CN114525277A - Nucleic acid sequence for detecting 17L397-1 in cotton and detection method thereof - Google Patents

Abstract

The present 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 the sequence shown in SEQ ID NO: 1 or its reverse complement, or SEQ ID The sequence shown in NO: 2 or its reverse complement. The 17L397-1 in the cotton of the present invention has the characteristics of yield trait improvement and glyphosate herbicide resistance, and the detection method can accurately and quickly identify whether the biological sample contains the DNA molecule of 17L397-1 in the transgenic cotton event.

Description

A kind of nucleic acid sequence for detecting 17L397-1 in cotton and its detection method

technical field

The present invention relates to the field of plant biotechnology. Specifically, it relates to a nucleic acid sequence for detecting 17L397-1 in cotton and a method for detecting the same, in particular to 17L397-1 in a transgenic cotton event with improved yield traits and tolerance to glyphosate herbicide application For detecting whether biological samples contain the nucleic acid sequence of 17L397-1 in a specific transgenic cotton event and a detection method thereof.

Background technique

Cotton is the most important natural fiber crop in the world and an important source of protein and oil. The global cotton planting area is about 490 million mu, and the output value exceeds 50 billion US dollars. China, India, the United States and other five countries accounted for more than 70% of the total production. Among them, the advanced cotton planting countries such as the United States and Australia have realized the whole process of mechanization, and their cotton industry has strong international competitiveness, leading the development direction of the cotton industry.

my country is a big cotton producing country. There are about 100 million cotton farmers in the country. The annual cotton planting area is about 6,000 to 80 million mu, and the annual output of raw cotton is 4.5 to 5.6 million tons, accounting for about 25% of the world's total cotton production. At the same time, China is the world's largest cotton importer. China's cotton imports are expected to continue to soar in the next few years. Chinese state media reports that China's annual cotton imports will soar to about 7 million metric tons in recent years. The reason is that China's textile and apparel industry is booming. Therefore, increasing the output of cotton in my country through biotechnology will have a great impact on the development of cotton planting and cotton spinning.

FCA and ABA abscisic acid have high affinity, stereospecificity, and conform to the principle of receptor kinetics. ABA binding directly controls the processing of mRNA precursors mediated by FCA, an ABA receptor involved in RNA metabolism. The study found that: the RRM2 domain that controls the FCA gene and the herbicide tolerance gene g10-epsps were transferred into the receptor, and the obtained transformants showed obvious changes in quality traits improvement and herbicide resistance. Therefore, it is considered that this method It can be widely used in the improvement of economic traits of dicotyledonous and monocotyledonous crops.

Field weeds compete with crops for water, fertilizer, light and growth space, which directly affects crop yield and quality. At the same time, many weeds are intermediate hosts of crop pathogens and pests, and are one of the important biological limiting factors for crop yield increase. According to the United Nations Food and Agriculture Organization, the annual loss of food production caused by weeds is as high as 95 billion US dollars, which is equivalent to the loss of 380 million tons of wheat, which is more than half of the global wheat production in 2009. Of the $95 billion in economic losses, poor developing countries bear about $70 billion (FAO. The lurking menace of weeds [J/OL]. (http://www.fao.org/news/story/en /item/29402/icode/), 2009-08-11.). Therefore, effective control of field weeds is one of the important measures to promote grain yield. With the acceleration of the migration of rural population to cities in my country, the large-scale and mechanization of corn planting is a predictable trend, which makes the traditional manual weeding method unrealistic. At present, the selective herbicides widely used in the market have large application rates and long residual periods, which can easily affect the normal growth of the next crop. Biocidal herbicides such as glufosinate-ammonium have the characteristics of high efficiency, low toxicity, easy degradation and no residue. But they are not selective for weeding and cannot be used directly in the growing season of crops. Breeding cotton that is resistant to this type of biocidal herbicide through transgenic technology can overcome this problem. Spraying 1-2 times during the cotton growing period can effectively solve the weed problem and reduce the dosage and input cost of herbicides. Therefore, herbicide-tolerant transgenic cotton has very broad application value and market potential.

The expression of exogenous genes in plants is known to be affected by their chromosomal location, possibly due to the proximity of chromatin structure (eg, heterochromatin) or transcriptional regulatory elements (eg, enhancers) to integration sites. To this end, it is usually necessary to screen a large number of events to be able to identify events that can be commercialized (ie, events in which the introduced target gene is optimally expressed). For example, it has been observed in plants and other organisms that the level of expression of the introduced gene may vary widely between events; there may also be differences in spatial or temporal patterns of expression, such as relative expression of transgenes between different plant tissues There is a difference in that the actual expression pattern may not be consistent with the expression pattern expected based on the transcriptional regulatory elements in the introduced gene construct, leading to differences in the performance of transformation events. Thus, it is often necessary to generate hundreds to thousands of different events and screen from these events for a single event with the desired transgene expression level and expression pattern for commercialization. Events with the expected amount and pattern of expression of the transgene can be used to introgress the transgene into other genetic backgrounds by sexual outcrossing using conventional breeding methods. Progeny produced by this crossing maintain the transgene expression characteristics of the original transformation event. Applying this strategic model ensures reliable gene expression in many varieties that are well adapted to local growing conditions. Therefore, more transformation events need to be characterized and screened to obtain excellent transformation events with excellent comprehensive performance and commercialization prospects.

It would be beneficial to be able to detect the presence of specific events to determine whether the offspring of a sexual cross contain the gene of interest. In addition, methods to detect specific events will also help to comply with regulations, such as the need for formal approval and labelling of foods derived from recombinant crops before they are 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 typically focus on commonly used genetic elements such as promoters, terminators, marker genes, and the like. Therefore, unless the sequence of the chromosomal DNA adjacent to the inserted transgenic DNA ("flanking DNA") is known, this method cannot be used to distinguish between different events, especially those generated with the same DNA construct event. Therefore, transgene-specific events are currently often identified by PCR using a pair of primers spanning the junction of the inserted transgene and flanking DNA, specifically a first primer containing the flanking sequence and a second primer containing the inserted sequence.

SUMMARY OF THE INVENTION

The object of the present invention is to provide an excellent cotton transformation event with excellent yield traits and herbicide tolerance traits, as well as a nucleic acid sequence for detecting 17L397-1 in cotton and a detection method thereof. 17L397-1 in the transgenic cotton event has excellent yield traits and good 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 a specific transgenic cotton event.

In order to achieve the above object, the present invention uses the pCAMBIA1300/EPSPS-csRRM2 expression vector to transform the cotton inbred line Zhongmian Institute 24 by the method mediated by Agrobacterium, and 13 positive transformed seedlings are obtained. And identified a transformation event 17L397-1 with better yield traits and glyphosate herbicide tolerance, which had better yield traits than the transformation event ICR24-397 (application number: 201811442263.1) containing the same gene and tolerance to the herbicide glyphosate, so 17L397-1 can be used to improve cotton yield and herbicide tolerance traits.

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

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

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

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

The present invention also provides a probe for detecting cotton transformation events, characterized in that it includes 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 The sequence shown in SEQ ID NO: 7 or a fragment or variant thereof or its reverse complement.

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

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

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

The present invention also provides a method for detecting cotton transformation events, which is characterized by comprising using the above-mentioned probe or the above-mentioned primer pair or the above-mentioned probe and primer pair or the above-mentioned kit or microarray to detect whether the sample is tested. The conversion event exists.

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

1) obtaining cotton comprising the above-mentioned nucleic acid molecule;

2) Cotton plants, seeds, plant cells, progeny plants or plant parts are obtained from the cotton obtained in step 1) through pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or hybridization or a combination of the above. ; and optionally,

3) Carry out identification of yield traits and/or herbicide resistance on the progeny plants obtained in step 2), and use the above-mentioned method to detect whether the transformation event exists therein.

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

The SEQ ID NO: 1 is a sequence of 22 nucleotides in length near the insertion junction at the 5' end of the insertion sequence in 17L397-1 in the transgenic cotton event, and the SEQ ID NO: 1 spans the cotton The genomic DNA sequence of the left flank of the insertion site and the DNA sequence of the 5' end of the left border of the insertion sequence, including the SEQ ID NO: 1 or its reverse complement, can be identified as the presence of 17L397-1 in the transgenic cotton event . Said SEQ ID NO:2 is a sequence of 22 nucleotides in length near the insertion junction at the 3' end of the insertion sequence in 17L397-1 in the transgenic cotton event, said SEQ ID NO:2 spanning the insertion The DNA sequence at the 3' end of the right border of the sequence and the genomic DNA sequence at the right flank of the cotton insertion site, including 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 contiguous polynucleotides (the first nucleic acid sequence) of any part of the transgenic insertion sequence in the described SEQ ID NO: 3 or its reverse complement, or At least 11 or more contiguous polynucleotides (second nucleic acid sequences) of any portion of the 5' left flanking cotton genomic DNA region in said SEQ ID NO: 3 or its reverse complement. Said nucleic acid sequence may further be homologous or reverse complementary to a portion of said SEQ ID NO:3 comprising said SEQ ID NO:1 or SEQ ID NO:6 in its entirety. When the first nucleic acid sequence and the second nucleic acid sequence are used together, these nucleic acid sequences include DNA primer pairs in a DNA amplification method that produces an amplification product. Transgenic cotton events can be diagnosed when the amplification product produced in a DNA amplification method using a 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 in the presence of 17L397-1 or its progeny.

The SEQ ID NO: 3 is a sequence of 586 nucleotides in length near the insertion junction at the 5' end of the insertion sequence in 17L397-1 in the transgenic cotton event, and the SEQ ID NO: 3 consists of 177 The nucleotides of the cotton left flank genomic DNA sequence (nucleotides 1-177 of SEQ ID NO: 3) and the 5' end DNA sequence of the first expression cassette of the 409-nucleotide glyphosate tolerance gene (SEQ ID NO: 3) ID NO: 3 nucleotides 178-586), including the SEQ ID NO: 3 or its reverse complement can be identified as the existence of 17L397-1 in the transgenic cotton event.

The nucleic acid sequence may be at least 11 or more contiguous polynucleotides (third nucleic acid sequence) of any portion of the transgenic insert in the SEQ ID NO: 4 or its reverse complement, or the SEQ ID NO: 4 or the reverse complement thereof. At least 11 or more contiguous polynucleotides (fourth nucleic acid sequence) of any portion of the 3' right flank cotton genomic DNA region in ID NO: 4 or its reverse complement. Said nucleic acid sequence may further be homologous or reverse complementary to a portion of said SEQ ID NO:4 comprising said SEQ ID NO:2 or SEQ ID NO:7 in its entirety. When the third nucleic acid sequence and the fourth nucleic acid sequence are used together, these nucleic acid sequences include a DNA primer set in a DNA amplification method that produces an amplification product. Transgenic cotton 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 presence of 17L397-1 or its progeny in the event.

The SEQ ID NO: 4 is a sequence of 836 nucleotides in length near the insertion junction at the 3' end of the inserted sequence in 17L397-1 in the transgenic cotton event, and the SEQ ID NO: 4 consists of 134 nucleotides The 3'-end DNA sequence of the second expression cassette of the trait-improving gene of nucleotides (nucleotides 1-134 of SEQ ID NO: 4), the 254-nucleotide DNA sequence of the right border of the pCAMBIA1300/EPSPS-CSRRM2 construct (nucleotides 135-388 of SEQ ID NO: 4) and a 448 nucleotide flanking genomic DNA sequence to the right of the cotton integration site (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 SEQ ID NO: 5 is a sequence that characterizes the length of 17L397-1 in the transgenic cotton event with a length of 4544 nucleotides, and the specific genome and genetic elements included are shown in Table 1. The presence of 17L397-1 in a transgenic cotton event can be identified by the inclusion of said SEQ ID NO: 5 or its reverse complement.

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

1: 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 solely of DNA, but may also include RNA, a mixture of DNA and RNA, or DNA, RNA, or others that do not act as a single or A combination of nucleotides or analogs thereof for multiple polymerase templates. Furthermore, the probes or primers of the present 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 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 nucleotides set forth in SEQ ID NO: 11. When selected from the group consisting of 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 21 in length About 50 or more consecutive nucleotides.

The present invention also provides a method of protecting cotton plants from herbicide-induced damage, comprising applying an effective amount of a glyphosate-containing herbicide to a field planted with at least one transgenic cotton plant, said The transgenic cotton plant sequentially comprises SEQ ID NO: 1, SEQ ID NO: 5 nucleotide sequence 178-3842 and SEQ ID NO: 2 in its genome, or the genome of the transgenic cotton plant comprises SEQ ID NO: 5; The transgenic cotton plants are tolerant to glyphosate herbicide.

The present invention also provides a method for improving the yield of cotton plants, which is characterized by comprising planting at least one transgenic cotton plant, wherein the transgenic cotton plant sequentially comprises SEQ ID NO: 1 and SEQ ID NO: 5 in its genome. The nucleic acid sequence at positions 178-3842 and SEQ ID NO: 2, or SEQ ID NO: 5 is included in the genome of the transgenic cotton plant; the transgenic cotton plant has an improved yield trait.

In the nucleic acid sequence for detecting cotton plants and the detection method thereof of the present invention, the following definitions and methods can better define the present invention and guide those of ordinary skill in the art to implement the present invention, unless otherwise specified, according to ordinary skills in the art People's regular usage to understand the term.

By "cotton" is meant upland cotton (Gossypium Hirsutum L) and includes all plant species with which cotton can mate, including wild cotton species.

The "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, and plants or plant parts intact in plants Cells, such as parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, shoots, fruits, stems, roots, root tips, anthers, and the like. Parts of transgenic plants that are understood to be within the scope of the present invention include, but are not limited to, plant cells, protoplasts, tissues, callus, embryos, as well as flowers, stems, fruits, leaves and roots derived from prior use of the present invention. A transgenic plant or progeny thereof transformed by a DNA molecule and thus consisting at least in part of transgenic cells.

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 found in nature with its own regulatory sequences. A "chimeric gene" refers to any gene that is not a native gene and that contains regulatory and coding sequences that are not found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign gene" is a foreign gene that exists in the genome of an organism and did not originally exist, and also refers to a gene introduced into a recipient cell through a transgenic step. A 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 transformation procedures. The site in the plant genome where recombinant DNA has been inserted may be referred to as the "insertion site" or "target site".

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

Transformation procedures that result in random integration of exogenous DNA will result in transformation events containing distinct flanking regions that are specifically contained in each transformation event. When recombinant DNA is introduced into a plant by conventional crossing, 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 genomic DNA or between two segments of heterologous DNA. A "junction" is the point at which two specific DNA fragments join. For example, junctions exist where the insert DNA joins the flanking DNA. Junctions are also present in transformed organisms, where two DNA fragments are joined together in a manner modified from those found in the native organism. "Conjugated DNA" refers to DNA that contains a junction.

The present invention provides a transgenic cotton event called Zhong 17L397-1 and its progeny, the transgenic cotton event Zhong 17L397-1 is cotton Zhong 17L397-1, including plants and seeds of the transgenic cotton event 17L397-1 and the same Plant cells or regenerable parts thereof, plant parts of 17L397-1 in said transgenic cotton event, including but not limited to cells, pollen, ovules, flowers, shoots, roots, stems, leaves and products from 17L397-1 in cotton, Examples are cottonseed, cottonseed oil, cotton clothing, quilts, batting, cotton cloth, and biomass left in cotton crop fields.

17L397-1 in the transgenic cotton events of the present invention comprises a DNA construct which, when expressed in plant cells, achieves improved yield traits and/or tolerance to glyphosate herbicides . The DNA construct comprises an expression cassette comprising a suitable promoter for expression in plants and a suitable polyadenylation signal sequence operably linked to csRRM2 with cell enlargement function gene, the nucleic acid sequence of the csRRM2 protein can improve cotton yield. The DNA construct comprises another expression cassette comprising a suitable promoter for expression in plants and a suitable polyadenylation signal sequence operably linked to encode 5-enolacetone The gene g10-epsps of acid shikimate-3-phosphate synthase (EPSPS), the nucleic acid sequence of the EPSPS protein, is tolerant to glyphosate herbicide. Further, the promoter can be a suitable promoter isolated from plants, including constitutive, inducible and/or tissue-specific promoters, and the suitable promoters include, but are not limited to, cauliflower mosaic virus (CaMV) 35S Promoter, Scrophulariaceae mosaic virus (FMV) 35S promoter, Ubiquitin promoter, Actin promoter, Agrobacterium tumefaciens nopaline synthase (NOS) promoter, Octopine synthase (OCS) promoter, Cestrum yellow leaf curl virus promoter, potato tuber storage protein (Patatin) promoter, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) promoter, glutathione sulfur transferase (GST) promoter, E9 promoter, GOS promoter, alcA/alcR promoter, Agrobacterium rhizogenes RolD promoter and Arabidopsis thaliana) Suc2 promoter. The polyadenylation signal sequence may be a suitable polyadenylation signal sequence functioning in plants including, but not limited to, derived from Agrobacterium tumefaciens nopal The polyadenylation signal sequence of the alkali synthase (NOS) gene, the 35S terminator derived from the cauliflower mosaic virus (CaMV), the polyadenylation signal sequence derived from the protease inhibitor II (PINII) gene, and the The polyadenylation signal sequence of the 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 can enhance the expression level of the gene, and the enhancer includes, but is not limited to, Tobacco Etching Virus (TEV) translation activator, CaMV35S enhancer and FMV35S enhancer. The signal peptide/transit peptide can direct the transport of EPSPS proteins to specific organelles or compartments outside or within the cell, for example, targeting the chloroplast with a sequence encoding a chloroplast transit peptide, or targeting the endoplasmic reticulum with a 'KDEL' retention sequence.

Cultivated transgenic cotton event 17L397-1 with yield trait-improving characteristics and tolerance to glyphosate herbicide by first sexually crossing a first parent cotton plant with a second parent cotton plant, thereby producing a Diverse first-generation progeny plants consisting of cotton plants grown from 17L397-1 and its progeny in a transgenic cotton event produced by utilizing the present invention A second parental cotton plant lacking the yield trait-improving trait or tolerant to glyphosate herbicide, obtained by transformation of an expression cassette with improved yield traits and tolerance to glyphosate herbicide; Glyphosate-tolerant progeny plants can be bred to glyphosate-tolerant cotton plants. These steps may further include backcrossing the yield trait-improved and glyphosate-tolerant progeny plants with a second parent cotton plant or a third parent cotton plant, followed by application with a glyphosate herbicide or by a trait-related Identification of molecular markers (such as DNA molecules comprising junction sites identified at the 5' and 3' ends of the insert sequence in 17L397-1 in transgenic cotton events) to select progeny resulting in yield trait improvement traits and resistance to grass Glyphosate herbicide tolerant cotton plants.

It is also understood that two different transgenic plants can also be crossed to produce progeny containing two separate, segregatingly added exogenous genes. Selfing of appropriate progeny can result in progeny plants that are homozygous for both added foreign genes. Backcrossing to parental plants and outcrossing to non-transgenic plants as previously described are also contemplated, as is vegetative propagation.

As used in the present invention, a substantially homologous sequence is a nucleic acid molecule that is capable of specifically hybridizing under highly stringent conditions to the complementary strand of another nucleic acid molecule that matches. Suitable stringent conditions to promote DNA hybridization, for example, treatment with 6.0x sodium chloride/sodium citrate (SSC) at approximately 45°C, followed by a wash with 2.0x SSC at 50°C, these conditions are very useful to those skilled in the art. is known. For example, the salt concentration in the wash step can be selected from about 2.0×SSC, 50°C under low stringency conditions to about 0.2×SSC, 50°C under highly stringent conditions. In addition, the temperature conditions in the wash steps can be increased from about 22°C at room temperature for low stringency conditions to about 65°C for high stringency conditions. Both the temperature conditions and the salt concentration can be changed, or one can be kept constant while the other variable. Preferably, a nucleic acid molecule of the invention can interact with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4. One or more nucleic acid molecules in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 or their complementary sequences, or any fragment of the above sequences specifically hybridize. More preferably, a nucleic acid molecule of the present invention interacts with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 under highly stringent conditions Specific hybridization occurs with one or more nucleic acid molecules in SEQ ID NO: 7 or their complementary sequences, or any fragment of the above-mentioned sequences. In the present invention, the preferred marker nucleic acid molecule has 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 its complementary sequence, or any fragment of the above sequence.

Another preferred marker nucleic acid molecule of the present invention is 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 complementary sequence thereof , or any fragment of the above sequence having 80% to 100% or 90% to 100% sequence identity. 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 the progeny of genetic crosses . Hybridization of probes to target DNA molecules can be detected by any method known to those skilled in the art, including, but not limited to, fluorescent labeling, radiolabeling, antibody-based labeling, and chemiluminescent labeling.

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

DNA detection kits can be developed using the compositions described herein and methods described or known in the art of DNA detection. The kit is useful for identifying whether the DNA of 17L397-1 in the transgenic cotton event exists in the sample, and can also be used to breed cotton plants containing the DNA of 17L397-1 in the transgenic cotton event. The kit may contain DNA primers or probes that are 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 , which are homologous to or complementary to DNA contained in transgenic genetic elements of DNA, and these DNA sequences can be used in DNA amplification reactions or as probes in DNA hybridization methods. The DNA structure at the junction of the transgene insert contained in the cotton genome and illustrated in Figure 1 and Table 1 with the cotton genome comprises: the genomic region on the left flank of 17L397-1 in cotton located at the 5' end of the transgene insert, from the An expression cassette consisting of two tandem cauliflower mosaic virus 35S promoters (2×P35S) operably linked to the glyphosate resistance gene sequence (g10-epsps) and operably linked to cauliflower flowers The second expression cassette consists of the 35S promoter (P35S) of the cauliflower mosaic virus, operably linked to the cell enlargement gene csRRM2, and operably linked to nopal Alkaline synthase gene terminator (Tnos), a part of the insert sequence from the right border region (RB) of Agrobacterium, and the right flank genomic region of 17L397-1 in cotton located at the 3' end of the transgene insert sequence (SEQ ID NO: 5). In the DNA amplification method, the DNA molecules used as primers can be derived from any part of the transgene insertion sequence in 17L397-1 in the transgenic cotton event, or can be derived from the DNA region of the flanking cotton genome in 17L397-1 in the transgenic cotton event any part of it.

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

The invention provides a nucleic acid sequence for detecting cotton plants and a detection method thereof. In transgenic cotton events, 17L397-1 has the effects of improving yield traits and tolerance to glyphosate herbicides. Cotton plants of this trait express csRRM2 protein and 5-enolpyruvate shikimate-3-phosphate synthase (EPSPS) protein, which confer improved yield traits and tolerance to glyphosate in plants. Meanwhile, in the detection method of the present invention, SEQ ID NO: 1 or its reverse complement, SEQ ID NO: 2 or its reverse complement, SEQ ID NO: 3 or its reverse complement, SEQ ID NO: 4 or its reverse Amplification to the complementary sequence, SEQ ID NO: 6 or its reverse complement, or SEQ ID NO: 7 or its reverse complement can be used as DNA primers or probes to generate amplification of 17L397-1 or its progeny in events diagnosed as transgenic cotton. The product can be increased rapidly, accurately and stably, and the existence of plant material derived from 17L397-1 in the transgenic cotton event can be identified.

Although the insertion sequence of Zhong 17L397-1 was incomplete and the quality traits were not significantly improved compared with the control, its glyphosate tolerance was better and its yield traits were more prominent. These characteristics make the transformant Zhong 17L397-1 can be used to improve the glyphosate herbicide tolerance and yield traits of cotton, so as to develop new high-yield herbicide-tolerant cotton varieties.

The technical solutions of the present invention will be further described in detail below through the accompanying drawings and embodiments.

Description of drawings

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

Figure 2 Physical map of the recombinant expression vector pCAMBIA1300/EPSPS-CSRRM2. The English and abbreviation meanings of each element are listed as follows:

T-DNA left border sequence of LB Agrobacterium.

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

g10-epsps encodes the EPSPS protein and detoxifies glyphosate.

Ch-L signal peptide

P2 x 35S tandem 35S promoter of cauliflower mosaic virus (CaMV).

Terminator for the Tnos nopaline synthase gene.

CsRRM2 CsRRM2 gene CDS.

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

RB T-DNA right border sequence of Agrobacterium.

PVS1 sta Plasmid stabilization site of the pVS1 plasmid.

The origin of replication of the pVS1 rep pVS1 plasmid.

pBR322 bom bom site of pBR322 plasmid.

pBR322 ori origin of replication of the pBR322 plasmid.

kanamycin(R) encodes an aminoglycoside phosphotransferase protein that confers kanamycin resistance to bacteria.

In Figure 3, the yield traits of the 17L397-1 event and the recipient control C. chinensis 24. Zhong Cotton Institute 24: non-transgenic recipient control cotton plant; Zhong 17L397-1: Zhong 17L397-1 transformation event cotton plant. A is a leaf; B is a young boll; C is a plant; D is an adult boll.

Figure 4. Glyphosate herbicide resistance of the 17L397-1 event and the recipient control in the cotton plant 24. Zhong Cotton Institute 24: non-transgenic recipient control cotton plant; Zhong 17L397-1: Zhong 17L397-1 transformation event cotton plant.

17L397-1 transformation event-specific PCR verification results in FIG. 5 . M: Marker, the size is marked next to it (unit: bp); N: blank control; P: vector pCAMBIA1300/EPSPS-csRRM2; C: genomic DNA of recipient control Zhongqianso 24; T: transformation event containing Zhong17L397-1 of cotton material genomic DNA. A: The expected size of the left border PCR fragment is 295 bp; B: The expected size of the right border PCR fragment is 445 bp.

Figure 6 Southern hybridization digestion and probe positions.

Southern blot hybridization diagram of the inserted copy number of 17L397-1 target gene csRRM2 in Figure 7

A: BamHI enzyme-digested DNA hybridization diagram; B: EcoRI enzyme-digested DNA hybridization diagram; C: Probe position and restriction enzyme cleavage site, the horizontal line indicates the probe position, and the vertical line indicates the enzyme cleavage site.

A1: DNA Marker, the band size is marked next to it, the unit is bp;

A2: BamHI digestion plasmid;

A3: BamHI enzyme cut China Cotton Research Institute 24;

A4: 17L397-1 in BamHI digestion;

B1: DNA Marker, the band size is marked next to it, the unit is bp;

B2: EcoRI digestion plasmid;

B3: EcoRI digestion of China Cotton Research Institute 24;

B4: 17L397-1 digested by EcoRI.

Southern blot hybridization diagram of the inserted copy number of 17L397-1 target gene g10-epsps in Figure 8

A: BamHI enzyme-digested DNA hybridization map; B: EcoRI enzyme-digested DNA hybridization map; C: g10-epsps probe position and restriction enzyme cleavage site, the horizontal line indicates the probe position, and the vertical line indicates the restriction enzyme cleavage site.

A1: BamHI digestion plasmid;

A2: blank control;

A3: BamHI enzyme cut China Cotton Research Institute 24;

A4: blank control;

A5: 17L397-1 in BamHI digestion;

A6: DNA Marker, the band size is marked next to it, the unit is bp;

B1: DNA Marker, the band size is marked next to it, the unit is bp;

B2: EcoRI digestion plasmid;

B3: EcoRI digestion of China Cotton Research Institute 24;

B4: blank control;

B5: 17L397-1 digested by EcoRI.

Southern blot hybridization diagram of inserted copy number of 17L397-1 vector backbone ① region and ④ region in Figure 9

A: The hybridization diagram of DNA digested by BamHI enzyme; B: The hybridization diagram of DNA digested by EcoRI enzyme; C: The probe positions and restriction endonuclease cleavage sites of regions ① and ④.

A1: DNA Marker, the band size is marked next to it, the unit is bp;

A2: BamHI digestion plasmid;

A3: blank control;

A4: BamHI enzyme cut China Cotton Research Institute 24;

A5, A6: 17L397-1 in BamHI digestion;

A7, A8: R1 (the PCR amplification product of the primer in the hybridization region of probe ①);

A9, A10: R4 (the PCR amplification product of the primer in the hybridization region of probe ④);

B1: DNA Marker, the band size is marked next to it, the unit is bp;

B2: EcoRI digested plasmid;

B3: blank control;

B4: EcoRI digestion of China Cotton Research Institute 24;

B5, B6: 17L397-1 in EcoRI digested transformants;

B7: R1 (the PCR amplification product of the primer in the probe ① hybridization region);

B8: R4 (PCR amplification product of the primer in the hybridization region of probe ④).

Figure 10. Southern blot hybridization diagram of inserted copy number in region ② and ③ region of vector backbone

A: BamHI enzyme-digested DNA hybridization map; B: EcoRI enzyme-digested DNA hybridization map; C: Probe positions and restriction endonuclease cleavage sites in the 2 and 3 regions of the backbone.

A1: DNA Marker, the band size is marked next to it, the unit is bp;

A2: plasmid digested with BamHI;

A3: blank control;

A4: BamHI enzyme cut China Cotton Research Institute 24;

A5, A6: 17L397-1 in BamHI digestion;

A7, A8: R2 (the PCR amplification product of the primer in the hybridization region of the probe ②);

A9, A10: R3 (the PCR amplification product of the primer in the hybridization region of the probe ③);

B1: DNA Marker, the band size is marked next to it, the unit is bp;

B2: EcoRI digested plasmid;

B3: blank control;

B4: EcoRI digestion of China Cotton Research Institute 24;

B5, B6: EcoRI digested 17L397-1;

B7: R2 (the PCR amplification product of the primer in the hybridization region of the probe ②);

B8: R3 (PCR amplification product of the primer in the hybridization region of probe ③).

Detailed ways

In the transformation events involved in this application, 17L397-1 refers to cotton inbred with cotton inbred line 24 as the receptor to obtain a cotton with a foreign gene insert (T-DNA insert) inserted between specific genome sequences after genetic transformation. plant. In a specific embodiment, the expression vector used for the transgene has the physical map shown in FIG. 2 , and the obtained 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, or to the T-DNA insert in the genome resulting from this process, or to a combination of T-DNA insert and flanking sequence, or to the Cotton plants obtained from the transgenic process. In a specific example, this event also applies 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 can also refer to progeny plants obtained by vegetative reproduction, sexual reproduction, doubling or doubling reproduction or a combination of the above plants.

Example 1 Acquisition and Character Identification of Transformation Events

csRRM2 gene is the RRM2 domain that controls FCA gene and is closely related to plant quality traits; g10-epsps gene encodes 5-enolpyruvate shikimate-3-phosphate synthase, which can improve plant tolerance to glyphosate herbicide ability. The previously obtained transformation event ICR24-397 (application number: 201811442263.1) has better quality traits than the receptor after being transformed into the above two expression cassettes. The fiber length, specific strength and uniformity of ICR24-397 cotton are all good. has been significantly improved. However, in the identification of herbicide tolerance, nearly 10% of the plants in ICR24-397 suffered phytotoxicity when glyphosate was applied at 2 times the recommended field concentration; When glyphosate was used, nearly 20% of the plants in ICR24-397 showed phytotoxicity. Therefore, the herbicide resistance performance of ICR24-397 is not very satisfactory.

In order to obtain a transformation event with more excellent herbicide tolerance, the present invention uses the pCAMBIA1300/EPSPS-csRRM2 expression vector (see Figure 2 for the physical map of the vector, including the csRRM2 gene expression cassette and the g10-epsps gene expression cassette), through the method mediated by Agrobacterium 13 positive transformed seedlings were obtained by transforming the recipient material Zhongmian Institute 24, and the herbicide resistance and quality-related agronomic traits of these transformed seedlings were screened and identified.

1. Screening of transformants with excellent herbicide resistance

Taking the receptors Zhongtian Institute 24 and transformant ICR24-397 as reference, the transformants with better herbicide tolerance were screened from the above 13 positive transformed seedlings by spraying glyphosate at twice the recommended concentration in the field. . The results showed that only 3 transformation events (Zhong17L397-1, Zhong17L397-2 and Zhong17L397-3) had significantly higher tolerance to glyphosate herbicide than the control, and not lower than the transformant ICR24-397.

Subsequently, the seeds of the above-mentioned four cotton transformants and the receptor control Zhongmian Institute 24 were sown in the test base of the China Cotton Institute in Anyang City, Henan Province, and the Zhong 17L397-1 was systematically identified by spraying different concentrations of glyphosate in the field. Herbicide tolerance of isotransformants. At the same time, the effectiveness of improving the quality traits of transgenic cotton was identified by examining cotton fiber length, uniformity, specific strength and other agronomic traits. Insertion positions were identified and analyzed.

2. Identification of herbicide tolerance

Herbicide tolerance identification was carried out in summer 2018. Glyphosate was sprayed with 1 times (82g/mu), 2 times (164g/mu) and 4 times (328g/mu) of the recommended concentration in the field when there were 4 to 6 true leaves in the cotton seedling stage. All control cotton plants withered yellow and died, the seedling rate was 0, and the phytotoxicity rate was 100%, while the four transformation events were able to grow seedlings normally, but showed different degrees of phytotoxicity rate (Table 2). When spraying glyphosate at twice the recommended concentration in the field, the glyphosate tolerance of Zhong 17L397-1 and Zhong 17L397-2 was significantly higher than that of Zhong 17L397-3 and ICR24-397; When the medium amount of glyphosate was 4 times, the glyphosate tolerance of medium 17L397-1 was significantly higher than that of the other three transformants. Therefore, the glyphosate herbicide resistance of 17L397-1 was the best.

Table 2 Herbicide Tolerance Performance

Values are from the mean ± standard deviation of 3 biological replicates. Statistical analysis uses LSD for multiple comparisons (α=0.05), and different letters indicate significant differences in the same column of data at the same herbicide concentration.

3. Analysis of the insertion positions of Zhong 17L397-1, Zhong 17L397-2 and Zhong 17L397-3 in the genome

100 mg of plant leaves were taken, rapidly ground in liquid nitrogen, and total DNA was extracted by CTAB method. The genomic DNA of the qualified sample is fragmented by ultrasonic wave, and then the fragmented DNA is purified, end repaired, A is added to the 3' end, and the sequencing adapter is connected. Fragment size selection was performed by agarose gel electrophoresis, and PCR amplification was performed to form a sequencing library. Qualified libraries were sequenced using the Illumina nova sequencing platform with a sequencing depth of 10×. The quality of the raw data obtained by sequencing was evaluated, and the filtered sequence Clean data was obtained, and a Perl script was written to convert the fq file of the Clean data into fa format, and then the filtered sequence was compared with the cotton Gh ZM24-CRI v1 reference genome sequence (https:// www.cottongen.org/blast/nucleotide/nucleotide) for alignment. Locate the position of the filtered sequence on the reference genome to obtain the insertion position information of the exogenous insert.

The sequencing data of Zhong 17L397-1, Zhong 17L397-2 and Zhong 17L397-3 were compared with the reference genome and exogenous T-DNA sequences, respectively. ) is aligned with the reference genome sequence, and the other end sequence (Reads) is aligned with the insert sequence; in the second category, a part of either end of the two ends is aligned with the reference genome sequence, and the other part is aligned with the insert sequence. Align the reference genome with Blast and assemble all the reads that align with the foreign insert. According to the assembled Contig, Blast was used to compare the exogenous insert sequence and the reference genome respectively, and the Contig sequence was selected to be aligned to the chromosomal region to obtain the insertion position information of the exogenous insert. Subsequently, forward and reverse primers were designed on the genomic flanking sequences and exogenous insertion sequences at the left and right borders of the insertion site, respectively, and the insertion sites of the three transformation events were verified by PCR amplification. The product was sequenced and analyzed.

The results showed that the exogenous fragments of the three transformants were integrated into different sites in the genome, which were the intergenic regions of the cotton genome and had no known functional genes. In addition, the expression cassettes of the exogenous sequences of Zhong 17L397-2 and Zhong 17L397-3 were intact, while the vector border sequences of the Zhong 17L397-1 transformation event were deleted during insertion, including part of the border sequences and the terminator T35S. Partial sequence (see Table 3 for details). Often, incomplete terminators lead to aberrant transcription and translation of genes, which in turn affects target traits. Unexpectedly, however, from the results of herbicide resistance trait identification, the glyphosate tolerance of the transformation event of Zhong 17L397-1 was significantly higher than that of the recipient control, and was significantly higher than that of ICR24-397, Zhong 17L397-2 and Zhong 17L397-3 transformation. body is more prominent.

Table 3 Insertion position information of exogenous fragments

4. Identification of quality traits

Quality traits such as cotton fiber length, uniformity and specific strength of the transformants such as Zhong 17L397-1 and the recipient control were investigated from cotton boll opening to final harvest. The results showed that the quality traits of cotton fiber length, uniformity and specific strength of Zhong 17L397-1, Zhong 17L397-2 and Zhong 17L397-3 were not significantly different from those of the recipient control Zhongmian Institute 24. , and weaker than ICR24-397.

5. Identification of yield traits

While the quality traits of these transformants were identified, their other traits (such as seed cotton weight, flower bud number, boll number, single boll weight, plant height, first fruit branch node height, fruit node number, The number of fruit branches and leaf size, etc.) were recorded in detail. When conducting data statistics, it was unexpectedly found that the total weight and single boll weight of medium 17L397-1 were not only much higher than the recipient control, but also significantly higher than the other 3 transformants including ICR24-397 (Table 4). 17L397-1's plant height and the height of the first fruit branch at the seedling stage, bud stage, flowering boll stage and boll opening stage were also significantly higher than those of the recipient control (Table 5).

Table 4 Seed cotton yield in boll opening period

Values are from the mean ± standard deviation of 3 biological replicates. Statistical analysis uses LSD for multiple comparisons (α=0.05), and different letters indicate significant differences in the same column data in the same period.

Table 5 Main agronomic characters

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

The above results showed that although the insertion sequence of Zhong 17L397-1 was incomplete and the quality traits were not significantly improved compared with the control, its glyphosate tolerance was better and its yield traits were more prominent. These characteristics make the transformant Zhong 17L397-1 can be used to improve the glyphosate herbicide tolerance and yield traits of cotton, so as to develop new high-yield herbicide-tolerant cotton varieties.

Example 2 Molecular Characterization of 17L397-1 in Transformation Events

In order to further clarify the identity characteristics of 17L397-1 in the transformation event, the present invention analyzes the flanking sequence and the inserted copy number of the insertion site of the exogenous sequence 17L397-1 in cotton genome.

1. Analysis of flanking sequences of foreign sequences in the cotton genome

As described in Example 1, the exogenous T-DNA sequence of 17L397-1 in the transformation event was inserted forward into the cotton genome at position Chr A11:7085972 or Chr D11:6406857. At the left border of the insertion site, cut off 500 bp upstream of the insertion site on the genome and 500 bp on the T-DNA sequence, and on the right border, take the 500 bp downstream of the genomic insertion site and 500 bp on the T-DNA sequence, and use the Primerblast software of the NCBI website ( https://blast.ncbi.nlm.nih.gov/Blast) designed primers for the cut sequence, and the amplified product fused a part of the cotton genome sequence and a part of the T-DNA sequence.

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

Based on the results of the flanking sequence and insertion position, amplification was performed using the genome upstream primer (SEQ ID NO:8) with the vector left border primer (SEQ ID NO:9) and the vector right border primer (SEQ ID NO:10) with the genome downstream primer ( SEQ ID NO: 11) PCR amplification of the 17L397-1 transformation event was performed to verify the insertion position of the exogenous fragment. The results are shown in Figure 5. The results showed that the exogenous fragment of Zhong17L397-1 was stably inserted into the cotton genome at Chr A11:7085972 or Chr D11:6406857, and the size of the inserted sequence was 3919bp.

By analyzing the border sequences on the left and right flanks, it can be seen that the insertion of the foreign sequence resulted in the deletion of 89 bp of the receptor genome, and the vector sequence was also deleted by 131 bp. The missing vector sequence includes part of the border sequence and part of the terminator T35S.

2. Insertion copy number analysis of foreign sequences

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

Southern hybridization selects two enzymes BamHI and EcoRI to digest the positive control plasmid, the acceptor control Zhongmian Institute 24 and Zhong 17L397-1 genomic DNA, and design probes covering the target gene and the full coverage of the vector backbone sequence, probes and restriction sites The schematic diagram is shown in Figure 6. The specific sequences of the probe primers are shown in Table 6.

Table 6 Southern hybridization test probe amplification primer positions and sequences

1: Unit bp.

Insertion copy number hybridization detection of the target gene csRRM2 Two restriction enzymes BamHI and EcoRI were selected to digest the positive control plasmid, the negative control genomic DNA of China Cotton Institute 24 and the genomic DNA of China 17L397-1 respectively. After the gel was transferred to the membrane, it was labeled with the csRRM2 gene probe, and the hybridization results were shown in Figure 7A and B. The probe position of the exogenous gene csRRM2 and the restriction endonucleases BamHI and EcoRI are shown in Figure 7C. From the hybridization results, the csRRM2 gene was inserted into the cotton genome as a single copy.

Insertion copy number hybridization detection of the target gene g10-epsps Two restriction enzymes BamHI and EcoRI were selected to digest the positive control plasmid, the negative control genomic DNA of Zhongmian Institute 24 and the genomic DNA of Zhong 17L397-1 transformant respectively. After the gel was transferred to the membrane, it was labeled with the g10-epsps gene probe, and the hybridization results were shown in Figure 8A and B. The probe position of the target gene g10-epsps and the restriction endonucleases BamHI and EcoRI are shown in Figure 8C. The g10-epsps gene was also inserted into the cotton genome as a single copy.

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

The detection method of 17L397-1 in the transformation event of embodiment 3

17L397-1 can be bred from the transgenic cotton event, and the bred new variety can be used to produce products such as agricultural products or commodities. The agricultural product or commodity is expected to contain a nucleotide sequence capable of diagnosing the presence of the 17L397-1 material in the agricultural product or commodity in the event of a transgenic cotton if a sufficient amount is detected in the agricultural commodity or commodity. The agricultural product or commodity includes, but is not limited to, cottonseed oil, cotton wool, quilts, cotton cloth, cotton clothing, and any other food products that are to be consumed by animals as a food source, or otherwise used for cosmetic purposes as leavening agents or ingredients in cosmetic compositions, etc. . Nucleic acid detection methods and/or kits based on probe or primer pairs can be developed to detect the 17L397-1 nucleotide sequence in a transgenic cotton event such as set forth 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, for example, 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 The sequences shown in to diagnose the presence of 17L397-1 in transgenic cotton events.

One of the detection methods is: using PCR method to detect the specific border sequences in the two Zhong 17L397-1 plants, and the PCR primer pairs used are SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 respectively. , SEQ ID NO: 11, PCR reaction system:

The reaction procedure is:

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

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

To sum up, 17L397-1 in the transgenic cotton event of the present invention can increase the yield of cotton bolls and has a high tolerance to glyphosate herbicide, and the detection method can accurately and quickly identify whether the biological sample contains the transgenic cotton event. DNA molecule of 17L397-1.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be The solutions can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

sequence listing

<110> Cotton Research Institute, Chinese Academy of Agricultural Sciences

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

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<213> Synthesis (unknown)

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ggaattagaa attttattga tagaagtatt ttacaaatac aaatacatac taagggtttc 240

ttatatgctc aacacatgag cgaaacccta taggaaccct aattccctta tctgggaact 300

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aactgagggt aggactttct gatgtggtgt gcaccggtga ttctaagtgg agcatctgct 480

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cctttccttt atcgcaatga tggcatttgt aggagccacc ttccttttcc actatcttca 60

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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 tgatttttttt 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

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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 aatatata 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

accacacatcag 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 (9)

1. a nucleic acid molecule, is characterized in that, comprises following any one: i) comprising the sequence shown in SEQ ID NO: 1 and/or SEQ ID NO: 2, or its reverse complement; ii) comprising the sequence shown in SEQ ID NO:3 and/or SEQ ID NO:4, or its reverse complement; iii) comprising the sequence shown in SEQ ID NO:6 and/or SEQ ID NO:7, or its reverse complement; iv) comprising the sequence shown in SEQ ID NO: 5, or its reverse complement. 2. a probe for detecting cotton transformation events, characterized in that, comprising SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: The sequence shown in 7 or a fragment or variant thereof or its reverse complement. 3. the primer pair that is used to detect cotton transformation event, it is characterised in that the amplification product of described primer pair comprises the sequence described in claim 2; Optionally, the primer pair is the sequence shown in SEQ ID NO:8 and SEQ ID NO:9; or the sequence shown in SEQ ID NO:10 and SEQ ID NO:11. 4. A kit or microarray for detecting cotton transformation events, characterized in that it comprises the probe according to claim 2 and/or the primer pair according to claim 3. 5. the method for detecting cotton transformation event, is characterized in that, comprises and utilizes any one of following to detect whether there is described transformation event in test sample: i) the probe of claim 2; ii) the primer pair of claim 3; iii) the probe described in claim 2 and the primer pair described in claim 3; iv) The kit or microarray of claim 4. 6. the method for breeding cotton, it is characterised in that the method comprises the following steps: 1) obtaining cotton comprising the nucleic acid molecule of claim 1; 2) Cotton plants, seeds, plant cells, progeny plants or plant parts are obtained from the cotton obtained in step 1) through pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or hybridization or a combination of the above ; and optionally, 3) Evaluate yield traits and/or identify herbicide resistance on the progeny plants obtained in step 2), and use the method of claim 5 to detect the presence or absence of the transformation event therein. 7. Articles made from cotton plants, seeds, plant cells, progeny plants or plant parts obtained by the method of claim 6, including food, feed or industrial raw materials. 8. A method of protecting cotton plants from herbicide-induced damage, comprising applying an effective dose of a glyphosate herbicide to a field planted with at least one transgenic cotton plant, the transgenic cotton plant The genome of the transgenic cotton plant comprises SEQ ID NO: 1, SEQ ID NO: 5, the nucleotide sequence 178-3842 and SEQ ID NO: 2 in sequence, or the genome of the transgenic cotton plant comprises SEQ ID NO: 5; the transgenic Cotton plants are tolerant to glyphosate herbicide. 9. A method for improving yield traits of cotton plants, comprising planting at least one transgenic cotton plant, wherein the transgenic cotton plant sequentially comprises SEQ ID NO: 1, SEQ ID NO: 5, 178-178 in its genome The nucleic acid sequence at position 3842 and SEQ ID NO: 2, or SEQ ID NO: 5 is included in the genome of the transgenic cotton plant; the transgenic cotton plant has significantly and stably improved yield traits.

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