CN114525277A - 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 the 17L397-1 in the cotton comprises a sequence shown in SEQ ID NO. 1 or a reverse complementary sequence thereof, or a sequence shown in SEQ ID NO. 2 or a reverse complementary sequence thereof. The 17L397-1 in the cotton has the characteristics of improved yield traits and glyphosate herbicide resistance, and the detection method can accurately and quickly identify whether a 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 events with improved yield traits and tolerance to glyphosate herbicide application, and for detecting whether a biological sample contains the 17L397-1 in a specific transgenic cotton event, 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 acres, and the yield value exceeds 500 hundred million dollars. The five countries of China, India, America and the like account for more than 70 percent of the total production. In advanced cotton-planting countries such as the United states and Australia, the cotton industry has strong international competitiveness due to the realization of whole-process mechanization, and the development direction of the cotton industry is led.

China is a big cotton production country, about 1 hundred million cotton farmers exist in China, the annual cotton planting area is about 6000-8000 ten thousand mu, the annual raw cotton yield is 450-560 ten thousand tons, and the annual raw cotton yield accounts for about 25% of the total cotton yield in the world. Meanwhile, China is the largest world import country, the import of the cotton in China is expected to continue to increase dramatically in the coming years, and the import of the cotton in China is increased to about 700 ten thousand metric tons every year in recent years according to official media reports of China. The reason is that the textile and apparel industry in china is developing vigorously. 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 accord with the receptor dynamics principle. Binding of ABA directly controls FCA-mediated processing of mRNA precursors, FCA being an ABA receptor involved in RNA metabolism. The research finds that: the RRM2 structural domain for controlling the FCA gene and the herbicide-tolerant gene g10-epsps are transferred into a receptor, and the obtained transformants all show obvious changes in the aspects of quality trait improvement, herbicide resistance and the like, so that the method can be widely applied to the improvement of the economic traits of dicotyledonous and monocotyledonous crops.

The weeds in the field compete with crops for water, fertilizer, light and growth space, and directly affect the yield and quality of the crops. Meanwhile, a plurality of weeds are also intermediate hosts of crop pathogenic bacteria and pests, and are one of important biological limiting factors for increasing the yield of crops. According to food and agricultural organization statistics of the united nations, the global food production loss caused by weeds is up to 950 hundred million dollars each year, which means that 3.8 million tons of wheat are lost, and the yield is about more than half of the global wheat yield in 2009. In an economic loss of $ 950 million, poverty developing countries bear approximately $ 700 million (FAO. the lurking company of waters [ J/OL ] (http:// www.fao.org/news/store/en/item/29402/icode /), 2009-08-11.). Therefore, effective control of weeds in the field is one of the important measures for promoting the yield increase of grains. With the increase of the migration speed of rural population in China to cities, the large-scale and mechanization of corn planting is a foreseeable trend, so that the traditional artificial 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 next-stubble crops. The biocidal herbicide such as glufosinate-ammonium has the characteristics of high efficiency, low toxicity, easy degradation, no residue and the like. However, they are not selective in weed control and cannot be used directly in the growing period of crops. The cotton which is resistant to the biocidal herbicide can be cultivated by a transgenic technology to overcome the problem. The problem of weeds can be effectively solved by spraying the herbicide for 1 to 2 times in the growth period of cotton, and the dosage and the input cost of the herbicide are reduced. Therefore, the herbicide-tolerant transgenic cotton has very wide application value and market potential.

It is known that expression of foreign genes in plants is influenced by their chromosomal location, possibly due to chromatin structure (e.g., heterochromatin) or the proximity of transcriptional regulatory elements (e.g., enhancers) to the integration site. For this reason, it is often necessary to screen a large number of events in order to be able to identify a commercializable event (i.e., an event in which the introduced gene of interest is optimally expressed). For example, it has been observed in plants and other organisms that the amount of expression of an introduced gene may 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, in that the actual expression pattern may not be consistent with the expression pattern expected from the transcriptional regulatory elements in the introduced gene construct, resulting in differences in the performance of the transformation event in the trait. Thus, it is often necessary to generate hundreds to thousands of different events and to screen those events for a single event with the amount and pattern of transgene expression expected for commercial purposes. Events with expected expression levels and patterns of transgenes can be used to introgress the transgenes into other genetic backgrounds by sexual outcrossing using conventional breeding methods. Progeny produced by this crossing pattern retain the transgene expression characteristics of the original transformation event. The use of this strategy ensures reliable gene expression in many varieties that are well adapted to local growth conditions. Therefore, there is a need for trait identification and screening of more transformation events to obtain superior transformation events with superior overall trait performance and commercial prospects.

It would be beneficial to be able to detect the presence of particular events to determine whether progeny of a sexual cross contain a gene of interest. Furthermore, methods of detecting specific events will also help to comply with relevant regulations, such as that foods derived from recombinant crops need to be officially approved and labeled before being placed on the market. It is possible to detect the presence of the transgene 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, and the like. Thus, unless the sequence of chromosomal DNA adjacent to the inserted transgenic DNA ("flanking DNA") is known, this method described above cannot be used to distinguish between different events, particularly those produced with the same DNA construct. Therefore, it is now common to identify specific events of a transgene 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 a cotton transformation event with excellent yield traits and herbicide tolerance traits, a nucleic acid sequence for detecting 17L397-1 in cotton and a detection method thereof. The 17L397-1 yield character in the transgenic cotton event is excellent, the glyphosate herbicide tolerance is good, and the detection method can accurately and quickly identify whether the biological sample contains the DNA molecule of the 17L397-1 in the specific transgenic cotton event.

In order to achieve the purpose, the invention uses pCAMBIA1300/EPSPS-csRRM2 expression vector to transform cotton seed 24 in cotton inbred line by Agrobacterium tumefaciens mediated method, and 13 positive transformation seedlings are obtained. And identified a transformation event with better yield traits and glyphosate herbicide tolerance, 17L397-1, which has better yield traits and herbicide glyphosate tolerance than transformation event ICR24-397 (application No.: 201811442263.1) containing the same gene, so that 17L397-1 can be used to improve the yield and herbicide tolerance of cotton.

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

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

Still 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.

Still 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 thereof or a variant or reverse complementary sequence thereof.

The invention also provides a primer pair for detecting cotton transformation events, which is characterized in that an 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 or reverse complementary sequence thereof.

In some embodiments, the primer pair is a sequence shown as SEQ ID NO. 8 and SEQ ID NO. 9; or the sequences shown in 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 cotton transformation events, which is characterized by detecting whether the transformation events exist in a sample to be detected by using the probe or the primer pair or the probe and primer pair or the kit or 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) subjecting the cotton obtained in step 1) to pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or crossing or a combination thereof to obtain a cotton plant, seed, plant cell, progeny plant or plant part; and optionally also (c) a second set of one or more of,

3) subjecting the progeny plants obtained in step 2) to yield trait and/or herbicide resistance identification and testing for the presence of said transformation event therein using the methods described above.

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

The SEQ ID NO. 1 is a sequence of 22 nucleotides in length located near the insertion junction at the 5 'end of the 17L397-1 insertion sequence in the transgenic cotton event, the DNA sequence of SEQ ID NO. 1 spanning the left flanking genomic DNA sequence of the cotton insertion site and the 5' end of the left border of the insertion sequence, comprising the SEQ ID NO. 1 or its reverse complement can be identified as the presence of 17L397-1 in the transgenic cotton event. The SEQ ID NO. 2 is a sequence of 22 nucleotides in length located near the insertion junction at the 3 'end of the insert sequence in 17L397-1 in the transgenic cotton event, the DNA sequence of SEQ ID NO. 2 spanning the right border 3' end of the insert sequence and the right flank genomic DNA sequence of the cotton insertion site, comprising the SEQ ID NO. 2 or its reverse complement can be identified as the presence of 17L397-1 in the transgenic cotton event.

In the present invention, the nucleic acid sequence may be at least 11 or more consecutive polynucleotides of any portion of the transgene insertion sequence in said SEQ ID NO. 3 or the reverse complement thereof (first nucleic acid sequence), or at least 11 or more consecutive polynucleotides of any portion of the 5' left flanking cotton genomic DNA region in said SEQ ID NO. 3 or the reverse complement thereof (second nucleic acid sequence). The nucleic acid sequence may further be homologous or reverse complementary to a portion of said SEQ ID NO. 3 comprising the entire said 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 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 the reverse complement thereof.

The SEQ ID NO 3 is a sequence which is 586 nucleotides in length and is positioned near an insertion junction part at the 5 'end of the 17L397-1 in the transgenic cotton event, the SEQ ID NO 3 consists of a cotton left flanking genomic DNA sequence (nucleotides 1 to 177 of the SEQ ID NO 3) of 177 nucleotides and a DNA sequence (nucleotides 178-586 of the SEQ ID NO 3) at the 5' end of a first expression cassette of a glyphosate-resistant gene of 409 nucleotides, and the existence of the SEQ ID NO 3 or a reverse complementary sequence thereof can be identified as the existence of the 17L397-1 in the transgenic cotton event.

The nucleic acid sequence may be at least 11 or more contiguous polynucleotides of any portion of the transgene insert sequence in SEQ ID No. 4 or its reverse complement (third nucleic acid sequence) or at least 11 or more contiguous polynucleotides of any portion of the 3' right flank cotton genomic DNA region in SEQ ID No. 4 or its reverse complement (fourth nucleic acid sequence). The nucleic acid sequence may further be homologous or reverse complementary to a portion of the SEQ ID NO. 4 comprising 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 comprise a set of DNA primers in a DNA amplification method that produces an amplification product. The presence of 17L397-1 or 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 the reverse complement thereof.

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

The SEQ ID NO 5 is a 4544 nucleotide sequence in length which characterizes 17L397-1 in the transgenic cotton event and specifically comprises the genome and genetic elements shown in Table 1. The presence of 17L397-1 in a transgenic cotton event can be identified by inclusion of the SEQ ID NO 5 or its reverse complement.

TABLE 1 genomic and genetic elements encompassed 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 analogs thereof that do not serve as templates for one or more polymerases. In addition, the probe or primer of the invention should be at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 contiguous nucleotides in length, which may be selected from the nucleotides set forth 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 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 a contiguous nucleotide of at least about 21 to about 50 or more in length.

The present invention also provides a method for protecting a cotton plant from herbicide-induced damage comprising applying to a field in which at least one transgenic cotton plant comprising in its genome the nucleic acid sequence SEQ ID NO 1, SEQ ID NO 5, position 178 and 3842 and SEQ ID NO 2 in that order is grown, an effective amount of a glyphosate herbicide, or the transgenic cotton plant comprising in its genome SEQ ID NO 5; the transgenic cotton plants have tolerance to glyphosate herbicides.

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

In the nucleic acid sequences for detecting cotton plants and the detection methods thereof according to the present invention, the following definitions and methods may better define the present invention and guide those of ordinary skill in the art in carrying out the present invention, unless otherwise specified, terms are understood according to their conventional usage by those of ordinary skill in the art.

The "cotton" refers to upland cotton (Gossypium Hirsutum L) and includes all plant species that can be mated 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, plant clumps (plant granules), and plant cells intact in plants or plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stalks, roots, root tips, anthers, and the like. It is to be understood that parts of transgenic plants within the scope of the present invention, which are derived from transgenic plants or progeny thereof which have been previously transformed with a DNA molecule of the invention and thus consist at least in part of transgenic cells, include, but are not limited to, plant cells, protoplasts, tissue, callus, embryos, and flowers, stems, fruits, leaves, and roots.

The term "gene" refers to a nucleic acid fragment that expresses a particular protein, including regulatory sequences preceding the coding sequence (5 'non-coding sequences) and regulatory sequences following the coding sequence (3' non-coding sequences). "native gene" refers to a gene that is naturally found to have its own regulatory sequences. "chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences not found in nature. "endogenous gene" refers to a native gene that is located in its natural location in the genome of an organism. "foreign gene" is a foreign gene that exists in the genome of an organism and does not originally exist, and also refers to a gene that has been introduced into a recipient cell through a transgenic step. The foreign gene may comprise a native gene or a 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 at which the recombinant DNA has been inserted may be referred to as an "insertion site" or "target site".

"flanking DNA" may comprise the genome as it occurs naturally in an organism such as a plant or foreign (heterologous) DNA introduced by the transformation process, such as a fragment associated with the transformation event. Thus, flanking DNA may include a combination of native and foreign DNA. In the present invention, a "flanking region" or "flanking sequence" or "genomic boundary region" or "genomic boundary 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 in length, which is located directly upstream or downstream of and adjacent to the originally exogenously inserted DNA molecule. When the flanking region is located upstream, it may also be referred to as "left border flanking" or "5 'genomic flanking region" or "genomic 5' flanking sequence" or the like. When the flanking region is located downstream, it may also be referred to as "right border flanking" or "3 'genomic flanking region" or "genomic 3' flanking sequence" or the like.

Transformation procedures that result in random integration of the exogenous DNA will result in transformation events that contain different flanking regions that are specific for each transformation event. When the recombinant DNA is introduced into a plant by conventional crossing, its flanking regions are not usually altered. Transformation events will also contain unique junctions between the heterologous insert DNA and segments of genomic DNA or between two segments of heterologous DNA. "junction" is the point at which two specific DNA fragments are joined. For example, the junction is present where the insert DNA joins the flanking DNA. A junction point is also present in a transformed organism where two DNA fragments are joined together in a manner that is modified in the way found in the native organism. "junction DNA" refers to DNA comprising a junction site.

The invention provides a transgenic cotton event named as middle 17L397-1 and progeny thereof, wherein the 17L397-1 in the transgenic cotton event is the 17L397-1 in cotton, which comprises plants and seeds of the 17L397-1 in the transgenic cotton event and plant cells or reproducible parts thereof, and plant parts of the 17L397-1 in the transgenic cotton event, including but not limited to cells, pollen, ovules, flowers, buds, roots, stems, leaves, and products derived from the 17L397-1 in cotton, such as cotton seeds, cotton coats, quilts, cotton battings, cotton cloths, and biomass left in cotton crop fields.

The 17L397-1 of the transgenic cotton events of the invention comprises a DNA construct that, when expressed in plant cells, results in improved yield traits and/or tolerance to glyphosate herbicide for 17L397-1 of the transgenic cotton events. The DNA construct comprises an expression cassette comprising a suitable promoter for expression in plants operably linked to a csRRM2 gene having cell enlarging function and a suitable polyadenylation signal sequence, the nucleic acid sequence of the csRRM2 protein increasing cotton yield. The DNA construct comprises a further expression cassette comprising a suitable promoter for expression in a plant operably linked to a gene g10-EPSPS encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) the nucleic acid sequence of which is tolerant to glyphosate herbicide and a suitable polyadenylation signal sequence. Further, the promoter may be a suitable promoter isolated from a plant, including constitutive, inducible and/or tissue specific promoters, suitable promoters include, but are not limited to, the cauliflower mosaic virus (CaMV)35S promoter, the Figwort Mosaic Virus (FMV)35S promoter, the Ubiquitin protein (Ubiquitin) promoter, the Actin (Actin) promoter, the Agrobacterium tumefaciens (Agrobacterium tumefaciens) nopaline synthase (NOS) promoter, the octopine synthase (OCS) promoter, the nocturnal (Cestrum) yellow leaf curly virus promoter, the potato tuber storage protein (Patatin) promoter, the ribulose-1, 5-bisphosphate carboxylase/oxygenase (rusco) promoter, the glutathione thiotransferase (GST) promoter, the E9 promoter, the GOS promoter, the alcA/alcR promoter, the Agrobacterium rhizogenes (Agrobacterium rhizogenes) RolD promoter, and the Arabidopsis thaliana (Arabidopsis thaliana) Suc2 promoter. The polyadenylation signal sequence may be a suitable polyadenylation signal sequence that functions in plants, including, but not limited to, polyadenylation signal sequence derived from the Agrobacterium tumefaciens nopaline synthase (NOS) gene, cauliflower mosaic virus (CaMV)35S terminator, polyadenylation signal sequence derived from the protease inhibitor II (PIN II) gene, and polyadenylation signal sequence derived 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/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 transportation of the EPSPS protein to a particular organelle or compartment outside or within the cell, for example, targeting the chloroplast using a sequence encoding a chloroplast transit peptide, or targeting the endoplasmic reticulum using a 'KDEL' retention sequence.

Culturing 17L397-1 in a transgenic cotton event having improved yield trait properties and tolerance to glyphosate herbicide by: first sexually crossing a first parent cotton plant consisting of a cotton plant grown from 17L397-1 and progeny thereof in a transgenic cotton event wherein 17L397-1 and progeny thereof are obtained by transformation using the yield trait improvement and glyphosate herbicide tolerant expression cassette of the present invention with a second parent cotton plant lacking yield trait improvement characteristics or glyphosate herbicide tolerance, thereby producing diverse first generation progeny plants; and then selecting the progeny plants with tolerance to the glyphosate herbicide to breed the cotton plants with tolerance to the glyphosate herbicide. These steps may further comprise backcrossing the yield trait-improved and glyphosate tolerant progeny plants with a second or third parent cotton plant, and then selecting the progeny by application with glyphosate herbicide or by identification of a trait-associated molecular marker, such as a DNA molecule comprising the junction sites identified at the 5 'and 3' ends of the 17L397-1 insert sequence in the transgenic cotton event, to produce cotton plants with yield trait-improved properties and tolerance to glyphosate herbicide.

It is also understood that two different transgenic plants may also be crossed to produce progeny containing two separate, separately added exogenous genes. Selfing of appropriate progeny can yield progeny plants that are homozygous for both added exogenous genes. Backcrossing of parental plants and outcrossing with non-transgenic plants as described above is also contemplated, as is asexual propagation.

As used herein, a substantially homologous sequence is a nucleic acid molecule that specifically hybridizes under highly stringent conditions to the complementary strand of a matched nucleic acid molecule. Suitable stringency conditions for promoting DNA hybridization include, for example, treatment with 6.0 XSSC/sodium citrate (SSC) at about 45 ℃ followed by a wash with 2.0 XSSC at 50 ℃, as is well known to those skilled in the art. For example, the salt concentration in the washing step can be selected from the group consisting of about 2.0 XSSC for low stringency conditions, 50 ℃ to about 0.2 XSSC for high stringency conditions, 50 ℃. In addition, the temperature conditions in the washing step can be raised from about 22 ℃ at room temperature for low stringency conditions to about 65 ℃ at high stringency conditions. Both the temperature conditions and the salt concentration may be varied, or one may be held constant while the other is varied. Preferably, a nucleic acid molecule of the invention can specifically hybridize 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 complements thereof, or any fragment thereof, under moderately stringent conditions, such as at about 2.0 XSSC and about 65 ℃. More preferably, a nucleic acid molecule of the invention specifically hybridizes 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 a complementary sequence thereof, or to a fragment of any of the foregoing. 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 their complementary sequences 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 any of the above. 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 progeny of a genetic cross. Hybridization of the probe to the target DNA molecule can be detected by any method known to those skilled in the art, including, but not limited to, fluorescent, radioactive, antibody-based, and chemiluminescent labels.

DNA detection kits based on DNA amplification methods contain DNA primer molecules that specifically hybridize to the target DNA and amplify the diagnostic amplicon under appropriate reaction conditions. The kit may provide an agarose gel based detection method 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 transgenic insert region of SEQ ID NO 5 are provided by the present invention. In particular, the primer pairs identified as 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' transgene/genomic region of 17L397-1 in the transgenic cotton event, wherein the amplicon includes SEQ ID NO 1. The primer pairs identified as useful in the DNA amplification method also included SEQ ID NO 10 and SEQ ID NO 11, which amplified a diagnostic amplicon homologous to a portion of the 3' transgene/genomic region of 17L397-1 in the transgenic cotton event, wherein the amplicon includes SEQ ID NO 2. Other DNA molecules used as DNA primers may be selected from SEQ ID NO 5.

The DNA detection kit can be developed using the compositions described herein and methods described or known in the DNA detection art. The kit is beneficial to identifying whether the DNA of 17L397-1 in the transgenic cotton event exists in a sample, and can also be used for cultivating cotton plants containing the DNA of 17L397-1 in 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 site where the transgene insert is incorporated into the cotton genome, as contained in the cotton genome and illustrated in figure 1 and table 1, comprises: the 17L397-1 left flanking genomic region in cotton located at the 5' end of the transgene insert sequence, was derived from a first expression cassette consisting of 2 tandem cauliflower mosaic virus 35S promoters (2 XP 35S), operably linked to a glyphosate resistance gene sequence (g10-epsps), and operably linked to a cauliflower mosaic virus 35S terminator (T35S); the second expression cassette consisted of the cauliflower mosaic virus 35S promoter (P35S), operably linked to the cell expansion gene csRRM2, and operably linked to the nopaline synthase gene terminator (Tnos), a portion of the insert from the Agrobacterium right border Region (RB), and the 17L397-1 right flanking genomic region in cotton located 3' of the transgene insert (SEQ ID NO: 5). In the DNA amplification method, the DNA molecule used as a primer may be derived from any part of the transgene insert sequence in 17L397-1 of the transgenic cotton event, or from any part of the DNA region of the 17L397-1 flanking cotton genome of the transgenic cotton event.

The 17L397-1 transgenic cotton event can be combined with other transgenic cotton varieties, such as herbicide (e.g., glyphosate, glufosinate, etc.) tolerant cotton, or transgenic cotton varieties carrying insect-resistant genes. Various combinations of all of these different transgenic events, when bred with 17L397-1 in the transgenic cotton event of the invention, can provide improved hybrid transgenic cotton varieties that are resistant to insects and to multiple herbicides. Compared with non-transgenic varieties and transgenic varieties with single characters, the varieties can show more excellent characteristics such as insect resistance, resistance to various herbicides and the like.

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

Although the 17L397-1 insertion sequence is incomplete, and the quality character is not obviously improved compared with that of a control, the glyphosate tolerance capability is better, and the yield character is more prominent. These characteristics allow the transformant of Zhong17L 397-1 to be used for improving the glyphosate herbicide tolerance and yield traits of cotton, thereby breeding new varieties of cotton with high yield and herbicide tolerance.

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

Drawings

FIG. 1 is a schematic diagram of the structure of the binding site of the transgene insert sequence to the cotton genome.

FIG. 2 physical map of recombinant expression vector pCAMBIA1300/EPSPS-CSRRM 2. English and abbreviation meanings of each element are listed as follows:

T-DNA left border sequence of LB Agrobacterium.

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

g10-EPSPS encodes an EPSPS protein, which detoxifies glyphosate.

Ch-L signal peptide

The 35S promoter of P2X 35S tandem cauliflower mosaic virus (CaMV).

Terminator of the Tnos nopaline synthase gene.

CsRRM2 CsRRM2 gene CDS.

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

The right border sequence of the T-DNA of RB Agrobacterium.

Plasmid stabilization site of the PVS1 sta pVS1 plasmid.

The replication initiation site of the PVS1 rep pVS1 plasmid.

The bom site of the PBR322 bom pBR322 plasmid.

Origin of replication of the PBR322 ori pBR322 plasmid.

kanamycins (R) encode aminoglycoside phosphotransferase proteins that confer kanamycin resistance to bacteria.

The 17L397-1 event in FIG. 3 is shown to compare the yield traits of cotton plant 24 in the recipient control. 24 of the middle cotton institute: a non-transgenic recipient control cotton plant; middle 17L 397-1: middle 17L397-1 transformation event cotton plants. A is a blade; b is young bells; c is a plant; d is a ringing bell.

In FIG. 4 the 17L397-1 event is compared to the glyphosate herbicide resistance of cotton plant 24 in the recipient control. 24 of the middle cotton institute: a non-transgenic recipient control cotton plant; middle 17L 397-1: medium 17L397-1 transformation event Cotton plants.

Results of 17L397-1 transformation event-specific PCR validation in FIG. 5. M: marker, marking the size beside (unit: bp); n: blank control; p: vector pCAMBIA1300/EPSPS-csRRM 2; c: genomic DNA of cotton 24 in the recipient control; t: genomic DNA from cotton material containing the medium 17L397-1 transformation event. A: the expected size of the left border PCR fragment is 295 bp; b: the right border PCR fragment was expected to be 445bp in size.

FIG. 6Southern hybridization cleavage and probe location.

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

A: BamHI enzyme digestion of DNA hybridization pattern; b: digesting the DNA hybridization diagram by EcoRI enzyme; c: the probe position and the restriction enzyme cutting site of the restriction enzyme, the horizontal line indicates the probe position, and the vertical line indicates the restriction enzyme cutting site.

A1: DNA Marker, the size of the strip is marked aside, unit bp;

a2: BamHI enzyme digestion plasmid;

a3: a BamHI enzyme digestion medium cotton plant 24;

a4: BamHI enzyme digestion of 17L 397-1;

b1: DNA Marker, the size of the strip is marked aside, unit bp;

b2: cutting plasmid with EcoRI;

b3: EcoRI enzyme digestion medium cotton institute 24;

b4: EcoRI enzyme cuts 17L 397-1.

Southern blot hybridization of 17L397-1 Gene of interest g10-epsps insert copy number in FIG. 8

A: BamHI enzyme digestion of DNA hybridization pattern; b: digesting the DNA hybridization diagram by EcoRI enzyme; c: g10-epsps probe position and restriction enzyme cutting site of restriction enzyme, the horizontal line indicates probe position, and the vertical line indicates cutting site.

A1: BamHI enzyme digestion plasmid;

a2: blank control;

a3: a BamHI enzyme digestion medium cotton plant 24;

a4: blank control;

a5: BamHI enzyme digestion of 17L 397-1;

a6: DNA Marker, the size of the strip is marked aside, unit bp;

b1: DNA Marker, the size of the strip is marked aside, unit bp;

b2: cutting plasmid with EcoRI;

b3: EcoRI enzyme digestion medium cotton institute 24;

b4: blank control;

b5: EcoRI enzyme cuts 17L 397-1.

Southern blot hybridization of copy number inserted in region(s) and region(s) of 17L397-1 vector backbone in FIG. 9

A: BamHI enzyme digestion of DNA hybridization pattern; b: digesting the DNA hybridization diagram by EcoRI enzyme; c: probe positions of the first and fourth regions and restriction sites of restriction enzymes.

A1: DNA Marker, marking the size of the strip by side, and the unit bp;

a2: BamHI enzyme digestion plasmid;

a3: blank control;

a4: a BamHI enzyme digestion medium cotton plant 24;

a5, a 6: BamHI enzyme digestion of 17L 397-1;

a7, A8: r1 (PCR amplification product of primer in the first hybridization region of probe);

a9, A10: r4 (PCR amplification product of primers in Probe (R) hybridization region);

b1: DNA Marker, the size of the strip is marked aside, unit bp;

b2: EcoRI digested plasmid;

b3: blank control;

b4: EcoRI enzyme digestion medium cotton institute 24;

b5, B6: EcoRI is used for digesting 17L397-1 in the transformant;

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

b8: r4 (Probe (R) PCR amplification product of primers in the hybridization region).

FIG. 10 Southern blot hybridization of inserted copy numbers of regions (c) and (c) of the vector backbone

A: BamHI enzyme digestion of DNA hybridization pattern; b: digesting the DNA hybridization diagram by EcoRI enzyme; c: the probe positions of the framework 2 region and the framework 3 region and the restriction enzyme cutting sites of restriction enzymes.

A1: DNA Marker, the size of the strip is marked aside, unit bp;

a2: a BamHI digested plasmid;

a3: blank control;

a4: a BamHI enzyme digestion medium cotton plant 24;

a5, A6: cutting 17L397-1 with BamHI enzyme;

a7, A8: r2 (Probe-PCR amplification product of primers in the hybridization region);

a9, A10: r3 (probe and PCR amplification product of primer in hybridization region);

b1: DNA Marker, the size of the strip is marked aside, unit bp;

b2: EcoRI digested plasmid;

b3: blank control;

b4: EcoRI enzyme digestion medium cotton institute 24;

b5, B6: cutting 17L397-1 by EcoRI enzyme;

b7: r2 (Probe-PCR amplification product of primers in the hybridization region);

b8: r3 (Probe-PCR amplification product of primers in the hybridization region).

Detailed Description

The transformation event 17L397-1 refers to the fact that cotton plants with foreign gene inserts (T-DNA inserts) inserted among specific genome sequences are obtained after genetic transformation by taking cotton plant 24 in a cotton inbred line as a receptor. 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 by nucleotide 178-4096 of SEQ ID NO: 5. 17L397-1 in the transformation event may refer to this transgenic process, to the T-DNA insert within the genome resulting from this process, or to the combination of the T-DNA insert with flanking sequences, or to the cotton plant resulting from this transgenic process. In a specific example, the event is also applicable to plants obtained by transforming other recipient varieties with the same expression vector and inserting the T-DNA insert into the same genomic position. 17L397-1 in a transformation event can also refer to progeny plants resulting from vegetative, sexual, doubling or doubling of the above plants, or a combination thereof.

Example 1 acquisition of transformation events and characterization

The csRRM2 gene is an RRM2 structural domain for controlling the FCA gene and is closely related to the quality traits of plants; g10-epsps gene encodes 5-enolpyruvylshikimate-3-phosphate synthase which can improve the tolerance of plants to glyphosate herbicides. The transformation event ICR24-397 (application number: 201811442263.1) obtained previously has better quality character 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 the plants in ICR24-397 became phytotoxic when glyphosate was applied at 2-fold the recommended field concentration; when glyphosate was applied at 4 times the recommended field concentration, approximately 20% of the plants in ICR24-397 developed phytotoxicity. Thus, the herbicide resistance 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 the vector comprises a csRRM2 gene expression box and a g10-EPSPS gene expression box), transforms cotton seed 24 in a receptor material by an agrobacterium-mediated method to obtain 13 positive transformed seedlings, and screens and identifies the herbicide resistance and the quality-related agronomic traits of the transformed seedlings.

1. Screening of transformants having Excellent herbicide resistance Properties

And (3) taking the cotton institute 24 in the receptor and the transformant ICR24-397 as references, and screening the transformant with better herbicide tolerance from the 13 positive transformed seedlings by a method of spraying glyphosate with the concentration 2 times of the recommended field concentration in the field. The results show that only 3 transformation events (medium 17L397-1, medium 17L397-2, and medium 17L397-3) were significantly more tolerant of glyphosate herbicide than the control and not less than the transformant ICR 24-397.

Subsequently, seeds of cotton 24 in the 4 cotton transformants and the receptor control were sown in a cotton institute test base in Anyang city, Henan province, and herbicide tolerance of the transformants such as 17L397-1 was systematically identified by spraying glyphosate with different concentrations in the field. Meanwhile, the effectiveness of improving the quality traits of the transgenic cotton is identified by a method for inspecting the agronomic traits such as the length, the uniformity, the specific strength and the like of cotton fibers, and the insertion positions of three transformation events of middle 17L397-1, middle 17L397-2 and middle 17L397-3 are identified and analyzed.

2. Herbicide tolerance identification

Herbicide tolerance identification was carried out in 2018 summer. When 4-6 main leaves are sprayed in the seedling stage of cotton according to the recommended field concentration of 1 time (82 g/mu), 2 times (164 g/mu) and 4 times (328 g/mu), after one week, all withered and yellow cotton plants of the receptor control die, the seedling rate is 0, the phytotoxicity rate is 100%, 4 transformation events can normally grow seedlings, but the phytotoxicity rates of different degrees are shown (Table 2). When glyphosate with the medium amount of 2 times of the recommended concentration in the field is sprayed, the glyphosate tolerance capacity of the medium 17L397-1 and the medium 17L397-2 is obviously higher than that of the medium 17L397-3 and the ICR 24-397; when 4 times the recommended field concentration of glyphosate was sprayed, the glyphosate tolerance of the medium 17L397-1 was significantly higher than the remaining 3 transformant materials. Thus, glyphosate herbicide resistance of medium 17L397-1 was best demonstrated.

TABLE 2 herbicide tolerance Performance

Values are from the mean ± standard deviation of 3 biological replicates. Statistical analysis multiple comparisons were performed using LSD (α ═ 0.05) with different letters indicating significance of differences between the data listed under the same herbicide concentration.

3. The positions of insertion of 17L397-1, middle 17L397-2 and middle 17L397-3 in the genome were analyzed

Taking 100mg plant leaves, quickly grinding the plant leaves by liquid nitrogen, and extracting total DNA by adopting a CTAB method. Fragmenting qualified sample genome DNA by using ultrasonic waves, and then purifying, repairing the tail end, adding A to the 3' end and connecting a sequencing joint to the fragmented DNA. And then agarose gel electrophoresis is carried out to select the size of the fragment, and PCR amplification is carried out to form a sequencing library. Sequencing the materials by utilizing an Illumina nova sequencing platform in the library qualified by quality inspection, wherein the sequencing depth is 10 x. Carrying out quality evaluation on the Raw data obtained by sequencing to obtain a filter sequence Clean data, independently writing a Perl script to convert an fq file of the Clean data into a fa format, and then comparing the filter sequence with a cotton Gh ZM24-CRI v1 reference genome sequence (https:// www.cottongen.org/blast/nucleotide/nucleotide). And (3) positioning the position of the filter sequence on the reference genome to obtain the position information of the exogenous insert insertion.

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

The results showed that the foreign fragments of 3 transformants integrated into different sites of the genome, which are cotton genomic intergenic regions, respectively, and there were no known functional genes. Furthermore, the expression cassettes for the exogenous sequences of intermediate 17L397-2 and intermediate 17L397-3 were intact, whereas the vector border sequence of the intermediate 17L397-1 transformation event was deleted during the insertion, including part of the border sequence and part of the sequence of terminator T35S (see Table 3 for details). In general, incompleteness of the terminator results in aberrant transcription and translation of the gene, which in turn affects the trait of interest. However, unexpectedly, from the results of the herbicide resistance trait identification, glyphosate tolerance of the medium 17L397-1 transformation event was significantly higher than that of the receptor control and was more prominent than the ICR24-397, medium 17L397-2 and medium 17L397-3 transformants.

TABLE 3 foreign fragment insertion position information

4. Quality trait identification

Quality traits such as cotton fiber length, uniformity and specific strength of the medium 17L397-1 transformant and the acceptor control were examined during the period from bolling to final harvest. The results showed that the cotton fibers of Medium 17L397-1, Medium 17L397-2 and Medium 17L397-3 did not differ greatly in quality properties such as fiber length, uniformity and specific strength, and did not show a significant difference compared to the cotton seed of the recipient control 24, and were weaker than ICR 24-397.

5. Identification of yield traits

While the quality traits of the several transformants are identified, other traits (such as seed cotton weight, flower bud number, boll forming number, single boll weight, plant height, first fruit branch node height, fruit node number, fruit branch number, leaf size and the like) of the several transformants are recorded in detail. Surprisingly, it was found that the total weight and the single boll weight of the 17L397-1 seed cotton were not only much higher than those of the recipient control, but also significantly higher than those of the other 3 transformants including ICR24-397 (Table 4), and that the plant height and the first fruit branch node height of the 17L397-1 were also significantly higher than those of the recipient control at the seedling stage, bud stage, boll stage and boll opening stage (Table 5)

TABLE 4 yield of seed cotton in boll opening period

Values are from the mean ± standard deviation of 3 biological replicates. Statistical analysis multiple comparisons were performed using LSD (α ═ 0.05), with different letters indicating significance of differences between the same period and the same column of data.

TABLE 5 major agronomic traits

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

The results show that although the 17L397-1 insertion sequence is incomplete and the quality character is not obviously improved compared with that of a control, the glyphosate tolerance capability is better and the yield character is more prominent. These characteristics allow the transformant of Zhong17L 397-1 to be used for improving the glyphosate herbicide tolerance and yield traits of cotton, thereby breeding new varieties of cotton with high yield and herbicide tolerance.

EXAMPLE 2 characterization of the 17L397-1 molecule in the transformation event

To further characterize the identity of 17L397-1 in transformation events, the present invention analyzed the flanking sequences and inserted copy number of the insertion site of the 17L397-1 foreign sequence in the cotton genome.

1. Analysis of flanking sequences of insertion sites of foreign sequences on the genome of cotton

As described in example 1, the exogenous T-DNA sequence of 17L397-1 was inserted forward in the cotton genome at position Chr A11:7085972 or Chr D11:6406857 in the transformation event. And (3) intercepting 500bp upstream of the insertion site on the genome and 500bp on the T-DNA sequence at the left border of the insertion site, and taking 500bp downstream of the insertion site of the genome and 500bp on the T-DNA sequence at the right border, and performing primer design on the intercepted sequence by using Primerlest software (https:// blast.ncbi.nlm.nih.gov/Blast) of an NCBI website, wherein an amplification product fuses a part of cotton genome sequence and a part of T-DNA sequence.

And (3) carrying out PCR amplification by using the genome DNA of the transgenic cotton strain as a template. The PCR reaction was carried out in a 20. mu.L system. The amplification cycle program was: pre-denaturation at 94 ℃ for 3 min; 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; extension at 72 ℃ for 5 min.

The insertion position of the foreign fragment was verified by PCR amplification of the 17L397-1 transformation event in the pair of the vector right border primer (SEQ ID NO:10) and the vector left border primer (SEQ ID NO:9) based on the results of the flanking sequence and the insertion position using the genome upstream primer (SEQ ID NO:8) and the vector left border primer (SEQ ID NO: 9). The results are shown in FIG. 5. The result proves that the 17L397-1 exogenous fragment is stably inserted into the cotton genome at the position of Chr A11:7085972 or Chr D11:6406857, and the size of the inserted sequence is 3919 bp.

By analyzing the boundary sequences of the left flank and the right flank, the insertion of the exogenous sequence causes 89bp sequence deletion of the receptor genome, and the vector sequence also deletes 131 bp. The deleted vector sequence included a partial border sequence and a partial sequence of terminator T35S.

2. Analysis of inserted copy number of exogenous sequences

The copy number of the exogenous sequence is determined by a Southern blot hybridization method. In the Southern hybridization detection, two restriction enzymes on a T-DNA region and not in a hybridization region are selected to digest genomic DNA, each insert copy in the genome is hybridized to display a single and specific band, and after the genomic DNA is digested by the restriction enzymes, a region to be detected is selected as a probe to carry out a Southern imprinting hybridization experiment.

Southern hybridization was performed by digesting the genomic DNA of the positive control plasmid, the genomic DNA of the acceptor control plasmid containing cotton 24 and the genomic DNA of the acceptor control plasmid containing 17L397-1 with BamHI and EcoRI enzymes, and designing probes covering the target gene and the backbone sequence of the full-covered vector, and the probe and the digestion position are schematically shown in FIG. 6. The specific sequences of the probe primers are shown in Table 6.

TABLE 6Southern 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 enzymes BamHI and EcoRI, and respectively enzyme-cuts the positive control plasmid, the cotton 24 genome DNA in the negative control and the middle 17L397-1 genome DNA. After running and transferring membranes, the membranes were labeled with csRRM2 gene probe, and the hybridization results are shown in FIG. 7A, B. The probe position of the foreign gene csRRM2 and the restriction sites of the restriction enzymes BamHI and EcoRI are shown in FIG. 7C. From the hybridization results, csRRM2 gene was inserted as a single copy into the cotton genome.

The inserted copy number hybridization detection of the target gene g10-epsps selects two restriction enzymes BamHI and EcoRI, and the restriction enzymes are used for respectively digesting the positive control plasmid, the cotton 24 genome DNA in the negative control and the 17L397-1 transformant genome DNA. The gel was run and membrane-transferred and labeled with g10-epsps gene probe, and the hybridization results are shown in FIG. 8A, B. The probe position of the target gene g10-epsps and the restriction sites of the restriction enzymes BamHI and EcoRI are shown in FIG. 8C. The g10-epsps gene was also inserted as a single copy into the cotton genome.

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

EXAMPLE 3 detection of 17L397-1 in transformation events

Breeding can be performed from 17L397-1 in a transgenic cotton event and the resultant new variety can be used to produce, for example, agricultural or commercial products. If a sufficient amount is detected in the agricultural or commodity product, the agricultural or commodity product is expected to contain a nucleotide sequence capable of diagnosing the presence of 17L397-1 material in the agricultural or commodity product during a transgenic cotton event. Such agricultural or commercial products include, but are not limited to, cottonseed oil, cotton wool, quilts, cotton cloths, cotton coats, and any other food product to be consumed by an animal as a food source, or otherwise used for cosmetic purposes as an ingredient in a bulking agent or cosmetic composition, and the like. A probe or primer pair based nucleic acid detection method and/or kit can be developed to detect the 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 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 detection method comprises the following steps: specific boundary sequences in the 17L397-1 plants in the two plants are detected by a PCR method, the used PCR primer pairs are respectively SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 11, and a PCR reaction system comprises:

the reaction procedure is as follows:

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

The PCR product was detected by electrophoresis on a 1% (w/v)1 XTAE agarose gel, and the results are shown in FIG. 5. The expected target band (SEQ ID NO:6 and SEQ ID NO:7, respectively) was amplified in the medium 17L397-1 transformation event. Moreover, the PCR method can track the existence of transformation events, thereby being applied to breeding work.

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

Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Sequence listing

<110> Cotton research institute of Chinese academy of agricultural sciences

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

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gttgaaaagt ctcaattgcc ctttggtctt ctgagactgt atctttgata tttttggagt 180

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tatcaacaca tttataatac tacatcaatt tgtcattttc aaatattact aaaaaaatta 600

gttaataaaa tcatagctat aatttgtgtc gagattagaa atttaagatt tgaaaaatat 660

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ttaattgcaa aatttagcca accaaaatta actactattg tttgcggtca aggttgaaat 780

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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> Artificial 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> Artificial 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> Artificial Synthesis (unknown)

<400> 9

accacatcag aaagtcctac cct 23

<210> 10

<211> 18

<212> DNA

<213> Artificial Synthesis (unknown)

<400> 10

gcgagttctg ttaggtcc 18

<210> 11

<211> 18

<212> DNA

<213> Gossypium hirsutum L.

<400> 11

tattgtgcgt gaagttgt 18

Claims (9)

1. A nucleic acid molecule comprising any one of:

i) comprises the sequence shown in SEQ ID NO. 1 and/or SEQ ID NO. 2, or the reverse complementary sequence thereof;

ii) comprises the sequence shown in SEQ ID NO 3 and/or SEQ ID NO 4, or the reverse complement thereof;

iii) comprises 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 the reverse complement thereof.

2. A probe for detecting cotton transformation events comprising the sequence shown in SEQ ID NO 1 or 2 or 3 or 4 or 6 or 7 or a fragment or variant or reverse complement thereof.

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

optionally, the primer pair is a sequence shown as SEQ ID NO. 8 and SEQ ID NO. 9; or the sequences shown in SEQ ID NO 10 and SEQ ID NO 11.

4. A kit or microarray for detecting cotton transformation events comprising the probe of claim 2 and/or the primer pair of claim 3.

5. 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:

i) the probe of claim 2;

ii) the primer pair of claim 3;

iii) the probe of claim 2 and the primer pair of claim 3;

iv) the kit or microarray of claim 4.

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

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

2) subjecting the cotton obtained in step 1) to pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or crossing or a combination thereof to obtain a cotton plant, seed, plant cell, progeny plant or plant part; and optionally also (c) a second set of one or more of,

3) evaluating the progeny plants obtained in step 2) for yield traits and/or herbicide resistance identification and detecting the presence or absence of the transformation event therein using the method of claim 5.

7. An article of manufacture made from a cotton plant, seed, plant cell, progeny plant or plant part obtained by the method of claim 6, including a food, feed or industrial material.

8. A method for protecting a cotton plant from herbicide induced damage comprising applying to a field in which at least one transgenic cotton plant comprising in its genome the nucleic acid sequence SEQ ID NO 1, SEQ ID NO 5, position 178-3842 and SEQ ID NO 2 in that order is grown, or comprising in its genome the nucleic acid sequence SEQ ID NO 5, an effective amount of a glyphosate herbicide; the transgenic cotton plants have tolerance to glyphosate herbicides.

9. A method for improving yield traits of a cotton plant, comprising growing at least one transgenic cotton plant comprising in its genome the nucleic acid sequence of SEQ ID NO 1, SEQ ID NO 5, position 178-3842 and SEQ ID NO 2 in that order, or comprising in its genome the nucleic acid sequence of SEQ ID NO 5; the transgenic cotton plants have significantly and stably improved yield traits.

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