Land cotton gene ghnrx16 and molecular marker for identifying salt tolerance of plants

Abstract

This invention relates to the field of bioengineering technology, and more particularly to a gene for identifying salt tolerance in upland cotton, GhNRX16, and its molecular marker. The CDS sequence of the upland cotton gene GhNRX16 is shown in SEQ ID NO.1 of the sequence listing. This invention uses RNA-seq technology to screen and identify this salt tolerance-related gene in the transcriptome data of upland cotton under salt stress treatment. Furthermore, the GhNRX16 sequence was obtained through gene cloning, and a transient silencing vector based on VIGS (virus-induced gene silencing) technology was successfully constructed. Experiments using VIGS-mediated gene silencing combined with salt stress treatment showed that silencing the GhNRX16 gene significantly reduced the plant's tolerance to salt stress, demonstrating that normal expression of this gene directly contributes to ensuring the plant's salt stress resistance. These results confirm the crucial role of the GhNRX16 gene in regulating cotton salt tolerance, providing important gene resources and theoretical basis for molecular breeding of salt-tolerant cotton.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a gene GhNRX16 for identifying salt tolerance in upland cotton and its molecular marker. Background Technology

[0002] Salt stress damages plants in a multi-dimensional and gradual process, primarily involving osmotic stress, ion toxicity, and nutrient imbalance. First, high concentrations of salt ions in the soil lower soil water potential, hindering root water absorption and triggering drought-like osmotic stress. This leads to loss of cell turgor pressure, stomatal closure, and inhibition of photosynthesis. Following this, ion toxicity, particularly from sodium (Na+), develops. + Massive invasion disrupts the homeostatic K+ in the cytoplasm. + / Na + Proportion, and K + Na+ is an essential cofactor for the activity of many enzymes, and its homeostasis can directly affect protein synthesis and metabolic activity. Meanwhile, high concentrations of Na+... + It will also be with Ca 2+ Divalent cations compete for binding sites on the plasma membrane, disrupting the integrity and selectivity of the membrane structure. Furthermore, salt stress can cause nutrient imbalances, such as inhibiting the plant's absorption of potassium. + Ca 2+ NO3 - The absorption and transport of essential elements further exacerbate the disorder of physiological metabolism.

[0003] To precisely regulate intracellular redox states, plants rely on a complex antioxidant network, with the thioredoxin (TRX) system playing a central role. TRX are conserved small proteins with a molecular weight of approximately 12 kDa. Their active site contains a highly conserved Cys-Gly-Pro-Cys motif, which directly regulates the activity of target proteins through reversible reduction / oxidation of disulfide bonds. In salt stress responses, TRX functions in multiple ways: First, it acts as an electron donor, directly reducing and activating a series of antioxidant enzymes (such as ascorbate peroxidase APX and peroxidoredoxin Prx), thereby enhancing ROS scavenging capacity. Second, TRX can regulate key enzymes in the Calvin cycle (such as fructose-1,6-bisphosphatase) to maintain photosynthetic efficiency under stress. More importantly, TRX directly affects the expression of stress-related genes by regulating the redox state of transcription factors or signaling proteins such as NF-YC2 and NPR1, thus acting as a "molecular switch" in redox signal transduction.

[0004] Within the TRX superfamily, the Nucleoredoxin (NRX) subfamily has become a research frontier due to its large molecular weight (typically 40-50 kDa) and unique domain composition (including other protein-protein interaction domains in addition to the TRX domain). NRX is believed to have specific functions in H2O2-mediated signal transduction. Studies in the model plant Arabidopsis thaliana have found that AtNRX1 can interact with the peroxidase protein PRXIIB and negatively regulate its peroxidase activity, thereby finely controlling H2O2 signal levels. In rice, OsNRX1 has been shown to be upregulated under salt stress, and overexpressing lines exhibit lower ROS accumulation and higher survival rates, demonstrating the positive regulatory role of NRX in salt tolerance in monocotyledonous plants. Furthermore, studies in animals (e.g., mouse NRX) and fungi have also shown that NRX plays a crucial role in maintaining redox homeostasis during cell cycle regulation and embryonic development, and its function is somewhat conserved evolutionarily. However, in tetraploid upland cotton (G. hirsutum L.), the systematic identification of the NRX gene family, analysis of its expression patterns, and its specific molecular targets and regulatory networks in salt stress response remain a gap. Therefore, in-depth functional research on cotton NRX genes is not only an important supplement to basic research in plant redox biology, but will also provide valuable genetic resources and theoretical basis for the genetic improvement of cotton salt tolerance.

[0005] To address the aforementioned issues, this application requires providing a gene for identifying salt tolerance in upland cotton, GhNRX16, and its molecular marker. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing a gene GhNRX16 and its molecular marker for identifying salt tolerance in upland cotton. By silencing the GhNRX16 gene, the plant's tolerance to salt stress is significantly reduced, demonstrating that normal expression of this gene has a direct effect on ensuring the plant's ability to resist salt stress.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A gene for identifying salt tolerance in upland cotton, GhNRX16, is disclosed. The CDS sequence of the upland cotton gene GhNRX16 is shown in SEQ ID NO.1. The gene number of this upland cotton gene GhNRX16 is GH_A03G1602.

[0009] A molecular marker for identifying the upland cotton gene GhNRX16, which determines salt tolerance in plants, is disclosed. The molecular marker comprises two reagents containing nucleotides with sequences SEQ ID NO.2 and SEQ ID NO.3. Here, the presence and normal expression of the GhNRX16 gene are used to determine cotton salt tolerance.

[0010] One method for regulating cotton salt tolerance is to silence the GhNRX16 gene sequence mentioned above, which can alter the salt tolerance of cotton.

[0011] An application of the above-mentioned gene GhNRX16 in the cultivation of salt-tolerant plants, wherein the plant is preferably cotton.

[0012] By employing the above-mentioned technical approach, the salt tolerance-related gene was screened and identified from the transcriptome data of upland cotton under salt stress treatment using RNA-seq technology. The GhNRX16 sequence was further obtained through gene cloning, and a transient silencing vector based on VIGS (virus-induced gene silencing) technology was successfully constructed. Experiments combining VIGS-mediated gene silencing with salt stress treatment showed that silencing the GhNRX16 gene significantly reduced the plant's tolerance to salt stress, demonstrating that normal expression of this gene directly contributes to ensuring the plant's salt stress resistance. These results confirm the crucial role of the GhNRX16 gene in regulating cotton salt tolerance, providing important gene resources and theoretical basis for molecular breeding of salt-tolerant cotton.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] This invention utilizes existing plant genetic engineering techniques, including gene cloning and RT-PCR, to isolate and identify cotton salt tolerance-related gene sequence information. The gene is then silenced using VIGS technology. Salt tolerance phenotype identification shows that the salt tolerance of the silenced plants is significantly reduced. Therefore, the molecular marker invented using this gene can serve as a molecular target for detecting the salt tolerance of cotton plants, and it is expected that salt tolerance can be improved by increasing the expression of this gene through genetic engineering. Attached Figure Description

[0015] Figure 1 This is a diagram showing the amplification results of the CDS fragment of the GhNRX16 gene in this invention;

[0016] Figure 2 This is a diagram showing the expression pattern of the GhNRX16 gene based on RPKM value under 300 mM salt treatment according to the present invention.

[0017] Figure 3The image shows the VIGS silencing effect of the GhNRX16 gene in this invention; (A) Phenotypic characteristics of the positive control group (showing an albino phenotype); (B) Relative expression levels of the GhNRX16 gene in the CK group, TRV2:00 empty vector group, and TRV2:GhNRX16 group; CK group: control group; TRV2:00 group: negative control; TRV2:GhNRX16 group: experimental group;

[0018] Figure 4 The phenotypic diagrams are of the CK group, TRV2:00 unloaded group, and TRV2:GhNRX16 group under 300mM salt treatment according to the present invention. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, thereby making a clearer definition of the scope of protection of the present invention. The embodiments described in this invention are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0020] Example 1: Cloning of the GhNRX16 gene

[0021] Using existing RNA-seq data from our laboratory, we discovered a gene called GhNRX16 that responds to salt stress.

[0022] 1.1 RNA Extraction

[0023] (1) After the cotton leaves were ground with liquid nitrogen, 100 mg of the sample was collected into a centrifuge tube and 500 μL of RCL was added.

[0024] Mix the Buffer / 10μL β-mercaptoethanol mixture immediately by vortexing.

[0025] (2) 55 ℃ water bath for 1-3 minutes, centrifuge at room temperature for 5 minutes at 10000×g.

[0026] (3) Insert the gDNA Filter column into a 2 mL collection tube, transfer the supernatant to the gDNA Filter column, and centrifuge at 14000×g for 2 minutes at room temperature.

[0027] (4) Add an equal volume of RCB Buffer to the filtrate and vortex mix for 20 s.

[0028] (5) Insert the HiBindRNA Mini binding column into a 2mL collection tube, transfer half of the mixture (<700μL) onto the HiBindRNA Mini binding column, centrifuge at 12000×g at room temperature for 1 min, and discard the filtrate.

[0029] (6) Repeat step 5 until all the mixture has been transferred through the column.

[0030] (7) Insert the HiBind RNA Mini binding column into the same 2 ml collection tube, add 400 μL RWF Wash Buffer to the HiBind RNA Mini binding column, centrifuge at 10000×g at room temperature for 30 s, and discard the filtrate.

[0031] (8) Insert the HiBind RNA Mini binding column into the same 2 mL collection tube, add 500 μL RNAWashBufferII (diluted with anhydrous ethanol beforehand) to the HiBind RNA Mini binding column, centrifuge at 10000×g at room temperature for 30 s, and discard the filtrate.

[0032] (9) Repeat step 8.

[0033] (10) Insert the HiBind RNA Mini binding column into the same 2 mL collection tube, centrifuge at 10000×g for 2 min at room temperature, and then dry the HiBind RNA Mini binding column matrix.

[0034] (11) Place the HiBind RNA Mini binding column into a new 1.5 mL centrifuge tube, take 50-100 μL LDPPC water, add it accurately to the center of the HiBind RNA Mini binding column membrane, incubate at room temperature for 2 min, centrifuge at 10000×g at room temperature for 1 min, and elute the RNA.

[0035] 1.2 cDNA Synthesis

[0036] (1) System:

[0037] The above RNA template 1 μg total RNA

[0038] 5×gDNA Remover Buffer 2 μL

[0039] gDNA Remover 1 μL

[0040] Nuclease-free Water (DEPC-treated) to a final volume of 10 μL

[0041] Mix well and centrifuge briefly. The reaction conditions are 37 °C for 5 min.

[0042] (2) Add the following components required for reverse transcription reaction directly to the above reaction tube to synthesize the first-strand cDNA.

[0043] system:

[0044] 10 μL of RNA sample after gDNA Remover treatment

[0045] 5xStarScript II Buffer (with Primer)4ul 4 μL

[0046] StarScript II Enzyme Mix 1 μL

[0047] Nuclease-free Water (DEPC-treated) to a final volume of 20 μL

[0048] (3) Mix gently and centrifuge briefly; incubate at 42 ℃ for 15-50 min.

[0049] (4) Heat at 85 ℃ for 5 min to deactivate StarScript II Enzyme Mix.

[0050] 1.3 Cloning of cDNA

[0051] Based on RNA-seq data from upland cotton in our laboratory, a gene GhNRX16 was identified that responds to salt stress. Figure 1 The complete ORF of the GhNRX16 gene was obtained by combining online analysis with NCBI.

[0052] Upstream primer 5'-GCCTCCATGGGGATCCATGGCAGATGCAGCAAACAA-3' (SEQ ID NO.4) and downstream primer 5'-CGAGACGCGTGAGCTCCAAGAGCACGAGGTGAGGTA-3' (SEQ ID NO.5) were designed at the first and second exons of the GhNRX16 gene ORF sequence. PCR was performed using upland cotton cDNA (synthesized in step 1.2) to obtain a 431 bp clone of the GhNRX16 gene with adapter. Figure 2 ), and reclaim the fragment.

[0053] 1.3.1 PCR reaction system

[0054] 2 μL cDNA template

[0055] 2 × Rapid Taq Master Mix 10 μL

[0056] 1 μL of upstream primer

[0057] 1 μL of downstream primer

[0058] Add ddH2O to a final volume of 20 μL.

[0059] 1.3.2 PCR Amplification Program

[0060] 95 ℃ for 5 min; 95 ℃ for 30 s, 58 ℃ for 30 s, 72 ℃ for 1 min, 35 cycles; 72 ℃ for 5 min; keep warm at 4 ℃.

[0061] Example 2: Construction of VIGS vector

[0062] 2.1 Vector digestion

[0063] (1) Enzyme digestion system:

[0064] 1 μL of endonuclease

[0065] 1 μL of endonuclease

[0066] 10×Buffer 5 μL

[0067] 15 μL of plasmid

[0068] Add ddH2O to a final volume of 50 μL.

[0069] (2) Incubate at 37 ℃ for 4 h, and gently stir to mix every 30 min.

[0070] 2.2 Ligation of the target fragment with the linearized vector

[0071] (1) Connection system:

[0072] 1 μL of enzyme digestion product

[0073] Target fragment 3 μL

[0074] Mix enzyme 1 μL

[0075] (2) Incubate at 37 ℃ for 30 min.

[0076] 2.3 Escherichia coli transformation

[0077] Take DH5α competent cells stored at -80 ℃ and thaw them on ice.

[0078] Add 5 μL of ligation product to 100 μL of competent cells, mix by tapping the bottom of the tube, and incubate on ice for 30 min.

[0079] Heat shock at 42 ℃ for 80 s.

[0080] Let it stand on ice for 3 minutes.

[0081] Add 700 μL of antibiotic-free LB liquid medium and incubate at 37 ℃ and 220 rpm for 1 h.

[0082] Centrifuge at 6000 rpm for 1 min, collect 100 μL of supernatant, resuspend the bacterial block by pipetting and spread it on LB agar plates containing the corresponding antibiotic, and incubate at 37 ℃ for 1-2 days.

[0083] Single colonies were picked and sequenced. The upstream and downstream primer sequences used are shown in SEQ ID NO.6 and SEQ ID NO.7. BLAST analysis confirmed that the sequence similarity between the sequencing results and the ligated target fragment was 100%.

[0084] 2.4 Plasmid Extraction

[0085] (1) Take 1-4 mL of overnight cultured bacterial solution, add it to a centrifuge tube, and centrifuge at 12000 rpm for 1 min using a conventional benchtop centrifuge. Remove the supernatant as much as possible.

[0086] (2) Add 150 μL of solution P1 to the centrifuge tube containing bacterial precipitate, and use a pipette or vortex mixer to completely suspend the bacterial precipitate.

[0087] (3) Add 150 μL of solution P2 to the centrifuge tube and gently invert it 6-8 times to fully lyse the bacteria.

[0088] (4) Add 350 μL of solution P5 to the centrifuge tube, immediately and quickly invert it 12-20 times to mix thoroughly, and centrifuge at 12000 rpm for 2 min.

[0089] (5) Transfer the supernatant collected in the previous step to the adsorption column CP3 using a pipette, trying not to aspirate the precipitate. Centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and put the adsorption column CP3 into the collection tube.

[0090] (6) Add 300 μL of washing solution PWT to the adsorption column CP3, centrifuge at 12000 rpm for 30 sec, discard the waste liquid in the collection tube, and put the adsorption column CP3 into the collection tube.

[0091] (7) Place the adsorption column CP3 into the collection tube and centrifuge at 12000 rpm for 1 min to remove the residual washing liquid in the adsorption column.

[0092] (8) Place the adsorption column CP3 in a clean centrifuge tube, add 50-100 μL of elution buffer TB to the middle of the adsorption membrane, and centrifuge at 12000 rpm for 30 sec to collect the plasmid solution into the centrifuge tube.

[0093] 2.5 Agrobacterium-mediated transformation

[0094] (1) Take the competent Agrobacterium cells stored at -80°C and let them partially melt at room temperature or in the palm of your hand. When they are in an ice-water mixture, insert them into ice.

[0095] (2) Add 0.01-1 μg of plasmid DNA to each 100 μL competent cells, mix by hand by tapping the bottom of the tube, and incubate on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes.

[0096] (3) Add 700 μL of antibiotic-free LB liquid medium and incubate at 28 °C with shaking for 3 hours.

[0097] (4) Centrifuge at 6000 rpm for one minute to collect the bacteria. Take about 100 μL of supernatant, gently pipette and resuspend the bacterial block, spread it on LB or YEB plates containing the corresponding antibiotics, and invert it in a 28°C incubator for 2-3 days.

[0098] Example 3: VIGS silencing of the GhNRX16 gene

[0099] (1) Streak activation of glycerol bacteria: TRV1 (containing pTRV1 vector), TRV2: target gene (containing pTRV2 recombinant vector with inserted target fragment), TRV2:00 (negative control), TRV:CLA (positive control), and cultured at 28 ℃ for 36–48 hours until single colonies appear.

[0100] (2) Pick a single colony and inoculate it into 5 mL of LB liquid medium (containing the corresponding antibiotic: Kan+Rif), and incubate at 28 ℃, 200–220 rpm with shaking for 12–16 hours (overnight).

[0101] (3) Transfer to 50 mL LB (containing antibiotics) at a ratio of 1:100, incubate at 28 ℃, 200–220 rpm until OD600≈ 1.0.

[0102] (4) Centrifuge at room temperature, 4000×g for 10 minutes, discard the supernatant and collect the bacterial cells.

[0103] (5) Resuspend the cells in immersion buffer (10 mM MgCl2, 10 mM MES, 200 μM acetylsalicylic acid, pH 5.6), adjust OD600 to 1.0, and let stand or gently shake at room temperature in the dark for 2–4 hours.

[0104] (6) Select TM-1 upland cotton with 4–6 leaves and vigorous growth. Water can be applied before injection to allow the leaves to fully absorb water, which is beneficial for soaking.

[0105] (7) Using a 1 mL needleless syringe, slowly push the bacterial solution into the interstitial space of the leaf from the back (far axis) of the leaf. Inject 2 cotyledons per plant. The water-soaked area will appear as a water stain and gradually spread.

[0106] (8) After injection, the plants were placed at 22–24 ℃ and cultured under 16 h light / 8 h darkness. Normal watering management was maintained, and the temperature was kept at 22–24 ℃.

[0107] (9) 7–10 days after injection, leaf whitening appeared in the TRV2:CLA positive control. Gene expression levels were verified by qRT-PCR. Figure 3 The upstream primer used was 5'-ATGGCAGATGCAGCAAACAA-3' (SEQ ID NO.2), and the downstream primer was 5'-TGGACCGCACCAAGATG-3' (SEQ ID NO.3), confirming that the GhNRX16 gene was successfully silenced.

[0108] Example 4: Salt Treatment Experiment

[0109] 7-10 days after the appearance of the phenotype, plants that had been silenced by VIGS were treated with 300 mM salt, and the phenotype was recorded. Figure 4 Phenotypic results show that silencing the GhNRX16 gene significantly reduced the salt tolerance of upland cotton, indicating that the GhNRX16 gene can significantly improve the salt tolerance of upland cotton.

[0110] In summary, based on the aforementioned RNA-seq experimental data, this invention preliminarily confirmed that the GhNRX16 gene plays a role under salt stress. Furthermore, by using the VIGS experiment, upland cotton plants with silenced GhNRX16 genes were obtained. Combined with salt treatment experiments, this demonstrates that the GhNRX16 gene plays a crucial role in cotton's resistance to salt stress.

[0111] The descriptions and practices disclosed in this invention are readily apparent and understandable to those skilled in the art, and various modifications and refinements can be made without departing from the principles of this invention. Therefore, any modifications or improvements made without departing from the spirit of this invention should also be considered within the scope of protection of this invention.

Claims

1. A gene GhNRX16 for identifying salt tolerance in upland cotton, characterized in that, The CDS sequence of the upland cotton gene GhNRX16 is shown in SEQ ID NO.

1.

2. The molecular marker for identifying the upland cotton gene GhNRX16, which is based on claim 1, is characterized in that, The molecular markers include two reagents containing nucleotides with sequences SEQ ID NO.2 and SEQ ID NO.

3.

3. A method for regulating the salt tolerance of cotton, characterized in that, Silencing the GhNRX16 gene sequence as described in claim 1 can alter the salt tolerance of cotton.

4. An application of the gene GhNRX16 described in claim 1 in the cultivation of salt-tolerant plants, characterized in that, The plant in question is cotton.

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