Osmanthus OfNHX2 gene as well as expression protein and application thereof

Abstract

The invention discloses an Osmanthus fragrans OfNHX2 gene as well as an expression protein and application thereof, and relates to the technical field of plant genetic engineering. The nucleotide sequence of the Osmanthus fragrans OfNHX2 gene disclosed by the invention is as shown in SEQ ID NO. 1, and the amino acid sequence of the expression protein of the Osmanthus fragrans OfNHX2 gene is as shown in SEQ ID NO. 2. The invention also comprises a recombinant vector containing the gene, a recombinant microorganism and a method for cultivating a salt-sensitive transgenic plant material by using the gene. Experiments show that the leaf relative conductivity, malondialdehyde content, superoxide anion content and hydrogen peroxide content of the transgenic nicotiana benthamiana overexpressed with the OfNHX2 gene are obviously higher than those of a control group under salt stress, and the transgenic nicotiana benthamiana shows a typical non-salt-tolerant phenotype. Therefore, the OfNHX2 gene as well as the expression protein and the application of the OfNHX2 gene provide a brand-new molecular tool and genetic material for plant stress resistance research.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, relates to an Osmanthus ofNHX2 gene, its expressed protein, and its applications. Background Technology

[0002] Soil salinization has become a major challenge for global agricultural production, and its formation is closely related to factors such as saline irrigation water and seawater. Saline soils, through the coordinated action of osmotic stress and ion toxicity, trigger mineral nutrient imbalances caused by sodium ion accumulation. This hinders the absorption of water and essential nutrients by plants from the soil, ultimately inducing a systemic salt stress response and severely interfering with normal plant physiological and biochemical processes. To cope with salt stress, plants have developed multi-level response systems, including transcriptional regulation, a process regulated by numerous genes. + / H + The reverse transport protein (NHX) family belongs to the monovalent cation / proton antitransporter family (CPAI), and it possesses Na+. + / H + Exchange protein domains. NHX has 10-12 transmembrane domains, whose main function is to facilitate the exchange of Na+ and Na+. + Separation, that is, Na+ separation driven primarily by the proton concentration difference across the membrane. + With H + Cross-membrane reverse transport.

[0003] Osmanthus fragrans, a native ornamental plant of my country, is renowned for its beautiful tree shape, evergreen foliage, and rich fragrance. However, some osmanthus varieties with excellent traits struggle to grow in saline-alkali soils, severely hindering their promotion and application. Therefore, identifying and screening functional genes in osmanthus that respond to salt stress is of great significance. In the future, these functional genes can be used in osmanthus transformation systems to cultivate salt-tolerant varieties through molecular breeding techniques (gene overexpression or gene knockout). Summary of the Invention

[0004] To address the aforementioned problems in existing technologies, the technical problem this invention aims to solve is to provide the Osmanthus fragrans OfNHX2 gene. Another technical problem this invention aims to solve is to provide the expression protein of the Osmanthus fragrans OfNHX2 gene. A further technical problem this invention aims to solve is to provide the application of the Osmanthus fragrans OfNHX2 gene in regulating plant salt tolerance, specifically for regulating the stress resistance of Osmanthus fragrans.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] A gene of osmanthus of NHX2, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0007] A protein encoded by the Osmanthus ofNHX2 gene, the amino acid sequence of which is shown in SEQ ID NO. 2.

[0008] A recombinant vector containing the Osmanthus OfNHX2 gene as described in claim 1.

[0009] A recombinant microorganism containing the Osmanthus OfNHX2 gene as described in claim 1 or the recombinant vector as described in claim 3.

[0010] A method for cultivating a transgenic plant, the method comprising introducing the Osmanthus ofNHX2 gene into plant cells and cultivating transgenic plants; wherein the transgenic plants have lower salt tolerance than wild-type plants.

[0011] A research model of a salt-intolerant plant, wherein the research model is a transgenic plant bred by the method described herein, the genome of which integrates the Osmanthus ofNHX2 gene shown in SEQ ID NO. 1, and exhibits more severe physiological damage than the wild-type plant under salt stress.

[0012] In some embodiments, in the salt-intolerant plant research model, the more severe physiological damage includes one or more of the following indicators being significantly higher than in the wild type: relative electrical conductivity of leaves, malondialdehyde content, superoxide anion content, and hydrogen peroxide content.

[0013] In some embodiments, the plant is Nicotiana benthamiana.

[0014] Application of any of the salt-intolerant plant research models described above in the study of plant salt stress mechanisms.

[0015] The application of any of the salt-intolerant plant research models described herein in screening plant salt stress alleviators or verifying the function of salt stress-related genes.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] 1) This invention is the first to clone the OfNHX2 gene from Osmanthus fragrans, the nucleotide sequence of which is shown in SEQ ID NO. 1, and the amino acid sequence of the expressed protein is shown in SEQ ID NO. 2. This invention constructs an expression vector for the Osmanthus fragrans OfNHX2 gene; transforms the constructed Osmanthus fragrans OfNHX2 gene expression vector into Nicotiana benthamiana; and cultivates, screens, and obtains transgenic Nicotiana benthamiana plants.

[0018] 2) The results of the embodiments of the present invention show that after being subjected to high salt stress, the relative conductivity, MDA, superoxide anion and hydrogen peroxide content in plants overexpressing the OfNHX2 gene increased, and the staining results showed that more superoxide anions and hydrogen peroxide accumulated in the leaves, indicating that overexpression of the OfNHX2 gene can reduce the salt tolerance of plants.

[0019] 3) The OfNHX2 gene and the salt-intolerant transgenic plant materials constructed from it provided by this invention have significant application value in scientific research: They can be used to elucidate the mechanism of plant salt stress response: This model amplifies signal responses and physiological disorders under salt stress, acting as a "signal amplifier," helping scientists to more clearly reveal the molecular pathways through which plants sense and transmit salt stress signals, as well as the cascade process of cell damage; They can also serve as a screening platform for agricultural technology innovation: This model can be used for high-throughput screening of chemical substances (such as novel water-retaining agents and antioxidants) or beneficial microorganisms that can alleviate salt stress, providing an efficient platform for developing stress-resistant agents. Simultaneously, it can be used to rapidly verify the function of candidate salt-tolerant genes—if the expression of a gene in this model reverses its salt-intolerant phenotype, it strongly proves that the gene has salt-tolerant function.

[0020] In summary, this invention provides indispensable tools, materials, and research platforms for plant stress resistance research, and has significant theoretical implications and application prospects. Attached Figure Description

[0021] Figure 1 Image of a Japanese cinnamon leaf used in this application;

[0022] Figure 2 The image shows the verification results of OfNHX2 gene amplification.

[0023] Figure 3 The image shows the results of double enzyme digestion verification of the OfNHX2 gene;

[0024] Figure 4 This is a graph showing the results of the bacterial test.

[0025] Figure 5 Image of qRT-PCR detection of positive OfNHX2 transgenic plants;

[0026] Figure 6 Histochemical staining of leaves from positive OfNHX2 transgenic plants. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art. Molecular biology experimental methods not specifically described can be performed according to the methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition) or conventional methods in the art, or according to the kit and product instructions.

[0028] The material used in this application is the leaves of Osmanthus fragrans var. rubrum, which grows in the Osmanthus National Germplasm Resource Bank. Figure 1 In April 2023, leaves of Osmanthus fragrans were placed in sterile centrifuge tubes, immediately flash-frozen in liquid nitrogen, and then stored at -80°C. The seedlings of Nicotiana benthamiana used were provided by the research group of Wang Lianggui at Nanjing Forestry University.

[0029] Example 1

[0030] 1. Total RNA extraction and cDNA acquisition

[0031] Total RNA was extracted from Osmanthus fragrans leaves using the Eastep® Super Total RNA Extraction Kit (Promega (Beijing) Biotechnology Co., Ltd.). Using the extracted total RNA as a template, reverse transcription experiments were performed using the HiScript Ⅲ 1st StrandcDNA Synthesis Kit (Nanjing Novizan Biotechnology Co., Ltd.) to obtain cDNA.

[0032] 2. Design primers

[0033] Based on the previously published Osmanthus fragrans genome database, a gene sequence was screened and named OfNHX2. The full-length nucleotide sequence of this gene was analyzed using BioXM software to determine restriction enzyme sites, and KpnI and SmaI enzymes were selected as the two restriction endonucleases. Primers were designed using CE design software. The required information was filled in, including the sequence near the restriction sites on the vector, the full length of the target gene, and the two restriction sites (5' and 3' ends) in sequence. The designed primers were then sent to Jereh Biotech for synthesis. The primer sequences are shown below:

[0034] OfNHX2-F:

[0035] 5'-aagcttctgcaggggcccgggATGGTGTTTGTTTTGGGAACTTTA-3',

[0036] OfNHX2-R:

[0037] 5'-gcccttgctcaccatggtaccTTCTCGCCATTGTTGCACAC-3'.

[0038] 3. Target gene amplification

[0039] Using cDNA diluted 10-fold as a template, the target gene was amplified by PCR.

[0040] The PCR reaction mixture consisted of: 1 μL Forward Primer, 1 μL Reverse Primer, 1 μL cDNA, 10 μL PrimeSTAR, and 7 μL ddH2O. Three 20 μL systems were prepared for each gene.

[0041] The PCR reaction conditions were: denaturation at 98℃ for 10 s; annealing at 58℃ for 15 s; extension at 72℃ for 1 min, for 35 cycles; total extension at 72℃ for 10 min; and termination of the reaction at 16℃.

[0042] The obtained amplification products were subjected to agarose gel electrophoresis, and then the gel was cut and recovered using a kit. Figure 2 The final sequencing yielded the nucleotide sequence of the OfNHX2 gene, as shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein, as shown in SEQ ID NO.2.

[0043] Example 2

[0044] 1. Constructing an overexpression vector for the OfNHX2 gene

[0045] 1) Vector double enzyme digestion

[0046] The Super1300 vector was taken out of the -80℃ ultra-low temperature freezer in advance for activation and culture. The Super1300 vector plasmid was extracted according to the kit and then double enzyme digestion experiment was performed.

[0047] The 20 μL enzyme digestion reaction system consisted of: KpnⅠ 1 μL, SmaⅠ 1 μL, Buffer 2 μL, vector plasmid X μL, and ddH2O 6 μL.

[0048] Where X (µL) = 1000 ng / vector plasmid concentration (ng / µL). Gently shake the centrifuge tube to mix, centrifuge briefly for 6 seconds, and incubate in a 37°C water bath for 1 hour. Perform agarose gel electrophoresis on the obtained double-digested vector, and then use a kit for gel extraction and recovery.

[0049] 2) Connection Conversion

[0050] The target gene fragment is recombined and ligated with the linearized vector fragment.

[0051] The ligation reaction system consisted of: 200 ng of target gene recovery product, 100 ng of plasmid double enzyme digestion recovery product, 2 μL of ligase, 4 μL of buffer, X μL of linearized vector, and 20 μL of ddH2O.

[0052] Gently shake the centrifuge tube to mix it, centrifuge briefly for 6 seconds, incubate in a 37°C water bath for 30 minutes, and then on ice for 2 minutes.

[0053] Transformation: In a clean bench, use a pipette to take 5 μL of the ligation product and add it to 50 μL of Trelief™ 5α competent cells. Gently mix, incubate on ice for 5 min, incubate in water at 42°C for 60 s, then incubate on ice for 2 min. Add 250 μL of LB liquid (without Kana) and incubate at 37°C and 200 rpm in a shaker for 30 min.

[0054] Spreading: Take 200 μL of the incubated bacterial solution, spread it evenly on LB solid medium (containing 50 mg / L Kana) with a sterile glass rod, and let it dry. After sealing with film, invert the plate and incubate in a 37°C constant temperature incubator for 12-14 h.

[0055] 3) Detection and sequencing of positive single colonies

[0056] After bacteria have grown on the culture medium, single colony detection is performed in a clean bench. Eight plump single colonies are selected for each gene and sequentially backed up on LB solid medium containing Kana resistance. The corresponding single colonies are then transferred to the following system for bacterial testing using a sterile toothpick:

[0057] The PCR reaction system consisted of: 1 μL 35sF, 1 μL Gene R, 10 μL Green Mix, and 8 μL ddH2O.

[0058] The PCR reaction conditions were as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; 72℃ total extension for 10 min; and termination of the reaction at 16℃. The obtained amplification products were subjected to agarose gel electrophoresis, and plasmids were extracted from 3 correct positive colonies.

[0059] 4) Double enzyme digestion verification

[0060] The plasmid with the correct sequence obtained from sequencing was verified by double enzyme digestion.

[0061] The double enzyme digestion system consisted of: KpnⅠ 1 μL, SmaⅠ 1 μL, Buffer 2 μL, vector plasmid X μL, and ddH2O 6 μL.

[0062] Where X (µL) = 1000 ng / vector plasmid concentration (ng / µL). Gently shake the centrifuge tube to mix thoroughly, centrifuge briefly for 6 seconds, and incubate in a 37℃ water bath for 1 hour. Perform agarose gel electrophoresis on the obtained double-digested vector to detect the double digestion status. Figure 3 ).

[0063] 2. Transformation of Agrobacterium GV3101

[0064] Remove the GV3101 competent cells stored in a -80℃ ultra-low temperature freezer and thaw them on ice. Add 1µL of plasmid to every 33µL of competent cells, mix thoroughly by pipetting, and then sequentially in an ice bath for 20min, quick freeze with liquid ammonia for 5min, in a 37℃ water bath for 5min, and in an ice bath for 5min. Add 500µL of antibiotic-free LB liquid medium and incubate at 28℃ on a shaker at 200rpm for 1h. After incubation, centrifuge the bacterial culture at 6000rpm for 1min, discard part of the supernatant, and keep 100µL to spread evenly on LB solid medium (containing 50mg / L Kana), seal with sealing film, and incubate upside down in a 28℃ incubator for 40-48h.

[0065] Microbial testing and backup: The target band in the microbial test is correct and has consistent brightness. Figure 4 If the corresponding colonies from the backup plate are picked into LB liquid medium (containing 50 mg / L Kana) and shaken, the bacterial solution and 50% glycerol are then kept in a volume ratio of 3:7. After being flash-frozen in liquid nitrogen, the culture is stored in an ultra-low temperature freezer at -80°C.

[0066] 3. Infection with Tobacco Benedict

[0067] Shaking culture: Remove the Super1300 empty vector and the vector containing the target gene from the bacterial culture and thaw them on ice. Use a pipette to add the bacterial culture to 20 mL of LB liquid medium (containing 50 mg / L Kana). Incubate in the dark on a shaker at 28°C and 200 rpm until the bacterial culture reaches OD. 600 =Between 0.4 and 0.45;

[0068] Infection: Using sterile forceps, transfer the cut tobacco leaves into the infection solution and infect for 10 minutes. Discard the bacterial solution and resuspend in an equal volume of buffer solution. The ratio of bacterial solution to buffer solution is 1:1. After standing in the dark for 2-3 hours, use a syringe to inject the bacterial solution into tobacco seedlings approximately 30 days old.

[0069] Screening of transgenic plants: RNA was extracted from leaves using a kit and reverse transcribed into cDNA. The cDNA was then diluted 10-fold and detected by real-time quantitative PCR (qRT-PCR). Figure 5 Transgenic seedlings with high expression levels and consistent growth status were selected for subsequent functional verification.

[0070] 4. Physiological indicators of OfNHX2 transgenic plants

[0071] Three transgenic Nicotiana benthamiana plants of identical condition were selected and transformed into an EV line containing the Super1300 empty vector as a transgenic control. After thorough watering, they were cultured in the dark for 10 hours, followed by normal light-dark alternation culture. Two days later, each plant was watered with 200 mL of 500 mmol / L saline solution.

[0072] 1) Leaf histochemical staining

[0073] Fresh plant tissue samples subjected to salt stress were immersed in 1 mmol / L NBT staining solution (protected from light, for 2 hours) and 0.1% DAB staining solution (protected from light, for 12-16 hours), respectively. After staining, the samples were transferred to 6000 lx light for 20 minutes to enhance the staining signal. The tissues were then destained using 95% ethanol at 95°C until completely destained, and subsequently fixed in 50% glycerol and photographed. Figure 6 ).

[0074] 2) Relative conductivity

[0075] Leaves from the same location in the control group and the transgenic Nicotiana benthamiana were taken, washed with water, and 0.1 g of each leaf was added to 20 mL of deionized water. The solution was soaked at room temperature for 24 hours, shaking 3-5 times during this period. The conductivity EC0 of the deionized water was measured using a conductivity meter. Then, the conductivity EC1 of each experimental group was measured. After heating in a 100℃ water bath for 30 minutes and cooling to room temperature, the solution was shaken well, and the conductivity EC2 was measured again. Relative conductivity = (EC1 - EC0 / EC2 - EC0) × 100%.

[0076] 3) MDA

[0077] The MDA content was determined using the TBA colorimetric method. 0.2 g of the solution was weighed and added to 5 mL of TCA solution. After mixing by inversion, the mixture was centrifuged at 4°C and 6000 rpm for 10 min. 2 mL of the supernatant was mixed with 2 mL of 0.67% TBA solution and reacted in a 100°C water bath for 30 min, followed by rinsing and cooling with cold water. After centrifuging the reaction solution at 4°C and 6000 rpm for 10 min, 200 μL of the supernatant was collected, and the absorbance at 532 nm, 450 nm, and 600 nm wavelengths was measured using a microplate reader.

[0078] 4) Determination of superoxide anion content

[0079] Weigh 0.2 g of the ground leaf tissue powder, add a small amount of phosphate buffer (pH 7.8), grind, and centrifuge. Take the supernatant and react it with hydroxylamine hydrochloride, p-aminobenzenesulfonic acid, and α-naphthylamine to generate a red azo product. Measure the absorbance at 530 nm and calculate the superoxide anion concentration based on the standard curve.

[0080] 5) Determination of hydrogen peroxide content

[0081] Similarly, weigh 0.2 g of leaf tissue powder, add 10 mL of cold acetone, grind, and centrifuge. Take the supernatant and react it with titanium sulfate solution to form a precipitate. After the precipitate dissolves, measure the OD. 415 The value was calculated using a standard curve to determine the hydrogen peroxide content.

[0082] The results are shown in Table 1. After salt stress treatment, the relative conductivity and MDA content in the leaves of the transgenic lines showed an increasing trend, increasing by 51.08% and 24.37% respectively compared with the unloaded line. At the same time, the superoxide anion and hydrogen peroxide contents in the leaves of the transgenic lines were significantly higher than those in the EV, being 1.21 and 3.59 times higher than those in the unloaded line, respectively.

[0083] Furthermore, NBT and DAB chemical staining results revealed that leaves overexpressing OfNHX2 showed deeper blue formazan precipitates and brown polymer precipitates than EVs.

[0084] Table 1 Physiological indicators of OfNHX2 transgenic plants

[0085]

[0086] In summary, under high salt stress, plants overexpressing the OfNHX2 gene showed increased relative conductivity, MDA, superoxide anion, and hydrogen peroxide levels. Staining results also revealed increased accumulation of superoxide anions and hydrogen peroxide in the leaves, indicating that overexpression of the OfNHX2 gene can reduce the salt tolerance of plants. Therefore, the OfNHX2 gene, its expressed protein, and its applications provided in this application offer a novel molecular tool and genetic material for plant stress resistance research.

[0087] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. An Osmanthus ofNHX2 gene, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A protein encoded by the Osmanthus ofNHX2 gene as described in claim 1, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that, It contains the Osmanthus OfNHX2 gene as described in claim 1.

4. A recombinant microorganism, characterized in that, It contains the Osmanthus OfNHX2 gene as described in claim 1 or the recombinant vector as described in claim 3.

5. A method for cultivating a transgenic plant, characterized in that, The method includes introducing the Osmanthus OfNHX2 gene of claim 1 into plant cells and cultivating transgenic plants; wherein the transgenic plants have lower salt tolerance than wild-type plants.

6. A research model for salt-intolerant plants, characterized in that, The research model is a transgenic plant bred using the method described in claim 5, whose genome integrates the Osmanthus OfNHX2 gene shown in SEQ ID NO. 1, and exhibits more severe physiological damage than the wild-type plant under salt stress.

7. The salt-intolerant plant research model according to claim 6, characterized in that, The more severe physiological damage includes one or more of the following indicators being significantly higher than the wild type: relative electrical conductivity of leaves, malondialdehyde content, superoxide anion content, and hydrogen peroxide content.

8. The salt-intolerant plant research model according to claim 6 or 7, characterized in that, The plant in question is Nicotiana benthamiana.

9. The application of the salt-intolerant plant research model described in any one of claims 6-8 in the study of plant salt stress mechanisms.

10. The application of the salt-intolerant plant research model according to any one of claims 6-8 in screening plant salt stress alleviators or verifying the function of genes related to salt stress response.

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