CN114525277B - Nucleic acid sequence for detecting 17L397-1 in cotton and detection method thereof - Google Patents

Abstract

The invention relates to a nucleic acid sequence for detecting 17L397-1 in cotton and a detection method thereof, wherein the nucleic acid sequence of 17L397-1 in cotton 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 17L397-1 in cotton has the characteristics of improved yield traits and glyphosate-tolerant herbicide, and the detection method can accurately and quickly identify whether the biological sample contains the DNA molecule of the 17L397-1 in a transgenic cotton event.

Description

Nucleic acid sequence for detecting 17L397-1 in cotton and detection method thereof

Technical Field

The invention relates to the technical field of plant biology. In particular to a nucleic acid sequence for detecting 17L397-1 in cotton and a detection method thereof, in particular to a nucleic acid sequence for detecting 17L397-1 in transgenic cotton event with improved yield traits and tolerance to glyphosate herbicide application and 17L397-1 in a specific transgenic cotton event in biological samples and a detection method thereof.

Background

Cotton is the most important natural fiber crop in the world and is also an important source of protein and grease. The global cotton planting area is about 4.9 hundred million mu, and the yield exceeds $500 hundred million. Five countries such as China, india and the United states account for more than 70% of the total yield. The cotton industry of advanced cotton-planting countries such as America and Australian and the like has extremely strong international competitiveness due to the realization of whole-process mechanization, and the development direction of the cotton industry is led.

The cotton production country is large, about 1 hundred million cotton farmers exist in the country, the annual cotton planting area is about 6000-8000 ten thousand mu, the annual cotton production is 450-560 ten thousand tons, and the annual cotton production area accounts for about 25% of the total cotton production amount in the world. Meanwhile, china is the largest cotton import country in the world, cotton importation in China is expected to continue to be increased rapidly in the next few years, china officially reports media, and in recent years, cotton importation in China is increased to about 700 ten thousand metric tons each year. The reason is that the China textile and clothing industries are vigorously developing. Therefore, the yield of cotton in China is improved through biotechnology, and the development of cotton planting industry and cotton spinning industry is greatly influenced.

FCA and ABA abscisic acid have high affinity and stereospecificity, and conform to the principle of receptor dynamics. ABA binding directly controls FCA-mediated processing of the pre-mRNA, an ABA receptor involved in RNA metabolism. The research finds that: the RRM2 domain for controlling the FCA gene and the herbicide-resistant gene g10-epsps are transferred into a receptor, and the obtained transformant shows obvious changes in aspects of quality property improvement, herbicide resistance and the like, so that the method can be widely applied to the improvement of economic properties of dicotyledonous and monocotyledonous crops.

Weeds in the field compete with crops for water, fertilizer, light and growth space, and the yield and quality of the crops are directly affected. Meanwhile, a plurality of weeds are intermediate hosts of crop pathogenic bacteria and pests, and are one of important biological limiting factors for crop yield increase. According to statistics of the combined national food and agricultural organization, the loss of food production caused by weeds in the world is up to 950 hundred million dollars each year, which is equivalent to the loss of 3.8 hundred million tons of wheat, and more than half of the global wheat yield in 2009. Of the economic losses of 950 million dollars, the developing world is bearing about 700 million dollars (FAO.The lurking menace of weeds[J/OL].(http://www.fao.org/news/story/en/item/29402/icode/),2009-08-11.). and therefore effective weed control in the field is one of the important measures to promote grain yield. Along with the acceleration of urban migration of rural population in China, the large-scale and mechanical corn planting is a foreseeable trend, so that the traditional manual weeding mode becomes unrealistic. At present, the selective herbicide widely applied in the market has large application amount and long residual period, and is easy to influence the normal growth of the following crops. The biocidal herbicide such as glufosinate has the characteristics of high efficiency, low toxicity, easy degradation, no residue and the like. However, they are not selective in weeding and cannot be used directly in the growing period of crops. This difficulty can be overcome by breeding cotton resistant to such biocidal herbicides by transgenic techniques. The weed problem can be effectively solved by spraying for 1-2 times in the cotton growing period, and the dosage and the input cost of herbicide are reduced. Therefore, the herbicide-resistant transgenic cotton has very wide application value and market potential.

Expression of exogenous genes in plants is known to be affected by their chromosomal location, possibly due to the proximity of chromatin structures (e.g., heterochromatin) or transcriptional regulatory elements (e.g., enhancers) to the integration site. For this reason, it is often necessary to screen a large number of events to make it 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 may vary greatly between events; there may also be differences in the spatial or temporal patterns of expression, such as differences in the relative expression of transgenes between different plant tissues, which differences may be manifested in actual expression patterns that are inconsistent with the expression patterns expected from the transcriptional regulatory elements in the introduced gene construct, resulting in differences in the performance of the transformation event. Thus, it is often desirable to generate hundreds or thousands of different events and screen those events for a single event having transgene expression levels and patterns that are expected for commercialization purposes. Events with expected transgene expression levels and expression patterns can be used to introgress transgenes into other genetic backgrounds by sexual outcrossing using conventional breeding methods. The progeny produced by this crossing retain the transgene expression characteristics of the original transformation event. The use of such a strategy can ensure reliable gene expression in many varieties that are well suited to the growth conditions of the locus. Therefore, more transformation events need to be identified and screened for superior transformation events with superior overall trait performance and commercial prospects.

It would be beneficial to be able to detect the presence of a particular event to determine whether the progeny of a sexual cross contain a gene of interest. In addition, methods of detecting specific events will also help to comply with relevant regulations, such as the need for formal approval and marking of foods derived from recombinant crops prior to their being put on the market. It is possible to detect the presence of the transgene by any well known polynucleotide detection method, such as the Polymerase Chain Reaction (PCR). These detection methods are generally focused on commonly used genetic elements such as promoters, terminators, marker genes, and the like. Thus, unless the sequence of chromosomal DNA adjacent to the inserted transgenic DNA ("flanking DNA") is known, such a method as described above cannot be used to distinguish between different events, particularly those generated with the same DNA construct. Therefore, it is common today to identify a transgene specific event by PCR using a pair of primers spanning the junction of the inserted transgene and flanking DNA, specifically a first primer comprising the flanking sequence and a second primer comprising the inserted sequence.

Disclosure of Invention

The invention aims to provide an excellent cotton transformation event with excellent yield characteristics and herbicide resistance characteristics, a nucleic acid sequence for detecting 17L397-1 in cotton and a detection method thereof. The 17L397-1 yield trait in the transgenic cotton event is excellent and has better tolerance to glyphosate herbicide, and the detection method can accurately and rapidly identify whether the biological sample contains the DNA molecule of 17L397-1 in the specific transgenic cotton event.

In order to achieve the aim, the invention uses pCAMBIA1300/EPSPS-csRRM2 expression vector to transform cotton seed 24 in cotton inbred line by agrobacterium-mediated method, to obtain 13 positive transformed seedlings. And identified 17L397-1 in a transformation event with better yield traits and tolerance to glyphosate herbicide, which event has better yield traits and tolerance to glyphosate herbicide than transformation event ICR24-397 (accession number 201811442263.1) containing the same gene, thus 17L397-1 can be used to improve cotton yield and herbicide tolerance traits.

To characterize the identity of 17L397-1, the present invention provides a nucleic acid molecule comprising the sequences shown in SEQ ID NO.1 and/or SEQ ID NO. 2, or the reverse complement thereof.

Further, the nucleic acid sequence comprises the sequence shown in SEQ ID NO. 3 and/or SEQ ID NO. 4, or the reverse complement thereof.

Further, the nucleic acid sequence comprises the sequence shown in SEQ ID NO.6 and/or SEQ ID NO. 7, or the reverse complement thereof.

Further, the nucleic acid sequence comprises the sequence shown in SEQ ID NO. 5 or the reverse complement thereof.

The invention also provides a probe for detecting cotton transformation events, which is characterized by comprising 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 or reverse complement thereof.

The invention also provides a primer pair for detecting a cotton transformation event, which is characterized in that an amplification product of the primer pair comprises a sequence shown as 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 or reverse complement thereof.

In some embodiments, the primer pair is the sequences set forth in SEQ ID NO.8 and SEQ ID NO. 9; or SEQ ID NO. 10 and SEQ ID NO. 11.

The invention also provides a kit or microarray for detecting cotton transformation events, which is characterized by comprising the probe and/or primer pair.

The invention also provides a method for detecting a cotton transformation event, which is characterized by comprising the step of detecting whether the transformation event exists in a sample to be detected by using the probe or the primer pair or the probe and primer pair or the kit or the microarray.

The invention also provides a method for breeding cotton, which is characterized by comprising the following steps:

1) Obtaining cotton comprising the nucleic acid molecule;

2) Obtaining cotton plants, seeds, plant cells, progeny plants or plant parts from the cotton obtained in step 1) by pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or hybridization or a combination thereof; optionally, the composition may be in the form of a gel,

3) Identifying yield traits and/or herbicide resistance of the progeny plant obtained in step 2) and detecting the presence or absence of said transformation event using the method described above.

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

The SEQ ID NO. 1 is a sequence of 22 nucleotides in length, which is positioned near the insertion junction at the 5 '-end of the insertion sequence in 17L397-1 in the transgenic cotton event, the SEQ ID NO. 1 spans the left flank genome DNA sequence of the cotton insertion site and the DNA sequence at the 5' -end of the left boundary of the insertion sequence, and the existence of 17L397-1 in the transgenic cotton event can be identified by the SEQ ID NO. 1 or the reverse complement sequence thereof. The SEQ ID NO. 2 is a sequence of 22 nucleotides in length, which is positioned near the insertion junction at the 3 '-end of the insertion sequence in 17L397-1 in the transgenic cotton event, the SEQ ID NO. 2 spans the DNA sequence at the 3' -end of the right border of the insertion sequence and the genomic DNA sequence on the right flank of the cotton insertion site, and the existence of 17L397-1 in the transgenic cotton event can be identified by the SEQ ID NO. 2 or the reverse complement thereof.

In the present invention, the nucleic acid sequence may be at least 11 or more contiguous polynucleotides (first nucleic acid sequence) of any portion of the transgene insert sequence in the SEQ ID NO. 3 or reverse complement thereof, or at least 11 or more contiguous polynucleotides (second nucleic acid sequence) of any portion of the 5' left-flanking cotton genomic DNA region in the SEQ ID NO. 3 or reverse complement thereof. The nucleic acid sequence may further be homologous or reverse complementary to a portion of the SEQ ID NO. 3 comprising 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 comprise a pair of DNA primers in a DNA amplification method that produces an amplification product. The presence of 17L397-1 or a progeny thereof in a transgenic cotton event can be diagnosed when the amplification product produced in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:6 or a reverse complement thereof.

The SEQ ID NO. 3 is a sequence of 586 nucleotides in length, which is positioned near the insertion junction at the 5 'end of the insertion sequence in 17L397-1 in the transgenic cotton event, the SEQ ID NO. 3 consists of 177 nucleotides of cotton left flank genomic DNA sequence (nucleotides 1-177 of SEQ ID NO. 3) and 409 nucleotides of 5' end DNA sequence of a first expression cassette of glyphosate-tolerant gene (nucleotides 178-586 of SEQ ID NO. 3), and the presence of 17L397-1 in the transgenic cotton event can be identified by comprising the SEQ ID NO. 3 or the reverse complement thereof.

The nucleic acid sequence may be at least 11 or more contiguous polynucleotides (third nucleic acid sequence) of any portion of the transgene insert sequence in the SEQ ID NO. 4 or reverse complement thereof, or at least 11 or more contiguous polynucleotides (fourth nucleic acid sequence) of any portion of the 3' right-flanking cotton genomic DNA region in the SEQ ID NO. 4 or reverse complement thereof. The nucleic acid sequence may further be homologous or reverse complementary to a portion of the SEQ ID NO. 4 comprising the complete 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 comprise a set of DNA primers in a DNA amplification method that produces an amplification product. The presence of 17L397-1 or a progeny thereof in a transgenic cotton event can be diagnosed when the amplification product produced in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:7 or a reverse complement thereof.

The SEQ ID NO. 4 is a sequence of 836 nucleotides in length near the insertion junction at the 3 '-end of the insertion sequence in 17L397-1 of the transgenic cotton event, the SEQ ID NO. 4 consists of the 3' -end DNA sequence of the second expression cassette of the trait improvement gene of 134 nucleotides (nucleotides 1-134 of SEQ ID NO. 4), the pCAMBIA1300/EPSPS-CSRRM2 construct right border DNA sequence of 254 nucleotides (nucleotides 135-388 of SEQ ID NO. 4) and the cotton integration site right flank genomic DNA sequence of 448 nucleotides (nucleotides 389-836 of SEQ ID NO. 4), comprising the SEQ ID NO. 4 or its reverse complement can be identified as the presence of 17L397-1 in the transgenic cotton event.

The sequence of SEQ ID NO. 5 is a sequence of 17L397-1 with a length of 4544 nucleotides in the transgenic cotton event, and the specific genome and genetic elements contained in the sequence are shown in Table 1. The inclusion of the SEQ ID NO. 5 or its reverse complement may be identified as the presence of 17L397-1 in a transgenic cotton event.

Table 1 genome and genetic elements comprised by SEQ ID NO:5

1: The unit bp.

It is well known to those skilled in the art that 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 analogues thereof that do not serve as templates for one or more polymerases. Furthermore, the probes or primers described herein should be at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides in length, which may be selected from 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 the nucleotides set forth in SEQ ID NO. 11. When selected from the group consisting of the nucleotides set forth in SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7, the primer may be at least about 21 to about 50 or more consecutive nucleotides in length.

The present invention also provides a method of protecting a cotton plant from injury caused by a herbicide comprising applying to a field where at least one transgenic cotton plant comprising in its genome the sequence of SEQ ID NO. 1, SEQ ID NO. 5, nucleotide sequence from position 178 to 3842 and SEQ ID NO. 2, or comprising in its genome SEQ ID NO. 5, an effective dose of a glyphosate herbicide; the transgenic cotton plants have tolerance to glyphosate herbicide.

The invention also provides a method for improving the yield of cotton plants, which is characterized by comprising the steps of planting at least one transgenic cotton plant, wherein the transgenic cotton plant sequentially comprises SEQ ID NO. 1, SEQ ID NO. 5 178-3842 nucleic acid sequences and SEQ ID NO. 2 in the genome of the transgenic cotton plant, or the genome of the transgenic cotton plant comprises SEQ ID NO. 5; the transgenic cotton plants have improved yield traits.

In the nucleic acid sequences and methods of the present invention for detecting cotton plants, the following definitions and methods may better define the present invention and direct one of ordinary skill in the art to practice the present invention, unless otherwise indicated, terms are understood in accordance with the ordinary usage by one of ordinary skill in the art.

The term "cotton" refers to upland cotton (Gossypium Hirsutum L) and includes all plant varieties that can mate with cotton, including wild cotton species.

The term "comprising" means "including but not limited to.

The term "plant" includes whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, clumps (plant clumps), and intact plant cells in plants or plant parts, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, and the like. It is to be understood that parts of transgenic plants within the scope of the present invention include, but are not limited to, plant cells, protoplasts, tissues, calli, embryos and flowers, stems, fruits, leaves and roots, which are derived from transgenic plants or their progeny which have been previously transformed with the DNA molecules of the present invention and thus at least partially consist of the transgenic cells.

The term "gene" refers to a nucleic acid fragment that expresses a particular protein, including regulatory sequences preceding (5 'non-coding sequences) and regulatory sequences following (3' non-coding sequences) the coding sequences. "native gene" refers to a gene that is found naturally to have its own regulatory sequences. By "chimeric gene" is meant any gene that is not a native gene, comprising regulatory and coding sequences found in a non-native manner. "endogenous gene" refers to a native gene that is located in its natural location in the genome of an organism. "exogenous gene" is a foreign gene that is present in the genome of an organism and that is not originally present, and also refers to a gene that has been introduced into a recipient cell by a transgenic procedure. The exogenous gene may comprise a native gene or chimeric gene inserted into a non-native organism. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. The site in the plant genome where the recombinant DNA has been inserted may be referred to as an "insertion site" or "target site".

"Flanking DNA" may comprise genomic or foreign (heterologous) DNA introduced by a transformation process, such as fragments associated with a transformation event, naturally occurring in an organism such as a plant. Thus, flanking DNA may include a combination of native and foreign DNA. In the present invention, a "flanking region" or "flanking sequence" or "genomic border region" or "genomic border sequence" refers to a sequence of at least 3, 5, 10, 11, 15, 20, 50, 100, 200, 300, 400, 1000, 1500, 2000, 2500, or 5000 base pairs or more that is immediately upstream or downstream of and adjacent to the initial exogenous inserted DNA molecule. When this flanking region is located upstream, it may also be referred to as a "left border flanking" or "5 'genomic flanking region" or "genomic 5' flanking sequence" or the like. When this flanking region is located downstream, it may also be referred to as a "right border flanking" or a "3 'genomic flanking region" or a "genomic 3' flanking sequence", etc.

Transformation procedures that cause random integration of exogenous DNA will result in transformation events that contain different flanking regions that each transformation event specifically contains. When recombinant DNA is introduced into plants by conventional hybridization, its flanking regions are generally not altered. Transformation events will also contain unique junctions between segments of heterologous insert DNA and genomic DNA or between two segments of heterologous DNA. "ligation" is the point at which two specific DNA fragments are ligated. For example, the junction exists where the insert DNA joins the flanking DNA. The junction point is also present in transformed organisms, where the two DNA fragments are joined together in a manner that modifies what is found in the native organism. "adapter DNA" refers to DNA that contains an adapter.

The present invention provides transgenic cotton events, designated as center 17L397-1, and progeny thereof, wherein 17L397-1 in the transgenic cotton event is 17L397-1 in cotton, including plants and seeds of 17L397-1 in the transgenic cotton event and plant cells thereof or regenerable parts thereof, and plant parts of 17L397-1 in the transgenic cotton event including, but not limited to, cells, pollen, ovules, flowers, shoots, roots, stems, leaves and products from 17L397-1 in cotton, such as cotton seed, cotton seed oil, cotton clothing, cotton quilt, cotton batting, cotton cloth and biomass left in the cotton crop field.

The 17L397-1 transgenic cotton event of the invention comprises a DNA construct that, when expressed in plant cells, results in improved yield traits and/or tolerance to glyphosate herbicide from the 17L397-1 transgenic cotton event. The DNA construct comprises an expression cassette comprising a suitable promoter for expression in a plant operably linked to a csRRM2 gene having a cell increasing function, and a suitable polyadenylation signal sequence, the nucleic acid sequence of the csRRM2 protein being capable of increasing cotton yield. The DNA construct comprises a further expression cassette comprising a suitable promoter for expression in plants operably linked to the gene g10-EPSPS encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), the nucleic acid sequence of which EPSPS protein is tolerant to glyphosate herbicide, and a suitable polyadenylation signal sequence. Further, the promoter may be a suitable promoter isolated from plants, including constitutive, inducible, and/or tissue-specific promoters, including, but not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, the Figwort Mosaic Virus (FMV) 35S promoter, ubiquitin protein (Ubiquitin) promoter, actin (action) promoter, agrobacterium (Agrobacterium tumefaciens) nopaline synthase (NOS) promoter, octopine synthase (OCS) promoter, night yellow leaf curly virus (cestrom) promoter, tuber storage protein (Patatin) promoter, ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) promoter, glutathione Sulphur Transferase (GST) promoter, E9 promoter, GOS promoter, xca/alcR promoter, agrobacterium (Agrobacterium rhizogenes) RolD promoter, and arabidopsis (Arabidopsis thaliana) sub 2 promoter. The polyadenylation signal sequence may be a suitable polyadenylation signal sequence for functioning in plants, including, but not limited to, polyadenylation signal sequences derived from the Agrobacterium tumefaciens (Agrobacterium tumefaciens) nopaline synthase (NOS) gene, from the cauliflower mosaic virus (CaMV) 35S terminator, from the protease inhibitor II (PIN II) gene, and from the alpha-tubulin (alpha-tubulin) gene.

In addition, the expression cassette may also include other genetic elements including, but not limited to, enhancers and signal peptides/transit peptides. The enhancer may enhance the expression level of a gene, including, but not limited to, tobacco Etch Virus (TEV) translational activator, caMV35S enhancer, and FMV35S enhancer. The signal peptide/transit peptide may direct the transit of the EPSPS protein to a specific organelle or compartment outside or inside the cell, for example, targeting to the chloroplast using a sequence encoding a chloroplast transit peptide, or targeting to the endoplasmic reticulum using a 'KDEL' retention sequence.

17L397-1 in a transgenic cotton event having yield trait improvement characteristics and tolerance to glyphosate herbicide is cultivated by the steps of: first sexually crossing a first parent cotton plant consisting of 17L397-1 and its progeny in a transgenic cotton event obtained by transformation with the yield trait improvement of the invention and an expression cassette tolerant to glyphosate herbicide to a second parent cotton plant lacking the yield trait improvement characteristic or tolerant to glyphosate herbicide to produce a plurality of first generation progeny plants; progeny plants that are tolerant to the glyphosate herbicide are then selected and cotton plants that are tolerant to the glyphosate herbicide can be grown. These steps may further include backcrossing the yield trait improved and glyphosate tolerant progeny plant with a second parent cotton plant or a third parent cotton plant and then selecting the progeny by application with a glyphosate herbicide or by identification of a molecular marker associated with the trait (e.g., a DNA molecule comprising the junction site identified at the 5 'and 3' ends of the insertion sequence in 17L397-1 in a transgenic cotton event), thereby producing a cotton plant with yield trait improved characteristics and tolerance to a glyphosate herbicide.

It will also be appreciated that two different transgenic plants can also be crossed to produce offspring containing two independent, separately added exogenous genes. Selfing of appropriate offspring can result in offspring plants that are homozygous for both added exogenous genes. Backcrossing of parent plants and outcrossing with non-transgenic plants as previously described are also contemplated, as are asexual propagation.

As used herein, a substantially homologous sequence is a nucleic acid molecule that is capable of specifically hybridizing under highly stringent conditions to the complementary strand of a matching other nucleic acid molecule. Suitable stringent conditions for promoting DNA hybridization, for example, treatment with 6.0 XSSC/sodium citrate (SSC) at about 45℃followed by washing with 2.0 XSSC at 50℃are well known to those skilled in the art. For example, the salt concentration in the washing step may be selected from about 2.0 XSSC at low stringency conditions, about 0.2 XSSC at 50℃to high stringency conditions, about 50 ℃. In addition, the temperature conditions in the washing step may be raised from about 22 ℃ at room temperature under low stringency conditions to about 65 ℃ under high stringency conditions. The temperature conditions and salt concentration may both be varied, or one may remain unchanged while the other variable is varied. Preferably, a nucleic acid molecule of the invention can specifically hybridize under moderately stringent conditions, e.g., at about 2.0 XSSC and about 65℃to one or more of 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, or to a complement thereof, or to any fragment of the foregoing. More preferably, a nucleic acid molecule of the invention hybridizes specifically under highly stringent conditions to one or more of 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 or to the complement thereof, or to any fragment of the above sequences. In the present invention, preferred marker nucleic acid molecules have SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 7 or the complement thereof, or any fragment of the above sequences.

Another preferred marker nucleic acid molecule of the invention has 80% to 100% or 90% to 100% sequence identity with SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 7 or the complement thereof, or any fragment of the above sequences. SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 6 and SEQ ID NO. 7 can be used as markers in plant breeding methods to identify offspring of genetic crosses. Hybridization of the probe to the target DNA molecule may be detected by any method known to those skilled in the art, including, but not limited to, fluorescent labels, radiolabels, antibody-based labels, and chemiluminescent labels.

DNA detection kits based on DNA amplification methods contain DNA primer molecules that hybridize specifically to the target DNA under appropriate reaction conditions and amplify the diagnostic amplicon. The kit may provide agarose gel-based detection methods or a number of methods known in the art for detecting diagnostic amplicons. Kits comprising DNA primers homologous or reverse complementary to any portion of the cotton genomic region of SEQ ID NO. 3 or SEQ ID NO. 4 and homologous or reverse complementary to any portion of the transgene insertion region of SEQ ID NO. 5 are provided by the invention. In particular, primer pairs identified as useful in DNA amplification methods are SEQ ID NO. 8 and SEQ ID NO. 9, which amplify a diagnostic amplicon homologous to a portion of the 5' transgene/genomic region of 17L397-1 in a transgenic cotton event, wherein the amplicon comprises SEQ ID NO. 1. Primer pairs useful in the DNA amplification method are also identified as SEQ ID NO. 10 and SEQ ID NO. 11, which amplify a diagnostic amplicon homologous to a portion of the 3' transgene/genomic region of 17L397-1 in a transgenic cotton event, wherein the amplicon comprises SEQ ID NO. 2. Other DNA molecules used as DNA primers may be selected from SEQ ID NO. 5.

DNA detection kits may be developed using the compositions of the present invention and methods described in or known to the DNA detection arts. The kit is useful for identifying the presence or absence of 17L397-1 of the transgenic cotton event in a sample, and can also be used to cultivate cotton plants that contain 17L397-1 of the transgenic cotton event. The kit may contain DNA primers or probes homologous 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 homologous or complementary to DNA contained in the transgenic genetic element of DNA, which DNA sequences may be used in DNA amplification reactions or as probes in DNA hybridization methods. The DNA structure of the binding site of the transgene insert sequence contained in the cotton genome and illustrated in fig. 1 and table 1 to the cotton genome comprises: the 17L397-1 left flanking genomic region in cotton at the 5' end of the transgene insert, from the first expression cassette consisting of 2 tandem cauliflower mosaic virus 35S promoters (2 XP 35S), operably linked to a glyphosate resistance gene sequence (g 10-epsps), and operably linked to a cauliflower mosaic virus 35S terminator (T35S); the second expression cassette consisted of the 35S promoter of cauliflower mosaic virus (P35S), operably linked to the cell enlargement gene csRRM2 and operably linked to the nopaline synthase gene terminator (Tnos), a portion of the insert sequence from the right border Region (RB) of Agrobacterium, and the 17L397-1 right flanking genomic region (SEQ ID NO: 5) in cotton at the 3' end of the transgenic insert sequence. In the DNA amplification method, the DNA molecule used as a primer may be any part derived from the transgene insert sequence in 17L397-1 of the transgenic cotton event, or any part derived from the DNA region of the flanking cotton genome in 17L397-1 of the transgenic cotton event.

17L397-1 in a transgenic cotton event may be combined with other transgenic cotton varieties, such as herbicide (e.g., glyphosate, glufosinate, etc.) tolerant cotton, or transgenic cotton varieties carrying an insect-resistant gene. Various combinations of all of these different transgenic events, when bred with 17L397-1 of the transgenic cotton events of the present invention, can provide improved hybrid transgenic cotton varieties that are resistant to insects and multiple herbicides. These varieties may exhibit superior characteristics of insect resistance, resistance to various herbicides, and the like, compared to non-transgenic varieties and transgenic varieties of single traits.

The invention provides a nucleic acid sequence for detecting cotton plants and a detection method thereof, and 17L397-1 in transgenic cotton events has the effects of improving yield traits and tolerating glyphosate herbicide. Cotton plants of this trait express csRRM2 protein and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein, which confers plant yield trait improvement and tolerance to glyphosate. Meanwhile, in the detection method, SEQ ID NO. 1 or a reverse complement thereof, SEQ ID NO. 2 or a reverse complement thereof, SEQ ID NO. 3 or a reverse complement thereof, SEQ ID NO. 4 or a reverse complement thereof, SEQ ID NO. 6 or a reverse complement thereof, or SEQ ID NO. 7 or a reverse complement thereof can be used as a DNA primer or a probe to generate an amplification product which is diagnosed as 17L397-1 or a progeny thereof in a transgenic cotton event, and the presence of plant material derived from 17L397-1 in the transgenic cotton event can be rapidly, accurately and stably identified.

Although the 17L397-1 insert was incomplete and the quality trait was not significantly improved over the control, it was better in glyphosate tolerance and more prominent in yield traits. These characteristics allow the intermediate 17L397-1 transformant to be used to improve glyphosate herbicide tolerance and yield traits of cotton, thereby breeding new varieties of high-yield herbicide-tolerant cotton.

The technical scheme of the invention is further described in detail through the drawings and the embodiments.

Drawings

FIG. 1 is a schematic structural diagram of a binding site between a transgene insert sequence and a cotton genome.

FIG. 2 physical map of recombinant expression vector pCAMBIA1300/EPSPS-CSRRM 2. The English and abbreviations of the elements are listed below:

T-DNA left border sequence of LB Agrobacterium.

35S terminator of T35S cauliflower mosaic virus (CaMV).

G10-EPSPS codes EPSPS protein, and the toxicity of glyphosate is relieved.

Ch-L signal peptide

35S promoter of Cauliflower mosaic Virus (CaMV) in P2X 35S tandem.

The terminator of Tnos nopaline synthase gene.

CsRRM2 CsRRM gene CDS.

35S promoter of P35S cauliflower mosaic virus (CaMV).

T-DNA right border sequence of Agrobacterium RB.

Plasmid stabilizing site of PVS1 sta PVS1 plasmid.

Replication initiation sites of PVS1 rep pVS1 plasmid.

The bom site of the PBR322 bom pBR322 plasmid.

The replication initiation site of the PBR322 ori PBR322 plasmid.

KANAMYCIN (R) encodes an aminoglycoside phosphotransferase protein, conferring kanamycin resistance on bacteria.

The 17L397-1 event in FIG. 3 is expressed in terms of yield of cotton 24 in the recipient control. Middle cotton station 24: non-transgenic recipient control cotton plants; 17L397-1: 17L397-1 transformation event cotton plants. A is a blade; b is young bell; c is a plant; d is a bell.

The case of glyphosate herbicide resistance of cotton 24 plants in the 17L397-1 event versus the recipient control in FIG. 4. Middle cotton station 24: non-transgenic recipient control cotton plants; 17L397-1: 17L397-1 transformation event cotton plants.

17L397-1 transformation event specific PCR validation results in FIG. 5. M: marker, size label side (unit: bp); n: blank control; p: vector pCAMBIA1300/EPSPS-csRRM2; c: genomic DNA of cotton site 24 in the recipient control; t: genomic DNA from cotton material containing the 17L397-1 transformation event. A: the expected size of the left border PCR fragment is 295bp; b: the right border PCR fragment was expected to be 445bp in size.

FIG. 6Southern hybridization cleavage and probe position.

FIG. 7 shows Southern blot hybridization of copy number of the 17L397-1 target gene csRRM2

A: bamHI enzyme digests the DNA hybridization map; b: digestion of DNA hybridization patterns with EcoRI enzyme; c: the probe position and restriction enzyme cleavage site, the horizontal line marks the probe position, and the vertical line marks the cleavage site.

A1: DNA MARKER, labeling the band size aside, and the unit bp;

A2: bamHI enzyme cutting plasmid;

A3: bamHI cuts cotton house 24;

A4: 17L397-1 in BamHI cleavage;

b1: DNA MARKER, labeling the band size aside, and the unit bp;

b2: ecoRI cleaves the plasmid;

b3: ecoRI, cut cotton 24;

b4: ecoRI was digested at 17L397-1.

FIG. 8 shows Southern blot hybridization of 17L397-1 target gene g10-epsps insertion copy number

A: bamHI enzyme digests the DNA hybridization map; b: digestion of DNA hybridization patterns with EcoRI enzyme; c: g10-epsps probe position and restriction enzyme cleavage site, wherein the horizontal line indicates probe position and the vertical line indicates cleavage site.

A1: bamHI enzyme cutting plasmid;

a2: blank control;

A3: bamHI cuts cotton house 24;

a4: blank control;

A5: 17L397-1 in BamHI cleavage;

A6: DNA MARKER, labeling the band size aside, and the unit bp;

b1: DNA MARKER, labeling the band size aside, and the unit bp;

b2: ecoRI cleaves the plasmid;

b3: ecoRI, cut cotton 24;

B4: blank control;

b5: ecoRI was digested at 17L397-1.

FIG. 9 shows hybridization patterns of 17L397-1 vector backbone ① and ④ insert copy number Southern blots

A: bamHI enzyme digests the DNA hybridization map; b: digestion of DNA hybridization patterns with EcoRI enzyme; c: ① The probe positions of the regions and ④ and the restriction enzyme cleavage sites.

A1: DNA MARKER, labeling the band size aside, and the unit bp;

A2: bamHI enzyme cutting plasmid;

a3: blank control;

A4: bamHI cuts cotton house 24;

a5, A6: 17L397-1 in BamHI cleavage;

A7, A8: r1 (PCR amplification product of primer in hybridization region of probe ①);

A9, a10: r4 (PCR amplification product of primer in hybridization region of probe ④);

b1: DNA MARKER, labeling the band size aside, and the unit bp;

b2: ecoRI digested plasmid;

B3: blank control;

B4: ecoRI, cut cotton 24;

B5, B6: 17L397-1 of EcoRI digested transformant;

b7: r1 (PCR amplification product of primer in hybridization region of probe ①);

b8: r4 (PCR amplification product of primer in hybridization region of probe ④).

FIG. 10 hybridization of the vector backbone ② and ③ regions by insertion copy number Southern blotting

A: bamHI enzyme digests the DNA hybridization map; b: digestion of DNA hybridization patterns with EcoRI enzyme; c: the probe positions of the 2 region and the 3 region of the skeleton and the restriction enzyme cleavage sites.

A1: DNA MARKER, labeling the band size aside, and the unit bp;

a2: bamHI digested plasmid;

a3: blank control;

A4: bamHI cuts cotton house 24;

a5, A6: 17L397-1 in BamHI cleavage;

a7, A8: r2 (PCR amplification product of primer in hybridization region of probe ②);

A9, a10: r3 (PCR amplification product of primer in hybridization region of probe ③);

b1: DNA MARKER, labeling the band size aside, and the unit bp;

b2: ecoRI digested plasmid;

B3: blank control;

B4: ecoRI, cut cotton 24;

b5, B6: 17L397-1 in EcoRI cleavage;

b7: r2 (PCR amplification product of primer in hybridization region of probe ②);

B8: r3 (PCR amplification product of primer in hybridization region of probe ③).

Detailed Description

17L397-1 in the transformation event related to the application refers to cotton plants obtained by genetic transformation with cotton 24 in cotton inbred lines as a receptor and inserting foreign gene inserts (T-DNA inserts) between specific genomic sequences. In a specific example, the expression vector used for the transgene has the physical map shown in FIG. 2, and the resulting T-DNA insert has the sequence shown in nucleotides 178-4096 of SEQ ID NO. 5. 17L397-1 in a transformation event may refer to this transgenic process, may refer to T-DNA inserts within the genome resulting from this process, or a combination of T-DNA inserts and flanking sequences, or may refer to cotton plants resulting from this transgenic process. In specific examples, the event is also applicable to plants obtained by transforming other recipient varieties with the same expression vector, thereby inserting a T-DNA insert into the same genomic location. 17L397-1 in a transformation event may also refer to progeny plants resulting from asexual, sexual, double or double propagation of the above plants, or combinations thereof.

Example 1 acquisition and characterization of transformation events

The csRRM2 gene is an RRM2 domain for controlling the FCA gene and is closely related to plant quality traits; the g10-epsps gene codes 5-enolpyruvylshikimate-3-phosphate synthetase, which can improve the tolerance of plants to glyphosate herbicide. The transformation event ICR24-397 (application number: 201811442263.1) obtained before has better quality characteristics than the receptor after being transferred into the two expression cassettes, and the fiber length, specific strength, uniformity and the like of the ICR24-397 cotton are remarkably improved. However, in identifying herbicide tolerance, it was found that approximately 10% of plants in ICR24-397 were phytotoxic when glyphosate was applied in an amount 2 times the field recommended concentration; when glyphosate in an amount 4 times the recommended concentration in the field was applied, approximately 20% of plants in ICR24-397 appeared phytotoxicity. Thus, the herbicide resistance performance of ICR24-397 is not very desirable.

In order to obtain a transformation event with more excellent herbicide tolerance, the invention uses a pCAMBIA1300/EPSPS-csRRM2 expression vector (the physical map of the vector is shown in figure 2, and comprises a csRRM2 gene expression cassette and a g10-EPSPS gene expression cassette) to transform cotton plants 24 in a receptor material by an agrobacterium-mediated method to obtain 13 positive transformation seedlings, and screening and identifying herbicide resistance and quality-related agronomic traits of the transformation seedlings.

1. Screening for transformants having excellent herbicide resistance

The transformants with better herbicide tolerance were screened from the 13 positive transformants by spraying glyphosate in the field at 2 times the recommended concentration in the field using cotton house 24 and transformant ICR24-397 in the recipients as references. The results indicate that only 3 transformation events (middle 17L397-1, middle 17L397-2, and middle 17L 397-3) were significantly more tolerant to glyphosate herbicide than the control and not lower than transformant ICR24-397.

Subsequently, seeds of cotton 24 in the 4 cotton transformants and the acceptor control were sown in the test base of cotton in the city of Anyang, henan province, and herbicide tolerance of transformants such as 17L397-1 was systematically identified by spraying glyphosate at different concentrations in the field. Meanwhile, the effectiveness of improvement of the quality traits of the transgenic cotton is identified by examining the agronomic traits such as the length, the uniformity, the specific strength and the like of the cotton fibers, and the insertion positions of three transformation events of 17L397-1, 17L397-2 and 17L397-3 are identified and analyzed.

2. Herbicide tolerance identification

Herbicide tolerance identification was performed in summer 2018. Glyphosate was sprayed at 1-fold (82 g/mu), 2-fold (164 g/mu) and 4-fold (328 g/mu) of the field recommended concentration at 4-6 true leaves of cotton in the seedling stage, and after one week, all the recipient control cotton plants died from being withered and yellow, the seedling rate was 0, the phytotoxicity rate was 100%, and 4 transformation events were able to normally seedling, but showed different degrees of phytotoxicity rate (table 2). When 2 times the amount of glyphosate in the recommended field concentration is sprayed, the glyphosate tolerance capacity of the middle 17L397-1 and the middle 17L397-2 is obviously higher than that of the middle 17L397-3 and the ICR24-397; when glyphosate in 4 times the field recommended concentration was sprayed, the glyphosate tolerance of the middle 17L397-1 was significantly higher than the remaining 3 transformant materials. Thus, the middle 17L397-1 glyphosate herbicide resistance performed best.

TABLE 2 herbicide resistance manifestation

Values are from the mean ± standard deviation of 3 biological replicates. Statistical analysis multiple comparisons (α=0.05) were made using LSD, with different letters indicating the significance of the same column data difference at the same herbicide concentration.

3. Insertion positions of 17L397-1, 17L397-2 and 17L397-3 on the genome were analyzed

100Mg plant leaves are taken, and after liquid nitrogen is rapidly ground, total DNA is extracted by adopting a CTAB method. And (3) fragmenting the detected qualified sample genome DNA by ultrasonic waves, purifying the fragmented DNA, repairing the tail end, adding A at the 3' end, and connecting a sequencing joint. And then agarose gel electrophoresis is used for fragment size selection, and PCR amplification is carried out to form a sequencing library. The qualified library was sequenced to a depth of 10× using an Illumina nova sequencing platform. And (3) carrying out quality evaluation on the Raw data obtained by sequencing to obtain a filtering sequence CLEAN DATA, independently writing a Perl script to convert the fq file of CLEAN DATA into a fa format, and then comparing the filtering sequence with a cotton Gh ZM24-CRI v1 reference genome sequence (https:// www.cottongen.org/blast/nucleic acid). The position of the filter sequence on the reference genome is located, and the information of the insertion position of the exogenous insert is obtained.

Comparing the sequencing data of the middle 17L397-1, the middle 17L397-2 and the middle 17L397-3 with the reference genome and the exogenous T-DNA sequence respectively, dividing the sequencing data into two types according to comparison results, wherein the type 1 is one end sequence (Reads) for comparing the upper reference genome sequence, and the other end sequence (Reads) for comparing the upper insertion sequence; class 2 is where one part of either end is compared to the upper reference genomic sequence and the other part is compared to the upper insert sequence. The entire Reads of the exogenous insert sequences were aligned using Blast alignment with the reference genome. And respectively comparing the exogenous insert sequence with a reference genome by using Blast according to the assembled Contig, and selecting a region of the chromosome to which the Contig sequence is compared to obtain the information of the insertion position of the exogenous insert fragment. Subsequently, forward and reverse primers were designed on the genomic flanking sequence and the foreign insert at the left and right boundaries of the insert, the insert sites for 3 transformation events were verified by PCR amplification, and the PCR products were sequenced.

The results showed that the exogenous fragments of 3 transformants were integrated into different loci of the genome, which are cotton genome gene spacers, without known functional genes. Furthermore, the expression cassettes for the foreign sequences of middle 17L397-2 and middle 17L397-3 were complete, whereas the vector border sequence of the middle 17L397-1 transformation event was deleted during insertion, including part of the border sequence and part of the terminator T35S sequence (see Table 3 for details). In general, incomplete terminators can lead to abnormal transcription and translation of genes, which in turn affect the trait of interest. Unexpectedly, however, from the identification of herbicide resistance traits, the glyphosate tolerance of the middle 17L397-1 transformation event was significantly higher than the receptor control and more prominent than the ICR24-397, middle 17L397-2 and middle 17L397-3 transformants.

TABLE 3 exogenous fragment insertion position information

4. Quality character identification

The quality traits such as length, uniformity and specific strength of cotton fibers of the medium 17L397-1 transformant and the receptor control were examined during the period from cotton opening to final harvest. The results show that the cotton fibers of middle 17L397-1, middle 17L397-2 and middle 17L397-3 have comparable quality traits such as length, uniformity and specific strength, do not differ significantly from the cotton of the recipient control 24, and are weaker than ICR 24-397.

5. Yield trait identification

The quality traits of the transformants are identified, and other traits (such as seed cotton weight, flower bud number, number of rings, single ring weight, plant height, first fruit branch position height, fruit node number, fruit branch number, leaf size and the like) of the transformants are recorded in detail. Unexpected findings in data statistics that the total weight and single bell weight of seed cotton of 17L397-1 in the middle were significantly higher than that of the recipient control, but also significantly higher than that of the other 3 transformants including ICR24-397 (Table 4), and that of 17L397-1 in the middle, first fruit branch height in seedling, bud, flower bell and boll period were also significantly higher than that of the recipient control (Table 5)

TABLE 4 seed cotton yield during the opening period

Values are from the mean ± standard deviation of 3 biological replicates. Statistical analysis multiple comparisons (α=0.05) were made using LSD, with different letters representing the significance of the same column data difference over the same period.

TABLE 5 major agronomic traits

Values are from the mean ± standard deviation of 3 biological replicates. Lower case letters between the different materials represent significant differences at the α=0.05 level.

The results show that although the 17L397-1 insert sequence is incomplete and the quality traits are not significantly improved compared with the control, the glyphosate tolerance capacity is better and the yield traits are more prominent. These characteristics allow the intermediate 17L397-1 transformant to be used to improve glyphosate herbicide tolerance and yield traits of cotton, thereby breeding new varieties of high-yield herbicide-tolerant cotton.

Example 2 characterization of 17L397-1 molecules in transformation event

To further define the identity of 17L397-1 in the transformation event, the present invention analyzed the flanking sequences and the insertion copy number of the 17L397-1 exogenous sequence at the insertion site on the cotton genome.

1. Insertion site flanking sequence analysis of exogenous sequences on cotton genome

As described in example 1, the exogenous T-DNA sequence of 17L397-1 was inserted forward into the cotton genome at positions chrA 11:7085972 or chrD 11:6406857 in the transformation event. On the left boundary of the insertion site, 500bp on the upstream 500bp and T-DNA sequence of the insertion site on the genome is intercepted, and on the right boundary, 500bp on the downstream 500bp and T-DNA sequence of the insertion site on the genome is taken, the intercepted sequence is subjected to primer design by utilizing Primerblast software (https:// blast.ncbi.lm.nih.gov/Blast) of NCBI website, and an amplification product fuses a part of cotton genome sequence and a part of T-DNA sequence.

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

PCR amplification was performed on the 17L397-1 transformation event with the genomic 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 genomic downstream primer (SEQ ID NO: 11) based on the results of flanking sequences and the insertion position to verify the foreign fragment insertion position. The results are shown in FIG. 5. The result proves that 17L397-1 exogenous fragment is stably inserted into the cotton genome at the position of chrA 11:7085972 or chrD 11:6406857, and the size of the inserted sequence is 3919bp.

By analyzing the border sequences on the left and right sides, the insertion of the exogenous sequence causes the deletion of 89bp of the acceptor genome, and the vector sequence is also deleted by 131bp. The deleted vector sequence includes a partial border sequence and a partial sequence of terminator T35S.

2. Insertion copy number analysis of exogenous sequences

The copy number of the exogenous sequence was determined by Southern blot hybridization. In the Southern hybridization detection, two restriction enzymes which are arranged on a T-DNA region and are not arranged in a hybridization region are selected to digest genome DNA, each insertion copy in the genome is hybridized to form a single and specific band, and after the genome DNA is subjected to restriction enzyme digestion, a region to be detected is selected as a probe to carry out Southern imprinting hybridization experiment.

Southern hybridization was performed by selecting BamHI and EcoRI enzymes to digest the positive control plasmid, cotton site 24 in the acceptor control, and 17L397-1 genomic DNA in the acceptor control, and designing probes covering the target gene and the whole vector backbone sequence, and schematic diagrams of the probes and the cleavage sites are shown in FIG. 6. The specific sequences of the probe primers are shown in Table 6.

TABLE 6 Southern hybridization assay probe amplification primer positions and sequences

1: The unit bp.

The inserted copy number hybridization detection of the target gene csRRM2 selects two restriction endonucleases BamHI and EcoRI, and respectively cleaves positive control plasmid, 24 genomic DNA of cotton in negative control and 17L397-1 genomic DNA in negative control. After running gel and transferring membrane, the membrane is marked by csRRM2 gene probe, and the hybridization result is shown in figure 7A, B. The probe position of the foreign gene csRRM2 and the cleavage sites of the restriction enzymes BamHI and EcoRI are shown in FIG. 7C. From the hybridization results, csRRM2 gene was inserted into cotton genome in single copy.

The inserted copy number hybridization detection of the target gene g10-epsps selects two restriction enzymes BamHI and EcoRI, and respectively enzyme-cuts positive control plasmid, 24 genome DNA of cotton in negative control and 17L397-1 transformant genome DNA. After running gel and transferring membrane, the membrane is marked by g10-epsps gene probe, and the hybridization result is shown in figure 8A, B. The probe positions of the target gene g10-epsps and the restriction enzyme sites of BamHI and EcoRI are shown in FIG. 8C. The g10-epsps gene was also inserted into the cotton genome in a single copy.

Similarly, the result of Southern hybridization of the vector backbone region showed that the sequence of the vector backbone region was not inserted into the cotton genome (see FIGS. 9 and 10).

Example 3 detection method of 17L397-1 in transformation event

The transgenic cotton event 17L397-1 can be used for breeding and producing agricultural products or commodity products from the new variety. If a sufficient amount is detected in the agricultural product or commodity, the agricultural product or commodity is expected to contain a nucleotide sequence capable of diagnosing the presence of 17L397-1 material in the agricultural product or commodity in a transgenic cotton event. Such agricultural products or commodities include, but are not limited to, cotton seed oil, cotton batting, cotton quilts, cotton cloths, cotton coats, any other food product to be consumed by animals as a food source, or otherwise used for cosmetic purposes, etc., as an ingredient in an expanding agent or cosmetic composition. Nucleic acid detection methods and/or kits based on probe or primer pairs can be developed to detect a 17L397-1 nucleotide sequence in a transgenic cotton event 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 group consisting of 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 17L397-1 in the transgenic cotton event.

One of the detection methods is as follows: the specific boundary sequences in 17L397-1 plants in the two plants are detected by a PCR method, the used PCR primer pairs are SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 11 respectively, and a PCR reaction system is adopted:

the reaction procedure is:

94 ℃ for 5min; (94 ℃,30sec;55 ℃,30sec;72 ℃,30 sec). Times.35 cycles; 72 ℃ for 5min;4 ℃ for 5min.

The PCR products were taken and electrophoretically detected in 1% (w/v) 1 XTAE agarose gel, and the results are shown in FIG. 5. The expected target bands (SEQ ID NO:6 and SEQ ID NO:7, respectively) can be amplified during the 17L397-1 transformation event. Moreover, the PCR method can track the existence of transformation events, so that the PCR method is applied to breeding work.

In conclusion, 17L397-1 in the transgenic cotton event can improve the yield of bolls and has higher tolerance to glyphosate herbicide, and the detection method can accurately and rapidly identify whether the biological sample contains the DNA molecule of 17L397-1 in the transgenic cotton event.

Finally, it should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.

Sequence listing

<110> Cotton institute of national academy of agricultural sciences

<120> A nucleic acid sequence for detecting 17L397-1 in cotton and a detecting method thereof

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ttcttgttgg tttaggaatt ag 22

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<213> Synthesis (unknown)

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<212> DNA

<213> Synthesis (unknown)

<400> 3

tttttattaa tattaataaa tatttacgga aaataaccaa tcaagttaaa aaaaatgtat 60

aaatcgacct taaaggataa tattggaagc taaattccta acgactacta gggccataag 120

ttgacggtgc tcatgcattt ataccggtct gtgggaccca aacaccttct tgttggttta 180

ggaattagaa attttattga tagaagtatt ttacaaatac aaatacatac taagggtttc 240

ttatatgctc aacacatgag cgaaacccta taggaaccct aattccctta tctgggaact 300

actcacacat tattatggag aaactcgagc ttgtcgatcg acagatccgg tcggcatcta 360

ctttaggcgg tagcctcagc gtattcgaat ctagcaccaa gagcttcaag gtgagcgaag 420

aactgagggt aggactttct gatgtggtgt gcaccggtga ttctaagtgg agcatctgct 480

ctgagaccaa gaagggtgag aagcatgatc attctgtggt caccgtgacc atcagcggtg 540

ataccaccag caaggtgagc agaaccagta acggagagag aatcgg 586

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<213> Synthesis (unknown)

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cctttccttt atcgcaatga tggcatttgt aggagccacc ttccttttcc actatcttca 60

caataaagtg acagatagct gggcaatgga atccgaggag gtttccggat attacccttt 120

gttgaaaagt ctcaattgcc ctttggtctt ctgagactgt atctttgata tttttggagt 180

agacaagtgt gtcgtgctcc accatgttga cgaagatttt cttcttgtca ttgagtcgta 240

agagactctg tatgaactgt tcgccagtct ttacggcgag ttctgttagg tcctctattt 300

gaatctttga ctccatgaag ctaaactgaa ggcgggaaac gacaatctga tccaagctca 360

agctgctcta gcattcgcca ttcaggctat tagactctgt attacgtgta tattattgat 420

ttaatcccta tactttaatt tgattttttt ttcatccttg tatttttcaa aattcaaaat 480

ttcaatcctg ttaaatttat taaattctat tgcttccaaa aaaatcttat gctactaaca 540

tatcaacaca tttataatac tacatcaatt tgtcattttc aaatattact aaaaaaatta 600

gttaataaaa tcatagctat aatttgtgtc gagattagaa atttaagatt tgaaaaatat 660

atatactaag aattgtcaaa atagagaata tggacttaag atacaacttc acgcacaata 720

ttaattgcaa aatttagcca accaaaatta actactattg tttgcggtca aggttgaaat 780

tttaaaattt gaaaattaca agtattaaaa ttgaccaaaa taaagtacaa aggcta 836

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tttttattaa tattaataaa tatttacgga aaataaccaa tcaagttaaa aaaaatgtat 60

aaatcgacct taaaggataa tattggaagc taaattccta acgactacta gggccataag 120

ttgacggtgc tcatgcattt ataccggtct gtgggaccca aacaccttct tgttggttta 180

ggaattagaa attttattga tagaagtatt ttacaaatac aaatacatac taagggtttc 240

ttatatgctc aacacatgag cgaaacccta taggaaccct aattccctta tctgggaact 300

actcacacat tattatggag aaactcgagc ttgtcgatcg acagatccgg tcggcatcta 360

ctttaggcgg tagcctcagc gtattcgaat ctagcaccaa gagcttcaag gtgagcgaag 420

aactgagggt aggactttct gatgtggtgt gcaccggtga ttctaagtgg agcatctgct 480

ctgagaccaa gaagggtgag aagcatgatc attctgtggt caccgtgacc atcagcggtg 540

ataccaccag caaggtgagc agaaccagta acggagagag aatcggcggt ctctcttgct 600

ctaagaccaa gtctttcaag ctcagctctg gtgtcagaga ttctatcgca ttccttgagt 660

ctaagagtag caacgttttc ccaggtggta tcaccctcag cgaaggcagc agcagcggta 720

agagcttgca cggcgtcggt gaaggaatca ccatctctag taacagcgtg gagaggtcta 780

ccacctctca cggtaagggt atcaccttct ctaacgatat cagcacccat ctctctaaga 840

acgttcacag cttccttctc accctggagg tcgtgttctc taaggttaga aagtctaacc 900

tcacctggga gaagagcagc ggcggtaagg atagcagcgg aaccagggta atcaccagga 960

acgagcactc tacctggtct gtacttctga ccaccaggga tggagattct tctaaggtca 1020

tcggaggcag tagctctaac accgaaatca gagagggtgt caagtgtctg tctaagagga 1080

gcgtgggact tgatatcacc ggtgagtcta agttcgagtc cgtcaggaag aagaggaccg 1140

aggaacataa gggcggaagc gtactgggag gatctttcgg cggaaacctc cactgtacca 1200

cctctaactg gaccggaaac ggagataggg agtctaccat cgttggagga cacccaagca 1260

ccaagtcttt cgagggcttc aagaaggtca ccctgaggtc tcttaccaag ggaatcaggg 1320

taatcggtaa cgaaagttgt accagaggtg agagcagcaa cacccataag gaatctggcc 1380

actgcaccag cgttacctgg gttaagggta acaccagcct gtggtctagc accgaaacct 1440

ctgatcacgg cgtcatcacc aacaagctca acaccagcac cccaatctct gaggcatctg 1500

agcatagctt cggcatcctc agaggtagcc acaccaacaa ctctggtttc accctcagcg 1560

agagcagcgg cgaggaggta tctagtggtg tagttcttgg atggctgtgc tctaagttca 1620

cctctgagtt ctctagctgg atgcacgata acgtcgaagg tagctggaag agcgtcggat 1680

cccttctccg ccgtggaaac agaagacatg accttaagag gacgaagctc agagccaatt 1740

aaagtcatcc cactcttctt caatccccac gatgaagaaa ttggataagc tcgtggatgc 1800

tgctgagtct tcagagaaac cgataaggga gatttccttt gactggattt agagagattg 1860

gagataagag atgggttctg cacaccattg cagattctgc taacttgagc catatctcat 1920

tgccccccgg gatctgcgaa agctcgagag agatagattt gtagagagag actggtgatt 1980

tcagcgtgtc ctctccaaat gaaatgaact tccttatata gaggaaggtc ttgcgaagga 2040

tagtgggatt gtgcgtcatc ccttacgtca gtggagatat cacatcaatc cacttgcttt 2100

gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc tcgtgggtgg gggtccatct 2160

ttgggaccac tgtcggcaga ggcatcttga acgatagcct ttcctttatc gcaatgatgg 2220

catttgtagg tgccaccttc cttttctact gtccttttga tgaagtgaca gatagctggg 2280

caatggaatc cgaggaggtt tcccgatatt accctttgtt gaaaagtctc aatagccctt 2340

tggtcttctg agactgtatc tttgatattc ttggagtaga cgagagtgtc gtgctccacc 2400

atgttatcac atcaatccac ttgctttgaa gacgtggttg gaacgtcttc tttttccacg 2460

atgctcctcg tgggtggggg tccatctttg ggaccactgt cggcagaggc atcttgaacg 2520

atagcctttc ctttatcgca atgatggcat ttgtaggtgc caccttcctt ttctactgtc 2580

cttttgatga agtgacagat agctgggcaa tggaatccga ggaggtttcc cgatattacc 2640

ctttgttgaa aagtctcaat agccctttgg tcttctgaga ctgtatcttt gatattcttg 2700

gagtagacga gagtgtcgtg ctccaccatg ttggcaagct gctctagcca atacgcaaac 2760

cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 2820

ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc 2880

aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat 2940

ttcacacagg aaacagctat gaccatgatt acgaattctg atagtttaat tcccgatcta 3000

gtaacataga tgacaccgcg cgcgataatt tatcctagtt tgcgcgctat attttgtttt 3060

ctatcgcgta ttaaatgtat aattgcggga ctctaatcat aaaaacccat ctcataaata 3120

acgtcatgca ttacatgtta attattacat gcttaacgta attcaacaga aattatatga 3180

taatcatcgc aagaccggca acaggattca atcttaagaa actttattgc caaatgtttg 3240

aacgatcggg gaaattcgag ctggtcaccc tctcatggta taagttccat tgagaccatc 3300

gatagctgcc atggccgtct ctttgcttga atatttaaca aacccgcatc cacgactctg 3360

tctatattca tcacgcatga gatagacatc ctccacgcga ccaaattgca aaaagagctc 3420

ctcaacctcg ttttcagtgg cttgcttgtt taaggaacca acaaaaagca gatctaccat 3480

ggtcaagagt cccccgtgtt ctctccaaat gaaatgaact tccttatata gaggaagggt 3540

cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc agtggagata tcacatcaat 3600

ccacttgctt tgaagacgtg gttggaacgt cttctttttc cacgatgctc ctcgtgggtg 3660

ggggtccatc tttgggacca ctgtcggcag aggcatcttc aacgatggcc tttcctttat 3720

cgcaatgatg gcatttgtag gagccacctt ccttttccac tatcttcaca ataaagtgac 3780

agatagctgg gcaatggaat ccgaggaggt ttccggatat taccctttgt tgaaaagtct 3840

caattgccct ttggtcttct gagactgtat ctttgatatt tttggagtag acaagtgtgt 3900

cgtgctccac catgttgacg aagattttct tcttgtcatt gagtcgtaag agactctgta 3960

tgaactgttc gccagtcttt acggcgagtt ctgttaggtc ctctatttga atctttgact 4020

ccatgaagct aaactgaagg cgggaaacga caatctgatc caagctcaag ctgctctagc 4080

attcgccatt caggctatta gactctgtat tacgtgtata ttattgattt aatccctata 4140

ctttaatttg attttttttt catccttgta tttttcaaaa ttcaaaattt caatcctgtt 4200

aaatttatta aattctattg cttccaaaaa aatcttatgc tactaacata tcaacacatt 4260

tataatacta catcaatttg tcattttcaa atattactaa aaaaattagt taataaaatc 4320

atagctataa tttgtgtcga gattagaaat ttaagatttg aaaaatatat atactaagaa 4380

ttgtcaaaat agagaatatg gacttaagat acaacttcac gcacaatatt aattgcaaaa 4440

tttagccaac caaaattaac tactattgtt tgcggtcaag gttgaaattt taaaatttga 4500

aaattacaag tattaaaatt gaccaaaata aagtacaaag gcta 4544

<210> 6

<211> 295

<212> DNA

<213> Synthesis (unknown)

<400> 6

ggacccaaac accttcttgt tggtttagga attagaaatt ttattgatag aagtatttta 60

caaatacaaa tacatactaa gggtttctta tatgctcaac acatgagcga aaccctatag 120

gaaccctaat tcccttatct gggaactact cacacattat tatggagaaa ctcgagcttg 180

tcgatcgaca gatccggtcg gcatctactt taggcggtag cctcagcgta ttcgaatcta 240

gcaccaagag cttcaaggtg agcgaagaac tgagggtagg actttctgat gtggt 295

<210> 7

<211> 445

<212> DNA

<213> Synthesis (unknown)

<400> 7

gcgagttctg ttaggtcctc tatttgaatc tttgactcca tgaagctaaa ctgaaggcgg 60

gaaacgacaa tctgatccaa gctcaagctg ctctagcatt cgccattcag gctattagac 120

tctgtattac gtgtatatta ttgatttaat ccctatactt taatttgatt tttttttcat 180

ccttgtattt ttcaaaattc aaaatttcaa tcctgttaaa tttattaaat tctattgctt 240

ccaaaaaaat cttatgctac taacatatca acacatttat aatactacat caatttgtca 300

ttttcaaata ttactaaaaa aattagttaa taaaatcata gctataattt gtgtcgagat 360

tagaaattta agatttgaaa aatatatata ctaagaattg tcaaaataga gaatatggac 420

ttaagataca acttcacgca caata 445

<210> 8

<211> 24

<212> DNA

<213> Gossypium hirsutum L.

<400> 8

ggacccaaac accttcttgt tggt 24

<210> 9

<211> 23

<212> DNA

<213> Synthesis (unknown)

<400> 9

accacatcag aaagtcctac cct 23

<210> 10

<211> 18

<212> DNA

<213> Synthesis (unknown)

<400> 10

gcgagttctg ttaggtcc 18

<210> 11

<211> 18

<212> DNA

<213> Gossypium hirsutum L.

<400> 11

tattgtgcgt gaagttgt 18

Claims (10)

1. A nucleic acid molecule comprising any one of the following:

i) Comprising the sequence shown in SEQ ID NO. 3 and/or SEQ ID NO.4, or the reverse complement thereof;

ii) comprises the sequence shown in SEQ ID No. 6 and/or SEQ ID No. 7, or the reverse complement thereof;

iii) Comprising the sequence shown in SEQ ID No. 5, or the reverse complement thereof.

2. A probe for detecting a cotton transformation event comprising the sequence set forth 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 or reverse complement thereof.

3. A primer pair for detecting a cotton transformation event, wherein the amplification product of the primer pair comprises the sequence of claim 2.

4. The primer pair of claim 3, wherein the primer pair is a sequence shown in SEQ ID NO. 8 and SEQ ID NO. 9; or SEQ ID NO. 10 and SEQ ID NO. 11.

5. Kit or microarray for detecting cotton transformation events, characterized in that it comprises a probe according to claim 2 and/or a primer pair according to any one of claims 3 to 4.

6. A method for detecting a cotton transformation event comprising detecting the presence or absence of said transformation event in a test sample using any one of the following:

i) The probe of claim 2;

ii) the primer pair of any one of claims 3-4;

iii) The probe of claim 2 and the primer pair of any one of claims 3-4;

iv) the kit or microarray of claim 5.

7. A method of breeding cotton, the method comprising the steps of:

1) Obtaining cotton comprising the nucleic acid molecule of claim 1;

2) Obtaining cotton plants, seeds, plant cells, progeny plants or plant parts from the cotton obtained in step 1) by pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or hybridization or a combination thereof; optionally, the composition may be in the form of a gel,

3) Assessing the yield traits and/or identifying herbicide resistance of the progeny plant obtained in step 2) and detecting the presence or absence of said transformation event using the method of claim 5.

8. A product made from a cotton plant, seed, plant cell, progeny plant or plant part obtained by the method of claim 6, comprising a food, feed or industrial feedstock.

9. A method of protecting a cotton plant from herbicide-induced injury comprising applying to a field where at least one transgenic cotton plant is grown, an effective dose of a glyphosate herbicide, said transgenic cotton plant comprising in its genome in sequence SEQ ID NO 3, the nucleic acid sequences at positions 178-3842 of SEQ ID NO 5, and SEQ ID NO 4, or said transgenic cotton plant comprising in its genome SEQ ID NO 5; the transgenic cotton plants have tolerance to glyphosate herbicide.

10. A method for improving yield traits in cotton plants, comprising growing at least one transgenic cotton plant comprising in its genome in sequence SEQ ID No.3, SEQ ID No. 5, nucleic acid sequence from position 178 to 3842, and SEQ ID No. 4, or comprising in its genome SEQ ID No. 5; the transgenic cotton plants have significantly and stably improved yield traits.