CN116926069A - Nucleic acid sequence for detecting cotton OE-3 and detection method thereof - Google Patents

Abstract

The invention relates to a nucleic acid sequence for detecting cotton OE-3 and a detection method thereof, wherein the nucleic acid sequence of the cotton OE-3 comprises a sequence shown as SEQ ID NO. 1 or a reverse complement sequence thereof, or a sequence shown as SEQ ID NO. 2 or a reverse complement sequence thereof. The cotton OE-3 has the characteristic of improving quality traits, and the detection method can accurately and rapidly identify whether the biological sample contains DNA molecules of the transgenic cotton event OE-3.

Description

A nucleic acid sequence for detecting cotton OE-3 and its detection method

Technical Field

The present invention relates to the field of plant biotechnology. Specifically, it relates to a nucleic acid sequence for detecting cotton OE-3 and a detection method thereof, and in particular to a transgenic cotton event OE-3 with improved quality traits and a nucleic acid sequence for detecting whether a biological sample contains a specific transgenic cotton event OE-3 and a detection method thereof.

Background Art

Cotton is the world's most important natural fiber crop and an important source of protein and oil. The global cotton planting area is about 490 million mu, with an output value of more than 50 billion US dollars. China, India, the United States and other five countries account for more than 70% of the total output. Among them, advanced cotton-growing countries such as the United States and Australia have achieved full mechanization, and their cotton industry has strong international competitiveness and leads the development direction of the cotton industry.

The quality of cotton cloth depends on the quality of cotton fibers. The longer and finer the staple fibers, the higher the air permeability, the better the gloss and the better the body feel. In the global cotton production, only 3% is the top-quality cotton with extra-long staple, which is the key material for spinning high-count yarns, and ultimately produces high-value-added textiles and clothing such as high-end yarn-dyed and home textiles. Therefore, high fiber quality traits are the main goal of cotton breeding. Worldwide, there are four cultivated species of cotton, of which the two diploid cultivated species of Asian cotton and grass cotton are no longer used in production. There are two cultivated species of allotetraploid cotton - Sea Island Cotton (Gossypium barbadense) and Upland Cotton (Gossypium hirsutum). The fiber yield of Upland Cotton accounts for more than 90% of the total fiber yield, while Sea Island Cotton cannot be widely cultivated due to the limitations of cultivation conditions, and the yield accounts for only 5%-8%. However, the fiber quality of Sea Island Cotton is better, which is specifically manifested in longer, thinner and stronger fibers. Such fibers can be processed into higher-quality textiles and can also improve production efficiency during the processing process. Through traditional hybridization and breeding methods, the hybrid offspring of sea island cotton are separated wildly, which brings great difficulties to breeding work. Therefore, introducing fiber quality-related genes from sea island cotton into upland cotton is an important breeding strategy for improving the fiber quality of upland cotton.

Cotton fiber is differentiated from the epidermal cells of the ovule and is a highly elongated, thickened, unbranched single-cell epidermal hair. The characteristics of the cotton fiber cell wall determine the fiber quality. The development of the fiber cell wall undergoes a series of complex physiological processes: biosynthesis and transport of cell wall components, cell wall relaxation, and deposition and distortion of cellulose. Among them, expansin proteins are expressed in high amounts and predominantly during the rapid elongation period of fibers, suggesting that they may play an important role in the fiber development process. GbEXPATR is an expansin gene unique to sea island cotton, which can control the length and strength of fibers by affecting the development of cotton fiber cell walls. Studies have found that overexpressing the GbEXPATR gene in upland cotton can obtain upland cotton materials with better fiber quality. Therefore, the use of the GbEXPATR gene can improve the fiber quality of upland cotton, obtain high-quality fiber cotton transformants with longer fibers and higher strength, and provide new germplasm resources for improving cotton fiber quality traits.

It is known that the expression of foreign genes in plants is affected by their chromosomal location, which may be due to chromatin structure (such as heterochromatin) or the proximity of transcriptional regulatory elements (such as enhancers) to the integration site. For this reason, it is usually necessary to screen a large number of events before it is possible to identify events that can be commercialized (i.e., events in which the introduced target gene is optimally expressed). For example, it has been observed in plants and other organisms that the expression level of the introduced gene can vary greatly between events; there may also be differences in the spatial or temporal pattern of expression, such as differences in the relative expression of the transgene between different plant tissues, which is manifested in the actual expression pattern may be inconsistent with the expression pattern expected based on the transcriptional regulatory elements in the introduced gene construct, resulting in differences in the trait expression of the transformation event. Therefore, it is usually necessary to generate hundreds or thousands of different events and screen them for a single event with the expected transgene expression level and expression pattern for commercialization purposes. Events with the expected transgene expression level and expression pattern can be used to introgress the transgene into other genetic backgrounds through sexual outcrossing using conventional breeding methods. The offspring produced by this hybridization method retain the transgene expression characteristics of the original transformation event. Applying this strategy model can ensure reliable gene expression in many varieties that are well adapted to local growing conditions. Therefore, more transformation events need to be identified and screened to obtain excellent transformation events with excellent comprehensive trait performance and commercial prospects.

It will be useful to be able to detect the presence of a specific event to determine whether the offspring of sexual hybridization includes the target gene. In addition, the method for detecting a specific event will also help to comply with relevant laws and regulations, such as the food derived from recombinant crops needs to be formally approved and marked before being put on the market. It is possible to detect the presence of transgenic by any well-known polynucleotide detection method, such as polymerase chain reaction (PCR). These detection methods usually focus on commonly used genetic elements, such as promoters, terminators, marker genes, etc. Therefore, unless the sequence of the chromosomal DNA ("flanking DNA") adjacent to the transgenic DNA inserted is known, this method just cannot be used to distinguish different events, particularly those events produced with the same DNA construct. Therefore, a pair of primers that often utilize the junction site of the transgenic and flanking DNA inserted to identify transgenic specific events by PCR, specifically the first primer comprising the flanking sequence and the second primer comprising the inserted sequence.

Summary of the invention

The purpose of the present invention is to provide an excellent cotton transformation event with excellent quality traits and a nucleic acid sequence and a detection method for detecting cotton OE-3. The transgenic cotton event OE-3 has excellent quality traits, and the detection method can accurately and quickly identify whether a biological sample contains a DNA molecule of a specific transgenic cotton event OE-3.

To achieve the above objectives, the present invention uses the pCambia2301-GbEXPATR expression vector to transform the cotton inbred line YZ1 through an Agrobacterium-mediated method, obtains more than 20 positive transformed seedlings, and identifies a transformation event OE-3 with longer fibers and higher strength, which can be used to improve the quality of cotton.

In order to characterize the identity of OE-3, the present invention provides a nucleic acid molecule comprising the sequence shown in SEQ ID NO: 1 and/or SEQ ID NO: 2, or the reverse complementary sequence thereof.

Furthermore, the nucleic acid sequence comprises the sequence shown in SEQ ID NO: 3 and/or SEQ ID NO: 4, or a reverse complementary sequence thereof.

Furthermore, the nucleic acid sequence comprises the sequence shown in SEQ ID NO: 6 and/or SEQ ID NO: 7, or the reverse complementary sequence thereof.

Furthermore, the nucleic acid sequence comprises the sequence shown in SEQ ID NO: 5 or its reverse complementary sequence.

The present invention also provides a probe for detecting cotton transformation events, characterized in that it comprises a sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 7 or a fragment or variant thereof or a reverse complementary sequence thereof.

The present invention also provides a primer pair for detecting cotton transformation events, characterized in that the amplification product of the primer pair comprises a sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 7 or a fragment thereof or a variant thereof or a reverse complementary sequence thereof.

In some embodiments, the primer pair is the sequence shown in SEQ ID NO:8 and SEQ ID NO:9; or the sequence shown in SEQ ID NO:10 and SEQ ID NO:11.

The present invention also provides a kit or microarray for detecting cotton transformation events, characterized in that it comprises the above-mentioned probe and/or primer pair.

The present invention also provides a method for detecting cotton transformation events, characterized in that it comprises using the above-mentioned probe or the above-mentioned primer pair or the above-mentioned probe and primer pair or the above-mentioned kit or microarray to detect whether the transformation event exists in the sample to be tested.

The present invention also provides a method for breeding cotton, characterized in that the method comprises the following steps:

1) obtaining cotton containing the above nucleic acid molecule;

2) subjecting the cotton obtained in step 1) to pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or hybridization or a combination thereof to obtain cotton plants, seeds, plant cells, offspring plants or plant parts; and optionally,

3) Improving the quality traits of the offspring plants obtained in step 2) and detecting whether the transformation event exists therein using the above method.

Furthermore, the present invention also provides products made from the cotton plants, seeds, plant cells, progeny plants or plant parts obtained by the above method, including food, feed or industrial raw materials.

The SEQ ID NO: 1 is a 22-nucleotide sequence located near the insertion junction at the 5' end of the insertion sequence in the transgenic cotton event OE-3. The SEQ ID NO: 1 spans the DNA sequence at the left flank of the cotton insertion site and the DNA sequence at the 5' end of the left border of the insertion sequence. The presence of the transgenic cotton event OE-3 can be identified by including the SEQ ID NO: 1 or its reverse complementary sequence. The SEQ ID NO: 2 is a 22-nucleotide sequence located near the insertion junction at the 3' end of the insertion sequence in the transgenic cotton event OE-3. The SEQ ID NO: 2 spans the DNA sequence at the 3' end of the right border of the insertion sequence and the DNA sequence at the right flank of the cotton insertion site. The presence of the transgenic cotton event OE-3 can be identified by including the SEQ ID NO: 2 or its reverse complementary sequence.

In the present invention, the nucleic acid sequence may be at least 11 or more continuous polynucleotides (first nucleic acid sequence) of any part of the transgenic insertion sequence in the SEQ ID NO:3 or its reverse complementary sequence, or at least 11 or more continuous polynucleotides (second nucleic acid sequence) of any part of the 5' left flank cotton genomic DNA region in the SEQ ID NO:3 or its reverse complementary sequence. The nucleic acid sequence may further be homologous to or reverse complementary to a part of the SEQ ID NO:3 that includes the complete SEQ ID NO:1 or SEQ ID NO:6. When the first nucleic acid sequence and the second nucleic acid sequence are used together, these nucleic acid sequences include a DNA primer pair in a DNA amplification method for producing an amplification product. When the amplification product produced in the DNA amplification method using the DNA primer pair is an amplification product including SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:6 or its reverse complementary sequence, the presence of the transgenic cotton event OE-3 or its progeny can be diagnosed.

The SEQ ID NO:3 is a sequence with a length of 1079 nucleotides located near the insertion junction site at the 5' end of the insertion sequence in the transgenic cotton event OE-3. The SEQ ID NO:3 consists of a 496-nucleotide cotton left flank genomic DNA sequence (nucleotides 1-496 of SEQ ID NO:3) and a 583-nucleotide 5' end DNA sequence of the first expression cassette of the marker gene (nucleotides 497-1079 of SEQ ID NO:3). The presence of the transgenic cotton event OE-3 can be identified by containing the SEQ ID NO:3 or its reverse complementary sequence.

The nucleic acid sequence may be at least 11 or more continuous polynucleotides (third nucleic acid sequence) of any part of the transgenic insertion sequence in the SEQ ID NO:4 or its reverse complementary sequence, or at least 11 or more continuous polynucleotides (fourth nucleic acid sequence) of any part of the 3' right flank cotton genomic DNA region in the SEQ ID NO:4 or its reverse complementary sequence. The nucleic acid sequence may further be homologous to or reverse complementary to a portion of the SEQ ID NO:4 that includes the entire SEQ ID NO:2 or SEQ ID NO:7. When the third nucleic acid sequence and the fourth nucleic acid sequence are used together, these nucleic acid sequences include a DNA primer set in a DNA amplification method for producing an amplification product. When the amplification product produced in the DNA amplification method using the DNA primer pair is an amplification product including SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:7 or its reverse complementary sequence, the presence of the transgenic cotton event OE-3 or its progeny can be diagnosed.

The SEQ ID NO:4 is a 1429 nucleotide sequence located near the insertion junction at the 3' end of the insertion sequence in the transgenic cotton event OE-3. The SEQ ID NO:4 consists of a 495-nucleotide 3' end DNA sequence of the second expression cassette of the trait improvement gene (nucleotides 1-495 of SEQ ID NO:4), a 23-nucleotide pCambia2301-GbEXPATR construct right border DNA sequence (nucleotides 496-518 of SEQ ID NO:4), and a 911-nucleotide cotton integration site right flank genomic DNA sequence (nucleotides 519-1429 of SEQ ID NO:4). The presence of the transgenic cotton event OE-3 can be identified by the presence of the SEQ ID NO:4 or its reverse complementary sequence.

The SEQ ID NO: 5 is a sequence of 4653 nucleotides in length that characterizes the transgenic cotton event OE-3, and the specific genome and genetic elements contained therein are shown in Table 1. The presence of the transgenic cotton event OE-3 can be identified by the presence of the SEQ ID NO: 5 or its reverse complementary sequence.

Table 1 Genome and genetic elements contained in SEQ ID NO:5

1: Unit: bp.

As is well known to those skilled in the art, the first and second nucleic acid sequences or the third and fourth nucleic acid sequences need not consist of only DNA, but may also include RNA, a mixture of DNA and RNA, or a combination of DNA, RNA or other nucleotides or their analogs that do not serve as templates for one or more polymerases. In addition, the probe or primer of the present invention should be at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides in length, which can be selected from the nucleotides described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11. When selected from the nucleotides shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, the primer can be at least about 21 to about 50 or more consecutive nucleotides in length.

The present invention also provides a method for improving the quality of cotton plants, characterized in that it includes planting at least one transgenic cotton plant, wherein the transgenic cotton plant comprises SEQ ID NO:1, the nucleic acid sequence at positions 497-3742 of SEQ ID NO:5 and SEQ ID NO:2 in sequence in its genome, or the genome of the transgenic cotton plant comprises SEQ ID NO:5; the transgenic cotton plant has improved quality traits.

In the present invention for detecting nucleic acid sequences of cotton plants and their detection methods, the following definitions and methods can better define the present invention and guide ordinary technicians in the field to implement the present invention. Unless otherwise specified, the terms are understood according to the conventional usage of ordinary technicians in the field.

The term "cotton" refers to Gossypium Hirsutum L. and includes all plant species that can be crossed with cotton, including wild cotton species.

"Flanking DNA" can comprise a genome naturally present in an organism such as a plant or an exogenous (heterologous) DNA introduced by a transformation process, such as a fragment relevant to a transformation event. Therefore, flanking DNA can comprise a combination of natural and exogenous DNA. In the present invention, "flanking region" or "flanking sequence" or "genomic border region" or "genomic border sequence" refers to at least 3, 5, 10, 11, 15, 20, 50, 100, 200, 300, 400, 1000, 1500, 2000, 2500 or 5000 base pairs or longer sequences, which are located directly upstream or downstream of the initial exogenous insertion DNA molecule and adjacent to the initial exogenous insertion DNA molecule. When the flanking region is located upstream, it can also be referred to as "left border flank" or "5 ' flank" or "5 ' genome flanking region" or "genome 5 ' flanking sequence" etc. When the flanking region is located downstream, it may also be referred to as the "right border flank" or "3' flank" or "3' genomic flanking region" or "genomic 3' flanking sequence" etc.

The transformation procedure that causes random integration of foreign DNA can lead to transformation events containing different flanking regions, and the different flanking regions are that each transformation event specifically contains.When recombinant DNA is introduced into plants by traditional hybridization, its flanking regions usually do not change.Transformation events also can contain the unique junction between the segment of heterologous insert DNA and genomic DNA or between two sections of genomic DNA or between two sections of heterologous DNA. "Joint" is the point where two specific DNA fragments connect.For example, the junction is present at the position where the insert DNA connects the flanking DNA.Jointing point is also present in the transformed organism, and wherein two DNA fragments are connected together in a way that is modified from finding in natural organisms. "Jointing DNA" refers to the DNA comprising the junction.

The present invention provides a transgenic cotton event called OE-3 and its progeny, wherein the transgenic cotton event OE-3 is cotton OE-3, which includes plants and seeds of the transgenic cotton event OE-3 and plant cells or their regenerable parts, and the plant parts of the transgenic cotton event OE-3 include but are not limited to cells, pollen, ovules, flowers, buds, roots, stems, leaves and products from cotton OE-3, such as cottonseed, cottonseed oil, cotton clothes, cotton quilts, cotton wool, cotton cloth and biomass left in the cotton crop field.

The transgenic cotton event OE-3 of the present invention comprises a DNA construct, and when expressed in a plant cell, the transgenic cotton event OE-3 obtains improved quality traits. The DNA construct comprises an expression cassette, the expression cassette comprises a suitable promoter and a suitable polyadenylation signal sequence for expression in a plant, the promoter is operably connected to the GbEXPATR gene that regulates the length and strength of cotton fibers, and the nucleic acid sequence of the GbEXPATR protein can improve the quality of cotton. The DNA construct comprises another expression cassette, the expression cassette comprises a suitable promoter and a suitable polyadenylation signal sequence for expression in a plant, the promoter is operably connected to the gene NPTII encoding neomycin phosphotransferase (NPTII), and the nucleic acid sequence of the NPTII protein is tolerant to aminoglycoside antibiotics (such as kanamycin, neomycin, G418, etc.).

The DNA construct is introduced into the plant using a transformation method, including but not limited to Agrobacterium-mediated transformation, biolistic transformation, and pollen tube pathway transformation.

Transgenic "events" are obtained by transforming plant cells with heterologous DNA constructs, i.e., including at least one nucleic acid expression cassette containing a target gene, inserted into the plant genome by transgenic methods to produce a plant population, regenerate the plant population, and select specific plants with the characteristics of insertion at a specific genomic site. The term "event" refers to the original transformation event that includes heterologous DNA and the offspring of the transformation event. The term "event" also refers to the offspring obtained by sexually crossing between the transformation event and other variety individuals containing heterologous DNA, even after repeated backcrossing with the recurrent parent, the inserted DNA and flanking genomic DNA from the parent of the transformation event are also present in the same chromosomal position in the hybrid offspring. The term "event" also refers to a DNA sequence from the original transformation event, which contains the inserted DNA and the flanking genomic sequence closely adjacent to the inserted DNA, which is expected to be transferred to the progeny, which is produced by sexually crossing the parental line containing the inserted DNA (e.g., the original transformation event and the progeny produced by its self-pollination) with the parental line that does not contain the inserted DNA, and the progeny receives the inserted DNA containing the target gene.

Cultivating a transgenic cotton event OE-3 with improved quality traits by the following steps: first sexually hybridizing a first parent cotton plant with a second parent cotton plant, thereby producing a variety of first generation progeny plants, wherein the first parent cotton plant consists of cotton plants cultivated from transgenic cotton event OE-3 and its progeny, wherein the transgenic cotton event OE-3 and its progeny are transformed using the quality trait improved expression cassette of the present invention, and the second parent cotton plant lacks the quality trait improved trait. These steps may further include backcrossing the progeny plants with improved quality traits with the second parent cotton plant or the third parent cotton plant, and then selecting the progeny by screening with antibiotics or by identifying molecular markers associated with the trait (such as DNA molecules containing the junction sites identified at the 5' end and 3' end of the inserted sequence in the transgenic cotton event OE-3), thereby producing cotton plants with improved quality traits.

It should also be understood that two different transgenic plants can also be crossed to produce offspring containing two independent, segregating added exogenous genes. Selfing of appropriate offspring can result in offspring plants that are homozygous for both added exogenous genes. Backcrossing of parental plants and outcrossing with non-transgenic plants as described above are also contemplated, as are asexual reproduction.

The term "probe" is an isolated nucleic acid molecule to which a conventional detectable label or reporter molecule, such as a radioisotope, a ligand, a chemiluminescent agent or an enzyme is bound. The probe is complementary to a strand of a target nucleic acid, and in the present invention, the probe is complementary to a strand of DNA from the genome of transgenic cotton event OE-3, whether the genomic DNA is from transgenic cotton event OE-3 or seeds or from plants or seeds or extracts of transgenic cotton event OE-3. The probes of the present invention include not only deoxyribonucleic acids or ribonucleic acids, but also polyamides and other probe materials that specifically bind to a target DNA sequence and can be used to detect the presence of the target DNA sequence.

The term "primer" is a separate nucleic acid molecule that anneals and binds to a complementary target DNA strand through nucleic acid hybridization, forms a hybrid between the primer and the target DNA strand, and then extends along the target DNA strand under the action of a polymerase (e.g., DNA polymerase). The primer pair of the present invention relates to its application in amplification of a target nucleic acid sequence, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods. The length of the primer is generally 11 polynucleotides or more, preferably 18 polynucleotides or more, more preferably 24 polynucleotides or more, and most preferably 30 polynucleotides or more. Such primers specifically hybridize with the target sequence under highly stringent hybridization conditions. Although primers that are different from the target DNA sequence and maintain hybridization ability to the target DNA sequence can be designed by conventional methods, preferably, the primers in the present invention have complete DNA sequence identity with the continuous nucleic acid of the target sequence.

As used herein, "kit" or "microarray" refers to a reagent set or chip for the purpose of identification and/or detection of cotton transformation events in biological samples. The kit or chip can be used, and its components can be specifically adjusted, for the purpose of quality control (e.g., purity of a seed batch), detection of events in plant material or materials containing or derived from plant material, such as but not limited to food or feed products.

Primers and probes based on the flanking genomic DNA and insert sequences of the present invention can be determined by conventional methods, for example, by isolating the corresponding DNA molecule from plant material derived from transgenic cotton event OE-3 and determining the nucleic acid sequence of the DNA molecule. The DNA molecule comprises the transgenic insert sequence and the flanking regions of the cotton genome, and fragments of the DNA molecule can be used as primers and probes.

The primers and probes of the present invention hybridize with the target DNA sequence under stringent conditions. Any conventional amplification method can be used to identify the presence of DNA derived from transgenic cotton event OE-3 in a sample. Nucleic acid molecules or fragments thereof are capable of specific hybridization with other nucleic acid molecules under certain circumstances. As used in the present invention, if two nucleic acid molecules can form an antiparallel double-stranded nucleic acid structure, it can be said that the two nucleic acid molecules can specifically hybridize with each other. If two nucleic acid molecules show complete complementarity, then one of the nucleic acid molecules is said to be the "complement" of the other nucleic acid molecule. As used in the present invention, when each nucleotide of one nucleic acid molecule is complementary to the corresponding nucleotide of another nucleic acid molecule, the two nucleic acid molecules are said to show "complete complementarity". If two nucleic acid molecules can hybridize with each other with sufficient stability so that they anneal and bind to each other under at least conventional "low stringency" conditions, the two nucleic acid molecules are said to be "minimally complementary". Similarly, if two nucleic acid molecules can hybridize with each other with sufficient stability so that they anneal and bind to each other under conventional "high stringency" conditions, the two nucleic acid molecules are said to have "complementarity". Deviations from complete complementarity are allowed as long as such deviations do not completely prevent the two molecules from forming a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe, it only needs to have sufficient complementarity in sequence to form a stable double-stranded structure under the specific solvent and salt concentration used.

As used herein, "amplified DNA" or "amplicon" refers to the nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template. For example, in order to determine whether a cotton plant is produced by sexual hybridization containing the transgenic cotton event OE-3 of the present invention, or whether a cotton sample collected from a field contains the transgenic cotton event OE-3, or whether a cotton extract, such as cotton wool or cottonseed oil, contains the transgenic cotton event OE-3, DNA extracted from a cotton plant tissue sample or extract can be subjected to a nucleic acid amplification method using a primer pair to produce an amplicon that is diagnostic for the presence of DNA of the transgenic cotton event OE-3. The primer pair includes a first primer derived from a flanking sequence adjacent to the inserted exogenous DNA insertion site in the plant genome, and a second primer derived from the inserted exogenous DNA. The amplicon has a certain length and sequence, and the sequence is also diagnostic for the transgenic cotton event OE-3.

Nucleic acid amplification reactions can be achieved by any nucleic acid amplification reaction method known in the art, including polymerase chain reaction (PCR). Various nucleic acid amplification methods are well known to those skilled in the art. PCR amplification methods have been developed to amplify 22kb of genomic DNA and 42kb of phage DNA. These methods and other DNA amplification methods in the art can be used in the present invention. The inserted exogenous DNA sequence and the flanking DNA sequence from the transgenic cotton event OE-3 can be amplified by using the provided primer sequences to amplify the genome of the transgenic cotton event OE-3, and after amplification, the PCR amplicon or cloned DNA is subjected to standard DNA sequencing.

The DNA detection kit based on the DNA amplification method contains DNA primer molecules that specifically hybridize to the target DNA under appropriate reaction conditions and amplify the diagnostic amplicon. The kit can provide agarose gel-based detection methods or many methods known in the art for detecting diagnostic amplicons. A kit containing DNA primers homologous or reverse complementary to any part of the cotton genomic region of SEQ ID NO:3 or SEQ ID NO:4 and homologous or reverse complementary to any part of the transgenic insertion region of SEQ ID NO:5 is provided by the present invention. In particular, the primer pairs useful in the DNA amplification method are SEQ ID NO:8 and SEQ ID NO:9, which amplify a diagnostic amplicon homologous to a portion of the 5' transgenic/genomic region of the transgenic cotton event OE-3, wherein the amplicon includes SEQ ID NO:1. The primer pairs useful in the DNA amplification method also include SEQ ID NO:10 and SEQ ID NO:11, which amplify a diagnostic amplicon homologous to a portion of the 3' transgenic/genomic region of the transgenic cotton event OE-3, wherein the amplicon includes SEQ ID NO:2. Other DNA molecules used as DNA primers can be selected from SEQ ID NO:5.

Taqman is described as a method for detecting and quantifying the presence of a DNA sequence and is described in detail in the instructions provided by the manufacturer. As a brief example, a FRET oligonucleotide probe is designed that spans the insert DNA sequence and the adjacent genomic flanking binding site. The FRET probe and PCR primers (one primer each in the insert sequence and the adjacent flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in the cleavage of the fluorescent and quenching portions of the FRET probe and the release of the fluorescent portion. The generation of a fluorescent signal represents the presence of the insert/flanking sequence, which indicates that amplification and hybridization were successful.

Based on the principle of hybridization, suitable techniques for detecting plant materials derived from transgenic cotton event OE-3 may also include Southern blot hybridization, Northern blot hybridization and in situ hybridization. In particular, the suitable techniques include incubating the probe and sample, washing to remove unbound probe and detecting whether the probe has hybridized. The detection method depends on the type of label attached to the probe, for example, radiolabeled probes can be detected by X-ray exposure and development, or enzyme-labeled probes can be detected by color change through substrate conversion.

A DNA detection kit can be developed using the compositions of the present invention and methods described or known in the art of DNA detection. The kit is useful for identifying the presence of DNA from transgenic cotton event OE-3 in a sample, and can also be used to grow cotton plants containing DNA from transgenic cotton event OE-3. The kit can contain DNA primers or probes that are homologous to or reverse complementary to at least a portion of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, or other DNA primers or probes that are homologous to or complementary to DNA contained in a transgenic genetic element of the DNA, and these DNA sequences can be used in DNA amplification reactions, or as probes in DNA hybridization methods. In the DNA amplification method, the DNA molecule used as a primer can be any portion of the transgenic insertion sequence derived from transgenic cotton event OE-3, or any portion of the DNA region flanking the cotton genome derived from transgenic cotton event OE-3.

The transgenic cotton event OE-3 can be combined with other transgenic cotton varieties, such as cotton tolerant to herbicides (such as glyphosate, glufosinate, etc.), or transgenic cotton varieties carrying insect-resistant genes. Various combinations of all these different transgenic events, bred together with the transgenic cotton event OE-3 of the present invention, can provide transgenic cotton varieties with improved insect-resistant or herbicide-resistant quality traits. These varieties can show better characteristics of insect resistance, herbicide resistance, and cotton fiber quality compared to non-transgenic varieties and single-trait transgenic varieties.

The present invention provides a nucleic acid sequence for detecting cotton plants and a detection method thereof. The transgenic cotton event OE-3 has the effect of improving the quality of cotton fibers. The cotton plant of this trait expresses the GbEXPATR protein, which confers improved plant quality traits. At the same time, in the detection method of the present invention, SEQ ID NO:1 or its reverse complementary sequence, SEQ ID NO:2 or its reverse complementary sequence, SEQ ID NO:3 or its reverse complementary sequence, SEQ ID NO:4 or its reverse complementary sequence, SEQ ID NO:6 or its reverse complementary sequence, or SEQ ID NO:7 or its reverse complementary sequence can be used as a DNA primer or probe to produce an amplification product diagnosed as the transgenic cotton event OE-3 or its progeny, and the presence of plant materials derived from the transgenic cotton event OE-3 can be identified quickly, accurately and stably.

The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 Schematic diagram of the structure of the binding site between the transgenic insertion sequence and the cotton genome.

Figure 2 Physical map of the recombinant expression vector pCambia2301-GbEXPATR. The English and abbreviations of each element are listed below:

LB T-DNA repeat The left border sequence of T-DNA of Agrobacterium.

CaMV 35S poly(A) Cauliflower mosaic virus 35S poly(A) terminator terminates transcription of NPTII.

NPTII neomycin phosphotransferase gene, used as a selection marker.

CaMV 35S The 35S promoter of the cauliflower mosaic virus initiates the transcription of the NPTII gene.

CaMV 35S The 35S promoter of the cauliflower mosaic virus initiates the transcription of the GbEXPATR gene.

GbEXPATR Cotton GbEXPATR protein encoding gene, regulating fiber quality.

NOS terminator The nopaline synthase gene terminator of Agrobacterium tumefaciens controls the transcription termination of GbEXPATR. RB T-DNArepeat The right border sequence of T-DNA of Agrobacterium.

pVS1 sta Plasmid stabilization site of the pVS1 plasmid.

pVS1 repA The replication origin of the pVS1 plasmid.

pVS1 oriV E. coli pBR322 replicon

bom The bom site of the pBR322 plasmid.

ori The replication origin of the pBR322 plasmid.

KanR encodes an aminoglycoside phosphotransferase protein that confers kanamycin resistance to bacteria.

Figure 3 Comparison of hand-combed length of cotton fibers. YZ1: non-transgenic recipient control cotton fiber; T5 generation OE-3: OE-3 transformation event cotton fiber.

Figure 4 Specific PCR verification results of OE-3 transformation event. M: Marker, size is marked next to it (unit: bp); 1: blank control; 2: genomic DNA of receptor control YZ1; 3: vector pCambia2301-GbEXPATR; 4: genomic DNA of cotton material containing OE-3 transformation event. A: The expected size of the left border PCR fragment is 884 bp; B: The expected size of the right border PCR fragment is 1063 bp.

Figure 5 Southern hybridization restriction enzyme cutting and probe position and inserted sequence information. The lower line segment indicates the position of the marker gene and target gene probes. The restriction sites of XbaI and HindⅢ are between the two probes. The CaMV 35S poly(A) terminator of the LB and NPTII genes was lost during the insertion process.

Figure 6 Southern blot hybridization of the insertion copy number of the target gene GbEXPATR in OE-3

A: XbaI enzyme digested DNA hybridization image; B: HindⅢ enzyme digested DNA hybridization image; C: Schematic diagram of probe and enzyme cutting position, the line segment below marks the probe position;

M: Molecular weight standard, with size marked next to it, unit: bp.

N: pure water;

1: positive control plasmid;

2: genomic DNA of T ₇ generation OE-3 transformants;

3: genomic DNA of T ₆ generation OE-3 transformants;

4-5: genomic DNA of other transformants with the same vector;

6: receptor control YZ1 genomic DNA;

Arrows on the right side of the image indicate exogenous bands.

Fig.7 Southern blot hybridization of the copy number of OE-3 marker gene NPTⅡ insertion

A: XbaI digestion; B: HindⅢ digestion; C: Schematic diagram of the probe and digestion position, the line below indicates the probe position.

M: Molecular weight standard, with size marked next to it.

N: pure water;

1: positive control plasmid;

2: genomic DNA of T ₇ generation OE-3 transformants;

3: genomic DNA of T ₆ generation OE-3 transformants;

4-5: genomic DNA of other transformants with the same vector;

6: receptor control YZ1 genomic DNA;

Arrows on the right side of the image indicate exogenous bands.

DETAILED DESCRIPTION

The transformation event OE-3 involved in the present application refers to a cotton plant in which an exogenous gene insert (T-DNA insert) is inserted between specific genomic sequences after genetic transformation using the cotton inbred line YZ1 as a recipient. In a specific embodiment, the expression vector used for transgenic has a physical map shown in Figure 2, and the obtained T-DNA insert has a sequence shown in nucleotides 497-3742 of SEQ ID NO:5. Transformation event OE-3 can refer to this transgenic process, or it can refer to the T-DNA insert in the genome obtained by this process, or a combination of the T-DNA insert and the flanking sequence, or it can refer to the cotton plant obtained by this transgenic process. In a specific example, this event is also applicable to plants obtained by transforming other recipient varieties with the same expression vector, thereby inserting the T-DNA insert into the same genomic position. Transformation event OE-3 can also refer to offspring plants obtained by asexual reproduction, sexual reproduction, doubling or doubling reproduction or a combination of the above plants.

Example 1 Acquisition of transformation events and character identification

The GbEXPATR gene is an Expansin gene unique to sea island cotton, which can control the length and strength of the fiber by affecting the development of the cotton fiber cell wall. The NPTII (neomycin phosphotransferase II) gene is a commonly used screening marker in plant genetic transformation. The gene encodes neomycin phosphotransferase, which can phosphorylate and inactivate aminoglycoside antibiotics (such as kanamycin, neomycin, G418, etc.). The present invention uses the pCambia2301-GbEXPATR expression vector (the vector physical map is shown in Figure 2, which contains the GbEXPATR gene expression cassette and the NPTII gene expression cassette), transforms the receptor material YZ1 by the Agrobacterium-mediated method, obtains more than 20 positive transformation seedlings, and backcrosses with the receptor cotton YZ1 as the recurrent parent in each generation to obtain BC ₅ F ₂ generation transgenic cotton seeds OE-1~OE-8, these 8 strains, and further investigates the quality traits of these transgenic cottons.

1. Screening of transformants with excellent cotton fiber quality

During the cotton-opening period, samples of transformants OE-1 to OE-8 and the control YZ1 were collected and ginned. The fiber quality was tested using a fiber quality measuring instrument to measure three indicators, namely, fiber length, micronaire value and breaking strength.

The Micronaire value is an indicator that comprehensively expresses the fineness and maturity of cotton fibers, measured by a Micronaire airflow meter. The Micronaire value is a measure of the flow rate of a certain amount of cotton fibers under specified conditions, expressed in Micronaire scales. The larger the value, the thicker the fiber and the higher the maturity of the fiber. The Micronaire value is divided into three grades: A, B, and C, with Grade B being the standard grade. The value range of Grade A is 3.7-4.2, with the best quality; the value range of Grade B is 3.5-3.6 and 4.3-4.9; the value range of Grade C is 3.4 and below and 5.0 and above, with the worst quality. The breaking strength refers to the thickness of textile fibers (yarns). The standard stipulates that tex is used as the unit. The specific strength usually refers to the tensile force that can be withstood when the thickness is 1tex. The unit is N/tex, and cN/dtex is commonly used. It is referred to as specific strength or weight specific strength (the strength of materials of the same weight).

As shown in Table 2, compared with the receptor control, the fiber quality-related traits such as cotton fiber length, micronaire value, and breaking strength of OE-3 to OE-8 were significantly improved compared with the control.

Table 2 Cotton fiber quality characteristics

Note: The values are expressed as the mean ± standard deviation of three biological replicates. Different letters indicate significant differences in the same column of data (LSD, α = 0.05).

2. Agronomic traits survey

While identifying the cotton fiber quality traits of several transformants, their agronomic traits (such as plant height and boll number per plant, etc.) were also recorded in detail. When conducting data statistics, it was unexpectedly found that the plant height and boll number per plant of transformants OE-5 to OE-8 were significantly lower than those of the control YZ1, and only the agronomic traits of transformants OE-3 and OE-4 were not significantly different from those of the control (Table 3).

Table 3 Main agronomic traits

Note: The values are expressed as the mean ± standard deviation of three biological replicates. Different letters indicate significant differences in the same column of data (LSD, α = 0.05).

3. Analysis of exogenous vector backbone sequences of transformants OE-3 and OE-4

100 mg of plant leaves were taken, quickly ground with liquid nitrogen, and then the total DNA was extracted using the CTAB method. After the concentration of genomic DNA was determined, the total amount of DNA was guaranteed to be >2 μg. Using genome resequencing (completed by Wuhan Biobank Co., Ltd.), each read length was 150 bp, at least 20 Gb of data was obtained, and the data quality indicator Q30 was guaranteed to be ≥80% (i.e.: the proportion of bases with a sequencing error rate greater than 0.1% was less than 20%). According to the genome resequencing results, the BWA software was used to use the transgenic vector backbone sequence as a template to perform sequence homology comparison with all the sequences obtained by sequencing (BWA, http://bio-bwa.sourceforge.net/, default settings). The comparison results showed that there was no backbone sequence insertion in OE-3; while there were multiple sequences homologous to the vector backbone in the OE-4 genome.

Forward and reverse primers were designed based on the sequences obtained by screening (Table 4), and the genome of transformant OE-4 was used as a template for verification by PCR amplification. The PCR products were sequenced and analyzed to confirm that a segment of the vector backbone sequence was inserted in transformant OE-4.

Table 4 Primer information for OE-4 backbone sequence identification

Backbone sequence sequencing results (933bp): shaded for primers, underlined for sequencing results

4. Analysis of the insertion site and flanking sequence of the exogenous sequence in the genome of transformant OE-3

In order to clarify the identity characteristics of the transformation event OE-3, the present invention analyzed the flanking sequences of the insertion site of the OE-3 exogenous sequence on the cotton genome.

FPNI-PCR was used to separate the T-DNA flanking sequence, clarify the insertion position of the exogenous sequence in the genome, and design specific primers to verify the insertion position and analyze the actual boundary sequence of the inserted sequence.

Using the genomic DNA of the leaves of OE-3 plants as a template, paired PCR was performed using three pairs of specific primers on the left border and random fusion primers and linker primers, and TA cloning sequencing was performed to obtain the flanking sequence of the left border. This partial sequence was compared with the reference genome, and it was found that the T-DNA sequence was inserted on the short arm of chromosome A10.

At the left border of the insertion site, 500 bp upstream of the insertion site on the genome and 500 bp on the T-DNA sequence were cut, and at the right border, 500 bp downstream of the genome insertion site and 500 bp on the T-DNA sequence were taken. Primers were designed for the cut sequences using the Primerblast software on the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast), and the amplified product was fused with a portion of the cotton genome sequence and a portion of the T-DNA sequence.

PCR amplification was performed using the genomic DNA of the transgenic cotton strain as a template. The PCR reaction was carried out in a 20 μL system. The amplification cycle program was: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, annealing for 30 s, 72°C extension for a certain time (set according to the product fragment size), 35 cycles; 72°C extension for 5 min.

According to the results of the flanking sequence and the insertion position, the OE-3 transformation event was PCR amplified using the genome upstream primer (SEQ ID NO: 8) and the vector left border primer (SEQ ID NO: 9) and the vector right border primer (SEQ ID NO: 10) and the genome downstream primer (SEQ ID NO: 11) to verify the insertion position of the exogenous fragment. The results are shown in Figure 4, proving that the OE-3 exogenous fragment is stably inserted into the cotton genome Chr A10: 18705326-18705351 position.

The full-length insertion sequence of OE-3 was further amplified and sequenced by overlapping PCR. The actual insertion sequence and the left and right flanking cotton genome sequences of transformant OE-3 are shown in SEQ ID NO: 5, and the insertion sequence size is 3246 bp.

By analyzing the border sequences of the left and right flanks, it can be seen that the insertion of the exogenous sequence caused the deletion of 24bp of the receptor genome, and the RB end of the T-DNA region of the vector sequence was deleted by 23bp, and the terminator CaMV 35S poly (A), LB and spacer sequence of the marker gene NPTⅡ were deleted by 283bp. At the same time, a base mutation (A110G) occurred in the target gene GbEXPATR, causing the amino acid sequence to mutate Q37R. Surprisingly, the mutation of amino acid Q37R did not affect the function of the GbEXPATR protein. From the results of the character identification of Example 1, the mutated exogenous protein still has the effect of improving the length and strength of cotton fibers.

5. Spatiotemporal expression analysis of target genes and marker genes

The expression of GbEXPATR and NPTⅡ in the main tissues of the transformant OE-3 at the seedling, bud and boll stages was detected at the transcription and translation levels by qRT-PCR and ELISA methods.

The results of transcriptional expression analysis are shown in Table 5, indicating that GbEXPATR and NPTⅡ genes were expressed in tissues at different stages of T ₆ and T ₇ transformants, and their expression was stable among different generations.

Table 5 Expression analysis of exogenous gene transcription level in transformant OE-3

The values are expressed as the mean ± standard deviation of three biological replicates, indicating the multiple of the expression of the reference gene (calculated according to the formula RQ = 2^(-ΔCt)). The differences in the same data were analyzed and compared using LSD (α = 0.05). "-" means not detected.

Table 6 Expression analysis of exogenous proteins (ng/g fresh weight)

The values are expressed as the mean ± standard deviation of 3 biological replicates. The data differences between different materials were compared using LSD (α = 0.05). "-" means not detected.

The results of translation expression analysis are shown in Table 6, indicating that GbEXPATR protein and NPTⅡ protein were expressed in the main tissues of _T6 and _T7 transformants, and the expression trend in various tissues in _T7 was similar to that in _T6 , indicating that their expression was stable among different generations.

The above analysis results show that although the terminator CaMV 35S poly(A) of the marker gene NPTⅡ was deleted and a base mutation (A110G) occurred in the target gene GbEXPATR, the above deletion and mutation did not affect the normal transcription and translation expression of the target gene and marker gene in the transformant OE-3.

In summary, the transformant OE-3 is an excellent transformant with excellent cotton fiber quality traits, the best agronomic traits, no vector backbone sequence insertion, and clear molecular characteristics.

Example 2 Systematic identification of cotton fiber quality of transformant OE-3

In the summer of 2022, the present invention sowed seeds of _T2 , _T4 and _T5 OE-3 strains and the control YZ1 at the cotton experimental base of Huazhong Agricultural University. A randomized block design was used with 3 replicates for each material. The cotton was planted according to the normal sowing time and field management method, and the appropriate humidity in the field was maintained. Before entering the rainy season, the field humidity was maintained, and other management was the same as that of the field.

The test results of the target traits of different generations of OE-3 and the control are shown in Table 7. The fiber length of OE-3 is between 28.87 and 29.79 mm, which is 5.86-6.96% higher than that of the control; the specific strength at break of OE-3 is between 26.70 and 28.20 cN/tex, which is 5.74-7.09% higher than that of the control; the micronaire value of OE-3 is B, while that of the control is C. The target traits of each generation of transformants are significantly enhanced compared with those of the control, and the differences between different generations are not significant, indicating that the target traits of the transformants are stable.

Table 7 Performance of target traits of OE-3 in different generations

Note: The values are expressed as the mean ± standard deviation of three biological replicates. Different letters indicate the significant difference between transformants and controls of the same generation (LSD, α = 0.05).

In addition, the length of mature fibers of T ₅ generation OE-3 was measured by hand combing method. The results are shown in FIG3 . The length of YZ1 was about 2.7 cm, while the length of OE-3 was about 3.0 cm, an increase of about 11%.

Example 3 Analysis of the Insertion Copy Number of Exogenous Sequences in Transformation Event OE-3

The Southern blot hybridization method is used to determine the copy number of the exogenous sequence. In the Southern blot test, two restriction endonucleases are selected on the T-DNA region and not in the hybridization region to digest the genomic DNA. After hybridization, each inserted copy in the genome will appear as a single and specific band. After the genomic DNA is digested by the restriction endonuclease, the region to be tested is selected as a probe for the Southern blot hybridization experiment.

Southern hybridization selected XbaI and HindⅢ enzymes to digest the positive control plasmid, receptor control YZ1 and OE-3 genomic DNA, and selected the sequence fragments of the target gene and marker gene as probes. The schematic diagram of the probe and enzyme cutting position is shown in Figure 5. The specific sequences of the probe primers are shown in Table 8.

Table 8 Southern hybridization test probe amplification primer position and sequence

The insertion copy number of the target gene GbEXPATR was detected by hybridization. Two restriction endonucleases, XbaI and HindⅢ, were used to digest the positive control plasmid, negative control YZ1 genomic DNA and OE-3 genomic DNA, respectively. After gel transfer, the GbEXPATR gene probe was used for labeling, and the hybridization results are shown in Figures 6A and 6B. The probe position of the exogenous gene GbEXPATR and the restriction endonuclease cutting sites of XbaI and HindⅢ are shown in Figure 6C. From the hybridization results, the GbEXPATR gene was inserted into the cotton genome as a single copy.

The insertion copy number of the marker gene NPTⅡ was detected by hybridization. Two restriction endonucleases, XbaI and HindⅢ, were used to digest the positive control plasmid, the negative control YZ1 genomic DNA, and the OE-3 transformant genomic DNA, respectively. After gel transfer, the NPTⅡ gene probe was used for labeling. The hybridization results are shown in Figures 7A and 7B. The probe position of the marker gene NPTⅡ and the restriction endonuclease cutting sites of XbaI and HindⅢ are shown in Figure 7C. The NPTⅡ gene is also inserted into the cotton genome as a single copy.

Example 4 Detection method of transformation event OE-3

Transgenic cotton event OE-3 can be used for breeding, and the new varieties bred can be used to produce agricultural products or commodities. If a sufficient amount is detected in the agricultural product or commodity, the agricultural product or commodity is expected to contain a nucleotide sequence that can diagnose the presence of transgenic cotton event OE-3 material in the agricultural product or commodity. The agricultural product or commodity includes, but is not limited to, cottonseed oil, cotton wool, cotton quilts, cotton cloth, cotton clothing, and any other food that will be consumed as a food source for animals, or as a bulking agent or an ingredient in a cosmetic composition for cosmetic purposes. A nucleic acid detection method and/or a kit based on a probe or primer pair can be developed to detect a transgenic cotton event OE-3 nucleotide sequence such as shown in SEQ ID NO: 1 or SEQ ID NO: 2 in a biological sample, wherein the probe sequence or primer amplification sequence is selected from the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 to diagnose the presence of transgenic cotton event OE-3.

One of the detection methods is: using PCR method to detect the specific border sequences in two OE-3 plants, the PCR primer pairs used are SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11, and the PCR reaction system is:

The reaction procedure is:

94°C, 5 min; (94°C, 30 sec; 55°C, 30 sec; 72°C, 60 sec) × 35 cycles; 72°C, 5 min; 4°C, 5 min.

The PCR products were subjected to electrophoresis in 1% (w/v) 1×TAE agarose gel, and the results are shown in Figure 4. The expected target bands (SEQ ID NO: 6 and SEQ ID NO: 7, respectively) can be amplified in the OE-3 transformation event. Moreover, the PCR method can track the existence of transformation events, and thus be applied to breeding work.

In summary, the transgenic cotton event OE-3 of the present invention can significantly improve the quality of cotton fiber, and the detection method can accurately and quickly identify whether the biological sample contains the DNA molecule of the transgenic cotton event OE-3. Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and are not limited thereto. Although the present invention is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims (8)

1. A nucleic acid molecule, characterized in that it includes any one of the following: i) Contains the sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2, or its reverse complement; ii) Comprising the sequence shown in SEQ ID NO:3 and/or SEQ ID NO:4, or the reverse complement thereof; iii) Comprising the sequence shown in SEQ ID NO:6 and/or SEQ ID NO:7, or the reverse complement thereof; iv) Comprises the sequence shown in SEQ ID NO:5, or its reverse complement. 2. A probe for detecting cotton transformation events, characterized by comprising SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: The sequence shown in 7 or its fragment or its variant or its reverse complement sequence. 3. A primer pair for detecting cotton transformation events, characterized in that the amplification product of the primer pair includes the sequence of claim 2; Optionally, the primer pair is the sequence shown in SEQ ID NO:8 and SEQ ID NO:9; or the sequence shown in SEQ ID NO:10 and SEQ ID NO:11. 4. A kit or microarray for detecting cotton transformation events, characterized by comprising the probe of claim 2 and/or the primer pair of claim 3. 5. A method for detecting cotton transformation events, which is characterized by comprising using any of the following to detect whether the transformation event exists in the sample to be tested: i) The probe according to claim 2; ii) The primer pair according to claim 3; iii) The probe according to claim 2 and the primer pair according to claim 3; iv) The kit or microarray of claim 4. 6. A method for breeding cotton, characterized in that the method includes the following steps: 1) Obtain cotton containing the nucleic acid molecule of claim 1; 2) Using the cotton obtained in step 1) through pollen culture, unfertilized embryo culture, double culture, cell culture, tissue culture, selfing or hybridization or a combination thereof to obtain cotton plants, seeds, plant cells, progeny plants or plant parts ; and optionally, 3) Identify the quality traits of the progeny plants obtained in step 2), and use the method of claim 5 to detect whether there is the transformation event. 7. Articles made from cotton plants, seeds, plant cells, progeny plants or plant parts obtained by the method of claim 6, including food, feed or industrial raw materials. 8. A method for improving quality traits of cotton plants, characterized by including planting at least one transgenic cotton plant, which contains SEQ ID NO: 1, SEQ ID NO: 5 No. 497- in sequence in its genome. The nucleic acid sequence at position 3742 and SEQ ID NO: 2, or the genome of the transgenic cotton plant contains SEQ ID NO: 5; the transgenic cotton plant has improved quality traits.