CN109929940B - Nematode resistant cotton transformation event GHM3 - Google Patents

Abstract

The application provides an anti-nematode cotton transformation event GHM3, and provides a related creation method, a detection method and application, belonging to the technical field of plant biology. Specifically, cotton plant with exogenous gene insert inserted between specific genome sequences is obtained by using cotton variety material YZ-1 as receptor through transgenosis, wherein the exogenous gene insert comprises nematode-resistant gene. According to the transformation event GHM3 obtained by the application, the inserted exogenous gene is located at a non-functional site of a cotton genome, so that the expression of other genes of a plant is not influenced, and the transgenic cotton plant can obtain insect-resistant characteristics and maintain good agronomic characters.

Description

Nematode resistant cotton transformation event GHM3

Technical Field

The application relates to the technical field of plant biology, in particular to a creation method, a detection method and application of a nematode-resistant cotton transformation event.

Background

Cotton, as a special commercial crop, is easily affected by external factors during the planting process. Besides the influences of factors such as temperature, humidity and fertilization, the cotton is easily affected by plant diseases and insect pests in the planting process, if necessary plant disease and insect pest prevention measures are not taken in time, the overall yield of cotton is affected, and meanwhile, the cotton planting is adversely affected.

Cotton diseases are divided into two major categories, infectious diseases and non-infectious diseases. Wherein the infectious disease is caused by pathogenic organisms, such as fungi, bacteria, nematodes, viruses, and the like. Although the method of spraying pesticide can be adopted to prevent the cotton pests, pesticide residue has certain hidden trouble on the safety of cotton products, so that the development of disease and pest resistant cotton varieties is the basis for preventing the insect pest outbreak from roots.

The transgenic technology can be applied to the development of cotton varieties, so that the cotton has the insect-resistant constitution, and is a powerful means for preventing and controlling insect pests which is popular at present. Insect-resistant genes for cotton are mainly against lepidopteran pests and homopteran insects, such as endotoxin protein genes of Bacillus thuringiensis (Bt), Cowpea Trypsin Inhibitor genes (CpTI), and lectin genes (1 lectin). For nematodes that harm cotton in the soil, there is still a need to develop effective transgenic resistant varieties.

Disclosure of Invention

In one aspect, the present application provides a nucleic acid molecule which is: i) comprises the sequence shown by nucleotides 374-1036 and/or nucleotides 3657-4356 of SEQ ID NO. 1, or a fragment or a variant or a complementary sequence thereof; ii) comprises the sequences shown at nucleotides 374-1036 and 3335-3722 of SEQ ID NO 1, or a fragment or variant thereof or the complement thereof; iii) comprises the sequences indicated by nucleotides 3335-3722 and 3657-4356 of SEQ ID NO 1 or a fragment or a variant or a complement thereof; or iv) comprises the sequence shown at nucleotides 374-1036, 3335-3722 and 3657-4356 of SEQ ID NO. 1, or a fragment or variant or complement thereof.

In one embodiment, the nucleic acid molecule provided herein comprises the sequence shown in SEQ ID NO. 1, or a fragment or variant thereof or the complement thereof.

In another embodiment, the nucleic acid molecules provided herein comprise an expression cassette for expression of an anti-nematode gene as represented by nucleotide numbers 2482-3991 of SEQ ID NO: 1.

In yet another embodiment, the nucleic acid molecule provided herein is obtained by introducing an expression cassette expressing an anti-nematode gene as depicted in nucleotides 2482-3991 of SEQ ID NO:1 into the genome of cotton.

In another embodiment, the nucleic acid molecules provided herein are present in a cotton plant, seed, plant cell, progeny plant or plant part.

In another aspect, the present application provides a probe for detecting a cotton transformation event comprising the sequence shown at nucleotides 374-1036 or 3657-4356 of SEQ ID NO. 1, or a fragment or variant or complement thereof.

In yet another aspect, the present application provides a primer pair for detecting a cotton transformation event, which is capable of specifically amplifying to produce a sequence comprising nucleotides 374-1036 or 3657-4356 of SEQ ID NO. 1, or a fragment or variant thereof or a complement thereof.

In one embodiment, the primer pairs provided herein for detecting a cotton transformation event are: i) a primer pair which specifically recognizes the sequence shown by the 374-1036 nucleotide of SEQ ID NO. 1; ii) a primer pair which specifically recognizes a sequence comprising the sequence shown by nucleotides 3657-4356 of SEQ ID NO. 1; iii) a forward primer which specifically recognizes a sequence comprising the sequence indicated by nucleotides 374 and 1036 of SEQ ID NO. 1 and a reverse primer which specifically recognizes a sequence comprising the sequence indicated by nucleotides 3335 and 3722 of SEQ ID NO. 1; or iv) a forward primer specifically recognizing the sequence comprising nucleotides 3335 and 3722 of SEQ ID NO. 1 and a reverse primer specifically recognizing the sequence comprising nucleotides 3657 and 4356 of SEQ ID NO. 1.

In another embodiment, the primer pair provided herein is the nucleotide sequence shown in SEQ ID No. 10 and SEQ ID No. 11 or the complementary sequence thereof; or the nucleotide sequences shown in SEQ ID No. 12 and SEQ ID No. 13 or the complementary sequences thereof.

In addition, the present application also provides a kit or microarray for detecting cotton transformation events comprising the above-described probe and/or the above-described primer pair.

In yet another aspect, the present application provides a method for detecting a cotton transformation event, comprising detecting the presence or absence of the transformation event in a test sample using: the above-mentioned probe; the above-mentioned primer pair; the above-mentioned probe and the above-mentioned primer pair; or a kit or microarray as described above.

The present application also provides a method of breeding cotton, the method comprising the steps of: 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 progeny plants, seeds, plant cells, progeny plants or plant parts; and optionally, 3) subjecting the progeny plant obtained in step 2) to nematode resistance identification and detecting the presence or absence of said transformation event therein using the method described above.

Further, the present application also provides cotton plants, seeds, plant cells, progeny plants or plant parts, etc. obtained by the above-described methods, as well as articles made from these cotton plants, seeds, plant cells, progeny plants or plant parts, etc., including industrial feedstocks and the like.

Drawings

FIG. 1 is a schematic diagram of the structure of a transformation vector pZZ00074, in which English and various abbreviations are as follows:

omega: omega sequence, enhancer

Kozak sequence: kozak sequence, enhancer

Cystatin: optimized Cystatin gene sequence

poly A: polyadenosine sequence

T-NOS: nopaline synthase terminator

T-BORDER (right): T-DNA right border sequence

CaMV35S promoter: cucumber mosaic virus 35S promoter

CaMV35S polyA: cucumber mosaic virus 35S polyadenylation sequence, terminator

T-BORDER (left): left border sequence of T-DNA

Bp: base pairing

kanamasin (r): kanamycin resistance sequence

pBR322 ori: pBR322 initiation region sequence

pBR322 born: pBR322 framework region sequence

pVS1 rep: pVS1 replicon

pVS1 sta: pVS1 transcriptional initiation region

Fig. 2 is a graph of the results of Southern blot detection of cotton transformation event GHM3 genomic DNA wherein: lane 1(GHM3) is transformation event GHM 3; lane 2(YZ-1) is a non-transgenic plant negative control; lane 3(P) is a positive plasmid control; lane 4(M) is a molecular weight marker.

FIG. 3 shows the result of detection of the insertion site of cotton transformation event GHM3 and a photograph of DNA electrophoresis, wherein: lane 1(M) is a molecular weight marker; lanes 2 and 5(WT) are wild type plant DNA template PCR results; lane 3 (ddH) ₂ O) and lane 6 (ddH) ₂ O) are blank template PCR results respectively; lanes 4(L) and 7(R) are PCR results using different primer pairs with transformation event GHM3 plant DNA template, respectively.

Detailed Description

The following definitions and methods are provided to better define the present application and to guide those of ordinary skill in the art in the practice of the present application. Unless otherwise indicated, terms are to be understood in accordance with their ordinary usage by those of ordinary skill in the relevant art. All patent documents, academic papers, industry standards and other publications, etc., cited herein are incorporated by reference in their entirety.

It is well known to those skilled in the art that expression of foreign genes in plants has a positional effect, i.e., is influenced by the location of insertion into the chromosome, which may be due to transcriptional regulatory elements near the chromosomal structure or integration site. Therefore, it is usually necessary to produce hundreds of different transformation events and to screen out excellent transformation events with desired exogenous gene expression levels and patterns for commercial production applications.

An excellent transformation event can be achieved by transferring exogenous genes into germplasm of other genetic backgrounds by means of conventional breeding methods, i.e., sexual crosses, whose progeny retain the transgene expression characteristics of the original transformants.

The present application relates to superior cotton transformation event GHM3 by screening out from a number of transformation events.

In this application, "transformation event GHM 3" refers to a cotton plant transgenic for cotton variety material YZ-1 as recipient to obtain a foreign gene insert (T-DNA insert) inserted between specific genomic sequences, wherein the foreign gene insert comprises a nematode-resistant gene. According to the transformation event GHM3 obtained by the application, the inserted exogenous gene is located at a non-functional site of a cotton genome, so that the expression of other genes of a plant is not influenced, and the transgenic cotton plant can obtain insect-resistant characteristics and maintain good agronomic characters.

In a specific example, the T-DNA insert obtained after the transgene has the sequence shown by nucleotides 512 and 4016 of SEQ ID NO: 1. Transformation event GHM3 may refer to this transgenic process, it may also refer to the T-DNA insert within the genome resulting from this process, or the combination of a T-DNA insert and flanking sequences, or it may refer to the cotton plant resulting from this transgenic process. Transformation event GHM3 may also refer to progeny plants resulting from vegetative, sexual, doubling or doubling of the above plants or a combination thereof.

In other embodiments, the event is also applicable to plants obtained by transforming other plant recipient varieties with the same foreign gene (sequence shown in nucleotides 512-4016 of SEQ ID NO: 1) to insert the T-DNA insert into the same genomic position. Suitable plants include dicotyledonous plants such as soybean, peanut, sunflower, and the like.

In the present application, a T-DNA insert was obtained with nucleotides 1 to 511 of SEQ ID NO:1 as the left flank sequence and nucleotides 4017-4517 of SEQ ID NO:1 as the right flank sequence (nucleotides 512-4016 of SEQ ID NO: 1). The flanking sequences are not limited to nucleotides 1-511 and 4017-4517 of SEQ ID NO. 1, and since the flanking sequences are listed only to indicate the position of the T-DNA insert in the genome, i.e., the insertion site of the T-DNA insert is located on chromosome 9 90496812 of cotton, the flanking sequences of the present application may be extended bilaterally depending on the genome sequence.

Since transformation event GHM3 produces a T-DNA insert that is inserted into a specific site in the genome, its insertion site is specific and can be used to detect whether a biological sample contains transformation event GHM 3. In particular embodiments, any sequence comprising the junction site of the T-DNA insert of transformation event GHM3 with the flanking sequences may be used to detect transformation event GHM3 of the present application, including, but not limited to, one or more of the following sequences or fragments thereof or variants thereof or complements thereof comprising an upstream insertion site (the junction site of the left flanking sequence with the T-DNA insert) or a downstream insertion site (the junction site of the right flanking sequence with the T-DNA insert): i) comprises the sequence shown as nucleotides 374 and 1036 of SEQ ID NO. 1; ii) comprises the sequence shown as nucleotides 1 to 1036 of SEQ ID NO. 1; iii) comprises the sequence indicated by nucleotides 3335 and 3722 of SEQ ID NO. 1; iv) comprises the sequence shown as nucleotide 512-4016 of SEQ ID NO. 1; v) comprises the sequence shown as nucleotide 3657-4356 of SEQ ID NO. 1; vi) comprises the sequence shown by nucleotides 3657-4517 of SEQ ID NO. 1; vii) comprises the sequence shown as nucleotide 374 and 4356 of SEQ ID NO. 1; viii) comprises the sequence shown in SEQ ID NO 1.

In specific examples, sequences useful for detecting the transformation event GHM3 of the present application are sequences comprising an upstream insertion site or fragments or variants thereof or complements thereof, such as the sequence shown as nucleotides 374-1036 of SEQ ID NO:1 or the sequence shown as nucleotides 1-1036 of SEQ ID NO:1, or sequences comprising a downstream insertion site, e.g., the sequence shown as nucleotides 3657-4356 of SEQ ID NO:1 or the sequence shown as nucleotides 3657-4517 of SEQ ID NO:1, or a combination of a sequence comprising an upstream insertion site and a sequence comprising a downstream insertion site.

In another example, sequences useful for detecting the transformation event GHM3 of the present application are a combination of a sequence comprising an upstream insertion site or a fragment thereof or a variant thereof or a complement thereof and a sequence comprising a T-DNA insert or a fragment thereof or a variant thereof or a complement thereof, e.g., a combination of a sequence comprising nucleotides 374-1036 of SEQ ID NO:1 or a sequence comprising nucleotides 1-1036 of SEQ ID NO:1 and a sequence comprising nucleotides 3335-3722 of SEQ ID NO:1 or a sequence comprising nucleotides 512-4016 of SEQ ID NO: 1.

In another example, sequences useful for detecting the transformation event GHM3 of the present application are combinations of a sequence comprising a downstream insertion site or a fragment thereof or a variant thereof or a complement thereof with a sequence comprising a T-DNA insert or a fragment thereof or a variant thereof or a complement thereof, e.g., a sequence comprising nucleotide 3657-4556 of SEQ ID NO:1 or a sequence comprising nucleotide 3657-4517 of SEQ ID NO:1 in combination with a sequence comprising nucleotide 3335-3722 of SEQ ID NO:1 or a sequence comprising nucleotide 512-4016 of SEQ ID NO: 1.

In another example, the sequence that can be used to detect the transformation event GHM3 of the present application is a sequence comprising nucleotide 374-4356 of SEQ ID NO. 1 or a fragment thereof or a variant thereof or a complement thereof, or a sequence comprising nucleotide 374-4356 of SEQ ID NO. 1 or a fragment thereof or a variant thereof or a complement thereof.

Thus, primer pairs, probes, and combinations of primer pairs and probes that are capable of specifically detecting the junction site of the T-DNA insert of transformation event GHM3 with the flanking sequences can be used to detect transformation event GHM3 of the present application.

As used herein, "nucleotide sequence" includes reference to deoxyribonucleotide or ribonucleotide polymers in either single-or double-stranded form, and unless otherwise limited, nucleotide sequences are written from left to right in the 5 'to 3' direction.

In some embodiments, the present application also relates to fragments of nucleic acid sequences, which refer to portions of smaller fragments that are incomplete in the complete portion. For example, SEQ ID NO:1 comprises SEQ ID NO:1, at least about 10 nucleotides, at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, or at least about 50 nucleotides of the complete sequence or more.

In some embodiments, changes may be made to the nucleic acid sequences of the present application to make conservative amino acid substitutions. In certain embodiments, substitutions that do not alter the amino acid sequence of a nucleotide sequence of the present application can be made in accordance with dicot codon preferences, e.g., codons encoding the same amino acid sequence can be substituted with dicot preferred codons without altering the amino acid sequence encoded by the nucleotide sequence. In some embodiments, the present application also relates to variants of the nucleic acid sequences. Generally, variants of a particular nucleic acid fragment will have at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or more sequence identity, or a complement thereof, to the particular nucleotide sequence. Such variant sequences include additions, deletions or substitutions of one or more nucleic acid residues, which may result in the addition, removal or substitution of the corresponding amino acid residue. Sequence identity is determined by sequence alignment programs known in the art, including hybridization techniques. Nucleotide sequence variants of the embodiments may differ from the sequences of the present application by as little as 1-15 nucleotides, as little as 1-10 (e.g., 6-10), as little as 5, as little as 4, 3, 2, or even 1 nucleotide.

As used herein, a "probe" is an isolated polynucleotide, complementary to a strand of a target polynucleotide, to which is attached a conventional detectable label or reporter molecule, such as a radioisotope, ligand, chemiluminescent agent or enzyme.

In a specific embodiment, the DNA probe provided herein for detecting the transformation event GHM3, comprises a DNA sequence comprising SEQ ID NO:1 or a sequence that is fully complementary thereto, which DNA probe hybridizes under stringent hybridization conditions to a nucleotide sequence comprising an upstream insertion site or a downstream insertion site and does not hybridize under stringent hybridization conditions to a nucleotide sequence not comprising an upstream insertion site or a downstream insertion site.

In specific examples, the probes provided herein comprise the sequence shown at nucleotide 374-1036 or 3657-4356 of SEQ ID NO. 1, or a fragment or variant thereof or the complement thereof. As used herein, a "primer" is an isolated polynucleotide that anneals to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then stretches along the target DNA strand by means of, for example, a DNA polymerase. Primer pairs are directed to their target polynucleotide amplification use, for example, by Polymerase Chain Reaction (PCR) or other conventional nucleic acid amplification methods.

In particular embodiments, the primer pair for detecting transformation event GHM3 provided herein comprises a first DNA molecule and a second DNA molecule different from the first DNA molecule, wherein the first and second DNA molecules each comprise SEQ ID NO:1 or the complete complement thereof, and wherein the first DNA molecule is present in SEQ ID NO:1 and the second DNA molecule are present in SEQ ID NO:1 which when used in conjunction with DNA from the transformation event GHM3 in an amplification reaction, generates an amplicon for detecting the transformation event GHM3 DNA in a sample, and wherein the amplicon comprises the sequence shown at nucleotide 374-1036 or nucleotide 3657-4356 of SEQ ID No. 1, or a fragment or variant or complement thereof.

In a specific embodiment, the primer pair provided herein is a primer pair that specifically recognizes a sequence comprising nucleotides 374-1036 or 1-1036 of SEQ ID NO. 1.

In a specific embodiment, the primer pair provided herein is a primer pair that specifically recognizes a sequence comprising nucleotides 3657-4356 or 3657-4517 of SEQ ID NO. 1.

In specific embodiments, the primer pairs provided herein are: i) a primer pair which specifically recognizes the sequence shown by nucleotides 374-1036 or 1-1036 of SEQ ID NO. 1; and ii) a primer pair which specifically recognizes the sequence shown by the nucleotides 3657-4356 or 3657-4517 of SEQ ID NO. 1; alternatively, the primer pair comprises: a forward primer which specifically recognizes the sequence shown by nucleotides 374-1036 or 1-1036 of SEQ ID NO. 1, and a reverse primer which specifically recognizes the sequence shown by nucleotides 3657-4356 or 3657-4517 of SEQ ID NO. 1.

In another embodiment, the primer pairs provided herein are: i) a primer pair which specifically recognizes the sequence shown by nucleotides 374-1036 or 1-1036 of SEQ ID NO. 1; and ii) a primer pair which specifically recognizes the sequence shown by the nucleotides 3335-3722 or 512-4016 of SEQ ID NO. 1; alternatively, the primer pair comprises: a forward primer which specifically recognizes the sequence shown by nucleotides 374 and 1036 or 1 to 1036 of the SEQ ID NO. 1, and a reverse primer which specifically recognizes the sequence shown by nucleotides 3335 and 3722 or 512 and 4016 of the SEQ ID NO. 1.

In another embodiment, the primer pairs provided herein are: i) a primer pair which specifically recognizes the sequence shown by the 3335-3722 th site or the 512-4016 th site of the SEQ ID NO. 1; and ii) a primer pair which specifically recognizes the sequence shown by the nucleotides 3657-4356 or 3657-4517 of SEQ ID NO. 1; alternatively, the primer pair comprises: a forward primer which specifically recognizes the sequence represented by nucleotides 3335-3722 or 512-4016 of SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence represented by nucleotides 3657-4356 or 3657-4517 of SEQ ID NO. 1.

In another embodiment, the primer pairs provided herein are: i) a primer pair which specifically recognizes the sequence shown by nucleotides 1036 374-1036 or 1-1036 of SEQ ID NO. 1, ii) a primer pair which specifically recognizes the sequence shown by nucleotides 3335-3722 or 512-4016 of SEQ ID NO. 1, and iii) a primer pair which specifically recognizes the sequence shown by nucleotides 3657-4356 or 3657-4517 of SEQ ID NO. 1.

In another embodiment, the primer pair provided herein is a primer pair that specifically recognizes a sequence comprising SEQ ID NO. 1.

In a specific example, the primer pair is a nucleotide sequence shown as SEQ ID No. 10 and SEQ ID No. 11 or a complementary sequence thereof; or the nucleotide sequences shown in SEQ ID No. 12 and SEQ ID No. 13 or the complementary sequences thereof.

Methods of designing and using primers and probes are well known in the art, for example, in Joseph Sambrook, Molecular Cloning: a Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001) and Current Protocols in Molecular Biology, Wiley-Blackwell.

As used herein, "kit" or "microarray" refers to a set of reagents or chips for the purpose of identification and/or detection of cotton transformation event GHM3 in a biological sample. For the purpose of quality control (e.g. purity of seed lot), detection of event GHM3 in or comprising plant material or material derived from plant material, such as but not limited to food or feed products, kits or chips may be used and the components thereof may be specifically adjusted.

In particular embodiments, a kit or probe provided herein includes any one of the probes or any one of the primer pairs provided herein. In another specific embodiment, a kit or probe provided herein comprises any one of the probes provided herein or a combination of any one of the primer pairs.

In addition, transgenic cotton plants, progeny, seeds, plant cells or plant parts and preparations thereof, including but not limited to industrial feedstocks, are provided. The plants, progeny, seeds, plant cells, plant parts, and preparations thereof all comprise a nucleic acid molecule sequence that is detectable as a site of engagement of the T-DNA insert provided herein with the flanking sequences.

Further, the present application also provides a method of breeding cotton comprising the steps of: 1) obtaining cotton comprising a nucleic acid molecule sequence comprising a junction site of a T-DNA insert provided herein with flanking sequences; 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 progeny plants, seeds, plant cells, progeny plants or plant parts; and optionally step 3), performing nematode-resistant identification on the cotton plant obtained in step 2), and detecting the presence or absence of transformation event GHM3 therein using a probe, primer pair, kit or array provided herein.

In addition, the present application provides methods of controlling or killing nematodes in a field.

In a specific embodiment, the methods of controlling or killing nematodes provided herein comprise feeding to said nematodes an effective amount of a cotton plant of transformation event GHM 3. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, steps or conditions of the present invention may be made without departing from the spirit and substance of the invention and are intended to be included within the scope of the present application.

Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular cloning Manual of Sambrook et al (Sambrook J & Russell DW, Molecular cloning: a laboratory Manual,2001), or following the conditions suggested by the manufacturer's instructions.

Unless otherwise specified, the chemical reagents used in the examples are conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

The cotton variety material YZ-1 according to the following examples was widely used for genetic transformation of cotton due to its high callus induction ability, and was originally provided by Zhang Long teacher of China university of agriculture and was self-bred by Chinese seed group.

Examples

Example 1Acquisition and molecular detection of cotton transformation event GHM3

1. Gene optimization

Cotton codon preference optimization of the coding sequence of the cystatin gene (Roderick, H., Tripathi, L., Babirye, A., Wang, D., Tripathi, J., Urwin, P.E. & Atkinson, H.J. (2012) Generation of transgenic Plant (Musa spp.) with resistance to Plant parasitism protocols 13:842 @. 851.) was performed using Vector NTI software, and an expression enhancing element omega sequence (Daniel R.Gallie. the 5-leader of tobaco molar virus proteins transformation through nucleic acid modified vector of eIF4F. nucleic Acids Res.2002Aug 1; 30(15): 3401-3411) and a Kozak sequence (Kozak M. Position details a sequence transforming the AUG initiator code molecules. cell.1986Jan 31; 44(2):283-92.) are added to the 5' end, the modified DNA sequence is named MCgaymes, the sequence is shown as SEQ ID NO:1, 3335-3722 nucleotide, wherein 1-68bp is an omega sequence; 69-76bp is a Kozak sequence; 77-388bp is cystatin gene coding sequence.

2. Vector construction

The omega Cys gene (5 'end with BamHI enzyme cutting site, 3' end with SacI enzyme cutting site) was synthesized artificially.

The omegaMCYS fragment and the expression vector pBI121 were treated with the restriction enzymes BamHI and SacI. The treated omegamCys fragment was inserted between the BamHI and SacI sites of the vector pBI121(NCBI accession No. AF485783) and composed of the expression cassette PCaMV35s-omega MCYS-Tnos with the original CaMV35s promoter and NOS terminator on the vector, the sequence of which is shown in SEQ ID NO:1 at nucleotide 2482 and 3991.

The PCaMV35s-omega MCYS-Tnos expression cassette on pBI121 was excised using restriction enzymes HindIII and EcoRII, and then inserted into pUC19(Construction of improved M13 vectors using oligodeoxynucleotides-directed mutagenesis. Norrander J, Kempe T, Messing J. Gene.1983 Dec; 26(1):101-6.10.1016/0378-1119(83)90040-9) multiple cloning site, which was named pZZ 01108.

Carrying out EcoRI single enzyme digestion treatment pZZ 01108; T4-DNA polymerase fills in the sticky end generated by enzyme digestion, and the sticky end is purified and recovered; the recovered linear plasmid was treated with HindIII to obtain a PCaMV35s-omega MCYS-Tnos fragment.

The pCambia3300 vector carrying the P35S-NPTII-T35spolyA element was treated with restriction enzymes HindIII and PmeI, and the pCambia3300 vector was ligated to the treated PCaMV35s-omega MCYS-Tnos fragment at the cut to obtain a transformation vector containing the two expression elements P35S-NPTII-T35spolyA and PCaMV35s-omega MCYS-Tnos, named pZZ00074, which was successfully constructed for the first time in the present invention and the structural diagram of which is shown in FIG. 1.

3. Genetic transformation

The transformation vector pZZ00068 is transferred into agrobacterium EHA105 by an electric shock method, and agrobacterium is utilized to mediate cotton genetic transformation, and the specific operation is as follows.

1) And (3) planting aseptic seedlings:

cotton seeds were dehulled, sterilized with 75% alcohol for 30 seconds, then surface sterilized with 20% bleached aqueous solution for 30 minutes, then rinsed 4-5 times with sterile water and seeded in the seedling medium (1/2MS + 1.5% glucose + 0.3% phytagel, pH adjusted to 5.9) and cultured at 28 ℃ for 7 days (either light or dark or alternating dark and light).

2) Preparation of agrobacterium:

the vector strains required for transformation are firstly scratched on a single clone plate by an LB plate (LB culture medium: 10g/L NaCl +10g/L tryptone +5g/L yeast), and then single clones are picked and propagated on the corresponding LB plate for the next transformation.

3) Activating the agrobacterium liquid:

the propagated vector strain was cultured in MGL liquid medium (5g tryptone +5g NaCl +0.1 MgSO) containing 100 μm AS ₄ .7H ₂ O+0.25gKH ₂ PO ₄ +5g mannitol +1.16g sodium glycinate, pH adjusted to 5.9) activation, OD ₆₀₀ The activated inoculum solution was used for hypocotyl infestation at 0.3.

4) Infection of agrobacterium liquid:

cutting the hypocotyl of cotton with leaves and roots removed into 0.5cm segments, impregnating for 15min with activated Agrobacterium liquid, sucking dry the liquid, and adding into co-culture medium (MS + B5+ 3% glucose + 0.3% phytagel +1mg/l IBA +0.5mg/l kinetin +1 g/LMgCl) ₂ pH adjusted to 5.9) at 21 ℃ for 2-3 days.

5) Induction and screening of embryogenic callus:

after co-cultivationThe hypocotyls were transferred to a selection medium (MS + B5+ 3% glucose + 0.3% phytagel +1mg/l IBA +0.5mg/l kinetin +1 g/LMgCl) supplemented with kanamycin ₂ + (75/50mg/L) kanamycin +400mg/LCef, pH 5.9) embryogenic callus was induced, screened at 28 ℃ for 4-6 months in light culture, and medium was changed every 2-4 weeks.

6) Differentiation and rooting

When embryogenic callus appeared, it was transferred to differentiation medium (MS (No NH4NO3, KNO3 doubled) + B5+ 3% glucose + 0.3% phytagel +0.5mg/l IBA +0.15mg/l kinetin +2.0g/l glutamine +1.0g/l asparagines, pH adjusted to 5.9) to induce embryogenic callus shoot, and the shoot plants were transferred to rooting medium (1/2MS + B5+ 1.5% glucose + 0.3% phytagel, pH adjusted to 5.9) and were grown to root by light culture.

7) Transplanting

When the transgenic plant in the rooting culture medium grows to a certain size, transplanting the transgenic plant into a small pot for growth, and simultaneously sampling for molecular detection.

4. Molecular identification

For T ₀ And (3) transforming the seedlings, and realizing positive detection, copy number detection and skeleton detection by using a molecular means.

The DNA secure Plant Kit (Cat. # DP320) from Tiangen Biochemical technology (Beijing) Ltd was used for the T ₀ DNA extraction is carried out on 128 cotton transformation event genomes, and qRT-PCR detection is carried out by taking SAD1 as an internal reference gene (Gossypium hirsutum multivar TM-1chromosome 22, ASM98774v1 (12841100-12841304)).

The sequences of the primers are as follows:

the PCR reaction was performed on ABI 7900 and the real-time PCR reaction system is shown in the following table, with the following reaction procedure: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 10 s; renaturation and elongation at 60 ℃ for 55s for 30 cycles.

After the real-time PCR reaction is finished, according to the average Ct (amplification cycle number) value of the reference gene and the target gene generated by the instrument, the equation RQ is 2 ^-ΔCt The RQ value of the corresponding sample is calculated.

Because the reference gene SAD1 is a single copy gene of cotton, it can be concluded that, theoretically, when the RQ value of a transformation event is around 0.5, the event is a single copy heterozygous; when RQ values approach 1.0, the event is homozygous for double copies. Therefore, we can deduce the copy number of the target gene from the RQ value. Genotyping of transformation events of higher generation is performed as described above.

Because the frameless sample has no amplification curve in the whole amplification interval, namely the Ct value is 35cycles, the transgenic sample without the skeleton insertion can be screened according to the average Ct value of the ori of the skeleton element.

Molecular detection results show that 122 exogenous fragments containing the omega MCYS gene are transferred in 128 transformation events. Wherein T of transformation event GHM3 ₀ The RQ value of the exogenous gene omegamCys of the generation-transformed seedling is 0.41, the RQ value of a wild control sample is 0, and the Ct value of the amplification of the skeleton element Ori is 35, so that the transformed seedling is a positive, low-copy and skeleton-free high-quality transformation event.

5. Southern blot detection

Labeling of Southern hybridization probes and hybridization and development Using the DIG High Primer DNA Labeling and Detection Starter Kit I from Roche. The specific experimental method is as follows:

step 1.CTAB method extracts and transforms the total genome DNA of the plant.

Taking 0.5-1g of leaves, putting the leaves into a precooled mortar, adding liquid nitrogen to quickly grind the leaves into powder, and pouring the powder into a 2mL centrifuge tube. Adding 700uL of 1.5% CTAB extract preheated to 65 deg.C, shaking, and keeping in 65 deg.C water bath for 30-60min, while shaking for several times; cooling at room temperature, adding 700 μ L chloroform, shaking, turning upside down, and shaking for 10min, and centrifuging at room temperature at 8000rpm for 10 min; transferring the supernatant to another centrifuge tube, adding equal volume of precipitation solution (isopropanol), precipitating at-20 deg.C for 30min, and centrifuging at room temperature at 8000rpm for 10 min; rinsing with 700 μ L75% ethanol for 2-3 times, air drying, dissolving in 50 μ L TE, and storing at-20 deg.C.

And 2, enzyme digestion of the genome DNA.

Restriction enzyme Hind III is selected to cut the total genome DNA, the cutting reaction system is as the following table, the total genome DNA is uniformly mixed and cut at 37 ℃ for about 24h, a small amount of DNA is firstly taken to carry out pre-electrophoresis to detect the cutting effect after cutting, and then the cut total genome DNA is subjected to low-voltage (30-40V) electrophoresis in 1% agarose gel overnight to fully separate the DNA.

And 3, transferring the film.

Trimming the gel, cutting off the lower right corner as a mark, soaking in 0.25mol/L HCl until bromophenol blue turns yellow, and washing with distilled water twice; denaturing in alkali denaturing solution [1.5M NaCl, 0.5M NaOH ] for 45min, and rinsing with deionized water; rinsing in neutralization solution [1M Tris-HCl (PH7.4), 1.5M NaCl ] for 30min, and replacing neutralization solution to rinse for 15 min; placing on a well-built membrane transferring table, using 10 XSSC solution as membrane transferring solution, rinsing Hybond-N + nylon membrane on the liquid surface of deionized water until the membrane is completely wet, and immersing in a transfer buffer solution; the DNA on the gel is transferred to a nylon membrane by capillary transfer for 16-20h using a10 XSSC solution. After the transfer is finished, the nylon membrane is simply rinsed by 2 XSSC solution, is crosslinked for 1min on an ultraviolet crosslinking instrument, is dried at room temperature, is wrapped by preservative film and is stored for standby at 4 ℃.

And 4, probe amplification and labeling.

And (3) probe amplification: the transformation vector screening marker NPT II gene is used for designing a probe, the primer sequence is shown as SEQ ID No. 8 (5'-CCCAATAGCAGCCAGTCCCTTCC-3') and SEQ ID No. 9 (5'-TCTGATGCCGCCGTGTTCCG-3'), the length of the probe is 191bp, and the probe sequence is shown as 1356-1546 nucleotides in SEQ ID No. 1.

Labeling a probe: taking 1. mu.g or (10 ng-3. mu.g) recovered probe DNA, adding sterilized ddH2O to 16. mu.l; the PCR instrument is quickly placed on ice at 95 ℃ for 10 minutes; adding 4. mu.l of DIG-High Prime for short-time centrifugation; PCR instrument or water bath at 37 deg.c for 1 hr or overnight; the reaction was stopped at 65 ℃ for 10 minutes by adding 2. mu.l of 0.2mol/L EDTA (pH 8.0).

And 5, detecting the efficiency of the probe.

Diluting the labeled probe into 8 concentration gradients; taking 1 mul of each dilution point on a nylon membrane, and drying for 30 minutes at 120 ℃; putting the membrane into a hybridization tube, adding 20ml of maleic acid into the hybridization tube, and rotating for 2 minutes at room temperature in a hybridization furnace; pouring off the maleic acid, adding 10ml of 1 × sealing solution, and rotating for 30 minutes at room temperature; pouring off 1 Xthe blocking solution, adding 10ml of antibody solution (Anti-Digoxigenin-AP Fab fragments), and rotating for 30 minutes at room temperature; pouring off the antibody solution, adding 20ml of washing solution, and rotating for 15 minutes at room temperature; pouring off the washing solution, adding 10ml of detection buffer solution, and rotating for 2-5 minutes at room temperature; the film was then gently removed with tweezers and placed in a sealed bag, and 2ml of developer (NBT/BCIP Stock Solution) was added to the bag and developed in the dark for 5-10 minutes, with no shaking. At the right time of color development, the film is placed on TE or ddH ₂ Soaking and washing in O. According to the color development result, the sample loading amount of the labeled probe was determined to be 40 ug.

And 6, hybridizing.

Heating the hybridization solution DIG Easy Hyb (10ml/100cm2), and prehybridizing at 42 ℃ for 30 minutes in a hybridization oven; probe (25ng/ml) was denatured at 95 ℃ and after 5 minutes placed on ice; adding the denatured probe to a previously heated DIG Easy Hyb (3.5ml/100cm2), and mixing well; pouring out the pre-hybridization solution, and adding the hybridization solution containing the denatured probe; hybridization was carried out in a hybridization oven at 42 ℃ for 14 hours.

And 7, washing the membrane and developing.

The hybridization solution was decanted and then washed twice with 2 XSSC, 0.1% SDS at room temperature for 5 minutes each; finally, the column was washed twice with 0.5 XSSC, 0.1% SDS at 65 ℃ for 15 minutes each. The color development method is the same as the probe efficiency detection operation.

Theoretically, the band obtained by HindIII enzyme digestion hybridization should be larger than 2Kb, the actually obtained hybridization band is about 3Kb in size, which is expected, and the transformation event GHM3Southern blot hybridization has only one positive band, so that the transformation event can be confirmed to be a single-site single copy insertion (as shown in FIG. 2). Thus, a single copy cotton transformation event GHM3 was obtained.

Example 2Breeding and field performance of cotton transformation event GHM3

1. Transformation event field breeding

As described in example 1, the recipient material for the transformation event of this example was cotton strain YZ1, T being obtained ₀ After the transformation event of generation (single copy insertion and no vector skeleton pollution), the transformation event is taken as a female parent and selfed continuously for 3 generations to obtain homozygous transgenic line seeds. During a period according to T ₀ The qRT-PCR result of generation differentiation seedling is the same as that of example 1 in the experimental method, and the transgenic line with RQ value smaller than 1.2 and no skeleton element inserted is selected for seed reproduction. Germinating the seeds to obtain T ₁ Plant generation, sampling and T extraction ₁ Generating plant genome DNA, carrying out qRT-PCR detection and selecting T ₁ Carrying out selfing on the plant lines with RQ values close to 1 in the generation plants until the characters are not separated; then, the homozygous seeds are planted, the resistance to cotton nematodes is identified in the seedling stage, and strains with good pest resistance are selected for continuous breeding.

That is, a cotton transformation event GHM3 is continuously bred for 3 generations in a greenhouse, whether the exogenous NPTII or omegaMCYS genes and the vector skeleton fragment exist in cotton plants is determined by RT-PCT detection aiming at each generation, the RQ values of the exogenous insertion genes and the cotton internal reference gene SAD1 are calculated, so that the transgenic strains with positive exogenous insertion genes and no skeleton residue are determined, and the homozygous strains with the RQ values close to 1 are selected. The examination result shows that the PCR results of the exogenous insertion genes of 3 consecutive generations of the transformation event GHM3 are all positive, the PCR results of the skeleton fragments are all negative, and the T is ₁ The RQ values at the beginning of generation were 1.2, 1.09, 0.95, respectively, all close to 1.

As can be seen, cotton transformation event GHM3 is a single copy backbone-free and genetically stable high-quality event.

2. Transformation event field Performance

Step 1, germplasm purification and investigation of transformation events. According to the result of RT-PCR detection of each generation transgenic line, calculating and recording the homozygous condition of the exogenous insertion gene of the transformation event, and selecting a high-quality frameless single-copy homozygous line by the aid of an RQ value.

And 2, identifying the cotton nematode resistance.

1) Culturing and separating the renal type reniform nematodes. Collecting nephrotic type nephrotic nematode in Hangzhou Zhejiang, detecting and purifying in greenhouse of plant protection institute of Chinese academy of agricultural sciences, culturing in soybean medium (yellow 13) root system in sand at 26 deg.C for 16h under light, and keeping soil moist in dark for 8 h. Irrigating the nutrient solution 1 time per week, culturing for 75 days, collecting root system of soybean, and separating ovum block of reniform nematode from root system by sodium hypochlorite method. Placing the collected egg mass in sterile water, incubating at 25 deg.C, collecting two-stage larvae (J2) 3d before and after incubation peak, quantifying to 3000 heads/ml under microscope, and storing at 4 deg.C for inoculation.

2) And (5) cotton culture and inoculation. Cotton plants were placed on moist filter paper and germinated in the dark at 25 ℃. After germination accelerating, selecting relatively strong plants and pots containing sterile sandy soil, covering a layer of sterile soil on the surface, and then placing the plants in a culture room at 25 ℃ for culture. After 14d, the cotton plants were transplanted into individual pots. After further culture for 4 days, the collected renal reniform nematodes were inoculated to the roots of the plants, and 1.4ml of nematode mixture (4000 heads) was inoculated to each plant. Spraying a small amount of sterile water for moisturizing by a watering can every day within 3 days after inoculation, and normally watering after 4 days. The number of females on the surface of the roots was measured 35d after inoculation.

3) And (6) detection and statistics. Carefully knocking the cotton out of the sandy soil, slightly swinging the cotton in clear water to wash off the sandy soil on the surface of the root, dyeing the cotton by adopting McCormick Schilling magenta, and observing and counting the number of female insects of each plant under a microscope. At present, the unified standard of kidney-free reniform nematode resistance detection at home and abroad only takes the unified female nematode index grading standard as reference. When the average number of the female insects of the test variety is obviously different from the comparison with the control, calculating the female insect decline rate.

The Nematode Index (Nematode Index) is the average female quantity of the test variety/the average female quantity of the control multiplied by 100 percent.

The female reduction rate is (average female quantity of the tested variety-average female quantity of the control)/average female quantity of the control x 100%

The cotton reniform nematode living body inoculation test shows that the average number of the female worms of the cotton transformation event GHM3(GHM3, sample number of 12) and the wild type cotton (WT, sample number of 13) is 12 and 25.54 respectively, and the mean value of the female worm reduction rate of the cotton transformation event GHM3 is 53 percent. Analysis of variance found a significant difference between transformation event GHM3 and WT at the 0.05 level (table 1).

Table 1: cotton transformation events and statistics of female insects on control material roots

*: analysis of variance (p <0.05)

Example 3Isolation of left (5 ') and right (3') flanking sequences of cotton transformation event GHM3

In general, DNA primer sequences and methods for detecting transgenic plants are simple and consistent, and these detection methods typically focus on frequently used gene expression elements, such as promoters, terminators, and marker genes, since for many transformation vectors, coding sequence regions are interchangeable. Thus, these methods cannot be used to distinguish between constructs that differ only in their coding sequences, nor between different transformation events, particularly those produced using the same transformation vector, unless the sequence of the chromosomal DNA, i.e., the "flanking DNA", adjacent to the inserted heterologous DNA is known. To this end, this example performed whole genome re-sequencing of cotton transformation event GHM3 by "Next-generation" sequencing technology, to precisely map the foreign insert and its flanking sequences.

Sequencing was committed to Jinwei Zhi corporation, Suzhou, at a sequencing depth of 30X. The reference genomic sources were: NCBI databases (see, in particular, https:// www.ncbi.nlm.nih.gov/genome/. After the sequencing data are compared with a reference genome, 5 ' flanking sequences and 3 ' flanking sequences of a cotton transformation event GHM3 and partial transformation vector sequences are obtained, wherein the total length is 4517bp and is shown as SEQ ID NO:1, and 1-511bp is a 5 ' flanking sequence (the sequence length is 511 bp); 4017 and 4517bp are 3' flanking sequences (the sequence length is 501 bp); the middle 512-channel 4016bp sequence is an exogenous insertion T-DNA sequence with the length of 3505 bp; compared with the expression vector pZZ00074, the right side of the inserted T-DNA sequence is deleted for 41bp of base including the right border. NCBI database analysis showed that the insertion site is located at 90496812 of chromosome 9 of cotton. Further analysis shows that the insertion sites are positioned at 4870bp downstream of the functional gene LOC107955236 and 4347bp upstream of the functional gene LOC107955234, and no influence on the cotton functional gene is inferred.

Example 4Application of flanking sequence of cotton transformation event GHM3

The method is characterized in that 3-5 pairs of primers are designed by utilizing the flanking genome sequence of the cotton transformation event and the NPTII and MCYS sequences in the exogenous fragment, and a qualitative PCR identification method of the transformation event and derived products thereof is established.

An upstream primer (csp-6386) designed according to a cotton genome at the 5 ' end of an integration site of an exogenous fragment of a transformation event GHM3 is 5'-GATACGTTATTGATTGGGTTTCGAC-3' (SEQ ID No:10), a downstream primer (csp-6387) designed according to an NPTII sequence is 5'-CGCTTTTCTGGATTCATCGA-3' (SEQ ID No:11), and the theoretical amplification length is 663 bp. The PCR procedure was 35cycles at 94 ℃ for 5min (94 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 1min), and 72 ℃ for 7 min.

The downstream primer (csp-6799) designed according to the cotton genome at the 3 ' end of the integration site of the exogenous fragment of the transformation event GHM3 is 5'-AAGCCATGGGAGAACTTCAAAGAGC-3' (SEQ ID No:12), the upstream primer (csp-6389) designed according to the NPTII sequence is 5'-CAAAGGACGAGGACTTAATTGATTG-3' (SEQ ID No:13), and the theoretical amplification length is 701 bp. The PCR reaction program is 95 ℃ for 5min, (94 ℃ for 30s, 60 ℃ for 30s, 72 ℃ for 1min)35 cycles, 72 ℃ for 7 min.

DNA samples of transformation event GHM3 were extracted by CTAB method, and amplified using the primers with water, non-transgenic plant DNA samples as negative controls. The results showed that none of the negative controls had amplified bands and that the DNA samples of transformation event GHM3 had amplified specific target fragments (as shown in figure 3). The DNA amplification sequence of the transformation event GHM3 has specific 663bp and 701bp target bands respectively.

This example demonstrates that the transformation event and its preparation can be specifically detected by PCR detection using the 5 'and/or 3' flanking sequences of transformation event GHM 3.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the technical principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Sequence listing

<110> China seed group Co., Ltd

<120> nematode resistant cotton transformation event GHM3

<160> 13

<170> SIPOSequenceListing 1.0

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gtgaattaat taatttaata tccttctaaa ttttaattat tttatatttc caaattattc 60

ttaatgaaat caactgaaat aactcatatt taataattat ctgcagaact taaatttctt 120

ttgattatat aatctcgaat catccatgct aagctaaaag caaagaaaaa attacttgtt 180

catcatttga gtaaataagt gatgaaaata ttttgagttt tgtttggcat gtaagatata 240

attgcttgat ctttgttttg ataaagaagt gatgaaaaaa ttgtaaagat ttttttcttt 300

aactttagct tagcatggaa atttaaagat gatataatca aaagaaaatt agaatttgta 360

gtcaattatt aaagatacgt tattgattgg gtttcgacat gattttgaat gttttaacga 420

ctagttttca actccaaggg tggttccaaa atatatatat acactaaaaa tatcgaaaca 480

ttataagtca atagctaaaa atatacacta aggatatatt gtggtgtaaa caaattgacg 540

cttagacaac ttaataacac attgcggacg tttttaatgt actgaattaa cgccgaatta 600

attcggggga tctggatttt agtactggat tttggtttta ggaattagaa attttattga 660

tagaagtatt ttacaaatac aaatacatac taagggtttc ttatatgctc aacacatgag 720

cgaaacccta taggaaccct aattccctta tctgggaact actcacacat tattatggag 780

aaactcgagt caaatctcgg tgacgggcag gaccggacgg ggcggtacct taattaacga 840

gctctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg 900

cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat 960

cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga 1020

tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatgag 1080

tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac agttcggctg 1140

gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc 1200

gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat 1260

caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg gcaggagcaa 1320

ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag tcccttcccg 1380

cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc agccacgata 1440

gccgcgctgc ctcgtcttga agttcattca gggcaccgga caggtcggtc ttgacaaaaa 1500

gaaccggcct cccctgcgct gacagccgga acacggcggc atcagagcag ccgattgtct 1560

gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa cctgcgtgca 1620

atccatcttg ttcaatcatg gccggccctt tcctgcagga gactcgagag agatagattt 1680

gtagagagag actggtgatt tcagcgtgtc ctctccaaat gaaatgaact tccttatata 1740

gaggaagggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc agtggagata 1800

tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc cacgatgctc 1860

ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg aacgatagcc 1920

tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac tgtccttttg 1980

atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat taccctttgt 2040

tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt cttggagtag 2100

acgagagtgt cgtgctccac catgttcaca tcaatccact tgctttgaag acgtggttgg 2160

aacgtcttct ttttccacga tgctcctcgt gggtgggggt ccatctttgg gaccactgtc 2220

ggcagaggca tcttgaacga tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc 2280

accttccttt tctactgtcc ttttgatgaa gtgacagata gctgggcaat ggaatccgag 2340

gaggtttccc gatattaccc tttgttgaaa agtctcaata gccctttggt cttctgagac 2400

tgtatctttg atattcttgg agtagacgag agtgtcgtgc tccaccatgt tggcaagaag 2460

cttgcatgcc tgcaggtccc cagattagcc ttttcaattt cagaaagaat gctaacccac 2520

agatggttag agaggcttac gcagcaggtc tcatcaagac gatctacccg agcaataatc 2580

tccaggaaat caaatacctt cccaagaagg ttaaagatgc agtcaaaaga ttcaggacta 2640

actgcatcaa gaacacagag aaagatatat ttctcaagat cagaagtact attccagtat 2700

ggacgattca aggcttgctt cacaaaccaa ggcaagtaat agagattgga gtctctaaaa 2760

aggtagttcc cactgaatca aaggccatgg agtcaaagat tcaaatagag gacctaacag 2820

aactcgccgt aaagactggc gaacagttca tacagagtct cttacgactc aatgacaaga 2880

agaaaatctt cgtcaacatg gtggagcacg acacacttgt ctactccaaa aatatcaaag 2940

atacagtctc agaagaccaa agggcaattg agacttttca acaaagggta atatccggaa 3000

acctcctcgg attccattgc ccagctatct gtcactttat tgtgaagata gtggaaaagg 3060

aaggtggctc ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt gaagatgcct 3120

ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag 3180

acgttccaac cacgtcttca aagcaagtgg attgatgtga tatctccact gacgtaaggg 3240

atgacgcaca atcccactat ccttcgcaag acccttcctc tatataagga agttcatttc 3300

atttggagag aacacggggg actctagagg atccgtattt ttacaacaat taccaacaac 3360

aacaaacaac aaacaacatt acaattacta tttacaatta caaaacaaca atggctgata 3420

ataccggaac tttggcgggt ggcatcaagg atgttccagg gaacgagaat gaccttcatc 3480

ttcaggaact cgcacgcttt gccgtcgatg agcacaacaa aaaggcaaat gctcttctgg 3540

gattcgagaa acttgtgaag gccaaaacac aagtggttgc tggtacgatg tactatctca 3600

ctattgaagt gaaggatggc gaagtgaaaa agctctatga agctaaagtc tgggagaagc 3660

catgggagaa cttcaaagag ctgcaggaat ttaagcctgt tgacgagggt gcaagcgcct 3720

aagagctcga atttccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat 3780

cctgttgccg gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta 3840

ataattaaca tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg 3900

caattataca tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta 3960

tcgcgcgcgg tgtcatctat gttactagat cgggaattaa actatcagtg tttgactgat 4020

acatggcaca atgttccggc acatcatcca tgatgtacag atacttggaa gcaaacatgg 4080

gccattacac tgttttgggc tttttgagcc taccaagacc cgtacttgat cccgaacccc 4140

tcggagcacc ctatactatt tccaaatgat tttaaaatgt tatgttaaat ttattttacg 4200

agatttttta tttgaattat tttctaactg ttgtttgcaa cttttaattt atttgattta 4260

ttatataatt caataattaa ttggattccc actcggaaac aacacacaat taagtcctca 4320

atcgaattta aacaatcaat taagtcctcg tcctttgatg tcgttaactt cgtcacgtgg 4380

catgtcagtc atttctgata tatttgttta aacaaattaa aaattctaaa aaatataaaa 4440

aaattagaat tttttatttt attaaaaata ttaaaataaa aatatttaaa ttaatagaaa 4500

atcatgtaaa atatttt 4517

<210> 2

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<213> Artificial Sequence (Artificial Sequence)

<400> 2

tgatgagaaa tgaggagcca agg 23

<210> 3

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<213> Artificial Sequence (Artificial Sequence)

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acgaagccca atacacaacc c 21

<210> 4

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<213> Artificial Sequence (Artificial Sequence)

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aggcaaatgc tcttctggga ttcg 24

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ccctcgtcaa caggcttaaa ttcc 24

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<213> Artificial Sequence (Artificial Sequence)

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aacaagacga actccaattc actg 24

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<213> Artificial Sequence (Artificial Sequence)

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tgttgattgt aacgatgaca gagc 24

<210> 8

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<213> Artificial Sequence (Artificial Sequence)

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cccaatagca gccagtccct tcc 23

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tctgatgccg ccgtgttccg 20

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<213> Artificial Sequence (Artificial Sequence)

<400> 10

gatacgttat tgattgggtt tcgac 25

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cgcttttctg gattcatcga 20

<210> 12

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

aagccatggg agaacttcaa agagc 25

<210> 13

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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caaaggacga ggacttaatt gattg 25

Claims (11)

1. A nucleic acid molecule which is a sequence shown as SEQ ID NO. 1 or a complementary sequence thereof.

2. The nucleic acid molecule of claim 1, comprising an expression cassette that expresses an anti-nematode gene.

3. The nucleic acid molecule as claimed in claim 2, wherein the expression cassette is the sequence shown as nucleotide 2482-3991 of SEQ ID NO. 1.

4. The nucleic acid molecule as claimed in claim 1, which is obtained by introducing an expression cassette for expressing a nematode-resistant gene as indicated by nucleotides 2482-3991 of SEQ ID NO. 1 into the genome of cotton.

5. The nucleic acid molecule of claim 1, which is present in a cotton plant, seed, plant cell, progeny plant or plant part.

6. A probe for detecting cotton transformation events, the sequence of which is shown as nucleotides 374-1036 or 3657-4356 of SEQ ID NO. 1, or the complementary sequence thereof.

7. A primer pair for detecting cotton transformation events, wherein the primer pair is a nucleotide sequence shown in SEQ ID No. 10 and SEQ ID No. 11 or a complementary sequence thereof; or the nucleotide sequences shown in SEQ ID No. 12 and SEQ ID No. 13 or the complementary sequences thereof.

8. A kit or microarray for detecting cotton transformation events comprising the probe of claim 6 and/or the primer pair of claim 7.

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

-the probe of claim 6;

-a primer pair according to claim 7;

-the probe of claim 6 and the primer pair of claim 6; or

-a kit or microarray according to claim 8.

10. A method of breeding cotton, the method 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 progeny plants, seeds, plant cells, progeny plants or plant parts.

11. The method of claim 10, further comprising: 3) subjecting the progeny plant obtained in step 2) to nematode resistance identification and detecting the presence or absence of said transformation event therein using the method of claim 9.

Images