Application of cotton salt tolerance related gene Gair.A03G043700

Abstract

The invention belongs to the technical field of plant genetic engineering, and relates to an application of a cotton salt tolerance related gene Gair.A03G043700. The invention discloses a molecular characteristic that the gene Gohir.A03G043700 maintains stable and high expression under stress in salt-tolerant cotton and a key biological function of the gene Gohir.A03G043700 serving as a basic defense. Specifically, the invention discloses application of the gene Gohir.A03G043700 in identification of salt tolerance of cotton, molecular-assisted breeding of salt-tolerant cotton varieties and improvement of salt tolerance of cotton.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology and relates to the application of a cotton salt tolerance-related gene, Gohir.A03G043700. Background Technology

[0002] Cotton, as a typical subtropical plant, possesses strong adaptability and can survive in dry and high-temperature environments. However, it still responds to environmental changes. Prolonged exposure to adverse factors such as drought, salinity, and temperature stress can lead to ion toxicity, osmotic stress, and oxidative damage. Studies have shown that cotton is significantly more sensitive to salt stress during germination, emergence, and seedling stages than at other growth stages. Salt stress not only delays flowering and reduces the number of fruits, but can also cause fruit drop and decreased boll weight, ultimately affecting cotton yield.

[0003] Existing research indicates that salt tolerance in cotton is controlled by multiple genes, and researchers have made some progress in recent years using genome-wide association studies (GWAS). Zheng et al. analyzed 149 cotton samples using a 70K microarray and identified 27 SNP markers associated with salt tolerance during germination; another study screened 23 candidate SNPs related to salt stress through GWAS analysis of 419 upland cotton samples. At the physiological mechanism level, studies have found that exogenous nitric oxide can alleviate oxidative damage in cotton seedlings, and the ethylene signaling pathway plays an important role in salt tolerance regulation. However, existing research still has significant limitations: first, the genetic effects of identified salt tolerance loci are limited and difficult to directly apply to breeding practices; second, most candidate genes lack systematic functional verification; and third, research on seedling salt tolerance is relatively weak, especially the discovery of key genes and the elucidation of their molecular mechanisms. Therefore, identifying major genes with significant genetic effects on seedling salt tolerance is of great value for the genetic improvement of cotton salt tolerance, the development of efficient molecular markers, and the advancement of salt-tolerant breeding. Summary of the Invention

[0004] The purpose of this invention is to at least partially solve the above-mentioned technical problems and provide an application of the cotton salt tolerance-related gene Gohir.A03G043700.

[0005] The technical solution provided by this invention is as follows:

[0006] In a first aspect, the present invention provides the application of the gene Gohir.A03G043700 in improving the salt tolerance of cotton, the sequence of which is shown in SEQ ID NO.1.

[0007]

[0008] Secondly, this invention provides the application of the gene Gohir.A03G043700 in the molecular-assisted breeding of salt-tolerant cotton varieties.

[0009] Based on the molecular mechanism by which this gene acts as a "basic defense protector," it maintains stable high expression in salt-tolerant materials even under salt stress, continuously providing basic protection such as osmotic regulation or ion homeostasis. However, in sensitive materials, the expression of this gene is drastically downregulated, causing the plant to lose its basic salt tolerance. Therefore, in marker-assisted breeding, KASP markers can be developed targeting SNPs (such as the salt-tolerant allele G and the sensitive allele A) associated with the "stable high expression" trait in the coding region or promoter region of this gene. By using KASP genotyping at the seedling stage, individual plants carrying the GG genotype can be selected to enrich and fix strong allelic variations, thereby efficiently breeding new cotton materials with strong basic salt tolerance defense capabilities.

[0010] Thirdly, a molecular-assisted breeding method for salt-tolerant cotton varieties is provided. This method involves positively selecting allelic variants associated with stable high expression of the gene Gohir.A03G043700 in the breeding population for molecular-assisted breeding, including the following steps:

[0011] S1. Development of molecular markers: By screening for functionally enhanced non-synonymous single nucleotide polymorphisms in the coding region of the gene Gohir.A03G043700, or by identifying regulatory units with strong promoter effects in the promoter region, a SNP site significantly associated with the stable high expression trait of the gene Gohir.A03G043700 is obtained, where the salt-tolerant allele is defined as G and the sensitive allele is defined as A;

[0012] S2. Design KASP primers: Design KASP primers for this SNP;

[0013] S3. Breeding population screening: Seedling DNA extraction and KASP typing were performed on the breeding population, and single plants with genotype GG were selected as salt-tolerant candidate single plants;

[0014] S4. Field verification: The salt-tolerant candidate plants were planted in the field under controlled salt stress conditions, and then the salt tolerance phenotype was identified.

[0015] Fourthly, the application of the gene Gohir.A03G043700 in the identification of salt-tolerant cotton germplasm is provided. The sequence of the gene Gohir.A03G043700 is shown in SEQ ID NO.1. The application is as follows: the salt-tolerant cotton germplasm is identified by detecting whether there is a significant change in the expression level of the gene Gohir.A03G043700 in the sample under salt stress conditions. If there is no significant change, it is a salt-tolerant cotton germplasm; if it is significantly downregulated, it is a non-salt-tolerant cotton germplasm.

[0016] Fifthly, a method for identifying salt-tolerant cotton germplasm is provided, comprising the following steps:

[0017] S1. Sample acquisition: Samples of the cotton germplasm to be tested were obtained under salt stress conditions and under normal growth conditions, respectively;

[0018] S2. Detect and compare gene expression levels: Detect and compare the expression levels of gene Gohir.A03G043700 in samples under two conditions;

[0019] S3. Determine based on comparison results: If the expression level of gene Gohir.A03G043700 under salt stress conditions is not statistically significantly different from that under normal growth conditions, the cotton germplasm to be tested is determined to be salt-tolerant; if the expression level of gene Gohir.A03G043700 under salt stress conditions is statistically significantly downregulated compared to normal growth conditions, the cotton germplasm to be tested is determined to be non-salt-tolerant. The sequence of gene Gohir.A03G043700 is shown in SEQ ID NO.1.

[0020] Compared with existing technologies, this invention reveals for the first time the molecular characteristics of the gene Gohir.A03G043700, which maintains stable high expression under salt stress in salt-tolerant cotton, and its key biological function as a "basic defense guardian." By detecting the expression level of this gene under salt stress or the allelic genotype of its coding / promoter regions, the salt tolerance of cotton germplasm can be accurately identified; simultaneously, enhancing the expression of this gene can effectively improve the salt tolerance of cotton. This invention provides new functional molecular markers, efficient breeding methods, and important gene resources for salt-tolerant cotton breeding, and has broad application prospects in the selection and genetic improvement of salt-tolerant cotton varieties. Attached Figure Description

[0021] These and / or other aspects and advantages of the present invention will become apparent and readily understood from the following description of preferred embodiments taken in conjunction with the accompanying drawings, in which:

[0022] Figure 1 The expression levels of the gene Gohir.A03G043700 in the salt-tolerant strain XiangFZ031 and the salt-sensitive strain CRI12 before and after salt treatment are shown.

[0023] Figure 2 The albino phenotype of the VIGS experimental positive control (TRV2:CLA1) is shown;

[0024] Figure 3 The results of VIGS function validation of the Gohir.A03G043700 gene are shown. Detailed Implementation

[0025] The technical solution of the present invention will be further described in detail below through embodiments and in conjunction with the accompanying drawings. In this specification, the same or similar reference numerals indicate the same or similar components. The following description of the embodiments of the present invention with reference to the accompanying drawings is intended to explain the overall inventive concept of the present invention and should not be construed as a limitation thereof.

[0026] It should be noted that the terms used in this application are generally those commonly used by those skilled in the art. If there is any inconsistency with commonly used terms, the terms used in this application shall prevail.

[0027] Example 1: Identification of gene Gohir.A03G043700 by real-time quantitative PCR (RT-qPCR)

[0028] (1) Experimental materials RNA extraction

[0029] Two cotton varieties, XiangFZ031 (a salt-tolerant variety) and CRI12 (a salt-sensitive variety), were selected. Seeds with plump, uniform size were used as the growing medium, and sown using a 3:1 mixture of substrate and vermiculite. After sowing, the plants were placed in an artificial climate chamber with conditions set at 25℃ and a photoperiod of 16h / 8h (light / dark). Once the first true leaf had fully unfolded, the following treatments were administered:

[0030] Treatment groups (Salt-XiangFZ031 and Salt-CRI12): Irrigated with 200mM saline solution.

[0031] Control groups (CK-XiangFZ031 and CK-CRI12): Irrigated with the same amount of clean water.

[0032] After 24 hours of treatment, two groups of cotton leaf samples were collected. RNA was extracted from the control and treatment groups using the Tiangen Polysaccharide Polyphenol RNA Kit, strictly following the official instructions of the kit.

[0033] After obtaining RNA, a systematic quality assessment was performed on four samples: RNA integrity and degradation were detected using 1% agarose gel electrophoresis; RNA concentration was determined using the Qubit quantification system; RNA integrity was assessed using an Agilent 2100 bioanalyzer; and the RNA was evaluated using NanoDrop. TM One / OneC assay was used to measure the OD260 / 280 ratio to determine the presence of contamination. These comprehensive tests ensured that the RNA extracted from both the control and treatment groups met the requirements for subsequent experiments.

[0034] (2) Synthesis of the first strand of cDNA

[0035] For each of the four samples, take 1 μg of the RNA obtained in step 1), and follow the instructions of the M5 First Strand cDNA Synthesis Kit. React at 50°C for 50 minutes and inactivate at 85°C for 5 minutes to synthesize the first strand of cDNA, which will serve as the template for subsequent RT-qPCR analysis.

[0036] (3) RT-qPCR analysis

[0037] The cotton ubiquitination gene GhUBQ14 was used as an internal reference gene. Primers for the CDS region of GhUBQ14 and the gene Gohir.A03G043700 were designed using PrimerPremier 6 software. The RT-qPCR primer sequences are shown in Table 1.

[0038] Table 1. RT-qPCR Primer Information

[0039]

[0040]

[0041] The designed primer sequences were sent to Suzhou Hongxun Biotechnology Co., Ltd. for synthesis.

[0042] RT-qPCR reactions were performed using the ChamQ SYBR Qpcr Master Mix kit (Novizan, Nanjing, China) and detected on an ABI 7500 real-time fluorescence quantitative PCR system (ABI, USA). Each 15 μL RT-qPCR reaction system contained 7.5 μL of 2×ChamQ SYBR Qpcr Master Mix, 0.3 μL of upstream and downstream primers, and 1 μL of cDNA template.

[0043] The amplification program was set as follows: pre-denaturation: 95℃, 30 sec; cycling phase: 95℃, 15 sec; 60℃, 30 sec, for a total of 40 cycles; melting phase: 95℃, 15 sec; 60℃, 60 sec; 95℃, 15 sec. The relative expression level of the gene was measured using 2... ∧ The calculation is performed using the (-ΔΔCt) method.

[0044] The RT-qPCR experiments were performed with three biological replicates and four technical replicates. Experimental data were statistically analyzed using t-tests. In the results, an asterisk indicates a significant difference at the 0.05 significance level, * indicates P ≤ 0.05, and ** indicates P ≤ 0.01. Finally, GraphPadPrism9 software was used for data visualization.

[0045] The RT-qPCR analysis results of the expression levels of the gene Gohir.A03G043700 before and after treatment with XiangFZ031 and CRI12 salts are as follows: Figure 1 As shown.

[0046] Example 2: VIGS function verification of gene Gohir.A03G043700.

[0047] To investigate the molecular function of Gohir.A03G043700 in the salt stress response, this study used virus-induced gene silencing (VIGS) technology for loss-of-function analysis.

[0048] (1) Acquisition and retrieval of target fragments

[0049] Specific VIGS primers for the gene Gohir.A03G043700 were designed using PrimerPremier 6 and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0050] Primer sequences are detailed in Table 2.

[0051] Table 2. VIGS Experimental Primer Information

[0052]

[0053] Subsequently, using cDNA from the salt-tolerant strain XiangFZ031 as a template, PCR amplification was performed using high-fidelity DNA polymerase. The reaction system is shown in Table 3.

[0054] Table 3. PCR experimental reaction system

[0055]

[0056] The amplified products were identified by 1% agarose gel electrophoresis. If a single band was observed and its size matched that of the target fragment, the target fragment was recovered using the Tiangen Agarose Gel DNA Recovery Kit.

[0057] (2) Construct a recombinant vector containing the gene Gohir.A03G043700

[0058] To construct the VIGS recombinant vector, the pYL156(TRV2) vector sequence was first analyzed using SnapGene software, and SacI and BamHI located in the multiple cloning site region were selected as restriction enzyme sites. The pYL156(TRV2) vector was double-digested using these two restriction endonucleases (reaction system shown in Table 4), and the reaction was carried out in a water bath at 37°C for 1 hour to linearize the pYL156(TRV2) vector.

[0059] Table 4. TRV2 vector enzyme digestion system

[0060]

[0061] Subsequently, the target fragment recovered in step (1) was ligated to the linearized pYL156(TRV2) vector using the Clone Express II One Step Cloning Kit and reacted at 50°C for 30 minutes to finally construct the recombinant vector pYL156(TRV2)-Gohir.A03G043700.

[0062] (3) Transformation of recombinant vectors

[0063] Gently mix 10 μL of the recombinant product with 50 μL of LDH5α E. coli competent cells (purchased from Novizan), incubate on ice for 30 minutes, then heat shock at 42°C for 45 seconds, and quickly transfer to ice to cool for 2 minutes. Add 700 μL of antibiotic-free LB liquid medium and incubate at 37°C and 220 rpm with shaking for 1 hour. After incubation, collect the cells by centrifugation at 7000 rpm for 5 minutes at room temperature, discard the supernatant, resuspend the cell pellet, and spread it evenly on LB agar plates containing kanamycin. Finally, invert the plates and incubate at 37°C for 14-16 hours.

[0064] (4) Identification of positive single colonies and plasmid extraction

[0065] On the previously incubated LB solid medium containing kanamycin, use sterile yellow pipette tips to pick up multiple single colonies and streak them onto fresh LB solid medium containing kanamycin. Invert the petri dishes and incubate at 37°C for 12-14 hours to allow for full colony growth. Place the streaked yellow pipette tips in sterile water and mix thoroughly. Use this aqueous solution as a template for colony PCR amplification according to the system in Table 5. After amplification, use 1% agarose gel electrophoresis to detect the PCR products and screen for single *E. coli* colonies with the correct band size (meeting the expected positive clones).

[0066] Table 5. Colony PCR reaction system

[0067]

[0068] From the selected positive clones, single colonies were picked and inoculated into LB liquid medium containing kanamycin. The culture was incubated at 37°C (220 rpm) for 6-8 hours. 1 ml of the bacterial culture was sent to Suzhou Genewiz for sequencing. Then, under aseptic conditions, 500 μL of the verified bacterial culture was transferred to 15 mL of LB liquid medium containing kanamycin and cultured at 37°C and 220 rpm for 16 hours to obtain the expanded bacterial culture. Subsequently, the plasmid was extracted using a high-purity plasmid small-volume rapid extraction kit (Adley Biotech, Beijing, China) to obtain the recombinant plasmid verified by sequencing. The concentration was determined by spectrophotometer.

[0069] (5) Recombinant vector was transformed into Agrobacterium.

[0070] After thawing GV3101 Agrobacterium competent cells stored at -80℃ on ice, 100 ng of the recombinant plasmid obtained in step (4) was added and gently mixed by pipetting. The cells were then subjected to heat shock and freeze-thaw cycles sequentially: ice bath for 5 minutes, water bath at 37℃ for 5 minutes, liquid nitrogen flash freezing for 1 minute, and ice bath for 5 minutes. Subsequently, 700 μL of antibiotic-free LB liquid medium was added to the bacterial culture, and the cells were incubated in the dark at 28℃ and 220 rpm for 1 hour. Bacterial cells were collected by centrifugation at 7000 rpm for 5 minutes at room temperature. The supernatant was discarded, and the bacterial pellet was resuspended and evenly spread on LB solid medium containing kanamycin and rifampin. The medium was incubated upside down in a 28°C incubator for 2 to 3 days. Several single colonies were picked with sterile yellow pipette tips and streaked in fresh LB solid medium containing kanamycin and rifampin. The medium was incubated upside down in a 28°C incubator in the dark for about 24 hours. The streaked yellow pipette tips were then placed in sterile water and stirred to mix. The aqueous solution was used as a template for colony PCR amplification (reaction system as shown in Table 5 above). Positive colonies with the correct band size and single positive colonies were screened and inoculated into LB liquid medium containing kanamycin and rifampin. The medium was incubated at 28°C (220 rpm) for 16 hours to obtain Agrobacterium tumefaciens bacterial suspension containing the TRV2-Gohir.A03G043700 recombinant vector. A portion of the bacterial suspension was mixed with 50% glycerol at a 1:1 ratio and stored at -80°C.

[0071] (6) VIGS infection procedure

[0072] Plump and uniformly sized seeds of the salt-tolerant strain XiangFZ031 were selected and sown using a 3:1 mixture of substrate and vermiculite. Seedlings were transferred to an artificial climate chamber for cultivation at 25℃ with a photoperiod of 16 / 8h. When the seedlings had fully expanded cotyledons but before true leaves had sprouted, Agrobacterium-mediated infection was performed. Details are as follows:

[0073] Positive control (TRV2:CLA1): In this experiment, the CLA1 gene was silenced in cotton as a positive control. This gene encodes 1-deoxyfructose-5-phosphate synthase, which is involved in chloroplast development. The CLA1 gene is highly conserved throughout evolution, and its silencing will result in a distinct albinism phenotype.

[0074] Negative control (TRV2:00): Empty vector TRV2:00 serves as the negative control.

[0075] Experimental group (TRV2: Gohir.A03G043700): Gohir.A03G043700 was silenced in cotton.

[0076] Small amounts of Agrobacterium bacterial suspensions containing different plasmids (RNA1 helper plasmid, TRV2:00 negative control, TRV2:CLA1 positive control, TRV2:Gohir.A03G043700) stored at -80℃ were inoculated into 1 mL of double-antibiotic LB liquid medium containing kanamycin and rifampicin, and cultured overnight at 28℃ and 220 rpm in the dark with shaking to activate Agrobacterium.

[0077] The activated Agrobacterium bacterial suspension was added to 10 ml of LB liquid medium containing kanamycin and rifampin, and cultured overnight at 28°C and 220 rpm in the dark until the OD600 reached 1.5–2.0, obtaining the expanded culture. The expanded culture was centrifuged at 7000 rpm for 10 minutes, and the Agrobacterium cell pellet was collected. The pellet was then resuspended using VIGS resuspension buffer (formulation shown in Table 6), and the OD600 value was adjusted to 1.5–2.0. The resuspended culture was incubated at 25°C in the dark for 3 hours. Then, the culture containing RNA1 helper plasmid was mixed with TRV2:00 (negative control), TRV2:GhCLA1 (positive control), and TRV2:Gohir.A03G043700 culture at a 1:1 volume ratio. A micro-hole was gently punctured on the back of a cotton leaf with a sterile needle, and the mixed culture was slowly injected into the leaf tissue using a sterile syringe. After infection, the plants were placed in a dark environment for 24 hours, and then transferred to normal culture conditions (temperature 25℃, photoperiod 16 hours light / 8 hours dark) for continued culture.

[0078] Table 6. Preparation of VIGS experimental resuspension

[0079]

[0080] (7) VIGS plant treatment and phenotypic biomass determination

[0081] Fourteen days after infection with VIGS bacterial solution, plants injected with TRV:CLA1 were observed to exhibit chlorosis due to chlorophyll inhibition caused by CLA1 gene silencing, while other plants grew normally. Figure 2 At this point, RNA was extracted from leaves of seedlings in the negative control group and experimental group, and RT-qPCR was performed to verify whether the target gene was effectively silenced. After confirming that the target gene was effectively silenced in the experimental group, the remaining plants in the experimental group and negative control group were treated with 200 mmol NaCl for 14 consecutive days. During the salt stress treatment, the growth status and phenotypic changes of the plants were closely observed, and relevant indicators were measured. The specific results are as follows: Figure 3 As shown. Figure 3In the figures, Figure A shows the phenotype of plants 14 days after Agrobacterium infection, with the left figure being TRV2:00 (negative control) and the right figure being TRV2:Gohir.A03G043700 (silent plant); Figure B shows a phenotypic comparison between negative control plants and silent plants after salt treatment; Figure C shows a phenotypic comparison between negative control plants and gene-silent plants after salt treatment (top view); Figure D shows a comparison of expression levels between negative control plants and gene-silent plants; Figure E shows a comparison of the aboveground fresh weight of negative control plants and gene-silent plants after salt treatment.

[0082] The results showed that the leaf fresh weight of TRV2:Gohir.A03G043700 was significantly lower than that of the negative control. Combined with RT-qPCR results, it can be concluded that the expression level of Gohir.A03G043700 in the salt-tolerant line XiangFZ031 did not change significantly, while its expression level significantly decreased after salt stress in the salt-sensitive line CRI12. Silencing this gene weakened the salt tolerance of cotton, specifically manifested as a decrease in leaf fresh weight, indicating that this gene may have a positive regulatory role under salt stress and plays an important role in the salt tolerance of cotton. Figure 3 This gene acts as a "basic defense mechanism," similar to an osmotic regulator synthase or ion transporter. In salt-tolerant materials, it forms a stable and robust basic defense, maintaining a high level of stable expression even under salt stress, continuously providing protection for the cells. However, in sensitive materials, this defense is very fragile; salt stress directly leads to a catastrophic downregulation of its expression, causing the plant to lose its basic protective ability and thus exhibit salt sensitivity. The VIGS experiment directly demonstrates that this gene is a positive effector of salt tolerance, and its presence is crucial for salt tolerance.

[0083] While some embodiments of the present general inventive concept have been shown and described, those skilled in the art will understand that changes may be made to these embodiments without departing from the principles and spirit of the present general inventive concept, the scope of which is defined by the claims and their equivalents.

Claims

1. Application of gene Gohir.A03G043700 in improving salt tolerance of cotton. The sequence of gene Gohir.A03G043700 is shown in SEQ ID NO.

1.

2. Application of gene Gohir.A03G043700 in molecular-assisted breeding of salt-tolerant cotton varieties.

3. A molecular-assisted breeding method for salt-tolerant cotton varieties, characterized in that, Molecular-assisted breeding was performed by positively selecting allelic variants associated with stable high expression of the gene Gohir.A03G043700 in the breeding population, including the following steps: S1. Development of molecular markers: By screening for functionally enhanced non-synonymous single nucleotide polymorphisms in the coding region of the gene Gohir.A03G043700, or by identifying regulatory units with strong promoter effects in the promoter region, a SNP site significantly associated with the stable high expression trait of the gene Gohir.A03G043700 is obtained, where the salt-tolerant allele is defined as G and the sensitive allele is defined as A; S2. Design KASP primers: Design KASP primers for this SNP; S3. Breeding population screening: Seedling DNA extraction and KASP typing were performed on the breeding population, and single plants with genotype GG were selected as salt-tolerant candidate single plants; S4. Field verification: The salt-tolerant candidate plants were planted in the field under controlled salt stress conditions, and then the salt tolerance phenotype was identified.

4. Application of gene Gohir.A03G043700 in the identification of salt-tolerant cotton germplasm. The sequence of gene Gohir.A03G043700 is shown in SEQ ID NO.

1. The application is as follows: the salt-tolerant cotton germplasm is identified by detecting whether there is a significant change in the expression level of gene Gohir.A03G043700 in the sample under salt stress conditions. If there is no significant change, it is a salt-tolerant cotton germplasm; if it is significantly downregulated, it is a non-salt-tolerant cotton germplasm.

5. A method for identifying salt-tolerant cotton germplasm, characterized in that, Includes the following steps: S1. Sample acquisition: Samples of the cotton germplasm to be tested were acquired under salt stress conditions and under normal growth conditions, respectively; S2. Detect and compare gene expression levels: Detect and compare the expression levels of gene Gohir.A03G043700 in samples under two conditions; S3. Determine based on comparison results: If the expression level of gene Gohir.A03G043700 under salt stress conditions is not statistically significantly different from that under normal growth conditions, the cotton germplasm to be tested is determined to be salt-tolerant; if the expression level of gene Gohir.A03G043700 under salt stress conditions is statistically significantly downregulated compared to that under normal growth conditions, the cotton germplasm to be tested is determined to be non-salt-tolerant. The sequence of gene Gohir.A03G043700 is shown in SEQ ID NO.1.

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