Cotton event cI55-3 and primer and method for detecting same

Abstract

The invention provides a cotton transformation event cI55-3 as well as a primer pair and a method for detecting the cotton transformation event cI55-3. A cotton plant carrying the cI55-3 transformation event comprises the event, namely the joint of an exogenous insertion DNA sequence and a cotton genome DNA sequence, on a group A chromosome 9. A detection primer can be designed by using an exogenous insertion DNA sequence and a DNA sequence of a junction region on a flanking cotton genome, and is used for specific detection of a cI55-3 event. The detection method aiming at the cI55-3 event can provide a convenient means for tracking the specific gene insertion event for the application of breeding by using the plant event, and the specific gene insertion event can be used as a molecular marker to improve the working efficiency of fiber improvement breeding by using the transformation event.

Description

Technical Field

[0001] The invention belongs to the field of plant molecular biology, especially the field of transgenic crop breeding in agricultural biotechnology research, and particularly relates to a cotton transformation event with improved cotton fiber quality, and a unique method for detecting whether the transformation event exists. Background Art

[0003] With the improvement of people's living standards and the innovation of textile technology, the requirements for cotton fiber quality have also increased accordingly, especially since air-spinning replaced ring spinning, requiring longer, stronger, finer and neater fibers.

[0004] Improving yield and improving cotton fiber quality are both important goals of cotton breeding in my country. In terms of yield, cotton lint percentage (the proportion of fiber weight to seed cotton weight) is an important yield factor. When seed cotton yield is similar, an increase in lint percentage means an increase in fiber yield. Lint percentage is a quantitative trait controlled by multiple genes and is greatly affected by the environment. It is very difficult to improve the lint percentage of excellent cotton varieties using conventional breeding methods. In terms of quality, the micronaire value is very important and is a comprehensive indicator of fiber fineness and maturity. Qualified fibers have a micronaire value between 3.5 and 5.0. Spinning fibers with a micronaire value of more than 5.0 is prone to breakage. The micronaire value is greatly affected by climate, and the micronaire value of the same variety will increase at high temperatures. As the global climate warms, the micronaire value of cotton is on the rise.

[0005] In recent years, with the rapid development of multi-omics research methods such as genome sequencing, transcriptome, translationome, proteome, degradation group, metabolome, single cell sequencing, and epigenetic modification, the widespread application of whole genome association analysis has accelerated the discovery of key genes for cotton fiber quality. At the same time, the increasing improvement of transgenic technology has laid a solid foundation for the biological function verification of a large number of cotton fiber development-related genes in cotton and the creation of new high-quality cotton materials. At present, many key genes involved in fiber development have been reported, and a batch of new cotton materials with significantly improved fiber quality have been obtained, which has greatly promoted the study of fiber development mechanism and the process of fiber quality breeding. Transgenic technology has shown that hormones, functional genes and transcription factors form a complex regulatory network to coordinate the dynamic development process of fibers (Huang et al 2021). With the development of contemporary molecular biology, transgenic breeding technology provides a feasible way to solve this problem. Summary of the invention

[0006] In one aspect, the present invention provides a cotton transformation event, characterized in that a partial genomic sequence of cotton is replaced with a T-DNA insertion sequence on chromosome 9 of group A of the cotton, wherein the T-DNA insertion sequence is the 1529-6021 bp sequence of the sequence shown in SEQ ID NO: 13, and the partial genomic sequence is as shown in SEQ ID NO: 18.

[0007] In some embodiments, the upstream flanking cotton genomic sequence of the T-DNA insertion sequence is: 1) The 1-1528 bp sequence of the sequence shown in SEQ ID NO: 13; or 2) The 3' end sequence of the 1-1528 bp sequence of the sequence shown in SEQ ID NO: 13 with a length of not less than 100 bp.

[0008] In some embodiments, the downstream flanking cotton genomic sequence of the T-DNA insertion sequence is: 1) The 6022-6309 bp sequence of the sequence shown in SEQ ID NO: 13; or 2) The 5' end sequence of the 6022-6309 bp sequence of the sequence shown in SEQ ID NO: 13 with a length of not less than 100 bp.

[0009] In some embodiments, the cotton transformation event results in the inclusion of a DNA sequence as shown in SEQ ID NO: 13 on chromosome 9 of group A of the cotton, and the DNA sequence is composed of a 1529-6021 bp T-DNA insertion sequence, a 1-1528 bp upstream flanking cotton genomic sequence, and a 6022-6309 bp downstream flanking cotton genomic sequence.

[0010] In some embodiments, the DNA fragment sequentially includes from the 5' end to the 3' end: 1) Upstream flanking cotton genomic sequence: The 1-1528 bp sequence of the sequence shown in SEQ ID NO: 13; or the 3' end sequence of the 1-1528 bp sequence of the sequence shown in SEQ ID NO: 13 with a length of not less than 100 bp; 2) T-DNA insertion sequence: The 1529-6021 bp sequence of the sequence shown in SEQ ID NO: 13; 3) Downstream flanking cotton genomic sequence: The 6022-6309 bp sequence of the sequence shown in SEQ ID NO: 13; or the 5' end sequence of the 6022-6309 bp sequence of the sequence shown in SEQ ID NO: 13 with a length of not less than 100 bp.

[0011] On the other hand, the present invention provides primer pairs for detecting the above-mentioned cotton transformation event or the above-mentioned DNA fragment, and the primer pairs are composed of a first primer that specifically recognizes the upstream flanking cotton genomic sequence or the downstream flanking cotton genomic sequence and a second primer that specifically recognizes the T-DNA insertion sequence.

[0012] In some embodiments, the sequence of the first primer is as shown in SEQ ID NO: 14, and the sequence of the second primer is as shown in SEQ ID NO: 15; or the sequence of the first primer is as shown in SEQ ID NO: 16, and the sequence of the second primer is as shown in SEQ ID NO: 17.

[0013] On the other hand, the present invention provides a method for identifying whether the above-mentioned cotton transformation event exists in a cotton biological sample, including: (a) extracting a DNA sample from the cotton biological sample to be identified; (b) using the above-mentioned primer pairs to perform PCR amplification with the extracted DNA sample as a template; and (c) detecting the PCR amplification product. If the length of the PCR amplification product is the same as that obtained by amplifying a DNA template with the sequence shown in SEQ ID NO: 13, it indicates that the cotton transformation event exists in the cotton biological sample.

[0014] On the other hand, the present invention provides a method for obtaining a transgenic high-quality cotton material, including: hybridizing a cotton material containing the above-mentioned cotton transformation event with other cotton breeding materials, and then further performing backcrossing to obtain a new material containing the cotton transformation event (such as obtaining a pure-line plant); wherein during the hybridization and / or backcrossing process, the above-mentioned identification method is used to screen and identify in the offspring population to confirm the existence of the cotton transformation event.

[0015] On the other hand, the present invention provides the use of the above-mentioned DNA fragment for improving cotton fiber quality and / or yield, for cotton breeding, and as a molecular marker. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a map of the plant expression vector E6-AtCel1-pBIOT1101.

[0017] Figure 2 It is a schematic diagram of the sequence flanking the left border (LB).

[0018] Figure 3 It is a schematic diagram of the sequence flanking the right border (RB).

[0019] Figure 4It shows the result of amplifying an amplification product of about 797 bp using primer pairs with sequences SEQ ID NO:11 and SEQ ID NO:12 respectively with the DNA of receptor material Jimian 14 as the template, and comparing the sequence after sequencing with the flanking sequence of cI55-3 event.

[0020] Figure 5 It is a schematic diagram of the inserted sequence and the flanking sequence.

[0021] Figure 6 It shows the result of amplifying cotton samples of cI55-3 (T1 generation), cI55-3 (T2 generation), cI55-3 (T3 generation), and Jimian 14 using primer pairs cI55-3LB5 / cI55-3LB3 respectively. In the figure: M, marker, DL2000 plus; 1: cI55-3-1 (the - after cI55-3 indicates the generation, and the number indicates the plant number, the same below), 2: cI55-3-2, 3: cI55-3-1-1, 4: cI55-3-1-2, 5: cI55-3-1-1-1, 6: cI55-3-1-1-1, 7: Jimian 14, the positive amplification band is about 255 bp.

[0022] Figure 7 It shows the result of amplifying cotton samples of cI55-3 (T1 generation), cI55-3 (T2 generation), cI55-3 (T3 generation), and Jimian 14 using primer pairs cI55-3RB5 / cI55-3RB3 respectively. In the figure: M, marker, DL2000 plus; 1: cI55-3-1, 2: cI55-3-2, 3: cI55-3-1-1, 4: cI55-3-1-2, 5: cI55-3-1-1-1, 6: cI55-3-1-1-1, 7: Jimian 14, the positive amplification band is about 315 bp. Detailed implementation mode

[0023] Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0024] The present invention provides a cotton transformation event cI55-3, as well as primer pairs and methods for its detection. Cotton plants carrying the cI55-3 transformation event contain the junction of the exogenous inserted DNA sequence and the cotton genomic DNA sequence at chromosome 9 of Group A. By using the exogenous inserted DNA sequence and the DNA sequences of the junction regions on its flanking cotton genome, detection primers can be designed for specific detection of the cI55-3 event. The detection method for the cI55-3 event can provide a convenient means for tracking the specific gene insertion event in breeding applications using this plant event. This specific gene insertion event can be used as a molecular marker to improve the efficiency of fiber improvement breeding using this transformation event.

[0025] In the present invention, a "transformation event" refers to an event in which an exogenous target gene is transformed into cotton cells by an Agrobacterium-mediated genetic transformation method (well-known to those skilled in the art), and the exogenous DNA sequence is inserted and integrated at a specific position in the cotton genome in the obtained transgenic cotton plants; the preferred cotton transformation event cI55-3 of the present invention has molecular characteristics with unique genetic information and exogenous cel1 gene expression patterns, and is suitable for the cultivation and application of high-quality cotton.

[0026] A "transformation event" is not a plant cell or plant. A plant cell or plant is merely the carrier for the existence of the transformation event. The core feature of a transformation event is a characteristic DNA sequence formed by the connection of the exogenous insertion sequence and a specific cotton genomic sequence resulting from the insertion of the exogenous gene at a specific locus in the plant genome, and it exhibits specific application attributes. Although the exogenous target gene vectors are the same, different transformation events exhibit different genetic traits due to the different insertion positions of the exogenous gene vectors. This difference comes from two aspects. On the one hand, the exogenous gene has different expression patterns due to the position effect of insertion. On the other hand, the insertion of the exogenous gene changes the gene structure of the recipient plant, resulting in changes in the genetic traits of the recipient plant. Seeking intellectual property protection for transgenic crops through transformation event-related patents is a new trend internationally. Because a commercialized transformation event is a unique biological creation obtained by the inventor through screening and identification, the integration of the exogenous gene with a specific position in the recipient crop genome not only endows the transformation event with unique molecular characteristics but also has a unique exogenous gene expression pattern, differentiating it from other transformation events obtained during the research process and having characteristics more suitable for commercial application, thus having obvious novelty, practicality, and creativity. Currently, there are already many transformation events internationally that have obtained intellectual property protection, all owned by some multinational companies, and some have also applied for patents in China, such as Chinese Patent ZL02802047.2.

[0027] "T-DNA insertion sequence" refers to an exogenous DNA sequence (including the cel1 protein coding sequence) that can be transferred into a plant with the help of the T-DNA sequence of Agrobacterium tumefaciens. Naming this insertion sequence as the T-DNA insertion sequence only indicates that the method of the present invention uses Agrobacterium tumefaciens to achieve plant transformation, and does not limit the cotton transformation event of the present invention to only being achievable through the T-DNA sequence.

[0028] "Upstream flanking cotton genomic sequence" refers to a sequence in the cotton's own genome. In the plant genome including the cotton transformation event of the present invention, it is located upstream (5' end) of the T-DNA insertion sequence and is adjacent to the 5' end of the T-DNA insertion sequence. In this way, the insertion position of the T-DNA insertion sequence in the cotton genome can be determined. In some embodiments, the upstream flanking cotton genomic sequence is the 1-1528 bp sequence of the sequence shown in SEQ ID NO: 13. In other embodiments, the upstream flanking cotton genomic sequence is the 3' end sequence of the 1-1528 bp sequence of the sequence shown in SEQ ID NO: 13 with a length of not less than 100 bp (such as 200 bp, 300 bp, 500 bp, 1000 bp, etc.). Those skilled in the art can understand that as long as the length of this 3' end sequence is sufficient to determine the genomic position in the cotton genome, especially in chromosome A9 (and thus determine the insertion position of the T-DNA insertion sequence), therefore, its length may even be less than 100 bp.

[0029] "Downstream flanking cotton genomic sequence" refers to a sequence in the cotton's own genome. In the plant genome including the cotton transformation event of the present invention, it is located downstream (3' end) of the T-DNA insertion sequence and is adjacent to the 3' end of the T-DNA insertion sequence. In this way, the insertion position of the T-DNA insertion sequence in the cotton genome can also be determined. In some embodiments, the downstream flanking cotton genomic sequence is the 6022-6309 bp sequence of the sequence shown in SEQ ID NO: 13. In other embodiments, the upstream flanking cotton genomic sequence is the 5' end sequence of the 6022-6309 bp sequence of the sequence shown in SEQ ID NO: 13 with a length of not less than 100 bp (such as 200 bp, 230 bp, 260 bp, etc.). Those skilled in the art can understand that as long as the length of this 5' end sequence is sufficient to determine the genomic position in the cotton genome, especially in chromosome A9 (and thus determine the insertion position of the T-DNA insertion sequence), therefore, its length may even be less than 100 bp.

[0030] When referring to a primer pair, the "specifically recognize" used herein means that the first primer in the primer pair is complementary to or identical to a partial upstream sequence of the given template, and the second primer in the primer pair is identical to or complementary to a partial downstream sequence of the given template, so as to perform PCR amplification to obtain an amplicon including the partial upstream sequence, the partial downstream sequence and the sequence therebetween.

[0031] The inventor of the present invention obtained a cotton plant transformation event that improves cotton fiber quality through a transgenic method, named cI55-3. The cotton of this plant transformation event (Gossypium hirsutum, Gossypium hirsutum ), the representative seeds of which were deposited on March 21, 2025 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (CGMCC, Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101), and the deposit number is: CGMCC No. 30702.

[0032] In the first aspect of the present invention, there is provided a cotton transformation event cI55-3, which is characterized in that a DNA sequence on chromosome 9 of Group A is as shown in SEQ ID NO: 13, which is composed of a T-DNA insertion sequence from 1529 to 6021 bp, an upstream flanking cotton genomic sequence from 1 to 1528 bp, and a downstream flanking cotton genomic sequence from 6022 to 6309 bp.

[0033] In the second aspect of the present invention, there is provided a DNA sequence, which at least comprises part or all of the T-DNA insertion sequence and part or all of the flanking cotton genomic sequence. This characteristic DNA sequence fragment can be detected by the method provided in the fourth aspect of the present invention.

[0034] In the third aspect of the present invention, there is provided a primer pair for detecting the cotton transformation event described in the first aspect of the present invention and the characteristic DNA sequence fragment described in the second aspect of the present invention, which is composed of a first primer that specifically recognizes the flanking sequence on any side described in the first aspect of the present invention and a second primer that specifically recognizes the T-DNA insertion sequence described in the first aspect of the present invention. In some embodiments, the sequence of the first primer is SEQ ID NO: 14, and the sequence of the second primer is SEQ ID NO: 15; or the sequence of the first primer is SEQ ID NO: 16, and the sequence of the second primer is SEQ ID NO: 17.

[0035] In the fourth aspect of the present invention, there is provided a method for identifying the transformation event cI55-3 in a cotton biological sample, which includes: (a) Extracting a DNA sample from the cotton biological sample to be identified; (b) Using the extracted DNA sample as a template, perform PCR amplification using the primer pair described in the fifth aspect of the present invention; and (c) Detect the PCR amplification product. If the length of the amplification product is consistent with the theoretical length between the sequences of the PCR primer pair described in SEQ ID NO: 13, it indicates the presence of the cI55-3 transformation event in the cotton biological sample.

[0036] The fifth aspect of the present invention provides a method for obtaining a fiber-improved transgenic cotton material, including: Hybridize the cotton material containing the cotton transformation event described in the first aspect of the present invention with other cotton breeding materials, and preferably perform backcrossing further to obtain a new material containing the transformation event described in the first aspect of the present invention; during the hybridization or backcrossing process, use the identification method described in the fourth aspect of the present invention to screen and identify in the offspring population to confirm the presence of the transformation event described in the first aspect of the present invention.

[0037] The sixth aspect of the present invention provides the use of the transformation event described in the first aspect of the present invention, the fragment of the characteristic DNA sequence of the transformation event described in the second aspect of the present invention, and the methods described in the fourth and fifth aspects of the present invention for improving cotton fiber quality and yield traits, for plant breeding, and as molecular markers.

[0038] This new cotton transformation event for improving fiber quality of the present invention can express the Cel1 protein and can be used to improve cotton fiber quality and yield.

[0039] The present invention will be further described below in conjunction with non-limiting examples.

[0040] Example 1 Genetic transformation of Gossypium hirsutum Gossypium hirsutum ) Using the Agrobacterium-mediated genetic transformation method, transform the hypocotyls of Jimian 14 (obtained from the National Cotton Medium-Term Storage of the Chinese Academy of Agricultural Sciences, unified number: ZM-30270) with the E6-AtCel1-pBIOT1101 vector ( Figure 1 ).

[0041] Agrobacterium tumefaciens LBA4404 containing the E6-AtCel1-pBIOT1101 vector (purchased from Biovector ScienceLab, Inc) was picked and inoculated into LB liquid medium containing 50 mg / L kanamycin (km), 50 mg / L rifampicin (rif), and 50 mg / L streptomycin (S / Sm). It was cultured overnight in the dark with shaking at 28 °C until the logarithmic growth phase of the bacteria. The bacterial solution was diluted with LB or YEB liquid medium at a ratio of bacterial solution:medium of 1:50 - 1:100, and then cultured with shaking for 4 - 6 h to dilute the bacterial solution to an OD600 value of 0.8 - 1.0.

[0042] The seeds of the receptor material Jimian 14 were germinated, and the hypocotyls of sterile seedlings grown for 3 - 4 days were taken and cut into segments 0.6 - 0.8 cm in length, immersed for 10 - 15 min, and then the hypocotyl segments were taken out and placed on the co-culture medium (MSB + KT 0.1 mg / L + 2,4-D 0.1 mg / L) and co-cultured at 22 °C - 25 °C for 2 days. It was transferred to the callus induction medium (MSB + KT 0.1 mg / L + 2,4-D 0.1 mg / L + km 50 mg / L) and subcultured once every 20 - 30 days. After 90 days, it was transferred to the callus proliferation medium (MSB + KT 0.1 mg / L + 2,4-D 0.05 mg / L + km 50 mg / L) and subcultured once every 20 - 30 days. After embryogenic callus grew, the embryogenic callus was subcultured to the germination medium (MSB + KT 0.1 mg / L + km 50 mg / L). After about 40 days, the germinated green shoots were picked and transferred to the rooting medium (SH + km 50 mg / L) for screening, and then 310 small plants and graftable resistant plants were obtained. The resistant plants were identified by PCR to have the target foreign gene ( cel1 ) and then grafted and transplanted to obtain 290 transgenic cotton plants with the starting number "cI", and 269 survived.

[0043] Example 2 Cultivation and Management of Transgenic Cotton in Field Experiments All the research work in the intermediate test stage of transgenic cotton was completed in Pingdi, Longgang, Shenzhen, Guangdong. Due to differences in climate conditions and other factors at the test site, the cultivation and management of field experiments were different from those in the main cotton-growing areas. The specific cultivation and management methods are as follows.

[0044] Seedling Raising and Transplanting Seedlings are raised in the greenhouse in mid-May, and it takes about 15 days from seeding to transplanting. Insert labels for marking during seeding. Before seeding, soak the substrate pots in water until the water content of the substrate pots reaches saturation, then take out the substrate pots and place them in the holes of the corresponding-sized plastic seedling trays, with one pot corresponding to one hole. Use smooth seeds for seeding. Use the tip of a chopstick to insert two small holes on the surface of the pot, with a hole distance of about 1 cm and a hole depth of about 0.8 cm. Sow one seed in each hole. When sowing, the pointed end of the seed should face down and be inserted into the small hole. The seeding depth should be such that the thick end of the seed is level with the surface of the pot. Then cover the soil, and control the soil covering thickness at 1.5 - 1.7 cm. After covering the soil, spray a 500-fold dilution solution of Likuling to prevent diseases such as damping-off. Then place the seedling tray in the tray bed and perform tidal irrigation, with the water level being 3 / 4 of the height of the seedling tray being appropriate. After one day, when the soil covering on the seedling tray is wet, drain the water in the tray bed. If the cotton seedlings show mild wilting after emergence, irrigation can be carried out, with the method being the same as above. The cotton seedlings emerge evenly 4 - 5 days after sowing. Spray a mixed solution (500-fold Likuling, 1000-fold omethoate, 500-fold acetamiprid, and 1000-fold imidacloprid) on the leaves to prevent diseases such as damping-off, aphids, whiteflies, and leaf miners. After one week, continue to spray the above pesticide dilution solution on the leaves, and spray the above pesticide dilution solution on the leaves one day before transplanting to prevent cotton seedlings from carrying pests and diseases into the field.

[0045] The cotton seedlings are transplanted in early June when they have grown 3 - 4 true leaves. Complete the land preparation and ridging work before the end of May, with ridging at 2 meters. The cotton seedlings are transplanted in a wide-narrow row arrangement, with a wide row of 1.1 meters, a narrow row of 1.0 meters, and a plant spacing of 0.5 meters. When transplanting, the soil covering should not be higher than the cotyledon node position of the cotton seedlings.

[0046] Management during the seedling stage (early June - late June) Apply seedling fertilizer about 10 days after transplanting (mid-June), with 30 catties of compound fertilizer per mu, and the method is to apply it by digging holes. After the cotton seedlings are transplanted, heavy rain often occurs. After the rain, promptly support the seedlings and clear the ditches to drain the accumulated water. The main pests during the seedling stage are leafhoppers and beet armyworms, and the pesticide used is a mixed solution (500-fold cotton and tobacco spirit, 1000-fold omethoate, and 500-fold acetamiprid), and the method is to spray on the back of the leaves. When the cotton seedlings have grown 7 - 9 true leaves, vegetative branches will grow in the axils of these leaves. When these vegetative branches grow to 2 - 3 cm, promptly remove them to reduce nutrient consumption. Remove the buds once during the seedling stage. Weeding during the seedling stage is mainly manual weeding, generally 2 times.

[0047] Management during the budding stage (early July - mid-late July) In the test location, the rainfall is large and frequent in summer, and soil erosion is serious, so the fertilizers applied to the cotton fields are easily lost. Therefore, the budding fertilizer is applied in two times, with an interval of about 15 days. Apply the initial budding fertilizer after leaving the first fruiting branch in early July, with 60 catties of compound fertilizer per mu and 600 catties of organic fertilizer per mu. Apply the fertilizer on the ridged surface, and then combine fertilization with intertillage and soil heaping on the ridged surface; apply the second budding fertilizer when the cotton plants have 6 - 7 fruiting branches in mid-late July, with 50 catties of compound fertilizer per mu and 4 catties of boron and zinc micronutrient fertilizer per mu. Scatter the fertilizer in the furrow.

[0048] To prevent the test cotton plants from growing too tall, mepiquat chloride was used for control. At the beginning of July, when the cotton plants had 10 - 12 true leaves, 0.8 grams of mepiquat chloride was used per mu, diluted with 15 kilograms of water, and evenly sprayed on the leaf surface to control the large seedlings but not the small ones. In the middle of July, when the cotton plants had 6 - 7 nodes, 1.2 - 1.5 grams of mepiquat chloride was used per mu, diluted with 15 kilograms of water and sprayed on the leaf surface to promote the steady growth of the cotton plants. From the end of June to the beginning of July, when the cotton seedlings grew 10 - 12 true leaves, fruiting branches would grow in the axils of the true leaves and start budding. Since the growth positions of these fruiting branches were relatively low and their growth vigor was weak, and with the wet and rainy weather, the flower buds on the fruiting branches developed poorly and were prone to falling off, forming empty branches. Therefore, these 3 fruiting branches were usually removed. The 4th fruiting branch was reserved as the first fruiting branch in the true sense. After entering the budding stage, every about 15 days, the redundant buds growing in the axils of the main stem leaves or fruiting branch leaves were removed in time. Generally, the buds were removed 2 - 3 times during the budding stage.

[0049] Management during the flowering and boll - setting stage (from the end of July to the beginning of September) In the first ten - day period of August, when the cotton plants bloomed to 4 - 5 nodes, flower - boll fertilizers were applied, 30 catties each of urea and potassium fertilizer, and the fertilizers were scattered in the furrows of the first - time hoed ridges. At the beginning of August, when the cotton entered the initial flowering stage (the cotton plants had 9 - 10 fruiting branches), chemical control was carried out again, using 2 grams of mepiquat chloride per mu, diluted with 15 kilograms of water. About one week after topping the cotton plants in the middle and late ten - day period of August, 2.5 grams of mepiquat chloride was used per mu, diluted with 15 kilograms of water, and evenly sprayed on the leaf surface. When the cotton plants grew 15 - 16 fruiting branches in the first and middle ten - day periods of August, the main stem top was topped. During the flowering and boll - setting stage, every about 15 days, the redundant buds growing in the axils of the main stem leaves or fruiting branch leaves were removed in time. The buds were removed 2 - 3 times during the flowering stage. Insect pests were controlled as appropriate according to the occurrence of insect pests.

[0050] Management during the boll - opening and lint - shedding stage (from the beginning of September to the end of October) After the cotton entered the boll - opening and lint - shedding stage, the cotton plants began to enter the decline stage, and the occurrence of insect pests was less, so basically no fertilization and pest control were carried out, but the pruning work still needed to be done well. The buds were removed 1 - 2 times during the boll - opening and lint - shedding stage. The seeds were harvested after they matured.

[0051] Through multiple generations of planting and identification, 269 homozygous strain materials of transgenic cotton plant offspring were obtained respectively. The specific method was as follows: In the early - generation plant rows, the single plants with the target exogenous gene were determined by PCR detection and self - pollinated seeds were harvested; the self - pollinated seeds were used to continue planting 10 - 20 plant rows, and the separation of the target exogenous gene in each plant row was detected by PCR; the plant rows with no separation of the target exogenous gene detected by PCR were the pure - line materials (if no non - separating plant rows were obtained, the self - pollination of the cotton single plants containing the exogenous gene and the offspring identification process were continued until pure lines were obtained).

[0052] Example 3 Analysis of the fiber quality of transgenic cotton Using the method of Example 2, homozygous lines of 269 different cotton transformation events obtained in Example 2 and the recipient control cotton variety Jimian 14 were planted. Each experimental material had three plant row replicates, and the seed cotton of 12 cotton bolls was harvested respectively.

[0053] Among the 269 transgenic cotton lines tested, only several lines were found to be significantly superior to the control cotton variety Jimian 14 in some fiber qualities. Among them was the transgenic cotton line numbered cI55-3 (hereinafter the transformation event existing therein is also referred to as the cI55-3 transformation event). Only the test results related to cI55-3 and the recipient control Jimian 14 are described below.

[0054] After weighing to obtain the weight of the seed cotton, ginning was carried out using a SY-201 roller gin produced by Jianghe Machinery Equipment Factory in Xinxiang County, Henan Province. The weight of the lint obtained after ginning the test seed cotton samples was weighed and recorded, and the boll weight and lint percentage of different test materials were calculated. The calculation formula was: sample lint percentage = lint weight / seed cotton weight × 100%. The test results are shown in Table 1.

[0055] The test results showed that the average boll weight of the transformation event cI55-3 was 3.31 grams, which was 30.3% lower than the average boll weight of 4.75 grams of the recipient control Jimian 14; the average lint percentage of the transformation event cI55-3 was 34.42%, which was significantly lower by 14.68% than the average lint percentage of 40.34% of the recipient control Jimian 14.

[0056] Table 1 Lint percentage of test cotton materials Test sample Weight of seed cotton (g) Weight of lint cotton (g) Weight per boll (g) Lint percentage (%) cI55-3 (Plant row 1) 41 15 3.42 36.59% cI55-3 (Plant row 2) 39 12 3.25 30.77% cI55-3 (Plant row 3) 39 14 3.25 35.90% cI55-3 (Average) 39.67 13.67 3.31±0.10 34.42%±3.18% Jimian 14 (Plant row 1) 60.00 25.00 5.00 41.67% Jimian 14 (Plant row 2) 50.00 20.00 4.17 40.00% Jimian 14 (Plant row 3) 61.00 24.00 5.08 39.34% Jimian 14 (Average) 57.00 23.00 4.75±0.41 40.34%±0.98% The HFT cotton tester produced by Prime (Jiangsu) Textile Electronic Instrument Co., Ltd. was used to detect the cotton fiber quality of the test lint samples, and fiber length, micronaire value, and specific strength data were obtained respectively. The test data and analysis are shown in Table 2.

[0057] The test results showed that the average fiber length of the transformation event cI55-3 was 32.51 mm, which was 5.27% higher than the fiber length of 30.89 mm of the recipient control Jimian 14; the micronaire value of the transformation event cI55-3 was 4.45, which was 28.82% lower than the micronaire value of 6.26 of the recipient control Jimian 14; the specific strength of the transformation event cI55-3 was 33.4, which was 11.83% higher than the specific strength of 29.9 of the recipient control Jimian 14.

[0058] Table 2 Fiber quality of test cotton materials Test sample Length (mm) Micronaire value (ug / inch) Specific strength (cN / tex) cI55-3 (Plant row 1) 32.10 4.56 34.0 cI55-3 (Plant row 2) 33.03 4.51 32.3 cI55-3 (Plant row 3) 32.41 4.29 33.9 cI55-3 (Average) 32.51±0.47 4.45±0.14 33.4±0.95 Jimian 14 (Plant row 1) 31.00 6.25 29.3 Jimian 14 (Plant row 2) 30.28 6.14 29.1 Jimian 14 (Plant row 3) 31.38 6.38 31.2 Jimian 14 (Average) 30.89±0.46 6.26±0.10 29.9±0.95 In the later stage, we also conducted repeated experiments for many years, and the results were basically the same. In summary, the impact of cotton transformation event cI55-3 on cotton fiber quality is that the three fiber quality indicators of cotton fiber length, micronaire value, and specific strength are simultaneously improved significantly, but the lint percentage and lint yield also decrease significantly.

[0059] Example 4 Flanking Sequence Analysis Sample Preparation: Extract the genomic DNA of cotton materials containing the cI55-3 transformation event using the plant DNA extraction method well-known to those skilled in the art. Take 2.5 µg of DNA and digest it with Stu I Sca I for 6 - 8 hours. After purification by alcohol precipitation, dissolve it in an appropriate amount of water.

[0060] Ligation of Adaptors: According to the analysis of the vector restriction sites, design and synthesize two pairs of adaptors respectively: GenomeWalker Adaptor +, GenomeWalker Adaptor – (the sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively). The 5' end of SEQ ID NO: 2 was phosphorylated and the 3' end was added with an amino group.

[0061] Mix GenomeWalker Adaptor + and GenomeWalker Adaptor – in equal amounts, incubate at 70°C for 10 minutes, and then slowly cool to room temperature. Take 4 μl of digested and purified DNA and add it to a tube containing 1.9 μl of GenomeWalker Adaptor (25 μM), 1.6 μl of 10X ligation buffer, and 0.5 μl of T4 DNA ligase (6 units / μl). Incubate overnight at 16°C to stop the reaction, incubate at 70°C for 5 min. In each tube, add 72 μl of TE (10 / 1, pH 7.5) and shake at low speed for 5 - 10 s.

[0062] LB End Sequence Analysis, Using Clontech GenomeWalker TMUniversal kit, using primers AP1 and GSP1 (the sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively), with the ligation product as the template, perform the first round of amplification: 7 cycles: 94°C for 25 s, 72°C for 4 min; 32 cycles: 94°C for 25 s, 67°C for 4 min; after the last cycle, incubate at 67°C for 7 minutes. After diluting the PCR product 50-fold, use primers AP2 and GSP2 (the sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6 respectively) for the second round of PCR amplification. The PCR program is as follows: 5 cycles: 94°C for 25 s, 72°C for 5 min; 20 cycles: 94°C for 25 s, 67°C for 4 min; after the last cycle, incubate at 67°C for 10 minutes. Recover the product and sequence it.

[0063] SEQ ID NO: 1 GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT SEQ ID NO: 2 ACCAGCCC SEQ ID NO: 3 GTAATACGACTCACTATAGGGC SEQ ID NO: 4 TGGCGATGCCTGCTTGCCGAATATCA SEQ ID NO: 5 ACTATAGGGCACGCGTGGT SEQ ID NO: 6 CGGGACTCTGGGGTTCGAAATGACCG The sequence analysis of the left border (LB) end of the cI55-3 event is as Figure 2 shown. A total of 2235 bp of nucleotide sequence (the sequence is shown in SEQ ID NO: 7) was obtained, including 1 bp - 1523 bp as the cotton sequence, 1524 bp - 1591 bp as the ligation sequence, 1592 bp - 1846 bp as the Tnos terminator sequence, and 1847 bp - 2235 bp as the ligation sequence.

[0064] SEQ ID NO: 7 Sequence description: 1 - 1523 Cotton DNA 1524 - 1591 Ligation sequence 1592 - 1846 Tnos terminator sequence 1847 - 2235 Ligation sequence 1 AACCTTTATT TTAGAAAGAT GAGGGAAAAA ACAGGATTTT TGGGGGCTCC TAGGGGCGGA 61 GGACCAACGG TCCGGTGATG TCCCCTTCAT CGGAACCCAA AATTCGATTC CTCATGACAT 121 GCCTGTGGCA AAAAAAAAAG TAGAAGAAAA GAAAAAAAAA GAAATAATAG ACTAAAATCG 181 GGTGGGCTTC GAACTATTCA AGAGAGAACA TAATTTAAAT GGGCCCCCAA TCGAGCTCGG 241 ACAAAAATGG GTGTCTACAG CTATAGAACT AAATGAATGT TTGGTTAGTT TGGAGTAAGG 301 TTGAAGTTGG TTTGAATGGT TAATGTTTTG ATATTTTATC ATTTGGAATT ATTGATAGGT 361 TGTGAATTGG TTGTTTGGAA ATGCATAAGT AAAATTTTAG GTCTTCTAAA TTTGAGCAGG 421 TTGAATTTTG ATTGTTACAA ATTGTCATGT CGCAACGACG TACCCTTGGT GTTGCGACGA 481 GGAACAGAGT AAGATTCTAG TGGTGATGTC TTTGCTTTGA GGTCACGACG AGCACCGAAC 541 AGAAAGTTGT GTTACGACGA GGAGCCTCTA GTATTGTGAC ATCAATCTAA AATTTTAAAA 601 TTTTTACAAT TTGGTCTTAT TTCAACCTTG GGTCAACAAA AGAGCTTTTG TAAGCCTGTA 661 TAAGACCCAA AAATGATTAT GTATGTTTTA TCCACTTGTT TTTGTTACAT TGGAATGTTT 721 AATGGTTTGT AATTGAACTT AATTTGACCG TAATTACTTC GGCAATGAAT ATAGTATCTT 781 ATAACTCAAA CCCGACAATC AAGTCGAGTA AAAAGGTGTA ACAGATAGAT TCTACTAACA 841 CCTTACAAAA CCATTTTCAT CGATGCAGTC GAATCCAATT CTATAATAAC TGATTTAATA 901 CCAGCATCCC AAGAAATGGT GCAACCATAA AACAATCCCC ACAATTCAGC TAAACAGTTT 961 CCCAACTTTG CCGCAAACCC AACAACCCAG TTTCCAAGAC TATTTCAGAC CACCCCATTG 1021 GCTCCAACAG TCTCTCTTTC CCGATCCCAC CTACCATTAA CATTGAGTTT AAACCAACCA 1081 TCCGTCGGCT TCCCTCGTGA CGGCAACACC AACTATTTTG GTTTGTTTGG CGTTTGAGAA 1141 ACCAGTCGTG AAGCACTAGT CTCCTTTTAG CTGTCACACA TAGCTTACAT TTTCATGTAG 1201 AGCGGCCAAA GTTTATCTGC CATGACAACA TCATTCCTCC ATTTTCAAAG CCGTCGGTGC 1261 ACTACTAGAA ACACCAAAGA CCAACTCCAC TCACTAACTC GCCTGTAAAG TAAAAGAACA 1321 AGAATATTCC ACCCCTTATT TGCACCCCTT CACGTGCCAT ATCAATCAAA TTTTGACATA 1381 TGTATTTTTC TAAAATACCA TTTATTTTTC GTTAACTTTG ATATCCCCAT TCCTTTTACT 1441 TAAAACAGAA AAAAACCCCA ACCAAATTCT AAATAACAAA TGAAAATTAA TGGTAAAATC 1501 TGCATCCACC TAAGTTTCTC AAT TGCCCTT ATACGAAGTT ATGTCGAGCACCAGAACCAC 1561 CACCAGAGCC GCCGCCAGCA TTGACAGGAG GCCCGATCTA GTAACATAGATGACACCGCG 1621 CGCGATAATT TATCCTAGTT TGCGCGCTAT ATTTTGTTTT CTATCGCGTATTAAATGTAT 1681 AATTGCGGGA CTCTAATCAT AAAAACCCAT CTCATAAATA ACGTCATGCATTACATGTTA 1741 ATTATTACAT GCTTAACGTA ATTCAACAGA AATTATATGA TAATCATCGCAAGACCGGCA 1801 ACAGGATTCA ATCTTAAGAA ACTTTATTGC CAAATGTTTG AAAGATCGGGGATCATCCGG 1861 GTCTGTGGCG GGAACTCCAC GAAAATATCC GAACGCAGCA AGATATCGCGGTGCATCTCG 1921 GTCTTGCCTG GGCAGTCGCC GCCGACGCCG TTGATGTGGA CGCCGGGCCCGATCATATTG 1981 TCGCTCAGGA TCGTGGCGTT GTGCTTGTCG GCCGTTGCTG TCGTAATGATATCGGCACCT 2041 TCGACCGCCT GTTCCGCAGA GATCCCGTGG GCGAAGAACT CCAGCATGAGATCCCCGCGC 2101 TGGAGGATCA TCCAGCCGGC GTCCCGGAAA ACGATTCCGA AGCCCAACCTTTCATAGAAG 2161 GCGGCGGTGG AATCGAAATC TCGTGATGGC AGGTTGGGCG TCGCTTGGTCGGTCATTTCG 2221 AACCCCAGAG TCCCG (SEQ ID NO: 7) RB end sequence analysis: Using Clontech GenomeWalker TMUniversal kit, using primers AP1 and GSP3 (sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 8 respectively), with the ligation product as the template, perform the first round of amplification: 7 cycles: 94°C for 25 s, 72°C for 4 min; 32 cycles: 94°C for 25 s, 67°C for 4 min; after the last cycle, incubate at 67°C for 7 minutes. After diluting the PCR product 50-fold, use primers AP2 and GSP4 (sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 9 respectively) for the second round of PCR amplification. The PCR program is as follows: 5 cycles: 94°C for 25 s, 72°C for 5 min; 20 cycles: 94°C for 25 s, 67°C for 4 min; after the last cycle, incubate at 67°C for 10 minutes. Recover the product and sequence it. SEQ ID NO: 8 TCCTCCGTCAAATCGCCAAGCGTCAG SEQ ID NO: 9 TGGATTACATTCTCGGAGACAACCCG Analysis of the right border (RB) end sequence of the cI55-3 event is as Figure 3 shown. A total of 1142 bp of nucleotide sequence (sequence shown in SEQ ID NO: 10) was obtained, including the cel1 partial gene sequence from 1 bp to 332 bp, the ligation sequence from 333 bp to 338 bp, the PS sequence from 339 bp to 527 bp, the ligation sequence from 528 bp to 533 bp, the Tnos terminator sequence from 534 bp to 789 bp, the ligation sequence from 790 bp to 854 bp, and the cotton DNA sequence from 855 bp to 1142 bp. SEQ ID NO: 10 Sequence description: 1-332 cel1 Partial gene sequence 333-338 Ligation sequence 339-527 PS sequence 528-533 Ligation sequence 534-789 Tnos terminator sequence 790-854 Ligation sequence 855-1142 Cotton DNA sequence 1 TGGATTACAT TCTCGGAGAC AACCCGATGG GACTGTCTTA CATGGTTGGA TACGGTCAAA 61 AGTTTCCACG TAGGATTCAT CACCGTGGTA GCTCGGTTCC TTCGGTTTCA GCCCATCCAA 121 GCCACATAGG GTGCAAAGAA GGCTCTCGCT ATTTCCTAAG CCCAAATCCT AACCCAAACC 181 TTTTGGTTGG TGCTGTAGTC GGTGGACCTA ATGTCACTGA TGCCTTTCCG GATTCAAGAC 241 CTTACTTTCA GCAGTCTGAG CCCACGACTT ATATCAATGC ACCACTAGTG GGCCTTCTCG 301 GTTACTTCTC CGCCCATTCT ACTTGGCGAT GAGTCGAGGC TGAGTGGTTA ACTTTGAGTA 361 TTATGGCATT GGAAAAGCCA TTGTTCTGCT TGTAATTTAC TGTGTTCTTT CAGTTTTGTT 421 TTCGGACATC AAGTTAACAA AAAAAAAAAA AAAAAAAAAA AAATTTAACA AAAAAAAAAA 481 AAAAAAAAAA AATTTAACAA AAAAAAAAAA AAAAAAAAAA ATTTAAAGAG CTCGAATTTC 541 CCCGATCGTT CAAACATTTG GCAATAAAGT TTCTTAAGAT TGAATCCTGT TGCCGGTCTT 601 GCGATGATTA TCATATAATT TCTGTTGAAT TACGTTAAGC ATGTAATAAT TAACATGTAA 661 TGCATGACGT TATTTATGAG ATGGGTTTTT ATGATTAGAG TCCCGCAATT ATACATTTAA 721 TACGCGATAG AAAACAAAAT ATAGCGCGCA AACTAGGATA AATTATCGCG CGCGGTGTCA 781 TCTATGTTAC TAGATCGGGA ATTCGATACT AGTGATCAGA TTGTCGTTTC CCGCCTTCAG 841 TTTAAACTAT CAGT ATAAAG ACCGAATCTC ATTGAGAATA TTATATACAA TAGATTTCTT 901 CAGTTCATTT ATTTTTGTTA AAAATAAAGA AACTAAAAAA GGGTTTTCAT TATTTTCCCT 961 ATAATTTTAT TTAATCAAAT AAAATGATTA AATTTTAACA AATAAATGCA GTTGGAAAGT 1021 TGGGAGTTTA ATTTCACAAT GTGTATATGT TGTTGCTACC CCTTACCACC TTTTTGGTGG 1081 AGCTATTGAT GTGATTGTTG TTATGCAACA AGAGGGACAT TTCGAAGCAC GCCATGTTAT 1141 GT ( SEQ ID NO: 10 ) Using the recipient material Jimian 14 DNA as a template, primers cI55-3J14F (sequence as shown in SEQ ID NO: 11) and cI55-3J14R (sequence as shown in SEQ ID NO: 12) were designed on the cotton genome sequence flanking the LB and RB ends of the insertion sequence, respectively. An amplification product of about 797 bp was obtained and sequenced. Comparison with the flanking sequence of cI55-3 showed that the event was obtained by replacing 82bp bases on the original genome sequence (TTATATTTCACTTAAAGCTAGAAAGCAACGACGTTAAATTTGGAATTAGGTTCTAATTGGGAAAAAAAAATTTTGACAAAAA, SEQ ID NO: 18) by the insertion sequence.

[0065] See the screenshot of the comparison results. Figure 4 .

[0066] SEQ ID NO: 11 CGGCAACACCAACTATTTTGGTT SEQ ID NO: 12 CATAACATGGCGTGCTTCGA Based on the above results, a person skilled in the art can easily derive the characteristic DNA sequence of the cI55-3 event (SEQ ID NO: 13), as shown below. The underlined portion shows the T-DNA insertion sequence, and the ununderlined portion shows the flanking cotton genomic DNA sequence of the insertion sequence. The schematic diagram of the insertion sequence and the flanking sequence is shown in FIG. Figure 5 shown.

[0067] SEQ ID NO: 13: 1 AACCTTTATT TTAGAAAGAT GAGGGAAAAA ACAGGATTTT TGGGGGCTCC TAGGGGCGGA 61 GGACCAACGG TCCGGTGATG TCCCCTTCAT CGGAACCCAA AATTCGATTC CTCATGACAT 121 GCCTGTGGCA AAAAAAAAAG TAGAAGAAAA GAAAAAAAAA GAAATAATAG ACTAAAATCG 181 GGTGGGCTTC GAACTATTCA AGAGAGAACA TAATTTAAAT GGGCCCCCAA TCGAGCTCGG 241 ACAAAAATGG GTGTCTACAG CTATAGAACT AAATGAATGT TTGGTTAGTT TGGAGTAAGG 301 TTGAAGTTGG TTTGAATGGT TAATGTTTTG ATATTTTATC ATTTGGAATT ATTGATAGGT 361 TGTGAATTGG TTGTTTGGAA ATGCATAAGT AAAATTTTAG GTCTTCTAAA TTTGAGCAGG 421 TTGAATTTTG ATTGTTACAA ATTGTCATGT CGCAACGACG TACCCTTGGT GTTGCGACGA 481 GGAACAGAGT AAGATTCTAG TGGTGATGTC TTTGCTTTGA GGTCACGACG AGCACCGAAC 541 AGAAAGTTGT GTTACGACGA GGAGCCTCTA GTATTGTGAC ATCAATCTAA AATTTTAAAA 601 TTTTTACAAT TTGGTCTTAT TTCAACCTTG GGTCAACAAA AGAGCTTTTG TAAGCCTGTA 661 TAAGACCCAA AAATGATTAT GTATGTTTTA TCCACTTGTT TTTGTTACAT TGGAATGTTT 721 AATGGTTTGT AATTGAACTT AATTTGACCG TAATTACTTC GGCAATGAAT ATAGTATCTT 781 ATAACTCAAA CCCGACAATC AAGTCGAGTA AAAAGGTGTA ACAGATAGAT TCTACTAACA 841 CCTTACAAAA CCATTTTCAT CGATGCAGTC GAATCCAATT CTATAATAAC TGATTTAATA 901 CCAGCATCCC AAGAAATGGT GCAACCATAA AACAATCCCC ACAATTCAGC TAAACAGTTT 961 CCCAACTTTG CCGCAAACCC AACAACCCAG TTTCCAAGAC TATTTCAGAC CACCCCATTG 1021 GCTCCAACAG TCTCTCTTTC CCGATCCCAC CTACCATTAA CATTGAGTTTAAACCAACCA 1081 TCCGTCGGCT TCCCTCGTGA CGGCAACACC AACTATTTTG GTTTGTTTGGCGTTTGAGAA 1141 ACCAGTCGTG AAGCACTAGT CTCCTTTTAG CTGTCACACA TAGCTTACATTTTCATGTAG 1201 AGCGGCCAAA GTTTATCTGC CATGACAACA TCATTCCTCC ATTTTCAAAGCCGTCGGTGC 1261 ACTACTAGAA ACACCAAAGA CCAACTCCAC TCACTAACTC GCCTGTAAAGTAAAAGAACA 1321 AGAATATTCC ACCCCTTATT TGCACCCCTT CACGTGCCAT ATCAATCAAATTTTGACATA 1381 TGTATTTTTC TAAAATACCA TTTATTTTTC GTTAACTTTG ATATCCCCATTCCTTTTACT 1441 TAAAACAGAA AAAAACCCCA ACCAAATTCT AAATAACAAA TGAAAATTAATGGTAAAATC 1501 TGCATCCACC TAAGTTTCTC AATTGCCC TT ATACGAAGTT ATGTCGAGCA CCAGAACCAC 1561 CACCAGAGCC GCCGCCAGCA TTGACAGGAG GCCCGATCTA GTAACATAGA TGACACCGCG 1621 CGCGATAATT TATCCTAGTT TGCGCGCTAT ATTTTGTTTT CTATCGCGTA TTAAATGTAT 1681 AATTGCGGGA CTCTAATCAT AAAAACCCAT CTCATAAATA ACGTCATGCA TTACATGTTA 1741 ATTATTACAT GCTTAACGTA ATTCAACAGA AATTATATGA TAATCATCGC AAGACCGGCA 1801 ACAGGATTCA ATCTTAAGAA ACTTTATTGC CAAATGTTTG AAAGATCGGG GATCATCCGG 1861 GTCTGTGGCG GGAACTCCAC GAAAATATCC GAACGCAGCA AGATATCGCG GTGCATCTCG 1921 GTCTTGCCTG GGCAGTCGCC GCCGACGCCG TTGATGTGGA CGCCGGGCCC GATCATATTG 1981 TCGCTCAGGA TCGTGGCGTT GTGCTTGTCG GCCGTTGCTG TCGTAATGAT ATCGGCACCT 2041 TCGACCGCCT GTTCCGCAGA GATCCCGTGG GCGAAGAACT CCAGCATGAG ATCCCCGCGC 2101 TGGAGGATCA TCCAGCCGGC GTCCCGGAAA ACGATTCCGA AGCCCAACCT TTCATAGAAG 2161 GCGGCGGTGG AATCGAAATC TCGTGATGGC AGGTTGGGCG TCGCTTGGTC GGTCATTTCG 2221 AACCCCAGAG TCCCGCTCAG AAGAACTCGT CAAGAAGGCG ATAGAAGGCG ATGCGCTGCG 2281 AATCGGGAGC GGCGATACCG TAAAGCACGA GGAAGCGGTC AGCCCATTCG CCGCCAAGCT 2341 CTTCAGCAAT ATCACGGGTA GCCAACGCTA TGTCCTGATA GCGGTCCGCC ACACCCAGCC 2401 GGCCACAGTC GATGAATCCA GAAAAGCGGC CATTTTCCAC CATGATATTC GGCAAGCAGG 2461 CATCGCCATG GGTCACGACG AGATCATCGC CGTCGGGCAT GCGCGCCTTG AGCCTGGCGA 2521 ACAGTTCGGC TGGCGCGAGC CCCTGATGCT CTTCGTCCAG ATCATCCTGA TCGACAAGAC 2581 CGGCTTCCAT CCGAGTACGT GCTCGCTCGA TGCGATGTTT CGCTTGGTGG TCGAATGGGC 2641 AGGTAGCCGG ATCAAGCGTA TGCAGCCGCC GCATTGCATC AGCCATGATG GATACTTTCT 2701 CGGCAGGAGC AAGGTGAGAT GACAGGAGAT CCTGCCCCGG CACTTCGCCC AATAGCAGCC 2761 AGTCCCTTCC CGCTTCAGTG ACAACGTCGA GCACAGCTGC GCAAGGAACG CCCGTCGTGG 2821 CCAGCCACGA TAGCCGCGCT GCCTCGTCCT GCAGTTCATT CAGGGCACCG GACAGGTCGG 2881 TCTTGACAAA AAGAACCGGG CGCCCCTGCG CTGACAGCCG GAACACGGCG GCATCAGAGC 2941 AGCCGATTGT CTGTTGTGCC CAGTCATAGC CGAATAGCCT CTCCACCCAA GCGGCCGGAG 3001 AACCTGCGTG CAATCCATCT TGTTCAATCA TGCGAAACGA TCCAGATCCG GTGCAGATTA 3061 TTTGGATTGA GAGTGAATAT GAGACTCTAA TTGGATACCG AGGGGAATTT ATGGAACGTC 3121 AGTGGAGCAT TTTTGACAAG AAATATTTGC TAGCTGATAG TGACCTTAGG CGACTTTTGA 3181 ACGCGCAATA ATGGTTTCTG ACGTATGTGC TTAGCTCATT AAACTCCAGA AACCCGCGGC 3241 TGAGTGGCTC CTTCAACGTT GCGGTTCTGT CAGTTCCAAA CGTAAAACGG CTTGTCCCGC 3301 GTCATCGGCG GGGGTCATAA CGTGACTCCC TTAATTCTCC GCTCATGAGG ATCTATAACT 3361 TCGTATAGCA TACATTATAC GAAGTTATGT TAACAAGCTT AAATTATAGC ATACCTCACG 3421 ATGTGGGTGA AGTAAAATTA TTTAACAAAT ATATTTTGAA AAATTGATAA AAATACTAAA 3481 TGAGGTTTTG GTTGAATAGT AAGATATAAT TATTACAAAT TATAAATATG TAGGTTCAAA 3541 ATCTATCATG TGTATATTTG TACTATTATT CTATATAAAT TGATAACCTT ATAAAAGTAT 3601 CTAATTTAGT TTATGGTTGA TTGATCGATA ATACCAAATT TATTAAAAAT TAATATTAGT 3661 AAAGATATAT AGTACAAAAC TAAACATAAA ATTTTATATG TTAAGGAAAT AGCGGAAAAA 3721 ATATCATATT TGTAGAACTG TTTAGCAGTG TGGGAGAATG GGATCATTAC AAGGAAAAAT 3781 GAAATATATA TCATTAATAC CAAACATAAA AGAAAGCGTC TTTTGATAAA GTTGTTATTG 3841 GTGTAATGTG AAGGGACCAC AATCATCACC ATTCACCACT TGCTCCTAAT TGAGTTGAAA 3901 TCTTTTTACA ACATAGAAAA CTAGAAGATC GCCCTTTCTT GCTTCATATA TATAGATTTT 3961 GTATCATCGC AATTTCACAT CACACACACA AGTAAAGCAT TAGCAACCAT AGCCTCTAGA 4021 ATGGCGCGAA AATCCCTAAT TTTCCCGGTG ATTTTGCTCG CCGTTCTTCT CTTCTCTCCG 4081 CCGATTTACT CCGCCGGTCA CGATTACCGC GACGCTCTCC GTAAAAGCAT TCTCTTCTTC 4141 GAAGGTCAAC GTTCCGGTAA ACTCCCTCCA GATCAACGCT TAAAATGGCG CCGTGACTCA 4201 GCATTACGCG ACGGTTCCTC CGCCGGCGTT GACTTATCCG GTGGTTACTA CGACGCCGGA 4261 GACAACATCA AGTTCGGTTT TCCGATGGCG TTCACAACAA CGATGCTTTC ATGGAGTATA 4321 ATCGATTTCG GTAAAACCAT GGGACCTGAG CTTAGAAACG CCGTGAAAGC TGTTAAATGG 4381 GGAACAGATT ACCTCCTTAA AGCGACGGCG ATTCCCGGAG TAGTCTTCGTCCAAGTCGGA 4441 GACGCTTACT CCGATCATAA CTGTTGGGAA AGGCCTGAAG ATATGGACAC TCTCCGTACT 4501 GTTTACAAAA TCGATAGAGC TCATCCTGGT TCTGACGTCG CTGGTGAAAC CGCAGCCGCT 4561 TTAGCCGCCG CTTCAATCGT TTTTAGAAAA CGCGATCCTG CTTATTCCAG ACTTCTACTT 4621 GACCGTGCCA CTAGGGTATT CGCGTTTGCT AACAGATATC GCGGCGCGTA TAGTAACAGT 4681 CTCTACCACG CGGTTTGTCC TTTTTACTGT GATTTCAACG GTTACCAGGA TGAGTTACTG 4741 TGGGGAGCGG CATGGCTACA CAAAGCCTCG AGGAAACGAG CGTACAGAGA ATTCATTGTG 4801 AAGAACGAGG TCATTCTTAA GGCTGGAGAT ACCATTAATG AGTTTGGTTG GGACAATAAG 4861 CATGCTGGGA TTAATGTCTT AATCTCCAAG GAAGTGTTAA TGGGAAAAGC AGAGTATTTT 4921 GAGTCTTTCA AGCAGAACGC AGATGGGTTT ATCTGTTCTA TATTGCCTGG AATTTCTCAC 4981 CCCCAAGTCC AATACTCTCG AGGAGGGCTA CTAGTGAAGA CTGGAGGGAG TAACATGCAA 5041 CATGTAACAT CACTATCTTT CCTCCTATTG GCTTACTCTA ATTATCTGAG CCATGCCAAA 5101 AAGGTTGTGC CTTGTGGCGA ATTAACTGCT TCCCCATCTC TCCTCCGTCA AATCGCCAAG 5161 CGTCAGGTGG ATTACATTCT CGGAGACAAC CCGATGGGAC TGTCTTACAT GGTTGGATAC 5221 GGTCAAAAGT TTCCACGTAG GATTCATCAC CGTGGTAGCT CGGTTCCTTC GGTTTCAGCC 5281 CATCCAAGCC ACATAGGGTG CAAAGAAGGC TCTCGCTATT TCCTAAGCCC AAATCCTAAC 5341 CCAAACCTTT TGGTTGGTGC TGTAGTCGGT GGACCTAATG TCACTGATGC CTTTCCGGAT 5401 TCAAGACCTT ACTTTCAGCA GTCTGAGCCC ACGACTTATA TCAATGCACC ACTAGTGGGC 5461 CTTCTCGGTT ACTTCTCCGC CCATTCTACT TGGCGATGAG TCGAGGCTGA GTGGTTAACT 5521 TTGAGTATTA TGGCATTGGA AAAGCCATTG TTCTGCTTGT AATTTACTGT GTTCTTTCAG 5581 TTTTGTTTTC GGACATCAAG TTAACAAAAA AAAAAAAAAA AAAAAAAAAA TTTAACAAAA 5641 AAAAAAAAAA AAAAAAAAAT TTAACAAAAA AAAAAAAAAA AAAAAAAATT TAAAGAGCTC 5701 GAATTTCCCC GATCGTTCAA ACATTTGGCA ATAAAGTTTC TTAAGATTGA ATCCTGTTGC 5761 CGGTCTTGCG ATGATTATCA TATAATTTCT GTTGAATTAC GTTAAGCATG TAATAATTAA 5821 CATGTAATGC ATGACGTTAT TTATGAGATG GGTTTTTATG ATTAGAGTCC CGCAATTATA 5881 CATTTAATAC GCGATAGAAA ACAAAATATA GCGCGCAAAC TAGGATAAAT TATCGCGCGC 5941 GGTGTCATCT ATGTTACTAG ATCGGGAATT CGATACTAGT GATCAGATTG TCGTTTCCCG 6001 CCTTCAGTTT AAACTATCAG T ATAAAGACC GAATCTCATT GAGAATATTATATACAATAG 6061 ATTTCTTCAG TTCATTTATT TTTGTTAAAA ATAAAGAAAC TAAAAAAGGGTTTTCATTAT 6121 TTTCCCTATA ATTTTATTTA ATCAAATAAA ATGATTAAAT TTTAACAAATAAATGCAGTT 6181 GGAAAGTTGG GAGTTTAATT TCACAATGTG TATATGTTGT TGCTACCCCTTACCACCTTT 6241 TTGGTGGAGC TATTGATGTG ATTGTTGTTA TGCAACAAGA GGGACATTTCGAAGCACGCC 6301 ATGTTATGT Example 5 Transformation Event Detection Whether the cotton material has the unique signature DNA information of the cI55-3 transformation event can identify whether the cotton material is obtained by using the technology of the present invention. Therefore, specific DNA primer pairs can be used for PCR amplification to detect the cI55-3 event. The primer pair consists of a first primer that specifically recognizes either flank sequence of the insertion sequence and a second primer that specifically recognizes the T-DNA insertion sequence of the present invention. For example, when the first primer is cI55-3LB5 (SEQ ID NO: 14), the second primer can be cI55-3LB3 (SEQ ID NO: 15); when the first primer is cI55-3RB5 (SEQ ID NO: 16), the second primer can be cI55-3RB3 (SEQ ID NO: 17). The results of amplifying the cotton samples of the cI55-3 event and Jimian 14 using the primer pairs cI55-3LB5 / cI55-3LB3 and cI55-3RB5 / cI55-3RB3 are as Figure 6 and Figure 7 shown.

[0068] The PCR system is as follows: 10×Ex buffer 5.0 μl dNTP (both 2.5 mmol / L) 1.0 μl 5’ primer (10 μmol / L) 2.0 μl 3' primer (10 μmol / L) 2.0 μl Template DNA 100 ng Ex Taq 1 U Add sterile water to a total volume of 50 μl cI55-3LB5 / cI55-3LB3, cI55-3RB5 / cI55-3RB3, PCR program: 94.0°C for 5 min; 35 cycles: 94.0°C for 30 s, 62.0°C for 30 s, 72°C for 30 s; 72.0°C for 5 min.

[0069] SEQ ID NO: 14 ACCCCTTCACGTGCCATATC SEQ ID NO: 15 GATCGGGCCTCCTGTCAATG SEQ ID NO: 16 CAGATTGTCGTTTCCCGCCTT SEQ ID NO: 17 CGTGCTTCGAAATGTCCCTC Example 6 Chromosome Localization According to the obtained cotton flanking sequences, using the published tetraploid cotton ( Gossypium hirsutum chromosome group genome sequence (https: / / phytozome-next.jgi.doe.gov / blast-search) for comparative analysis, it can be known that in the cI55-3 event, the cotton flanking DNA sequence joined to the inserted sequence is highly homologous to the sequence on chromosome A9. Therefore, it can be known that the integration site of the exogenous DNA inserted sequence in the cI55-3 event is located on the 9th chromosome of the A group of the recipient tetraploid cotton. The representative seeds of this plant transformation event cotton (Gossypium hirsutum, Gossypium hirsutum ) were deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (CGMCC, Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101) on March 21, 2025, and the deposit number is: CGMCC No. 30702.

Claims

1. A cotton transformation event, characterized in that, The partial genomic sequence of cotton is replaced with a T-DNA insertion sequence on chromosome 9 of Group A of the cotton, wherein the T-DNA insertion sequence is the 1529-6021 bp sequence of the sequence shown in SEQ ID NO: 13, and the partial genomic sequence is as shown in SEQ ID NO:

18.

2. The cotton transformation event according to claim 1, characterized in that, The upstream flanking cotton genomic sequence of the T-DNA insertion sequence is: 1) The 1-1528 bp sequence of the sequence shown in SEQ ID NO: 13; or 2) The 3' end sequence of the 1-1528 bp sequence of the sequence shown in SEQ ID NO: 13 with a length of not less than 100 bp.

3. The cotton transformation event according to claim 1 or 2, characterized in that, The downstream flanking cotton genomic sequence of the T-DNA insertion sequence is: 1) The 6022-6309 bp sequence of the sequence shown in SEQ ID NO: 13; or 2) The 5' end sequence of the 6022-6309 bp sequence of the sequence shown in SEQ ID NO: 13 with a length of not less than 100 bp.

4. The cotton transformation event according to any one of claims 1-3, characterized in that, The DNA sequence shown in SEQ ID NO: 13 is included on chromosome 9 of Group A of the cotton, and the DNA sequence is composed of a T-DNA insertion sequence of 1529-6021 bp, an upstream flanking cotton genomic sequence of 1-1528 bp, and a downstream flanking cotton genomic sequence of 6022-6309 bp.

5. A DNA fragment, characterized in that, The DNA fragment sequentially includes from the 5' end to the 3' end: 1) Upstream flanking cotton genomic sequence: the 1-1528 bp sequence of the sequence shown in SEQ ID NO: 13; or the 3' end sequence of the 1-1528 bp sequence of the sequence shown in SEQ ID NO: 13 with a length of not less than 100 bp; 2) T-DNA insertion sequence: the 1529-6021 bp sequence of the sequence shown in SEQ ID NO: 13; 3) Downstream flanking cotton genomic sequence: the 6022-6309 bp sequence of the sequence shown in SEQ ID NO: 13; or the 5' end sequence of the 6022-6309 bp sequence of the sequence shown in SEQ ID NO: 13 with a length of not less than 100 bp.

6. A primer pair for detecting the cotton transformation event according to any one of claims 1-4 or the DNA fragment according to claim 5, characterized in that, It is composed of a first primer that specifically recognizes the upstream flanking cotton genomic sequence or the downstream flanking cotton genomic sequence and a second primer that specifically recognizes the T-DNA insertion sequence.

7. The primer pair according to claim 6, wherein the sequence of the first primer is as shown in SEQ ID NO: 14, and the sequence of the second primer is as shown in SEQ ID NO: 15; or the sequence of the first primer is as shown in SEQ ID NO: 16, and the sequence of the second primer is as shown in SEQ ID NO:

17.

8. A method for identifying whether the cotton transformation event described in claim 1 is present in a cotton biological sample, characterized in that, Includes: (a) Extracting a DNA sample from the cotton biological sample to be identified; (b) Using the primer pair according to claim 6 or 7 for PCR amplification with the extracted DNA sample as a template; and (c) Detect the PCR amplification product. If the length of the PCR amplification product is consistent with the length obtained by amplifying the DNA template of the sequence shown in SEQ ID NO: 13, it indicates the presence of the cotton transformation event in the cotton biological sample.

9. A method for obtaining transgenic high-quality cotton materials, characterized in that, Comprising: After hybridizing the cotton material containing the cotton transformation event according to any one of claims 1-4 with other cotton breeding materials, further backcrossing is carried out to obtain a new material containing the cotton transformation event; wherein during the hybridization and / or backcrossing process, the method according to claim 8 is used to screen and identify in the offspring population to confirm the presence of the cotton transformation event.

10. Use of the DNA fragment according to claim 5 for improving cotton fiber quality and / or yield, for cotton breeding, and as a molecular marker.

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