Application of the Citrus aurantium transporter gene CtrNPF2.1 in regulating plant salt sensitivity and / or root growth

Abstract

The present invention belongs to the field of plant genetic engineering and discloses a trifoliate orange transporter protein CtrNPF2.1 and its application in genetic improvement of plant salt resistance and root growth. CtrNPF2.1 The gene was derived from the citrus rootstock Poncirus trifoliata ( Citrus trifoliata ) was isolated and cloned and named CtrNPF2.1 , whose sequence is shown in SEQ ID NO.1. The gene was constructed into overexpression and interference vectors respectively, and introduced into trifoliate orange through Agrobacterium-mediated genetic transformation. The obtained transgenic plants were verified by biological function, indicating that the cloned gene of the present invention CtrNPF2.1 The gene has the function of controlling plant salt resistance and regulating root growth. The development and utilization of this genetic resource will help reduce agricultural production costs and achieve environmental friendliness.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering, and specifically relates to the application of the trifoliate orange transporter gene CtrNPF2.1 in regulating plant salt sensitivity and / or root growth. The applicant isolated and cloned the transporter protein CtrNPF2.1 from trifoliate orange (Citrus trifoliata), overexpressed the gene in trifoliate orange plants, and obtained transgenic plants with significantly improved salt resistance and root growth. Background Art

[0002] Salt stress is one of the major abiotic stresses affecting plant growth and development, severely restricting the increase in crop yields. Excessive salt ion accumulation can cause ion toxicity, disrupt membrane stability, induce the production of reactive oxygen species, and ultimately disrupt cellular metabolism, ultimately affecting normal plant growth and crop yield (Yang & Guo, 2018). To cope with the harm caused by soil salinization, plants have evolved a variety of perception and response mechanisms, activating different signal transduction pathways to adapt to salt stress environments (Zhu, 2002). Among them, regulating ion transport is one of the key strategies plants use to alleviate salt stress. By regulating the accumulation of toxic ions such as sodium and chloride ions, it can effectively reduce the damage caused by salt stress to plants (Zhu, 2016).

[0003] Transporters and ion channels play a key role in plant salt stress response mechanisms. These proteins maintain normal physicochemical homeostasis of the cytoplasm by increasing salt ion efflux and promoting salt ion compartmentalization to the vacuole, thereby ensuring the normal operation of plant metabolic activities (Wu & Li, 2019). Citrus, a typical chloride-sensitive crop, is crucial for improving its salt tolerance by identifying efficient chloride ion transporters (Brumós, Colmenero-Flores et al., 2009). Currently, research on salt stress-responsive transporters primarily focuses on sodium ion transporters, while relatively little research has been conducted on anion transporters, particularly chloride ion transporters (Rajappa, Krishnamurthy et al., 2024).

[0004] The NRT (nitrate transporter) family of proteins possesses broad substrate transport activity. Sixty-four NRT members have been identified in trifoliate orange (Zhao, Li et al., 2022). Traditional research has suggested that NRTs are primarily involved in the absorption and utilization of nitrate in plants (Wang, Hsu et al., 2012). Given that nitrate and chloride are both monovalent anions, in-depth study of NRT family members with dual nitrate and chloride transport functions is crucial for simultaneously regulating plant salt stress responses and nitrogen use efficiency (Li, Tester et al., 2017). This research direction holds significant potential for crop improvement, particularly the genetic improvement of rootstock varieties.

[0005] As a close relative of citrus, trifoliate orange is widely used as a rootstock in citrus production and is an important genetic resource for improving the adaptability of citrus cultivation. Therefore, systematically exploring key genes in trifoliate orange related to salt tolerance and root development will not only help clarify the mechanisms of plant salt tolerance but also provide an important theoretical basis and genetic resources for salt tolerance genetic engineering. Summary of the Invention

[0006] The present invention aims to provide a trifoliate orange transporter protein CtrNPF2.1, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] Another object of the present invention is to provide the use of the trifoliate orange transporter CtrNPF2.1 in regulating plant salt sensitivity and / or root growth. Overexpression or silencing of this gene in plants can produce plants with enhanced or weakened salt resistance and root growth.

[0008] In order to achieve the above purpose, the present invention adopts the following technical measures

[0009] The applicant cloned a new gene CtrNPF2.1 from the trifoliate orange based on plant gene cloning technology, wherein the protein encoded by the CtrNPF2.1 gene is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.1. The gene contains an open reading frame of 1767 bp, encoding 588 amino acids, with an isoelectric point of 9.36 and a predicted molecular weight of 65.37 kDa.

[0010] The protection scope of the present invention includes:

[0011] The applicant used qRT-PCR technology to analyze the relative expression of the CtrNPF2.1 gene after treatment with salt stress and high nitrogen environment. The results showed that the expression of CtrNPF2.1 was induced by salt stress and high nitrogen environment. In addition, the phenotype and related physiological indicators of the overexpressing CtrNPF2.1 transgenic plants were analyzed. The results showed that: compared with the wild-type plants, the nitrogen utilization efficiency of the roots of the CtrNPF2.1 overexpressing plants was increased, and they had stronger salt resistance under salt stress. In addition, compared with the wild type, the total length, surface area, number of branches and nitrate absorption of the roots of the transgenic plants were higher, and the Fv / Fm and O2 under salt stress were higher. ·- However, the phenotypic and physiological data of CtrNPF2.1-interference plants showed the opposite, indicating that CtrNPF2.1 gene is a gene that positively regulates salt tolerance and root nitrogen use efficiency.

[0012] The protection content of the present invention also includes:

[0013] A nucleic acid molecule containing the CtrNPF2.1 gene, or an expression frame thereof, a recombinant vector, or a recombinant microorganism. The protein encoded by the CtrNPF2.1 gene is shown in SEQ ID NO.2.

[0014] The application of the trifoliate orange transporter gene CtrNPF2.1 in regulating trifoliate orange salt sensitivity and / or root growth, wherein the protein encoded by the gene CtrNPF2.1 is shown in SEQ ID NO.2.

[0015] The applications mentioned above are:

[0016] Increasing the expression of the CtrNPF2.1 gene to improve salt resistance and / or root growth ability of trifoliate orange;

[0017] The above application is preferably to introduce a substance that increases the expression of the CtrNPF2.1 gene into the trifoliate orange; preferably, the substance is a nucleic acid molecule containing the CtrNPF2.1 gene, or its expression cassette, recombinant vector, or recombinant microorganism.

[0018] In the above-mentioned application, the improvement of root growth ability is achieved by increasing the total root surface area, total root length and / or the number of root branches.

[0019] Reducing the expression of the CtrNPF2.1 gene can weaken the salt resistance and / or root growth ability of trifoliate orange;

[0020] The above application is preferably to weaken the salt resistance and / or root growth ability of trifoliate orange by knocking out, inhibiting or silencing the CtrNPF2.1 gene.

[0021] In the above-mentioned application, the vector used in the silencing method is the pTRV2 vector.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] The applicant used qRT-PCR to analyze the relative expression of the CtrNPF2.1 gene after treatment with salt stress and a high nitrogen environment. The results showed that the expression of CtrNPF2.1 was induced by salt stress and a high nitrogen environment. In addition, the phenotypic and physiological indicators of transgenic plants overexpressing CtrNPF2.1 were analyzed. The results showed that compared with wild-type plants, the CtrNPF2.1 overexpressing plants had increased nitrogen use efficiency in their roots and stronger salt tolerance under salt stress.

[0024] The successful cloning of the salt-resistant gene CtrNPF2.1 of trifoliate orange provides new genetic resources for the design and breeding of plant stress-resistant molecules and the promotion of root growth, and provides new genetic resources for the implementation of green agriculture and high-yield agriculture. The development and utilization of this genetic resource is conducive to reducing agricultural production costs and achieving environmental friendliness. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a technical flow chart of the present invention.

[0026] Figure 2 Schematic diagram of the expression pattern of CtrNPF2.1 in response to salt stress and nitrogen treatment and in different tissue locations;

[0027] Among them: A is the relative expression level of CtrNPF2.1 gene under high nitrogen (20 times nitrate content) and salt stress (200 mM NaCl) treatments; B is the relative expression level of CtrNPF2.1 gene in root, stem and leaf tissues.

[0028] Figure 3 This is the tissue expression and subcellular localization analysis of CtrNPF2.1 of the present invention.

[0029] Among them: A is the blank negative control of in situ hybridization of the cross section of the trifoliate orange root system; B is a schematic diagram of in situ hybridization of the CtrNPF2.1 gene in the root; C is a local enlargement of Figure B, as shown in the figure, CtrNPF2.1 is mainly expressed in the cortex; D is a schematic diagram of the subcellular localization of the CtrNPF2.1 gene.

[0030] Figure 4 This is a schematic diagram of the determination of the relative expression level and root growth index of the CtrNPF2.1 overexpressing transgenic trifoliate orange (abbreviated as OE-CtrNPF2.1) of the present invention;

[0031] Among them: A is the relative expression level of CtrNPF2.1 in CtrNPF2.1-overexpressing transgenic trifoliate orange (OE-1, OE-2) and control EV; B is the scanning image of trifoliate orange in the OE-CtrNPF2.1 group and the control group after 1 month of growth in a normal hydroponic environment; C is the total root surface area counted by the root scanner of the OE-CtrNPF2.1 group and the control group; D is the total root length of the OE-CtrNPF2.1 group and the control group; E is the number of root forks.

[0032] Figure 5 Schematic diagram of the salt resistance analysis of the transgenic trifoliate orange overexpressing CtrNPF2.1 of the present invention

[0033] Among them: A is the chlorophyll fluorescence phenotype of CtrNPF2.1 overexpressing trifoliate orange (OE-1, OE-2) and wild-type trifoliate orange before and after salt stress treatment; B is the chlorophyll green fluorescence ratio of overexpressing trifoliate orange (OE-1, OE-2) and wild-type trifoliate orange after salt stress; C is the hydrogen peroxide content of overexpressing trifoliate orange (OE-1, OE-2) and wild-type trifoliate orange after salt stress; D is the superoxide anion content of overexpressing trifoliate orange (OE-1, OE-2) and wild-type trifoliate orange after salt stress; E is the chloride ion content in the aboveground part of overexpressing trifoliate orange (OE-1, OE-2) and wild-type trifoliate orange after salt stress; F is the chloride ion content in the underground part of overexpressing trifoliate orange (OE-1, OE-2) and wild-type trifoliate orange after salt stress.

[0034] Figure 6 This is a schematic diagram of the determination of relative expression levels and root growth indicators in CtrNPF2.1 gene silenced plants of Poncirus trifoliata (abbreviated as TRV-CtrNPF2.1);

[0035] Among them: A is the relative expression level of NPF2.1 (TRV-1, TRV-2) and control EV in trifoliate orange with CtrNPF2.1 gene interference; B is the scanning image of trifoliate orange in the TRV-CtrNPF2.1 group and the control group after 1 month of growth in a normal hydroponic environment; C is the total root surface area of ​​trifoliate orange in the TRV-CtrNPF2.1 group and the control group counted by the root scanner; D is the total root length of trifoliate orange in the TRV-CtrNPF2.1 group and the control group; E is the number of root forks of trifoliate orange in the TRV-CtrNPF2.1 group and the control group.

[0036] Figure 7 This is a schematic diagram of the salt tolerance analysis of CtrNPF2.1 gene silenced plants in Poncirus trifoliata;

[0037] Among them: A is the chlorophyll fluorescence phenotype of CtrNPF2.1 interference system trifoliate orange (TRV-1, TRV-2) and empty vector control trifoliate orange EV before and after salt stress treatment; B is the chlorophyll green fluorescence ratio of trifoliate orange in the TRV-CtrNPF2.1 group and the control group after salt stress; C is the hydrogen peroxide content of trifoliate orange in the TRV-CtrNPF2.1 group and the control group after salt stress; D is the superoxide anion content of trifoliate orange in the TRV-CtrNPF2.1 group and the control group after salt stress; E is the chloride ion content of the aboveground part of trifoliate orange in the TRV-CtrNPF2.1 group and the control group after salt stress; F is the chloride ion content of the underground part of trifoliate orange in the TRV-CtrNPF2.1 group and the control group after salt stress. DETAILED DESCRIPTION

[0038] The present invention is described in detail below with reference to specific embodiments. Based on the following description and examples, those skilled in the art can ascertain the essential features of the present invention and, without departing from the spirit and scope of the present invention, can make various changes and modifications to the present invention to adapt it to various uses and conditions.

[0039] Example 1:

[0040] Cloning of the full-length cDNA of CtrNPF2.1 gene from Poncirus trifoliata

[0041] Using Prunus trifoliata cDNA as a template, amplification was performed using a high-fidelity enzyme. The primer sequences for CtrNPF2.1 gene amplification were as follows: forward primer: 5'-aaaggaaccaattcagtcgacATGGAGAAGAACAATAAGACTGCAGT-3' and reverse primer: 5'-tggaaaagggaattcggtaccTACTGGAGATTTTTCAGGATTCACC-3'; lowercase letters represent vector linker sequences;

[0042] The amplified product was purified and recovered using the AxyPrep-96 DNA Gel Extraction Kit. Using seamless DNA cloning technology, the purified product was ligated into the pENTER1A vector. The ligated product was then transformed into DH5α competent cells, plated, and shaken for positive identification. Positive clones were obtained and sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. Based on the sequencing results, CtrNPF2.1 was obtained.

[0043] Sequencing results showed that the ORF of CtrNPF2.1 sequence was 1767 bp, encoding 588 amino acids. The molecular weight of the protein was 65.37 kDa, the isoelectric point was 9.36, the nucleotide sequence was shown in SEQ ID NO.1, and the amino acid sequence was shown in SEQ ID NO.2.

[0044] Example 2:

[0045] Analysis of the expression pattern of CtrNPF2.1

[0046] Two-month-old wild-type trifoliate orange seedlings of similar growth vigor were hydroponically cultured in a growth chamber for seven days under salt stress (200 mM NaCl) and high nitrogen (20x nitrate) conditions. Leaves were harvested at each time point and quickly frozen in liquid nitrogen before being stored at -80°C for subsequent gene expression analysis.

[0047] Hydroponic nutrient solution formula

[0048]

[0049] The expression pattern of CtrNPF2.1 gene under salt stress and high nitrogen was analyzed by real-time fluorescence quantitative PCR (qRT-PCR). AceQ qPCR SYBR Green Master Mix reagent was used for real-time fluorescence quantitative PCR. The method was referred to the instruction manual. The prepared reaction system was used with QuantStudio TM The reaction was performed using a 7Flex Real-Time PCR fluorescence quantitative analyzer.

[0050] Actin from Prunus trifoliata was used as the internal reference gene (forward primer: 5'-CCGACCGTATGAGCAAGGAAA-3'; reverse primer: 5'-TTCCTGTGGACAATGGATGGA-3'). -ΔΔCt The gene expression was calculated by the algorithm. CtrNPF2.1 real-time quantitative primers (forward primer: 5'-TGGAGAAGAACAATAAGACTGCAG-3'; reverse primer: 5'-AATTTTGCTACTTTGCTCGGAG-3').

[0051] The results of this experiment showed that CtrNPF2.1( Figure 2 Middle A) The expression level is induced by salt stress and high nitrogen environment, and the highest expression level is found in the root system among the three parts of the root, stem and leaf ( Figure 2 Middle B), Taken together, these results indicate that CtrNPF2.1 is a salt- and nitrogen-induced gene and may play an important role in plant resistance to salt stress and nitrogen-promoted development.

[0052] Example 3:

[0053] Tissue expression analysis and subcellular localization of CtrNPF2.1

[0054] In situ hybridization was used to detect the expression preference of CtrNPF2.1 in root tissues. The results showed that CtrNPF2.1 was mainly expressed in the cortical tissue of the root ( Figure 3AC). The ORF region of CtrNPF2.1 (excluding the stop codon) was amplified and fused to the vector pYFP101 (containing the YFP protein), driven by the CaMV35S promoter. The control 35S:YFP and 35S:CtrNPF2.1-YFP were then transiently transformed into leaf epidermal cells of Nicotiana benthamiana, and protoplasts were isolated and vacuoles extracted {Li, 2025#63}. Laser confocal microscopy revealed that the fluorescence of the control filled the entire epidermal cell, including the cytoplasm and nucleus, while the fluorescence of the transformed 35S:CtrNPF2.1-YFP was only detected in the vacuole membrane ( Figure 3 Middle D) confirmed that CtrNPF2.1 is a tonoplast-localized protein.

[0055] Example 4:

[0056] Construction of plant transformation vectors, identification of positive seedlings of the Agrobacterium-mediated rooting system of trifoliate orange, and root system scanning analysis

[0057] 1. Plant transformation vector construction

[0058] Using the cDNA of Prunus trifoliata as a template, primers were designed to amplify the full length of the CtrNPF2.1 gene. The primer sequences were as follows:

[0059] pENTER1A-CtrNPF2.1-F:

[0060] 5'-aaaggaaccaattcagtcgacATGGAGAAGAACAATAAGACTGCAGT-3';

[0061] p pENTER1A-CtrNPF2.1-R:

[0062] 5'-tggaaaagggaattcggtaccTACTGGAGATTTTTCAGGATTCACC-3'.

[0063] The linearized pENTER1A vector and the cloned NPF2.1 fragment were inserted into the pENTER1A vector in one step using the One Step Cloning Kit (Novozymes, China).

[0064] The pENTER1A vector (JP6869530B2) carrying the CtrNPF2.1 fragment was ligated with the pK7GW2D vector (CN117126868A) by LR reaction. Following the BP Clonase™ II kit instructions, positive clones that had been sequenced were shaken. Plasmids were then extracted using the AxyPrep Plasmid DNA Miniprep Reagent (Axygen, USA). Transformation into competent E. coli was then performed. After positive identification, the clones were shaken and the plasmids were extracted to obtain the final overexpression vector, pK7GW2D-CtrNPF2.1. The procedures for amplified fragment recovery, positive clone detection, and sample submission for sequencing were similar to those in Example 1. Finally, the vector was transformed into competent Agrobacterium K599 for later use.

[0065] 2. Genetic Transformation of Trifoliate Orange

[0066] 1) Plant material preparation

[0067] Soak the seeds in 1 mol / L NaOH for approximately 15 minutes to remove the pectin. Rinse thoroughly with water. Place the seeds in a clean bench and sterilize them by soaking them in 2% NaClO for 15 minutes. Discard the NaClO and rinse three to four times with sterile water. Place the sterilized seeds in a sterilized flask with a small amount of water. Store in a refrigerator at 4°C.

[0068] The seeds were sown in a moist vermiculite substrate and cultured in the dark at 28°C. They germinated after about two weeks and were transferred to a hydroponic environment and grown to 2-month-old seedlings. The trifoliate orange seedlings with uniform growth were selected and waited for infection.

[0069] 2) Preparation of Agrobacterium infection solution

[0070] On a sterile operating table, use a sterilized inoculation loop to pick up the pK7GW2D-CtrNPF2.1 Agrobacterium stored at -80°C, streak it on a culture medium containing 50mg / L Spec antibiotics, and place the culture medium in a 28°C incubator for dark culture for 2 days. Pick a single clone and inoculate it on a new culture medium containing 50mg / L Spec antibiotics, then place it in an incubator and continue to culture for 2 days. Take a sterilized 100mL small triangular flask and pour 50mL of MT liquid culture medium containing 20mg / L AS (Acetosyringone). Scrape the grown Agrobacterium and dissolve it in MES buffer containing 20mg / L AS. Oscillate at 200r / min for 20min at 28°C. During this period, use scissors to cut off the roots of the trifoliate orange seedlings, ensuring that the cut is oblique to increase the contact area. Add MES buffer to the bacterial solution to adjust the concentration to OD 600 The value is 0.4-0.6.

[0071] 3) Infection and co-cultivation

[0072] Soak the cut ends of the freshly cut Trifoliate Orange seedlings in the prepared bacterial solution. Vacuum the solution for 15 minutes, return the pressure to normal, and let it sit for 30 minutes. Once completed, incubate in the dark, moisturizing the soil for two days. Wild-type control plants were not soaked; they were directly cut and planted in a vermiculite substrate.

[0073] 4) Screening of root-positive seedlings

[0074] Infected trifoliate orange seedlings were planted in a moist vermiculite substrate, maintained at 28 degrees Celsius with ample water. After one month, new roots showing GFP fluorescence were identified using ultraviolet light. Roots without fluorescence were removed, and this process was repeated several times until all newly emerged roots were GFP-positive.

[0075] 3. Identification of positive seedlings

[0076] The positive seedlings with fluorescence signals were randomly divided into two groups, and the roots were taken for real-time fluorescence quantitative analysis of the expression of CtrNPF2.1. The results showed that the relative expression of CtrNPF2.1 gene in CtrNPF2.1 transgenic trifoliate orange (OE-1, OE-2) was 1.37~1.38, compared with the wild type WT. Figure 4 The positive transgenic trifoliate oranges were hydroponically cultured for subsequent root growth trait evaluation and salt tolerance analysis.

[0077] 4. Root Scanning Analysis

[0078] The root system analysis system WinRHIZO (Shanghai Qianfeinuo Agricultural Technology Co., Ltd.) was used to scan the positive seedlings of trifoliate orange root infection and wild-type controls grown in a hydroponic environment for about 40 days, and relevant indicators were analyzed. The results showed that compared with the wild-type, the root system of trifoliate orange overexpressing the NPF2.1 gene grew more vigorously ( Figure 4 Middle B). Total root surface area of ​​OE-1 and OE-2 Figure 4 Middle C), total root length Figure 4 D) and the number of root branches Figure 4 In conclusion, overexpression of NPF2.1 promoted the root growth of trifoliate orange.

[0079] Example 5:

[0080] Analysis of salt resistance of overexpressed trifoliate orange

[0081] Transgenic trifoliate oranges (OE-1 and OE-2) and wild-type trifoliate oranges (WT) were used to characterize their salt stress resistance. Three-month-old trifoliate oranges infected with Agrobacterium rhizogenes were used as test materials and treated with 200 mM NaCl in hydroponics for seven days. Various parameters were then measured.

[0082] The chlorophyll fluorescence parameter Fv / Fm is used to characterize the efficiency of light energy conversion in the PSⅡ reaction center. When plants are subjected to external stress, this parameter decreases significantly. Before salt stress treatment, there was no obvious phenotypic difference between wild-type and transgenic trifoliate oranges. After salt stress treatment, the overexpressing trifoliate oranges showed stronger chlorophyll fluorescence ( Figure 5 A) and higher Fv / Fm values ​​( Figure 5 (B). This indicates that the transgenic trifoliate orange was less damaged. After salt stress treatment, the wild-type trifoliate orange had a higher hydrogen peroxide content than the overexpressed trifoliate orange ( Figure 5 C, the first group from the left is the WT group), and accumulated more superoxide anion content ( Figure 5 D, the first group from the left is the WT group), indicating that the wild type of trifoliate orange is more seriously damaged by salt stress. In addition, after salt stress treatment, the chloride ion content in the aboveground part of trifoliate orange seedlings in groups OE-1 and OE-2 was significantly lower than that in WT ( Figure 5 E, the first group from the left is the WT group), while the chloride ion content in the underground part is the opposite ( Figure 5 (F, the first group from the left is the WT group), indicating that the transgenic trifoliate orange can better restrict chloride ions to the underground part. In summary, phenotypic observations and physiological data measurements show that overexpressing the CtrNPF2.1 gene improves salt tolerance in transgenic trifoliate orange.

[0083] Example 6:

[0084] Identification of positive seedlings of trifoliate orange by VIGS and root scanning analysis

[0085] The VIGS-mediated method was used to interfere with the CtrNPF2.1 gene in trifoliate orange.

[0086] 1. Vector Construction

[0087] Using Prunus trifoliata cDNA as a template, specific primers were designed to amplify the 3' non-conserved region of the CtrNPF2.1 gene. The fragment was then inserted between the BamHI and SmaI restriction sites on the pTRV2 vector using the One Step Cloning Kit (Novozymes, China). The specific method is shown in Table 7. After sequencing, the constructed vector was transformed into GV3101 competent cells to construct pTRV2-CtrRAV2 Agrobacterium. At the same time, pTRV1 and pTRV2 were transformed into GV3101 competent cells, respectively.

[0088] The primers for constructing the vector are as follows:

[0089] pTRV2-CtrNPF2.1-F(BamHI):

[0090] 5'-AGAAGGCCTCCATGGGGATCC TGGAGAAGAACAATAAGACTGCAG-3';

[0091] pTRV2-CtrNPF2.1-R(SmaI):

[0092] 5'-TGTCTTCGGGACATGCCCGGG CTCCGAGCAAAGTAGCAAAATT-3'.

[0093] 2.VIGS Agrobacterium infection

[0094] Fresh seeds of trifoliate orange fruit were peeled, soaked in 1 mol / L NaOH solution, stirred with a magnetic stirrer for 15 minutes to remove pectin, washed 2-3 times with sterile water, then soaked and stirred in 2% NaClO for 15 minutes. The seeds were then washed 2-3 times with sterile water to remove excess sodium hypochlorite. The seeds were spread flat on moistened sterile gauze and placed in a 28°C incubator in the dark to accelerate germination. When the epicotyls germinated to 1.5 cm, they were ready for VIGS infection. Plants co-transformed with the helper plasmids TRV1 and TRV-CtrNPF2.1 served as the experimental group, while plants co-transformed with TRV2 and TRV1 served as the control group.

[0095] 1) Streak TRV1, TRV2, and TRV-CtrNPF2.1 Agrobacterium onto LB solid medium (containing 50 mg / L Rif and 50 mg / L Kan) and culture inverted at 28°C for 3 days to obtain single colonies.

[0096] 2) Pick one single colony from each strain and place it in 5 mL of LB liquid medium containing the same antibiotics. Incubate at 28°C, 220 rpm, and shake gently for 24 hours to fully activate the bacteria.

[0097] 3) The activated Agrobacterium culture liquid was inoculated into the same LB liquid medium at a ratio of 1:100, cultured at 28°C, 220 rpm, and expanded for 12 h. The cells were collected by centrifugation at 4000 rpm and suspended in MES buffer (10 mmol / LMES, 10 mmol / L MgCl2, 150 μmol / L AS, pH = 5.6-5.7). The OD 600 Adjust to 2.0;

[0098] 4) Mix the resuspensions of TRV1 and TRV2, and TRV1 and the target recombinant bacteria in a 1:1 ratio, mix well, and incubate in the dark at 28°C for 3 h;

[0099] 5) Use a syringe needle to lightly poke small holes in the hypocotyls of the buds. Completely immerse the seeds in the prepared Agrobacterium infection solution. Vacuum the solution for 40 minutes, releasing air every 10 minutes, and gently shake to mix. Remove the infected seeds and spread them flat on a large dish soaked in distilled water or MES buffer. Wrap with plastic wrap to retain moisture, poke small holes to allow ventilation, and incubate in a dark room for 3 days.

[0100] 6) Gently wash the dark-cultured seeds with distilled water to remove residual bacterial liquid, and sow them in a substrate (seedling soil: vermiculite:perlite = 3:1:1).

[0101] The intervention plants were randomly divided into two groups, and the expression of CtrNPF2.1 gene in VIGS intervention plants was detected by real-time fluorescence quantitative PCR (qRT-PCR). The results showed that compared with the control plants (EV), CtrNPF2.1 gene was suppressed to 30% to 10% ( Figure 6 A, TRV-1, and TRV-2) generally showed low expression levels. These results demonstrate that VIGS has a high interference efficiency and that the CtrNPF2.1 gene was successfully interfered with in VIGS plants. Positive VIGS plants were cultured hydroponically and analyzed for root growth phenotype and salt tolerance after approximately three months of age.

[0102] The positive seedlings TRV-1, TRV-2 and EV were scanned using a root scanner. The results showed that the root growth of wild-type trifoliate orange was more vigorous than that of trifoliate orange with NPF2.1 gene interference ( Figure 6 Middle B). Total root surface area of ​​TRV-1 and TRV-2 ( Figure 6 Middle C), total root length ( Figure 6 D) and the number of root branches ( Figure 6 In conclusion, silencing NPF2.1 inhibited the root growth of Poncirus trifoliata.

[0103] Example 7:

[0104] Identification of salt-tolerance traits of trifoliate orange using VIGS

[0105] Trifoliate orange seedlings cultured in hydroponics for 3 months after intervention and wild-type trifoliate orange were selected as test materials, transferred to a hydroponic environment and treated with 150 mM NaCl for 7 days, after which various indicators were tested.

[0106] Under normal conditions, there was no significant difference in the morphology of VIGS and TRV control plants. However, after salt stress treatment, VIGS plants showed a significant salt-sensitive phenotype compared to TRV controls ( Figure 7 A), the chlorophyll fluorescence values ​​of the two TRV groups were lower than those of the EV ( Figure 7In Figure B, the first group from the left is the EV control group). In addition, compared with EV, TRV-1 and TRV-2 plants contained higher levels of hydrogen peroxide and superoxide anions after salt stress treatment ( Figure 7 Middle C, Figure 7 (D, the first group from the left in each group is the EV control group). Furthermore, in the aboveground parts, the TRV group accumulated more chloride ions compared to the EV group. Furthermore, the TRV-intervention group accumulated less chloride ions in the underground parts of the trifoliate oranges compared to the EV group. In summary, interfering with the CtrNPF2.1 gene reduced the plant's ability to scavenge reactive oxygen species and weakened the root system's ability to restrict chloride ion transport to the aboveground parts, severely impairing the plant's salt tolerance, further demonstrating the important role of this gene in improving plant salt tolerance.

Claims

1. Use of the protein shown in SEQ ID NO. 2, a gene encoding the protein shown in SEQ ID NO. 2, or an expression cassette expressing the gene encoding the protein shown in SEQ ID NO. 2, a recombinant vector, or a recombinant microorganism in regulating salt sensitivity and / or root growth in trifoliate orange, wherein the root growth is defined as total root surface area, total root length, and / or number of root forks.

2. The use according to claim 1, wherein the regulation is to improve the salt resistance and / or root growth ability of trifoliate orange.

3. The use according to claim 2, wherein the use process comprises introducing a substance into the trifoliate orange to increase the expression of the gene encoding the protein shown in SEQ ID NO. 2; the substance is an expression cassette, a recombinant vector or a recombinant microorganism that expresses the gene encoding the protein shown in SEQ ID NO.

2.

4. The use according to claim 2, wherein the improvement of root growth ability is achieved by increasing the total root surface area, total root length and / or the number of root forks.

5. The use according to claim 1, wherein the regulation is to reduce the expression level of the gene encoding the protein shown in SEQ ID NO. 2 to weaken the salt resistance and / or root growth ability of the trifoliate orange.

6. The use according to claim 5, wherein the use process is to weaken the salt resistance and / or root growth ability of trifoliate orange by knocking out, inhibiting or silencing the gene encoding the protein shown in SEQ ID NO.

2.

7. The use according to claim 6, wherein the vector used in the silencing method in the use is a pTRV2 vector.

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