium wilt resistance include:

[0008] S1, Constructing a system for regulation GhMP Expression level regulation carriers;

[0009] S2. Introduce the expression regulation vector into cotton plants to obtain... GhMP Transgenic plants whose expression was upregulated or downregulated;

[0010] S3. The plants obtained in step S2 GhMP The transcriptional level was validated to determine the expression changes relative to the wild type;

[0011] S4. Phenotypic determination was performed under conditions of fiber development and pathogen stress evaluation: when GhMP When expression is increased, the cotton fiber length of the target plants increases and their resistance to Verticillium wilt improves; when GhMP When expression is reduced, the cotton fiber length of the target plant decreases and its resistance to Verticillium wilt is reduced.

[0012] Preferably, GhMP The open reading frame is 1695 bp long and encodes 564 amino acids.

[0013] Preferably, the expression regulation vector in step S1 includes:

[0014] Includes GhMP Overexpression vectors of coding sequences; and / or

[0015] Includes GhMP RNA interference vectors encoding specific region sequences.

[0016] Preferably, step S2 is introduced via Agrobacterium-mediated genetic transformation.

[0017] Preferably, the transcriptional level verification in step S3 is performed using real-time quantitative PCR.

[0018] Preferably, the phenotypic assessment of fiber length is carried out during the fiber development stage, 5–20 days after flowering, and compared with the wild-type control.

[0019] Preferably, the phenotypic assessment of Verticillium wilt resistance is performed 14–30 days after pathogen inoculation and compared with a wild-type control.

[0020] Preferably, GhMP Proteins are located on the cell membrane and interact with it. GhPIP2.7 and GhTIP1.1 The interaction occurred, and the interaction was verified by yeast two-hybrid, bimolecular fluorescence complementation, and dual luciferase complementation assays.

[0021] Preferably, GhMP , GhPIP2.7 and GhTIP1.1 It participates in the transmembrane transport of hydrogen peroxide, and its function was verified by expressing the protein in yeast cells and growing them under hydrogen peroxide treatment conditions.

[0022] A transgenic cotton plant, obtained by the above method, comprising components for regulating... GhMP The expression box of expression, making GhMP The expression changed relative to the corresponding wild type, and compared with the corresponding wild type, it showed differences in cotton fiber length and Verticillium wilt resistance. GhMP Changes in expression correspond to corresponding synergistic changes.

[0023] According to specific embodiments provided by the present invention, the present invention discloses the following technical effects:

[0024] This invention has linked a key gene controlling fiber length in upland cotton through genome-wide association analysis. GhMP This gene is also upregulated by Verticillium wilt. This invention, through overexpression and RNAi transgenic cotton, creatively discovered… GhMP Its synergistic function in regulating cotton fiber elongation and Verticillium wilt resistance provides important genetic resources for molecular breeding of high-quality, disease-resistant cotton.

[0025] This invention provides a new approach for breeding high-quality, disease-resistant cotton plants by overexpression, enabling the production of cotton plants with increased fiber elongation and resistance to Verticillium wilt. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 A schematic diagram of GWAS association analysis, expression patterns, and gene bioinformatics analysis provided in an embodiment of the present invention;

[0028] Figure 2 Provided for embodiments of the present invention GhMP A schematic diagram showing the location on the cell membrane;

[0029] Figure 3 Provided for embodiments of the present invention GhMP Schematic diagram of transcriptional level analysis in fiber and leaf tissues of overexpression and RNAi lines;

[0030] Figure 4 Provided for embodiments of the present invention GhMP Schematic diagram of positive regulation of fiber elongation;

[0031] Figure 5 Provided for embodiments of the present invention GhMP A schematic diagram illustrating the positive regulation of Verticillium wilt resistance.

[0032] Figure 6 Provided for embodiments of the present invention GhMP and GhPIP2.7 , GhTIP1.1 Interaction diagram.

[0033] Figure 7 Provided for embodiments of the present invention GhMP and GhPIP2.7 , GhTIP1.1 A schematic diagram of hydrogen peroxide transport. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] The purpose of this invention is to provide upland cotton GhMP Application of gene synergistic regulation in cotton fiber elongation and Verticillium wilt resistance, through regulation of upland cotton GhMP The gene expression level was positively regulated to improve cotton fiber length and resistance to Verticillium wilt, providing key gene resources and technical means for molecular breeding of high-quality cotton varieties.

[0036] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] This embodiment first performed a genome-wide association analysis on 419 upland cotton accessions, discovering 212 SNPs significantly associated with fiber length (FL) in a candidate region on chromosome Dt11, and identifying the gene for this project. GhMP Furthermore, SV-GWAS of 1081 upland cotton materials was used to further identify structural variations associated with FL at Dt11, one of which was located at... GhMP The gene is located 550 bp upstream. This was determined through cloning of varieties with differences in fiber length. GhMP The gene promoter sequence further confirms the presence of this SV site. These data suggest that this gene may affect fiber length.

[0038] GhMP Encoding a membrane protein, with an open reading frame of 1695 bp, encoding 564 amino acids, and a molecular weight of approximately 62.76 kDa. Conserved domain analysis revealed... GhMP The absence of known conservative structural domains indicates GhMP It is a novel gene, potentially possessing unique biological functions or molecular mechanisms. Transmembrane domain analysis revealed... GhMP The N-terminus has five transmembrane structures. Subcellular localization analysis indicates... GhMP It is located in the cell membrane. Tissue expression characterization analysis revealed... GhMP The gene is highly expressed in root tissues and differentially expressed after being induced by Verticillium wilt, suggesting that it may play a role in Verticillium wilt resistance.

[0039] To clarify GhMP To investigate the role of this gene in fiber development, this example constructed overexpression and RNAi transgenic lines for this gene. qRT-PCR analysis showed that, at 15 days post-flowering (10⁵ DPA) in fibers, the overexpression lines... GhMP Transcriptional levels were significantly increased by 20-40 times compared to wild-type (WT), while the interference efficiency of this gene in RNAi lines was around 60%. Fiber dynamic elongation analysis showed that during fiber development at 5-20 DPA and into mature fibers, the fiber length of overexpressing lines was consistently higher than that of wild-type, while the fiber length of RNAi lines was significantly lower than that of wild-type. This confirms... GhMP Positive regulation of fiber elongation.

[0040] Further clarification GhMPTo investigate the role of this gene in the response to Verticillium wilt, this study utilized overexpression of the gene and RNAi transgenic lines. qRT-PCR analysis showed that in the first true leaf, the overexpressing lines... GhMP The transcription level was significantly increased by 7-10 times compared to the wild type (WT), while the interference efficiency of this gene in the RNAi lines was around 70%. Continuous observation of the resistance response after Verticillium wilt infection showed that, 17-30 days after inoculation, the disease index of the overexpression lines was consistently lower than that of the wild type, while the disease index of the RNAi lines was consistently higher than that of the wild type.

[0041] To explore further GhMP The molecular function, in this embodiment will GhMP The CDS sequence was ligated into the pGBKT7 vector as bait, and yeast coryza libraries were used as prey for screening interacting proteins in yeast two-hybrid (Y2H) experiments. Two candidate interacting proteins were identified and named... GhPIP2.7 and GhTIP1.1 (Both encode aquaporins). To verify GhMP and GhPIP2.7 , GhTIP1.1 The interaction relationship between them was verified in this embodiment using experiments such as bimolecular fluorescence complementarity (BiFC) and dual luciferase complementarity (LUC), all of which proved that... GhMP and GhPIP2.7 , GhTIP1.1 There are interactions between them, suggesting that they may form a regulatory complex that works together in cotton fiber development and Verticillium wilt response.

[0042] Aquaporins are tetrameric channel proteins that transport small molecules (including H₂O₂) across the membrane during growth, development, and related stresses, maintaining intracellular homeostasis. To explore... GhMP Regarding the effect of hydrogen peroxide, this implementation will GhMP , GhPIP2.7 and GhTIP1.1 The CDS sequence was ligated into the pYES2 vector and expressed in yeast strain BY4742. The growth of yeast cells under hydrogen peroxide treatment was observed, indicating... GhMP and GhPIP2.7 , GhTIP1.1 It participates in hydrogen peroxide transport.

[0043] The biomaterials used in this embodiment are described below:

[0044] *E. coli* Trans1-T1 was purchased from Beijing TransGen Biotech Co., Ltd. (catalog number CD501); *Agrobacterium* GV3101 was purchased from Shanghai Weidi Biotechnology Co., Ltd. (catalog number AC1001); pGreen-GFP vector was used for transient expression of green fluorescent protein in tobacco, pGBKT7 and pGADT7 vectors were used for yeast two-hybrid validation experiments, pGreenII0800-Luc vector was used for transient expression of LUC in tobacco, and pYES2 vector was used for protein expression in yeast cells; all were previously preserved in the laboratory. All cotton germplasm materials used were from germplasm resources collected and preserved in the laboratory.

[0045] The nucleotide sequence of the gene or the amino acid sequence of the protein used in this embodiment are as follows:

[0046] GhMP Encoded sequence (SEQ ID NO.1):

[0047]

[0048] GhMP Amino acid sequence (SEQ ID NO.2):

[0049] MDAGNSNQAGWLRIVYVVFAFCSALFLGALKGLLVGPIAALILIIGNLGVILGLLPAHIAWTIYTVVKTNRFDAPLKVALLIALPALFGIWLGLSIAGTVIVSVCYGFFTPWVSSFEAFRLDDESDRFFHCVVDGTWDTIK GSCTVVRDFSDLCFHSYPLYLKELRESPVSNEVRTLRLIHVPGLIVVGLLGLIVHIPIYTIIAIVKSPYMLFRGWFRLTHDLISREGPFLETACIPVAGLTILLWPIVVIGSIIMAIFSSIFIGLYGSVIVYQERSFKRGV AYVIAMIAEFDEYTNDWLYLREGTIFPKPSYRKKNGSEIEYSVGGLGGRFSSTTGEPPAMLMPTLARSVREAIKEVKMVQVWTNVMKSCEIRGKELLEAKVITSLDLCEWLKAKGSNEGAIIGVGLPCYSFLQTLLVSI RSGSNGLLMLDNVEINSLNRPKDKLLDWFFNPIMVLKEQIRVIKLGDGEVKLLEKLVLFGTNLERMDAWDNGSIVPQDSLRAAQMEGISRRMIGIARSISKLPTYRRKFRQVVKELITHASDKQDIPKCGSIKSTSSYEQV

[0050] Example 1 GhMP Related to fiber elongation and resistance to Verticillium wilt

[0051] Based on SNP-GWAS analysis of 419 upland cotton samples and fiber length traits and SV-GWAS analysis of 1081 upland cotton samples, this invention discovered that the trait located on chromosome D11... GhMP The gene is significantly associated with cotton fiber length. Notably, one of the structural variation sites is located at... GhMP At a position 550 bp upstream of the gene, differences in fiber length were observed in cloning varieties. GhMP The gene promoter sequence further confirms the existence of this SV site. Figure 1 A). These data suggest that this gene may influence fiber length. (Based on measurements...) GhMP The expression levels of genes at different developmental stages in upland cotton varieties with differences in fiber quality were investigated. GhMP The expression level was higher in high-quality fiber varieties than in low-quality upland cotton varieties, mainly in fibers at 0 days after flowering (0 DPA) and after 15 days after flowering (>15 DPA). Figure 1 B), indicating GhMP It may play a key role in fiber initiation and secondary wall thickening. Tissue expression characterization analysis revealed... GhMP High expression in root tissue ( Figure 1 C), and differential expression after Verticillium wilt induction, as evidenced by a significant increase in gene expression after Verticillium wilt infection (C). Figure 1 (D) indicates that this gene is likely involved in the resistance response to Verticillium wilt.

[0052] GhMP It encodes a membrane protein with an open reading frame of 1695 bp, encoding 564 amino acids, and a molecular weight of approximately 62.76 kDa. Analysis of the protein's conserved domains revealed... GhMP No known conservative structural domains ( Figure 1 E), indicating GhMP It is a novel gene, potentially possessing unique biological functions or molecular mechanisms. Based on its encoding of a membrane protein, transmembrane domain analysis revealed… GhMP It has 5 transmembrane domains at its N-terminus ( Figure 1 F). Subcellular localization analysis showed GhMP Located in the cell membrane ( Figure 2 ). Foreshadowing GhMP It may integrate molecular networks of fiber elongation and disease resistance response through transmembrane transport and metabolic remodeling.

[0053] Example 2 GhMP Positive regulation of fiber elongation

[0054] To clarify GhMP To investigate the role of this gene in fiber development, this example constructed overexpression and RNAi transgenic lines for this gene. qRT-PCR analysis showed that, at 15 days post-flowering (10⁵ DPA) in fibers, the overexpression lines... GhMP Transcription levels were significantly increased by 20-40 times compared to wild-type (WT) (e.g. Figure 3 A), while the interference efficiency of this gene in RNAi lines is around 60% ( Figure 3 B). To clarify GhMP The role of transgenic cotton in fiber elongation was dynamically observed at 5, 10, 15, and 20 DPA after flowering. Results showed that during the 5-20 DPA period, the fiber length of the transgenic lines was consistently longer than that of the wild type, while the fiber length of the RNAi transgenic lines was significantly shorter than that of the wild type. This confirms... GhMP Positive regulation of fiber elongation ( Figure 4 ).

[0055] Example 3 GhMP Positive regulation of cotton Verticillium wilt resistance

[0056] To further clarify GhMP The role of overexpression and RNAi transgenic lines in Verticillium wilt resistance response was investigated by inoculating them with Verticillium wilt pathogen. qRT-PCR analysis showed that in the first true leaf, the overexpression lines... GhMP Transcription levels were significantly increased by 7-10 times compared to wild-type (WT). Figure 3 C), while the interference efficiency of this gene in RNAi lines is around 70% ( Figure 3 D). To clarify GhMP The role of overexpression in Verticillium wilt resistance response was investigated by continuously observing resistance performance after Verticillium wilt infection. From 17 to 30 days post-infection, the disease index of overexpression lines remained consistently lower than that of wild-type, while the disease index of RNAi lines remained consistently higher than that of wild-type. Figure 5 ).

[0057] Example 4 GhMP and GhPIP2.7 , GhTIP1.1 Interactions affecting hydrogen peroxide transport jointly regulate fiber elongation and Verticillium wilt resistance

[0058] To explore further GhMP The molecular function, in this embodiment will GhMP The CDS sequence was ligated into the pGBKT7 vector as bait, and yeast coryza libraries were used as prey for screening interacting proteins in yeast two-hybrid (Y2H) experiments. Two candidate interacting proteins were identified and named... GhPIP2.7 and GhTIP1.1 (Both encode aquaporins). To verify GhMP and GhPIP2.7 , GhTIP1.1 The interaction between them will GhPIP2.7 and GhTIP1.1 The CDS sequence was ligated to the pGADT7 vector, and then ligated to the CDS sequence ligated to the pGBKT7 vector. GhMP The recombinant vector was co-transformed into Y2H-gold cells and cultured on SD / -Leu / -Trp / -His / -Ade medium. Results showed colony growth on the positive control plate, indicating the effectiveness of the yeast experimental system, while no colony growth was observed on the corresponding negative control plate. pGBKT7- GhMP +pGADT7- GhPIP2.7 and pGBKT7- GhMP +pGADT7- GhTIP1.1 The presence of bacterial colony growth indicates GhMP and GhPIP1.1, GhTIP1.1There is an interaction relationship between them. Figure 6 A, B). To further verify GhMP and GhPIP2.7 , GhTIP1.1 To investigate whether the interaction exists within the plant, this example conducted a bimolecular fluorescence complementation (BiFC) experiment in tobacco leaves. The results showed that when... GhMP -nYFP and GhPIP2.7 -cYFP fusion protein or GhTIP1.1 When the -cYFP fusion protein is co-expressed in the lower epidermal cells of tobacco leaves, YFP fluorescence can be specifically observed in the cell membrane. Figure 6 C, D). Furthermore, the Dual-Luciferase Complementation Assay (LUC) yielded similar results. Figure 6 E, F). A review of experimental results for Y₂H, BiFC, and LUC indicates that... GhMP and GhPIP2.7 , GhTIP1.1 They interact on the cell membrane and may form a regulatory complex that plays a joint role in cotton fiber development and Verticillium wilt response.

[0059] Aquaporins are tetrameric channel proteins that transport small molecules (including H2O2) across the membrane during growth, development, and related stresses, maintaining intracellular homeostasis. To verify... GhMP Regarding the effect of hydrogen peroxide, this implementation will GhMP , GhPIP2.7 and GhTIP1.1 The CDS sequence was ligated into the pYES2 vector and expressed in yeast strain BY4742. In yeast growth assays, yeast transformants were diluted to specific OD values. 600 Values ​​(0.70, 0.07, and 0.007) were obtained and uniformly dotted on SD-URA+GAL solid medium containing 0 or 1.5 mmol / L H2O2. The activity of yeast transformants was recorded after culturing at 30°C for 3 days. The results showed... GhMP , GhPIP2.7 and GhTIP1.1 The growth of yeast transformants was inhibited on a medium containing H2O2, and this result was also confirmed by growth curves measured on liquid medium. Figure 7 The above results indicate that... GhMP , GhPIP2.7 and GhTIP1.1 It has the ability to transport H2O2.

[0060] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0061] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, those skilled in the art will recognize that, based on the ideas of the present invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method based on upland cotton GhMP A method for gene-coordinated regulation of cotton fiber elongation and Verticillium wilt resistance, characterized in that, include: S1, Constructing a system for regulation GhMP Expression level regulation carriers; among which, GhMP The nucleotide sequence is shown in SEQ ID NO. 1; S2. Introduce the expression regulation vector into cotton plants to obtain... GhMP Transgenic plants with upregulated expression; S3. The plants obtained in step S2 GhMP The transcriptional level was validated to determine the expression changes relative to the wild type; S4. Phenotypic determination was performed under conditions of fiber development and pathogen stress evaluation: when GhMP When expression is enhanced, the cotton fiber length of the target plant increases and its resistance to Verticillium wilt improves.

2. The method according to claim 1, characterized in that, GhMP The open reading frame is 1695 bp long and encodes 564 amino acids.

3. The method according to claim 1, characterized in that, The introduction method in step S2 is Agrobacterium-mediated genetic transformation.

4. The method according to claim 1, characterized in that, The transcriptional level was verified in step S3 using real-time quantitative PCR.

5. The method according to claim 1, characterized in that, Phenotypic assessment of fiber length was conducted during the fiber development stage, 5–20 days after flowering, and compared with the wild-type control.

6. The method according to claim 1, characterized in that, Phenotypic assessment of Verticillium wilt resistance was conducted 14–30 days after pathogen inoculation and compared with wild-type controls.

7. A transgenic cotton plant, characterized in that, The plant is obtained by the method of claim 1, and includes methods for regulating... GhMP The expression box of expression, making GhMP The expression changed relative to the corresponding wild type, and compared with the corresponding wild type, it showed differences in cotton fiber length and Verticillium wilt resistance. GhMP The corresponding synergistic changes in expression; among which, GhMP The nucleotide sequence is shown in SEQ ID NO. 1.

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