Application of GhDBB28 gene in regulating salt tolerance of upland cotton plants

Abstract

The application relates to the field of agricultural transgenic technology, and particularly discloses GhDBB28 application of a gene in regulating salt tolerance of upland cotton plants, the gene GhDBB28 The nucleotide sequence of the gene is shown as SEQ ID NO. 1; silencing the gene GhDBB28 increases the salt sensitivity of the upland cotton plants. The application reduces the activity of an antioxidant system in the upland cotton plants by silencing the gene GhDBB28 , so that the plants are more sensitive to salt stress, and a scientific basis is provided for constructing a transgenic upland cotton plant with new salt-sensitivity.

Description

Technical Field

[0001] This invention relates to the field of agricultural genetically modified technology, specifically to GhDBB28 Application of genes in regulating salt tolerance in upland cotton plants. Background Technology

[0002] The immobility of plants means they may experience abiotic stress throughout their growth and development. Salt stress harms plants through various means, including osmotic stress, ion toxicity, nutrient imbalance, and oxidative damage. Transcription factors play a crucial role in enhancing plant resilience to abiotic stress by regulating the expression of functional genes.

[0003] Cotton (Gossypium spp.) is a plant belonging to the genus Gossypium in the family Malvaceae. Its fiber production accounts for nearly 35% of the world's total natural fiber consumption. However, salt stress has a significant impact on the growth and development of cotton. Breeding salt-sensitive cotton is of great significance for studying salt tolerance pathways and developing transgenic cotton. Summary of the Invention

[0004] To develop the function of an existing gene in regulating salt tolerance in cotton plants, this invention provides... GhDBB28 Application of genes in regulating salt tolerance in upland cotton plants. This invention utilizes... GhDBB28 The silence of the substance reduces the activity of the antioxidant system in upland cotton plants, making the plants more sensitive to salt stress.

[0005] This invention provides GhDBB28 The application of genes in regulating salt tolerance in upland cotton plants, the aforementioned GhDBB28 The nucleotide sequence of the gene is shown in SEQ ID NO.1; silencing the gene... GhDBB28 Genes increase the salt sensitivity of upland cotton plants.

[0006] This invention uses silence GhDBB28 Transgenic upland cotton plants, after obtaining the gene, exhibited stunted growth under salt stress, with some leaves turning yellow and dying, and the plants becoming short and weak. Chlorophyll content, POD activity, and SOD activity were significantly lower than those of the control group, while MDA content and relative conductivity were significantly higher. GhDBB28 The silence of the substance reduces the activity of the antioxidant system in upland cotton plants, making the plants more sensitive to salt stress.

[0007] Furthermore, the biological product containing the target gene fragment shown in SEQ ID NO. 9 is used to silence the [specific gene]. GhDBB28 Gene.

[0008] Furthermore, the biological product includes: a recombinant plasmid or recombinant Agrobacterium containing the target gene fragment shown in SEQ ID NO.9.

[0009] Furthermore, the recombinant plasmid is obtained by inserting the gene fragment shown in SEQ ID NO.9 into the TRV2 vector.

[0010] Furthermore, the recombinant Agrobacterium is obtained by introducing the recombinant plasmid into Agrobacterium.

[0011] Furthermore, the silence stated GhDBB28 Genes, including: injecting recombinant Agrobacterium into the cotyledons of upland cotton plants.

[0012] Furthermore, after culturing recombinant Agrobacterium to obtain an infecting bacterial solution, during the cotyledon unfolding stage of upland cotton plants, the bacterial solution containing the helper plasmid TRV1 was mixed with the infecting bacterial solution in equal proportions and injected into the cotyledons of the upland cotton plants, thereby achieving the effect of infecting the upland cotton plants with the bacteria. GhDBB28 Gene silencing.

[0013] Furthermore, the improvement of upland cotton plants' salt sensitivity is manifested in: reducing the chlorophyll content, POD activity, and SOD activity of upland cotton plants, and increasing the MDA content and relative conductivity of upland cotton plants.

[0014] The present invention also provides the application of recombinant plasmids or recombinant Agrobacterium in regulating the salt tolerance of upland cotton plants, wherein the recombinant plasmids or recombinant Agrobacterium contain the target gene fragment shown in SEQ ID NO.9.

[0015] The present invention also provides GhDBB28 The application of gene-encoded proteins in regulating salt tolerance in upland cotton plants, the aforementioned GhDBB28 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.4, and is obtained by silencing the protein. GhDBB28 Genes, reduce GhDBB28 The level of the gene-encoded protein increases the salt sensitivity of upland cotton plants.

[0016] The present invention also provides a method for cultivating salt-sensitive upland cotton plants, comprising the following steps:

[0017] The recombinant plasmid TRV2:GhDBB28 was introduced into Agrobacterium and cultured to obtain an infectious bacterial solution. At the cotyledon unfolding stage of upland cotton plants, the helper plasmid TRV1 was mixed with the infectious bacterial solution in an equal ratio and injected into the cotyledons of the upland cotton plants to achieve control of the infection in the upland cotton plants. GhDBB28 Gene silencing was used to obtain salt-sensitive transgenic upland cotton plants.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention discovers transcription factors GhDBB28 Its expression is upregulated by salt, high osmotic pressure and exogenous ABA treatment, and it participates in the cotton response to salt stress and ABA signaling to improve the transcription of downstream functional proteins, thereby regulating the plant's ability to adapt to adversity.

[0020] This invention uses silence GhDBB28 Transgenic upland cotton plants, after obtaining the gene, exhibited stunted growth under salt stress, with some leaves turning yellow and dying, and the plants becoming short and weak. Chlorophyll content, POD activity, and SOD activity were significantly lower than those of the control group, while MDA content and relative conductivity were significantly higher. GhDBB28 The silence of the substance reduces the activity of the antioxidant system in upland cotton plants, making the plants more sensitive to salt stress. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Picture 1 The vector map of the recombinant vector PRI101-AN-GhDBB28 constructed in this invention.

[0023] Picture 2 This invention relates to different tissues of upland cotton plants. GhDBB28 The expression pattern.

[0024] Picture 3 The present invention relates to the root system of upland cotton plants under salt stress. GhDBB28 The expression pattern.

[0025] Picture 4 The present invention relates to the root system of upland cotton plants under osmotic stress. GhDBB28 The expression pattern.

[0026] Picture 5 This invention relates to the treatment of exogenous ABA stress in the leaves of upland cotton plants. GhDBB28 The expression pattern.

[0027] Picture 6 G in this invention hDBB28 Silent efficiency.

[0028] Picture 7 For silence in this invention GhDBB28 The influence of genes on the salt tolerance of upland cotton;

[0029] In the diagram, A represents silence under salt stress. GhDBB28The influence of genes on the phenotypic traits of upland cotton;

[0030] B represents silence under salt stress. GhDBB28 The effect of genes on chlorophyll content in upland cotton leaves;

[0031] C represents silence under salt stress. GhDBB28 The influence of genes on the relative electrical conductivity of upland cotton;

[0032] D represents silence under salt stress. GhDBB28 The influence of genes on malondialdehyde content in upland cotton;

[0033] E represents silence under salt stress. GhDBB28 The effect of genes on peroxidase activity in upland cotton;

[0034] F represents silence under salt stress. GhDBB28 The effect of genes on superoxide dismutase activity in upland cotton. Detailed Implementation

[0035] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0036] Example 1: GhDBB28 Application of genes in regulating salt tolerance in upland cotton plants.

[0037] I. Construction of the recombinant vector PRI101-AN-DBB28

[0038] High-fidelity enzyme amplification was performed on cDNA from leaves of upland cotton variety Lumianyan 37. GhDBB28 The coding sequence was obtained and sent to a company for sequencing to obtain the nucleotide sequence of the gene. The specific steps are as follows:

[0039] 1. Primer design

[0040] Based on the upland cotton gene transcript information and PRI101-AN vector information, suitable experimental primers were designed using Novizan's online primer design software CE Design, including the upstream primer shown in SEQ ID NO.2 and the downstream primer shown in SEQ ID NO.3, and submitted to the company for synthesis.

[0041] SEQ ID NO.2: 5'-cccgtcgaccccgggggtacATGAGGATACAGTGCAACGTGTG-3';

[0042] SEQ ID NO. 3: 5'-agagttgttgattcagaattcCTAGTGGAAAGGGTTTCCACACT-3'.

[0043] 2. Total RNA extraction and cDNA synthesis from upland cotton

[0044] Total RNA was extracted from plants using the OminiPlant RNA Kit (Dnase I) from Kangwei Century. The extraction method was performed according to the kit instructions.

[0045] RNA reverse transcription was performed using the HiFiScript cDNA Synthesis Kit from Kangwei Century to obtain plant cDNA. The amplification system is shown in Table 1.

[0046] Table 1 Reverse transcription amplification system

[0047]

[0048] Prepare samples according to the system in Table 1. After sample preparation, mix thoroughly by pipetting and centrifugation, incubate at 42℃ for 15 minutes, and then at 85℃ for 5 minutes. After reverse transcription, store the plant cDNA samples at -20℃ for later use.

[0049] 3. GhDBB28 Gene cloning

[0050] (1) GhDBB28 Gene amplification

[0051] Plant cDNA was used as a template for PCR amplification using primer pairs shown in SEQ ID NO.2–SEQ ID NO.3, with TOYOBO's KOD high-fidelity enzyme employed. The PCR amplification system is shown in Table 2, and the PCR amplification procedure is shown in Table 3. After PCR amplification, the PCR products were recovered by 1% agarose gel electrophoresis. The PCR products were recovered using Kangwei Century's Gel Extraction Kit. Specific experimental procedures were strictly followed according to the product instructions.

[0052] Table 2 PCR amplification system

[0053]

[0054] Table 3 PCR amplification program

[0055]

[0056] (2) Linearization of PRI101-AN vector

[0057] The linearized PRI101-AN vector was double-digested using Thermo Fisher Scientific's FastDigest KpnI and FastDigest EcoRI enzymes. Following the enzyme digestion system for plasmid DNA in Table 4, the reaction was carried out at 37°C for 15 min, followed by inactivation at 80°C for 5 min. The linearized vector was then recovered using a Gel Extraction Kit. The recovered PCR products were then digested under the same enzyme digestion system and reaction conditions as described in Table 4 to obtain... GhDBB28 Gene fragments.

[0058] Table 4 Enzyme digestion system

[0059]

[0060] (3) Using the ClonExpress Ultra One Step Cloning Kit V3 homologous recombination kit to... GhDBB28 The gene fragment and the linearized vector were ligated, following the instructions in the kit.

[0061] (4) The ligation product was transformed into competent DH5α Escherichia coli cells from Weidi Biotechnology. The specific method is described in the instructions for use with competent cells. Using kanamycin as the selection pressure, positive clones were picked to obtain the recombinant vector PRI101-AN-DBB28, which was sent to the company for sequencing. According to the company's sequencing results, the recombinant vector PRI101-AN-DBB28 contained… GhDBB28 The cDNA sequence was obtained, and the resulting cDNA sequence is shown in SEQ ID NO.1. The protein sequence it encodes is shown in SEQ ID NO.4. The recombinant vector PRI101-AN-DBB28 is shown in the image below. Picture 1 As shown.

[0062] SEQ ID NO.1:

[0063] ATGAGGATACAGTGCAACGTGTGCGAGGTGGCGGAGGCGAAGGTGCTGTGCTGCTCAGACGAGGCGGCGCTGTGTTTGGAGTGTGACGAGAAAGTTCATGCTGCCAACAAGTTGGCCAGCAAGCATCAGCGTGTTCCCCTCTCGTCTTCTTCTTCTCATATGCCTACATGTGACATTTGCCAGGAAACATCTGGGTTTTTCTTCTGTTTGCAAGACCGAGCTTTACTCTGCCGAAAATGTGATATTGCTATACATACAGCAAATCCTTATGTCTCTAGTCACCAGAGATTTGTGCTTACTGGAGTCAAAGTTGGTCTTGACACAACAACTGACCCTGTTGGATCTTCATCCTATATCAAATCACCTTCTAGTGAAAAGACTTCAGAAACTAAATCCGATTCTATGTCTAAAAGGGACGCTCCTATGTCATTTATTAGTGAATGCAATGAAGTTCTTCCGTCTATTATCGGAGTTGAGAACAGCGTGCCGACAAAAGTTTCATATGGTGGGGGTTCTACAGCTGGAGGCATTCAGTCATGGCATATGGATGATTTGTTTGGGCTAACTGGCTTAAACCAAAGCTTTGGTTACATGGATAATGAGTTATCTAAGGCTGATAGTGGCAAGTGCGGAGACTCCGATGGCTCACCGTTCTTGCGACCTGCTGAGGAAGAGGTCTTTGATGATGAATACATGGGTCAGGTGCCGGAATCCCACTGGGCAGTACCACAAGTGCCTTCACCTCCTACAGCCTCAGGGTTGTATTGGCCTAAAGATTCTCACAATCAATCTGATAGCGCGGTATTTGTCCCCGACATATCCTGGTCTAGTGTGGAAAACCCTTTCCACTAG。

[0064] SEQ ID NO.4:

[0065] MRIQCNVCEVAEAKVLCCSDEAALCLECDEKVHAANKLASKHQRVPLSSSSSHMPTCDICQETSGFFFCLQDRALLCRKCDIAIHTANPYVSSHQRFVLTGVKVGLDTTTDPVGSSSYIKSPSSEKTSETKSDSMSKRDAPM SFISECNEVLPSIIGVENSVPTKVSYGGGSTAGGIQSWHMDDLFGLTGLNQSFGYMDNELSKADSGKCGDSDGSPFLRPAEEEVFDDEYMGQVPESHWAVPQVPSPPTASGLYWPKDSHNQSDSAVFVPDISWSSVENPFH.

[0066] II. qPCR Identification GhDBB28 Gene expression patterns and their response to exogenous stress

[0067] 1. qPCR identification GhDBB28 Gene expression patterns

[0068] (1) qPCR primer design

[0069] The upstream primer shown in SEQ ID NO.5 and the downstream primer shown in SEQ ID NO.6 were designed and obtained as primers for real-time PCR, and were sent to the company for synthesis.

[0070] SEQ ID NO.5: 5'-ACTCCGATGGCTCACCGTTC-3';

[0071] SEQ ID NO. 6: 5'-GGCCAATACAACCCTGAGGC-3'.

[0072] (2) Different parts of cotton during its peak flowering period GhDBB28 Gene expression level

[0073] RNA was extracted from the roots, stems, leaves, sepals, petals, stamens, pistils, and ovules of cotton plants in full bloom and reverse transcribed into cDNA. Using cDNA from different sites as templates, qPCR reactions were performed using the primer pairs shown in SEQ ID NO.5–SEQ ID NO.6 on an ABI 7500 real-time PCR instrument to detect... GhDBB28 Gene expression characteristics in different tissues. The qPCR reaction system is shown in Table 5, and the qPCR reaction procedure is shown in Table 6.

[0074] Table 5 qPCR reaction system

[0075]

[0076] Table 6 qPCR reaction procedure

[0077]

[0078] The denaturation and annealing were set for 40 cycles. After the reaction was complete, 2... -ΔΔCT The method calculates gene expression levels, where CT is the cycle threshold. All data are averaged across three technical replicates to reduce error.

[0079] The results are as follows Picture 2 As shown, GhDBB28 The gene is expressed in all organs of cotton. The highest expression level is found in leaves, followed by relatively high levels in roots, stamens, pistils, petals, and sepals. Expression is relatively low in ovules. This indicates that the gene participates in regulating various stages of plant growth and development.

[0080] (3) Different parts of cotton under different environmental stresses GhDBB28 Gene expression level

[0081] Abiotic stress treatment was applied to cotton plants at the two-leaf-one-heart stage.

[0082] Exogenous ABA stress: Irrigate the soil with tap water until it is saturated, and spray the surface of cotton leaves with a 500uM ABA solution.

[0083] Salt stress: Irrigate with 250mM Nacl solution until the soil is saturated with water.

[0084] Drought stress: Irrigate with 10% PEG6000 solution until the soil is saturated with water.

[0085] Root and leaf tissues were collected at 0, 3, 6, 12 and 24 hours after different treatments, flash-frozen in liquid nitrogen and stored at -80°C. RNA was extracted and then reverse transcribed into cDNA.

[0086] qRT-PCR analysis was performed using the UltraSYBR Mixture Real-Time Fluorescence Kit from Kangwei Century. The reaction system is shown in Table 5, and the qPCR reaction procedure is shown in Table 6.

[0087] The results are as follows Picture 3 As shown, under salt stress, in the root tissue GhDBB28 The expression level gradually increased, reaching 5.59 times that of the 0-hour control at 12 hours after treatment, and then decreased to a level similar to that of the control plants. Salt stress is usually caused by osmotic stress pressure exerted on plants by ion osmosis. Picture 4 As shown, after osmotic stress treatment with PEG6000, GhDBB28Expression levels rose rapidly, peaking at 6 hours post-treatment, reaching 1.8 times that of the 0-hour control. After plants are subjected to drought and high salt stress, large amounts of ABA accumulate within them to regulate the expression of downstream stress-resistance genes, thereby regulating cellular homeostasis and enhancing their adaptability to stress environments. Picture 5 As shown, in this invention, after ABA treatment, the blade contains GhDBB28 The expression level increased, reaching a peak at 3 hours post-treatment, which was 1.58 times that of the 0-hour control.

[0088] In summary, transcription factors GhDBB28 Its expression is upregulated by salt, high osmotic pressure and exogenous ABA treatment, and it participates in the cotton response to salt stress and ABA signaling to improve the transcription of downstream functional proteins, thereby regulating the plant's ability to adapt to adversity.

[0089] III. VIGS Silence GhDBB28 Salt tolerance assessment of upland cotton

[0090] 1. VIGS Silence GhDBB28

[0091] Using the recombinant vector PRI101-AN-DBB28 as a template, the upstream primer shown in SEQ ID NO. 7, the downstream primer shown in SEQ ID NO. 8, and the KOD high-fidelity enzyme were used to amplify the array shown in SEQ ID NO. 9. GhDBB28 The silenced fragment, i.e. the target gene fragment, is inserted into the TRV2 vector to obtain the recombinant plasmid TRV2:GhDBB28.

[0092] SEQ ID NO.7: 5'-ggatccATGAGGATACAGTGCAACGTGTG-3';

[0093] SEQ ID NO. 8: 5'-gagctcAGTAAGCACAAATCTCTGGTGACTAGA-3'.

[0094] SEQ ID NO.9:

[0095] ATGAGGATACAGTGCAACGTGTGCGAGGTGGCGGAGGCGAAGGTGCTGTGCTGCTCAGACGAGGCGGCGCTGTGTTTGGAGTGTGACGAGAAAGTTCATGCTGCCAACAAGTTGGCCAGCAAGCATCAGCGTGTTCCCCTCTCGTCTTCT TCTTCTCATATGCCTACATGTGACATTTGCCAGGAAACATCTGGGTTTTTCTTCTGTTTGCAAGACCGAGCTTTACTCTGCCGAAAATGTGATATTGCTATACATACAGCAAATCCTTATGTCTCTAGTCACCAGAGATTTGTGCTTACT.

[0096] The recombinant plasmid TRV2:GhDBB28 was introduced into Agrobacterium GV3101 to obtain recombinant bacteria containing the recombinant plasmid TRV2:GhDBB28. The empty vector TRV2:00 was used as a negative control. Agrobacterium GV3101 containing the recombinant plasmid TRV2:GhDBB28, the negative control (empty vector TRV2:00), and the positive control (TRV2:GhCLA1) were cultured separately and resuspended as infection cultures.

[0097] TRV2:GhCLA1 was used as a positive control, and the successful infection was verified by monitoring the albinism phenotype of cotton leaves. At the cotyledon expansion stage, the auxiliary bacterial suspension TRV1 was mixed 1:1 with the infection suspensions of TRV2:GhCLA1, TRV2:00, and TRV2:GhDBB28, respectively. The three mixed bacterial suspensions were then injected into the cotton cotyledons through the back of the leaves. After injection, the plants were grown in the dark for 24 hours before normal culture. When the positive control plants showed the albinism phenotype, leaves from plants injected with TRV2:00 and TRV2:GhDBB28 were used for verification via qRT-PCR. GhDBB28 Silent efficiency in VIGS-treated plants.

[0098] TRV2:GhCLA1 vector construction method reference: Zhang, J.; Wang, F.; Zhang, C.; Zhang, J.; Chen, Y.; Liu, G.; Zhao, Y.; Hao, F.; Zhang, J. A novel VIGS method by agroinoculation of cotton seeds and application for elucidating functions of GhBI-1 in salt-stress response. PlantCell Reports2018, 37 ,1091-1100,doi:10.1007 / s00299-018-2294-5.

[0099] The results are as follows Picture 6 As shown, when the positive control showed an albino phenotype, leaves from TRV2:00 and TRV2:GhDBB28 plants were taken for qRT-PCR verification. The results showed that... GhDBB28 The expression level decreased by an average of 71%, indicating a significant silencing effect.

[0100] 2. Identification of salt tolerance in silent plants

[0101] Salt stress treatment involved continuous irrigation with a 300 mM NaCl solution for 14 days. Total chlorophyll content, relative conductivity (REC), superoxide dismutase (SOD) activity, peroxidase (POD) activity, and malondialdehyde (MDA) content were measured on days 0, 7, and 14 after salt treatment. Two cotton seedlings per pot represented one biological replicate, and each data point was the average of three biological replicates. The methods for measuring the above indicators were based on those described in the following literature:

[0102] Kotchoni,SO;Kuhns,C.;Ditzer,A.;Kirch,H.-H.;Bartels,D.Over-expression of different aldehyde dehydrogenase genes in Arabidopsis thalianaconfers tolerance to abiotic stress and protects plants against lipidperoxidation and oxidative stress. Plant, Cell & Environment 2006, 29 ,1033-1048.

[0103] The results are as follows Picture 7 As shown in Figure A, after continuous irrigation with 300 mM NaCl solution for 14 days, compared with plants injected with empty vectors, the silent plants showed better results. GhDBB28The cotton plants exhibited restricted growth, with some leaves turning yellow and dying, resulting in stunted and weak plants. After 7 days of salt treatment, there were no significant differences in chlorophyll content, relative conductivity, and MDA content in the leaf cells between the two groups. However, after 14 days of salt stress, the chlorophyll content in the silent group was significantly lower than that in the control group, while the MDA content and relative conductivity were significantly higher, indicating more severe cell damage in the silent group. Seven days after salt treatment, the intracellular POD activity in the silent group was significantly lower than that in the control group, and the SOD activity was also lower than that in the control group on both days 7 and 14 after salt treatment. These results suggest that... GhDBB28 The silencing of these substances reduces the activity of the plant's antioxidant system, making the plant more sensitive to salt stress.

[0104] This invention utilizes cloning GhDBB28 The gene was identified, and its expression was determined to be induced by salt, osmotic stress, and ABA stress. The VIGS technology was used to confirm that the gene plays a key role in regulating the salt tolerance of cotton, providing candidate genes and technical support for breeding salt-sensitive cotton varieties and stress-resistant cotton varieties.

[0105] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0106] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. GhDBB28 The application of genes in regulating salt tolerance in upland cotton plants is characterized by, The GhDBB28 The nucleotide sequence of the gene is shown in SEQ ID NO.1; silencing the gene... GhDBB28 Genes increase the salt sensitivity of upland cotton plants.

2. As described in claim 1 GhDBB28 The application of genes in regulating salt tolerance in upland cotton plants is characterized by, Silencing the target gene fragment shown in SEQ ID NO.9 using a biological product GhDBB28 Gene; The biological product includes: a recombinant plasmid or recombinant Agrobacterium containing the target gene fragment shown in SEQ ID NO.

9.

3. As described in claim 2 GhDBB28 The application of genes in regulating salt tolerance in upland cotton plants is characterized by, The recombinant plasmid was obtained by inserting the gene fragment shown in SEQ ID NO.9 into the TRV2 vector.

4. The method according to claim 3 GhDBB28 The application of genes in regulating salt tolerance in upland cotton plants is characterized by, The recombinant Agrobacterium was obtained by introducing the recombinant plasmid into Agrobacterium.

5. The method according to claim 4 GhDBB28 The application of genes in regulating salt tolerance in upland cotton plants is characterized by, The silence GhDBB28 Genes, including: injecting recombinant Agrobacterium into the cotyledons of upland cotton plants.

6. The method according to claim 5 GhDBB28 The application of genes in regulating salt tolerance in upland cotton plants is characterized by, Recombinant Agrobacterium was cultured to obtain an infecting bacterial suspension. During the cotyledon expansion stage of upland cotton plants, the bacterial suspension containing the helper plasmid TRV1 was mixed with the infecting bacterial suspension in equal proportions and injected into the cotyledons of the upland cotton plants to achieve control of the bacteria in the upland cotton plants. GhDBB28 Gene silencing.

7. The method according to claim 1 GhDBB28 The application of genes in regulating salt tolerance in upland cotton plants is characterized by, The improvement of upland cotton plants' salt sensitivity is manifested in the following ways: reducing chlorophyll content, POD activity, and SOD activity in upland cotton plants, and increasing MDA content and relative conductivity in upland cotton plants.

8. The application of recombinant plasmids or recombinant Agrobacterium in improving the salt sensitivity of upland cotton plants, characterized in that... The recombinant plasmid or recombinant Agrobacterium contains the target gene fragment shown in SEQ ID NO. 9; the target gene is GhDBB28 Genes, the ones mentioned GhDBB28 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

9. GhDBB28 The application of gene-encoded proteins in regulating salt tolerance in upland cotton plants, the aforementioned GhDBB28 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.4, characterized in that, By silencing the claims in claim 1 GhDBB28 Genes, reduce GhDBB28 The level of genes encoding proteins increases the sensitivity of upland cotton plants to salt.

Images