Ptnf-ya3 gene and its application in genetic improvement of plant drought resistance

Abstract

The present application belongs to the field of plant genetic engineering, and discloses a P. quinquefolium transcription factor gene and application thereof in drought resistance genetic improvement of plants. PtNF-YA3 The present application belongs to the field of plant genetic engineering, and discloses a P. quinquefolium transcription factor gene and application thereof in drought resistance genetic improvement of plants. PtNF-YA3 The present application belongs to the field of plant genetic engineering, and discloses a P. quinquefolium transcription factor gene and application thereof in drought resistance genetic improvement of plants. Poncirus trifoliata The present application belongs to the field of plant genetic engineering, and discloses a P. quinquefolium transcription factor gene and application thereof in drought resistance genetic improvement of plants. PtNF-YA3 The present application belongs to the field of plant genetic engineering, and discloses a P. quinquefolium transcription factor gene and application thereof in drought resistance genetic improvement of plants. The present application belongs to the field of plant genetic engineering, and discloses a P. quinquefolium transcription factor gene and application thereof in drought resistance genetic improvement of plants.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to the PtNF-YA3 gene of trifoliate orange and its application in the genetic improvement of drought resistance in plants. The applicant isolated and cloned a drought-negative regulatory gene from trifoliate orange (Poncirus trifoliata). Interference with this gene in trifoliate orange peel resulted in transgenic plants with significantly enhanced drought resistance, demonstrating the important role of this gene in drought resistance in citrus. Background Technology

[0002] Plants are immobile organisms that attach to plants and are constantly threatened by adverse external environments throughout their lives. Common stresses include biotic stress (drought, low temperature, high salinity, high osmotic pressure, nutrient deficiency, or ion toxicity). In recent years, global warming and frequent extreme weather events have led to drought stress, which has severely impacted plant growth and development. Water is a crucial component of plants, accounting for over 80% of their composition and forming the basis for normal physiological activities. However, drought increases transpiration in plant leaves, causing them to dry out and curl. Prolonged drought can lead to abnormal division of meristematic tissues, restricting plant growth and development, and even causing plant death. Although the geographical and ecological distribution of plants is severely affected by drought stress, the immobility of plants necessitates a wide range of adaptability. This, to some extent, leads to geographical divisions of plants and the gradual formation of unique phenological periods. However, plants do not simply sit idly by. They adapt to external drought stimuli and minimize the damage caused by stress through a series of adjustments. For example, drought affects root depth, density, and root-to-shoot ratio; prolonged drought increases wax accumulation on leaf surfaces to reduce water loss through transpiration (Seleiman et al 2021, Aslam et al 2022). Alternatively, they synthesize key antioxidant enzymes and reactive oxygen species scavengers, such as SOD, CAT, POD, APX, GST, and GPX, to maintain intracellular redox homeostasis and promptly eliminate oxidative stress caused by the accumulation of reactive oxygen species under plant stress (Choudhury et al. 2017, Qi et al. 2018, Shi et al. 2020). Furthermore, plants produce a large amount of osmotic substances under drought conditions, such as polyamines, betaine, sucrose, and proline, which maintain cell turgor pressure, stabilize key enzymes and proteins, and maintain the stability of the cell membrane system and the ability to scavenge reactive oxygen species under stress (Basu et al. 2016, Bharath et al. 2021, De Rossi et al. 2021, Yang et al. 2021). At the molecular level, various genes involved in signal transduction constitute a complex stress regulatory network. Elucidating the mechanisms by which plants respond to drought stress and discovering superior resistance genes are of great significance for breeding and improvement work.

[0003] Nuclear Factor Y (NF-Y) transcription factors are a multi-subunit family that play diverse roles in plant development and stress responses. NF-Y transcription factors are involved in the entire life cycle of plant growth, including the vegetative (including seed germination, root elongation, and leaf development) and reproductive (including floral organogenesis, fruit maturation, embryogenesis, and seed morphogenesis) stages (Hwang et al. 2016; Liu et al. 2016; Yan et al. 2019; Huang et al. 2020; Feng et al. 2022; Xu et al. 2022; Zhang et al. 2023a; Cai et al. 2024; Li et al. 2024b; Wang et al. 2024b). For example, Arabidopsis thaliana AtNF-YB9 regulates embryonic patterns by integrating light and hormone signals (Sorin et al. 2014), and overexpression of AtNF-YB2 accelerates plant cell division and elongation, thereby promoting taproot elongation (Hwang et al. 2016). Furthermore, AtNF-YC1, 3, 4, and 9 act as redundant repressors of light-mediated hypocotyl elongation through histone deacetylation (Tang et al. 2017). AtNF-YC3 / 4 / 9 synergistically interact with AtNF-YB2 / 3 to enhance the expression of the downstream flowering TF promoter (Kumimoto et al. 2010). In addition, the NF-Y complex plays a crucial role in various stress responses, including drought, salinity, low temperature, and high temperature (Bi et al., 2017; Li et al., 2024a; Manimaran et al., 2017; Nardone et al., 2017; Wang et al., 2021; Xu et al., 2022; Zanetti et al., 2017; Zhou et al., 2022). The response mechanisms of NF-Y transcription factors to various stresses may involve different plant hormone pathways. For example, the soybean GmNF-YC14-YA16-YB2 heterotrimer activates the ABA signaling pathway via GmPYR1 (Yu et al., 2021), while the Arabidopsis AtNF-YA1-YB2-YC9 complex enhances salt tolerance through the jasmonic acid signaling pathway (Li et al., 2024a). Despite these advances, the function of NF-Y genes in citrus remains largely unknown.

[0004] Trifoliate orange (Poncirus trifoliata (L.) Raf) thrives in light and warm conditions, is resistant to cold, humidity, drought, and disease, but is intolerant of saline-alkali soils. It exhibits strong grafting compatibility, early fruiting, and high yields, and is currently widely used for grafting onto citrus rootstocks. Therefore, identifying important drought-resistant genes and molecular regulatory networks is of significant practical importance for creating stress-resistant citrus germplasm resources through modern techniques such as gene editing. Summary of the Invention

[0005] The purpose of this invention is to provide a trifoliate orange transcription factor PtNF-YA3, wherein the protein encoded by the trifoliate orange transcription factor PtNF-YA3 is shown in SEQ ID NO.2.

[0006] Another objective of this invention is to provide the application of the trifoliate orange transcription factor PtNF-YA3 in controlling drought resistance traits in plants. Knocking out or suppressing this gene or its homologs in plants significantly enhances the drought resistance of the plants.

[0007] To achieve the above objectives, the present invention adopts the following technical measures.

[0008] The applicant identified and cloned a novel gene, PtNF-YA3, from *Citrus trifoliata* using plant gene cloning technology. Its sequence is shown in SEQ ID NO.1, and the protein it encodes is shown in SEQ ID NO.2. The gene is 615 bp in length, encoding 204 amino acids, and its sequence is shown in SEQ ID NO.2. The gene encoding this protein is shown in SEQ ID NO.1. Molecular weight prediction shows that the protein has a molecular weight of 22.77 kDa and an isoelectric point (pI) of 10.24.

[0009] The applicant constructed a PtNF-YA3 silent line and analyzed the drought resistance phenotype and related physiological indicators of PtNF-YA3 transgenic plants before and after drought treatment. The results showed that compared to the untreated plants, the PtNF-YA3 interference line exhibited increased drought resistance, improved photosynthetic intensity, and lower electrical conductivity and MDA content. This indicates that PtNF-YA3 is a potential breeding gene that negatively regulates drought resistance in trifoliate orange.

[0010] The scope of protection of this invention also includes:

[0011] The application of PtNF-YA3 in regulating drought resistance of trifoliate orange, wherein the protein encoded by the gene PtNF-YA3 is shown in SEQ ID NO.2.

[0012] The applications described above, specifically:

[0013] Application of knocking out or inhibiting the expression of the PtNF-YA3 gene in improving the drought resistance of trifoliate orange;

[0014] The above-described application involves introducing a substance that reduces or eliminates the expression of the PtNF-YA3 gene in trifoliate orange into the trifoliate orange. The substance is an expression cassette, recombinant vector, or recombinant microorganism that reduces the expression of the PtNF-YA3 gene.

[0015] The above-described applications involve knockout using homologous recombination or CRISPR gene editing methods. The knockout gene translates into a protein that has no original function or cannot be translated into a protein.

[0016] The above-described applications involve inhibition using antisense RNA technology or interfering RNA technology.

[0017] The interfering RNA technology described above utilizes VIGS technology.

[0018] The PtNF-YA3 gene described in the above applications is shown in SEQ ID NO.1.

[0019] Application of reagents for detecting the gene encoding the protein shown in SEQ ID NO.2 in the screening or breeding of drought resistance in trifoliate orange.

[0020] The method for determining the application described above is as follows: drought-resistant trifoliate orange is defined as one in which the gene is not detected or the expression level of the gene is significantly reduced compared to normal.

[0021] Applications of expression cassettes that reduce PtNF-YA3 gene expression, recombinant vectors, or recombinant microorganisms in the creation of drought-resistant trifoliate oranges.

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

[0023] The cloning and isolation of the drought-negative regulatory gene PtNF-YA3 in trifoliate orange provides a new genetic resource for molecular design breeding of plant stress resistance, and a new genetic resource for implementing green agriculture and water-saving agriculture. The development and utilization of this genetic resource is conducive to reducing agricultural production costs and achieving environmental friendliness. Attached Figure Description

[0024] Figure 1 This is a technical flowchart of the present invention.

[0025] Figure 2 This is a schematic diagram of the expression mode of PtNF-YA3 in response to drought stress treatment according to the present invention.

[0026] Where: A is the relative expression level of the PtNF-YA3 gene under treatment; B is GUS staining of callus transiently transformed by the PtNF-YA3 promoter; C is quantitative analysis.

[0027] Figure 3 This is a schematic diagram of the subcellular localization of PtNF-YA3 in this invention;

[0028] Wherein: A is a schematic diagram of the construction of the PtNF-YA3 subcellular localization vector of the present invention; B is the PtNF-YA3 subcellular localization detection of the present invention.

[0029] Figure 4 This is a schematic diagram of the PCR amplification detection results of the VIGS silencing material of the present invention.

[0030] TRV2-F is the forward primer for the vector, and PtNF-YA3-R is the reverse primer for the fragment.

[0031] Figure 5 This is a schematic diagram illustrating the quantitative expression of the PtNF-YA3 gene in the VIGS silencing material of this invention.

[0032] Figure 6 This is a schematic diagram illustrating the drought resistance analysis of the VIGS silent material of this invention;

[0033] Wherein: A is the drought resistance phenotype identification of PtNF-YA3 interferometric material (TRV-NF-YA3) and unloaded (TRV); B is the determination of chlorophyll fluorescence Fv / Fm values ​​of the two groups of plants; C and D are the leaf electrical conductivity and MDA content before and after drought treatment. Detailed Implementation

[0034] The present invention will now be described in detail with reference to specific embodiments. Based on the following description and embodiments, those skilled in the art can determine the basic features of the present invention, and various changes and modifications can be made to the present invention without departing from its spirit and scope to make it suitable for various uses and conditions.

[0035] Example 1:

[0036] Cloning of the full-length cDNA of the PtNF-YA3 gene of Citrus aurantium

[0037] Using trifoliate orange cDNA as a template, amplification was performed using a high-fidelity enzyme. The primer sequences were: forward primer: 5'-ATGGGCGTGCATTCAGCTAG-3', reverse primer: 5'-TTTGATGGTCAGAGCCCTGTGT-3'.

[0038] 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 pEASY-Blunt vector. The ligation product was then transformed into DH5α competent cells (Weidi Biotechnology, China), plated, and incubated upside down at 37°C. Spots were picked and shaken to detect bacterial growth. After PCR detection, positive clones were sent to Wuhan Tianyi Huayu Gene Technology Co., Ltd. for sequencing. Based on the sequencing results, the full-length gene sequence of PtNF-YA3 was obtained.

[0039] Sequencing results revealed that the gene is 615 bp in length, encoding 204 amino acids. Molecular weight prediction showed that the protein has a molecular weight of 22.77 kDa and an isoelectric point (pI) of 10.24. The gene was named PtNF-YA3, with the nucleotide sequence shown in SEQ ID NO.1 and the amino acid sequence shown in SEQ ID NO.2.

[0040] Example 2:

[0041] Expression analysis of PtNF-YA3 under drought treatment

[0042] Wild-type trifoliate orange seedlings of similar growth age (2 months old) were collected and placed in a light-controlled incubator. The seedlings were slowly removed from the soil substrate, and the soil around the roots was cleaned without damaging them. They were then placed on dry filter paper for dehydration. Samples were taken at 0h, 1h, 3h, 6h, 12h, 24h, and 48h. Leaves collected at each time point were immediately frozen in liquid nitrogen and then stored at -80℃ for later use in gene expression pattern analysis.

[0043] The drought expression pattern of the PtNF-YA3 gene was analyzed using real-time quantitative PCR (qPCR). The qPCR was performed using AceQ qPCR SYBR Green Master Mix reagent (Novizan, China), following the manufacturer's instructions. The prepared reaction mixture was processed using a QuantStudio 7Flex system (Applied Biosystems, USA) quantitative PCR analyzer.

[0044] Using Actin from Citrus aurantium as an internal reference gene, 2 -ΔΔCt The algorithm calculates gene expression. The primers used are as follows:

[0045] Actin-F: 5'-CCGACCGTARAVGCAAGGAAA-3'

[0046] Actin-R: 5'-TTCCTGTGGACAATGGATGGA-3'

[0047] PtNF-YA3-qPCR-F: 5'-AAGTAACCGAGCCCCAAGTG-3'

[0048] PtNF-YA3-qPCR-R: 5'-GCTGCCCTGACAAATCTCCT-3'

[0049] The results of this experiment indicate that PtNF-YA3 gene expression is strongly induced by drought, with the highest expression level occurring 3 hours after dehydration treatment, increasing approximately 60-fold compared to 0 hours. Figure 2 (A). This indicates that PtNF-YA3 is a drought-inducible gene and may play an important role in plant drought response.

[0050] Example 3:

[0051] GUS staining analysis of callus transiently transformed by the PtNF-YA3 gene promoter

[0052] 1. Carrier Construction

[0053] This experiment was based on the *Citrus aurantium* whole genome database CPBD (http: / / citrus.hzau.edu.cn / ). Specific primers were designed for the PtNF-YA3 (Pt9g015340) gene promoter sequence, and amplification was performed using *Citrus aurantium* cDNA as a template with a high-fidelity enzyme (Novozymes, China). The amplification reaction system and procedure are shown in Tables 1 and 2. After ligation into the pEASY intermediate vector, sequencing was performed to confirm the PtNF-YA3 promoter sequence. The amplification primer sequences are as follows:

[0054] pPtNF-YA3-F: 5'-GTATGCAACTCTTTTACTCCTGTT-3'

[0055] pPtNF-YA3-R: 5'-TTTGAGGATGGATCTAAACAAT-3'

[0056] The amplified products were purified and recovered using the AxyPrep-96 DNA gel extraction kit (Axygene, USA). The purified products were ligated into the pEASY-Blunt vector (see Table 3 for the ligation system). After incubation at room temperature for 5 min, the ligation was performed on competent *E. coli* DH5α cells. The cells were plated, single clones were picked, and PCR was performed for positive identification. Positive clones were then sent to Wuhan Tianyi Huayu Gene Technology Co., Ltd. for sequencing. Based on the sequencing results, the promoter sequence of the PtNF-YA3 gene was obtained.

[0057] Using the plasmid with correct sequencing results from the previous step as a template, specific primers were designed to amplify the 5' end of each vector by adding a 15-20 bp sequence from the linearized vector terminal as a homologous sequence. This primer pair was then used to amplify the insert fragment containing the homologous sequence. Using the OneStep Cloning Kit (Novizan, China), the insert was ligated between the PstⅠ and BamHI restriction sites on the DX2181G vector. After successful sequencing, the constructed vector was transformed into GV3101 competent cells. The primers used are as follows:

[0058] pPtNF-YA3-DX2181G-F:

[0059] 5'-CTACAGCGCTAAGCTTGGCTGCAGGTATGCAACTCTTTTACTCCTGTT-3'

[0060] pPtNF-YA3-DX2181G-R:

[0061] 5'-AAGGGACTGACCACCCGGGATCCGCTTTGAGGATGGATCTAAACAAT-3'

[0062] 2. Sweet orange wound healing instant transformation

[0063] (1) Suspension of callus: 4-5 days in advance, use a sterile spoon to transfer the prepared sweet orange callus to 50ml of MT liquid culture medium and shake it thoroughly on a shaker (120r / min) in a dark room at room temperature.

[0064] (2) Activation of Agrobacterium: Take Agrobacterium stored at -80℃, streak it in LB solid medium (containing antibiotics with carrier resistance) with a sterile inoculation loop, and incubate it upside down in a 28℃ incubator for 2 days until single colonies grow.

[0065] (3) Expanding culture: Use a sterile inoculation loop to pick up a single colony on the plate, inoculate it again onto LB solid medium with the same resistance, streak it twice, and incubate it again in an incubator at 28℃ for 2-3 days. After the colony has grown, it can be used for infection.

[0066] (4) Preparation of infection solution: On a clean bench, scrape off the Agrobacterium cells that have been streaked twice with a sterile knife and add them to 50 ml of suspension medium containing 50 mg / L AS (MT liquid + 0.5 g / L maltose + 1.5 g / L L-glutamine). Shake at 200 r / min for 20 min at 28℃ to fully disperse the cells. Then adjust the OD600 value of the bacterial solution to between 0.6 and 0.8 with MT suspension.

[0067] (5) Infection and culture: Let the shaken callus stand for several minutes until the callus settles to the bottom of the bottle. Carefully pour off the liquid on top. Use a sterile spoon or tweezers to transfer the callus to a glass dish lined with sterile filter paper. Drain the water and then transfer it to the prepared Agrobacterium infection solution. Shake at 200 r / min for 10 min at 30℃. Let it stand for 30 min. Similarly, spread the infected callus on multiple layers of filter paper until the callus dries (let it stand at low wind speed in a clean bench for about 40 min). Transfer the callus to co-culture medium (MT solid medium + 50 mg / L AS) with sterile filter paper and incubate at room temperature in the dark for 3-5 days.

[0068] (6) After co-culturing for 3 days, part of the callus tissue was kept in the co-culture medium, and the other part was transferred to MT medium containing 10% PEG (MT solid medium + 50 mg / L AS + 10% PEG) to simulate drought treatment, and then GUS staining was performed.

[0069] 3. GUS staining analysis

[0070] GUS staining of sweet orange callus was performed using a GUS staining kit (coolaber, SL7160, China), and the GUS staining results were quantified using ImageJ software.

[0071] The results of this experiment show that callus transformed by pPtNF-YA3:GUS without drought treatment is light yellow, while the blue color of callus transformed by pPtNF-YA3:GUS significantly deepened after drought treatment. Figure 2 (B) indicates that drought can enhance the activity of the PtNF-YA3 promoter ( Figure 2 (C) This further proves that PtNF-YA3 is strongly induced by drought.

[0072] Example 4:

[0073] Subcellular localization of PtNF-YA3 gene

[0074] Amplify the ORF region of PtNF-YA3 (excluding the stop codon) using the following primer sequences (underlined parts indicate restriction sites):

[0075] p101YFP-PtNF-YA3-F:5'-ATGGGATCTACTAGTGAATTCATGGGCGTGCATTCAGCTAG-3'

[0076] p101YFP-PtNF-YA3-R:5'-GGGGGTACCGTCGACGGATCCTTTGATGGTCAGAGCCCTGTGT-3';

[0077] The target gene was constructed into the p101YFP vector. The YFP protein is located at the 3' end of the gene, and its expression is driven by the CaMV35S promoter. Figure 3 (A) 35S:PtNF-YA3-YFP+mCherry and control 35S:YFP (i.e., empty p101YFP vector)+mCherry were transiently transformed into leaf epidermal cells of *Nicotiana benthamiana*. Laser confocal fluorescence observation revealed that the control cell fluorescence filled the entire epidermal cell, including the cytoplasm and nucleus, while the fluorescence of the 35S:PtNF-YA3-YFP transformed cell was concentrated only in the nucleus. This indicates that PtNF-YA3 is a nuclear localization protein (…). Figure 3(B)

[0078] Example 5:

[0079] VIGS Interference Identification of Trifoliate Orange and Positive Seedlings

[0080] 1. Carrier Construction

[0081] Using *Citrus aurantium* cDNA as a template, specific primers were designed to amplify a non-conserved region of approximately 500 bp in the CDS of the PtNF-YA3 gene. A one-step ligation method using the OneStep Cloning Kit (Novizan, China) was employed to insert the fragment between the BamHI and SmaI restriction sites on the pTRV2 vector, yielding the recombinant plasmid TRV2-PtNF-YA3. The empty vector was a vector plasmid containing no target fragment. Both the correctly sequenced recombinant plasmid and the empty vector plasmid were transformed into GV3101 competent cells. The primers used to construct the vector are as follows (underlined portions indicate restriction sites):

[0082] pTRV2-PtNF-YA3-F(BamH I):5'-AGAAGGCCTCCATGGGGATCCGCTTGATAGTCAAACTTCTAATGGC-3';

[0083] pTRV2-PtNF-YA3-R(Sma I):5'-TGTCTTCGGGACATGCCCGGGGATGGTCAGAGCCCTGTG-3'.

[0084] 2. VIGS infection

[0085] Seeds were isolated from the fruit of the trifoliate orange, soaked in 1 mol / L NaOH solution for 15 minutes to remove pectin, rinsed twice with sterile water, and then spread flat on a moistened clean gauze. The seeds were then placed in a 28℃ incubator in the dark to germinate. Once the seedlings had germinated to 1-2 cm in length, they were ready for VIGS infection. The procedure is as follows:

[0086] 1) Agrobacterium containing TRV1, TRV2, TRV2-PtNF-YA3, etc., were streaked onto LB solid medium (containing 50 mg / L Rif and 50 mg / L Kan) and cultured upside down at 28°C for 2-3 days to obtain single clones;

[0087] 2) Pick one single clone from each culture and place it in 5 mL of LB liquid medium containing the same antibiotic. Incubate at 28°C and 220 r / min for 24-48 h to fully activate the cells.

[0088] 3) Inoculate the activated Agrobacterium tumefaciens bacterial suspension at a ratio of 1:100 into LB liquid medium containing the same culture medium. Incubate at 28°C and 220 rpm for 10-12 hours. Centrifuge at 4000 rpm to collect the bacterial cells. Resuspend the cells in MES buffer (10 mmol / L MES, 10 mmol / L MgCl2, 150 μmol / L AS, pH 5.6-5.7). OD 600 Adjust to 1.0;

[0089] 4) Mix TRV1 and TRV2, and TRV1 and TRV2-PtNF-YA3 resuspensions in a 1:1 ratio respectively. After mixing, incubate in the dark at 28℃ for 2-3 hours to prepare the infection solution.

[0090] 5) Use a syringe needle to gently poke some small holes in the germinating seedlings, completely immerse them in the prepared Agrobacterium infection solution, vacuum for 10 minutes, then quickly release the air to allow the Agrobacterium to penetrate the germinating seeds. Repeat this process 3 times. After that, let them stand for 15 minutes, then remove the infected seeds and air them on dry filter paper. After standing for 2-3 minutes, spread them evenly in a large dish with filter paper moistened with sterile water. Incubate in the dark at room temperature for 2-3 days.

[0091] 6) Rinse the seeds after dark culture with clean water to remove residual bacterial solution, sow them in the substrate (soil:vermiculite = 3:1), and grow them in a room temperature light incubator for about one month before positive identification.

[0092] 3. Identification of positive materials

[0093] VIGS-silenced trifoliate orange positive plants were identified by PCR using extracted trifoliate orange DNA as a template and a reverse primer constructed from the TRV2 forward primer and the target gene recombinant vector. The sequences are as follows:

[0094] TRV2-F: 5'-ATTCACTGGGAGATGATACGCT-3'

[0095] PtNF-YA3-R: 5'-TGGTCAGAGCCCTGTG-3'.

[0096] The results are as follows Figure 4 As shown, the recombinant plasmid carrying the target gene in the VIGS vector was successfully transformed into the plant. The PtNF-YA3 gene in the positive plants was quantified using the real-time quantitative PCR (qPCR) method described in Example 2. The results are as follows. Figure 5 As shown, the expression level of the PtNF-YA3 gene in VIGS-silenced trifoliate orange plants was significantly lower than that in the control group.

[0097] Example 6:

[0098] Drought resistance identification of PtNF-YA3 trifoliate orange

[0099] Positive plants with high inhibition levels of TRV-PtNF-YA3 expression in the silent line were selected and subjected to drought treatment. Before treatment, there was no significant difference in growth between the empty vector plants (TRV2) and the silent line plants (TRV2-NF-YA3). However, after 25 days of natural drought treatment, the empty vector plants showed severe leaf curling, yellowing, drying, and even death, while the TRV2-PtNF-YA3 silent line plants only showed mild yellowing and wilting. Figure 6 (A) This indicates that TRV2-NF-YA3 interference can alleviate drought stress damage in trifoliate orange. Simultaneously, chlorophyll imaging showed that the photosynthetic intensity of the TRV2-NF-YA3 silent line plants after drought treatment was significantly higher than that of the untreated plants, and the Fv / Fm value was also significantly increased. Figure 6 (B) indicates that the inhibition of photosynthetic response in the TRV2-NF-YA3 silent line was alleviated under drought stress. The electrical conductivity and MDA content of the TRV2-NF-YA3 silent line after drought were also observed. Figure 6 The values ​​of C and D were significantly lower than those of the uninoculated plants, indicating that the TRV2-NF-YA3 silent line plants suffered less drought damage compared to the uninoculated plants.

[0100] The above results demonstrate that silencing PtNF-YA3 can significantly improve the drought resistance of trifoliate orange plants, further proving the important regulatory role of PtNF-YA3 in improving plant drought resistance.

Claims

1. Knockout PtNF-YA3 Gene or repression PtNF-YA3 The application of gene expression levels in improving the drought resistance of trifoliate orange, the aforementioned PtNF-YA3 The protein sequence encoded by the gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, wherein the application process involves reducing the amount of trifoliate orange. PtNF-YA3 The expression level of a gene or a substance that inhibits its expression is introduced into the trifoliate orange, wherein the substance is a substance that reduces the expression level of a gene. PtNF-YA3 Gene expression cassettes, recombinant vectors, or recombinant microorganisms.

3. In the application according to claim 2, the knockout is performed using homologous recombination or CRISPR gene editing methods, and the protein translated from the knocked-out gene has no original function or cannot be translated into a protein.

4. In the application according to claim 2, the inhibition is performed using antisense RNA technology or interfering RNA technology.

5. Application of reagents for detecting the gene encoding the protein shown in SEQ ID NO.2 in the screening or breeding of drought resistance in trifoliate orange. The determination method in the application is: the trifoliate orange is drought-resistant if the gene is not detected or the expression level of the gene is significantly reduced compared with the normal level.

6. Reduce PtNF-YA3 The application of gene expression cassettes, recombinant vectors, or recombinant microorganisms in the creation of drought-resistant trifoliate oranges. PtNF-YA3 The protein sequence encoded by the gene is shown in SEQ ID NO.2.

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