Genetic locus for regulating efficient utilization of nitrogen nutrients in apples and application thereof

Abstract

The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples MdMYB73L and application of an encoded protein thereof, and belongs to the technical field of molecular biology. MdMYB73L The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples The application discloses a gene for regulating and controlling high-efficiency utilization of nitrogen nutrients of apples

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to the application of the gene MdMYB73L, which regulates the absorption and utilization of nitrogen nutrients in apples, and its encoded protein. Background Technology

[0002] Nitrogen is one of the essential macronutrients for plant growth and development, and a crucial component of proteins, nucleic acids, chlorophyll, and various plant hormones. For perennial fruit trees such as apples, an adequate supply of nitrogen directly affects the tree's vegetative growth, flower bud differentiation, fruit set rate, and the yield and quality of the fruit. Rational application of nitrogen fertilizer can effectively promote new shoot growth, increase leaf photosynthetic rate, and increase single fruit weight and soluble solids content, thus serving as a vital guarantee for high-quality and high-yield orchards.

[0003] However, in current fruit production, the excessive application of nitrogen fertilizer is prevalent in pursuit of yield. Statistics show that nitrogen fertilizer application in my country's main apple-producing areas far exceeds the actual needs of fruit trees. This not only leads to a significant waste of fertilizer resources and a continuous increase in production costs, but also triggers a series of ecological and environmental problems such as soil acidification, compaction, groundwater nitrate pollution, and increased greenhouse gas emissions. More importantly, excessive nitrogen supply induces vigorous growth of apple shoots, intensifies competition between vegetative and reproductive growth, inhibits the allocation of photosynthetic products to the fruit, resulting in poor fruit coloring, decreased firmness, and a lower sugar-acid ratio. It also exacerbates physiological diseases such as bitter pit and watercore, ultimately affecting the commercial value and storage performance of the fruit. Therefore, exploring and utilizing key gene resources for regulating the efficient use of nitrogen nutrients in apples, cultivating nitrogen-efficient apple varieties, or developing supporting cultivation and regulation technologies have become urgent needs for promoting the green and sustainable development of the apple industry.

[0004] In recent years, significant progress has been made in the study of the molecular regulatory mechanisms of nitrogen absorption, translocation, and assimilation in plants. In the model plant Arabidopsis thaliana (… Arabidopsis thaliana ) and rice ( Oryza sativa In the study of nitrogen regulation, several important functional genes involved in nitrogen regulation have been identified, mainly including: nitrate transporter families (such as the NRT1 / PTR family), key nitrogen assimilation enzymes (such as nitrate reductase NR and nitrite reductase NiR), and upstream regulatory transcription factors (such as members of the NLP, TCP, and LBD families). These genes synergistically mediate the sensing, uptake, long-distance transport, and assimilation of nitrate nitrogen in the environment by plants, and participate in the fine regulation of nitrogen signaling pathways.

[0005] However, research on the molecular regulatory mechanisms of efficient nitrogen nutrient utilization in Rosaceae fruit trees such as apples is relatively lagging. While there are some reports on genes related to nitrogen absorption and assimilation in apples, systematic research on upstream transcription factors and their regulatory networks that regulate nitrogen use efficiency remains very limited. In particular, key gene resources with significant application value for genetic improvement are still scarce. Therefore, isolating and identifying key genes in apples that regulate efficient nitrogen nutrient utilization and elucidating their molecular mechanisms of nitrogen absorption and assimilation is of significant theoretical importance and application potential for overcoming the bottleneck in high-efficiency nitrogen breeding of apples and optimizing orchard nitrogen fertilizer management strategies. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide a gene that regulates the absorption and utilization of nitrogen nutrients in apples. MdMYB73L And the application of its encoded protein. This invention inhibits... MdMYB73L Gene expression can significantly improve the nitrogen absorption and assimilation capacity of apple plants under low nitrogen conditions, providing important gene resources and operational targets for high-efficiency nitrogen breeding of apples.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an apple gene. MdMYB73L Applications in (1) or (2) below: (1) Improve the nitrogen use efficiency of apples under low nitrogen conditions; (2) To cultivate transgenic apple varieties that utilize nitrogen nutrients efficiently under low-nitrogen conditions; The low-nitrogen condition refers to a condition containing a low concentration of nitrate nitrogen. The apple gene MdMYB73L It is a DNA molecule as shown in any of the following (I)-III): I) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.1, as follows: SEQ ID NO.1: Atggcggtgatcaggaaggatatggatcgaatcaagggtccatggagccccgaggaggacgactcgctccagaagctggtgcagaagcacggccccaggaactggtcgctgatcagcaagtcgattcctggtcggtccgggaagtcgtgccggctgcggtggtgcaaccagctgtcgccgcaggtggagcaccgcgctttcactcccgaggaggacgacacgattatccgggcccatgcccggttcgggaacaagtgggccaccatcgcccgcctcctcaacggccggaccgacaacgccatcaagaaccactggaactccaccctcaagcgaaaatgctccgacgtcggcggcgtcgttttgaatggaggatacgatggtcattatctccttgaccacgagcagccgcctctgaagcggtcggttagtgccgggtcgggcgtgcctgtttccaccgggctttacatgagcccgggtagcccatccgggtcggatgtgagcgactccagcgtgcaagtcatgtctcttcccgactgccacgtgtacagacccgtggcaagatctggcggggttttgcctcccgtggaaacgacgtcgtcttccaacaacagcagtaacggcgagaaggagaacgaccctccgacttcactgtctctgtcactgcccggagtcgacgccggggaggtgtcgaaccgtgttgctgcggcgaccgagtcgatccctgctccggctccggttccggttccgattccggctgctccggtgttgaatatgccctcggagttgctgggagtgatgcaggggatgataaggaaggaggtgaggagctacatggcgaggttggagcagggcggggtttgttttcagcaggctgcggctgctggcaatgacgggtttaggaatgttgggatgaatcgaattgggtatagcaggattgagtga。

[0008] II) DNA molecules encoding the amino acid sequence shown in SEQ ID NO.2, excluding those in I). The specific amino acid sequence is as follows: SEQ ID NO.2: MAVIRKDMDRIKGPWSPEEDDSLQKLVQKHGPRNWSLISKSIPGRSGKSCRLRWCNQLSPQVEHRAFTPEEDDTIIRAHARFGNKWATIARLLNGRTDNAIKNHWNSTLKRKCSDVGGVVLNGGYDGHYLLDHEQPPLKRSVSAGSGVPVSTGLYMS PGSPSGSDVSDSSVQVMSLPDCHVYRPVARSGGVLPPVETTSSSNNSSNGEKENDPPTSLSSLLPGVDAGEVSNRVAAATESIPAPAPVPVPIPAAPVLNMPSELLGVMQGMIRKEVRSYMARLEQGGVCFQQAAAAGNDGFRNVGMNRIGYSRIE.

[0009] (III) DNA molecules that have 80% or more identity with the DNA fragment defined in (I) and that encode a protein that is functionally equivalent to the protein shown in SEQ ID NO.2.

[0010] In the above scheme, improving the nitrogen use efficiency of apples under low-nitrogen conditions is achieved by inhibiting apple genes. MdMYB73L The expression of [the expression] can improve the nitrogen use efficiency of apples under low nitrogen conditions.

[0011] In the above scheme, the improvement of nitrogen use efficiency of apples under low nitrogen conditions is manifested in the following ways: the growth of apple plants under low nitrogen conditions and the improvement of physiological and biochemical indicators related to nitrogen absorption, translocation and assimilation capacity. The growth includes plant height and / or fresh weight; the physiological and biochemical indicators related to nitrogen absorption, translocation, and assimilation capacity include nitrate content, total nitrogen content, nitrate reductase activity, and nitrite reductase activity. These indicators are all important parameters for measuring plant nitrogen nutrition status and nitrogen assimilation capacity. A comprehensive evaluation of these indicators can objectively reflect... MdMYB73L The regulatory effect of genes on nitrogen use efficiency in apples.

[0012] In this invention, Agrobacterium-mediated genetic transformation is used to obtain... MdMYB73L Transgenic apple plants with suppressed nitrogen expression were used to verify nitrogen use efficiency. Experimental results showed that under low nitrogen (0.5 mM KNO3) conditions, MdMYB73LThe biomass (plant height, fresh weight) and nitrogen metabolism-related indicators of the suppressed expression lines were significantly higher than those of the wild type, while MdMYB73L Overexpression lines exhibited the opposite phenotype, thus confirming... MdMYB73L Genes play a negative regulatory role in nitrogen utilization in apples.

[0013] In a second aspect, the present invention provides a method for interfering with the aforementioned apple gene. MdMYB73L The recombinant vector for expression is used in the following (1) or (2): (1) Improve the nitrogen use efficiency of apples under low nitrogen conditions; (2) Develop transgenic apple varieties that utilize nitrogen nutrients efficiently under low nitrogen conditions.

[0014] The recombinant vector contains the apple gene shown in SEQ ID NO.3. MdMYB73L The specific nucleotide sequence information for the antisense fragment is as follows: SEQ ID NO.3: Ggaggtgtcgaaccgtgttgctgcggcgaccgagtcgatccctgctccggctccggttccggttccgattccggctgctccggtgttgaatatgccctcggagttgctgggagtgatgcaggggatgataaggaaggaggtgaggagctacatggcga ggttggagcagggcggggtttgttttcagcaggctgcggctgctggcaatgacgggtttaggaatgttgggatgaatcgaattgggtatagcaggattgagtgagggagaaaagtgcggattattccggacattttcgcaatttggaaggtggggatg.

[0015] After the recombinant vector was introduced into apple plants via Agrobacterium-mediated transformation, it could be transcribed into hairpin RNA within the plant, thereby inducing... MdMYB73L The mRNA of the gene undergoes specific degradation, thereby effectively inhibiting the expression of the gene.

[0016] In a third aspect, the present invention provides an apple gene. MdMYB73L Application of the encoded protein in improving nitrogen use efficiency in apples under low nitrogen conditions, the apple gene MdMYB73L The encoded proteins are shown in either (A1) or (A2): (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2 of the sequence listing; (A2) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0017] In a fourth aspect, the present invention provides a method for improving nitrogen use efficiency in apples by reducing nitrogen content in the target apple plant. MdMYB73L Improving the nitrogen utilization efficiency of apples can be achieved by adjusting the expression level of genes or the activity of their encoded proteins.

[0018] In the above method, the reduction of the target apple plant MdMYB73L Gene expression levels are achieved through, but not limited to, gene silencing, gene editing, RNA interference, promoter mutation, or transcriptional repression. Preferably, RNA interference technology is used. MdMYB73L Gene silencing via transcription in apple plants offers advantages such as high inhibition efficiency, ease of operation, and genetic stability.

[0019] The method is to improve the nitrogen use efficiency of apples under low nitrogen conditions, wherein the low nitrogen conditions are 0.5 mM KNO3.

[0020] In a fifth aspect, this invention provides a method for cultivating a transgenic apple variety with high nitrogen nutrient utilization under low-nitrogen conditions, comprising the following steps: (1) Construct an apple gene containing the one shown in SEQ ID NO.3. MdMYB73L Interference carriers for antonymous fragments; (2) The interference expression vector was transformed into Agrobacterium, and the transformation of apple explants was mediated by Agrobacterium. (3) Screening and identifying positive transgenic plants to obtain MdMYB73L Transgenic apple varieties with interference expression and high nitrogen nutrient utilization efficiency.

[0021] The beneficial effects of this invention are: This invention isolates and identifies from apples MdMYB73L The gene was found to be a negative regulator of nitrogen use efficiency in apples, and overexpression of this gene was found to be beneficial. MdMYB73L Inhibiting its expression reduces nitrogen use efficiency in apple plants, while suppressing its expression significantly improves nitrogen absorption and assimilation capacity under low-nitrogen conditions. This invention provides a new genetic target for optimizing orchard nitrogen fertilizer management and reducing excessive application of chemical nitrogen fertilizers, effectively mitigating environmental risks such as soil acidification and greenhouse gas emissions caused by nitrogen fertilizer abuse. Furthermore, this gene resource is significant for enhancing the adaptability of fruit trees under low-nitrogen conditions, reducing planting costs, and promoting the green and sustainable development of the apple industry, showing broad application prospects in nitrogen-efficient molecular breeding. Attached Figure Description

[0022] Figure 1 yesMdMYB73L Identification diagram of transgenic apple plants; where A and B are electrophoresis images of PCR amplification products; C is the qRT-PCR detection image. MdMYB73 The expression.

[0023] Figure 2 yes MdMYB73L Phenotypic diagram of transgenic apple plants under nitrogen treatment conditions.

[0024] Figure 3 yes MdMYB73L Biomass statistics of transgenic apple plants; where A is fresh weight and B is plant height.

[0025] Figure 4 yes MdMYB73L Physiological indicators related to nitrogen utilization in transgenic apple plants were detected; where A represents nitrate nitrogen content; B represents total nitrogen content; C represents nitrate reductase activity; and D represents nitrite reductase activity. Detailed Implementation

[0026] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0027] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0028] Example 1: Apple MdMYB73L Gene cloning.

[0029] 1. RNA extraction from 'GL-3' apple tissue culture seedlings Total RNA was extracted from apple leaves using the "FastPure Universal Plant Total RNA Isolation Kit" (Novizan) according to standard methods. The specific steps are as follows: (1) Take 100 mg of 'GL-3' apple tissue culture seedlings, put them in a mortar without RNase, add liquid nitrogen and grind them thoroughly, immediately add 600 μL of buffer PSL, vortex vigorously for 30 sec to mix the sample and lysis buffer thoroughly, and centrifuge at 12,000 rpm (13,400 × g) for 5 min. (2) Take 500 μL of supernatant into FastPure gDNA-Filter Columns III (FastPure gDNA-Filter Columns III has been placed in the collection tube), centrifuge at 12,000 rpm (13,400 × g) for 30 sec, and collect the filtrate; (3) Add 0.5 times the volume of the filtrate to the collection tube and shake thoroughly to mix. (4) Transfer the above mixture to FastPure RNA Columns V (FastPure RNA Columns V has been placed in the collection tube), centrifuge at 12,000 rpm (13,400 × g) for 30 sec, and discard the waste liquid in the collection tube; (5) Add 700 μL of Buffer RWA to FastPure RNA Columns V, centrifuge at 12,000 rpm (13,400× g) for 30 sec, and discard the waste liquid in the collection tube; (6) Add 500 μL of Buffer RWB to FastPure RNA Columns V (please check whether 48 mL of anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (13,400 × g) for 30 sec, and discard the waste liquid in the collection tube. (7) Repeat step 6; (8) Place the FastPure RNA Columns V back into the collection tube and centrifuge at 12,000 rpm (13,400 × g) for 2 min; (9) Transfer FastPure RNA Columns V to a new RNase-free Collection Tubes 1.5mL centrifuge tube, add 30-100 μL of RNase-free ddH2O to the center of the adsorption column membrane, and centrifuge at 12,000 rpm (13,400 × g) for 1 min; (10) The extracted RNA was stored at -80℃ or used directly in downstream experiments.

[0030] 2. Reverse transcription of cDNA Reverse transcription was performed using the "SPARKscript II All-in-one RT Super Mix for qPCR" kit (Cisco). Using RNA as a template, the following components (Table 1) were added to an RNase-free tube. The mixture was gently pipetted until fully combined, briefly centrifuged to collect the residue at the bottom of the tube, and then subjected to the following reaction in a PCR instrument: 50°C for 15 min; 85°C for 5 sec. The resulting cDNA product was stored at -20°C.

[0031] Table 1: Reaction System 3. MdMYB73L Obtaining the encoded sequence and the antisense sequence according to MdMYB73L Gene sequence, design specific primers MdMYB73L -F and MdMYB73L -R、 MdMYB73L (anti)-F and MdMYB73L (anti)-R was used for PCR amplification using cDNA obtained from reverse transcription as a template. The PCR reaction program used was: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min.

[0032] MdMYB73L -F:ttgatacatatgcccgtcgacATGGCGGTGATCAGGAAGG MdMYB73L -R:cttgctcaccatggatccCTCAATCCTGCTATACCCAATTCG Justice Fragments MdMYB73L (anti)-F1:agaacacgggggactctagaGGAGGTGTCGAACCGTGTTG MdMYB73L (anti)-R1:ggggttcccccggggtcgacCATCCCACCTTCCAAATTGC Antonyms MdMYB73L (anti)-F2:ggttcgaaatcgatggtaccCATCCCACCTTCCAAATTGC MdMYB73L (anti)-R2:gatcggggaaattcgagctcGGAGGTGTCGAACCGTGTTG PCR amplification system: 2 μL cDNA template, 1 μL each of forward and reverse primers (10 μM), 1 μL dNTP mix, 25 μL 2 × Phanta Max Buffer, and DNA Polymerase ddH2O to a final volume of 50 μL.

[0033] The obtained PCR products were separated by 1% agarose gel electrophoresis, and the target fragment was recovered and sequenced (Qingke Biotechnology). MdMYB73L Gene sequence. The open reading frame of this gene is 942 bp in length, encoding 313 amino acids, and its nucleotide and amino acid sequences are shown in SEQ.ID.NO.1 and SEQ.ID.NO.2, respectively. The nucleotide sequence of the antisense fragment is shown in SEQ.ID.NO.3. Single clones with correct sequencing were selected, and plasmid DNA was extracted using a plasmid extraction kit and stored at -20°C for subsequent functional verification.

[0034] Example 2: MdMYB73L Construction of overexpression and RNAi interference vectors 1. Vector double digestion and linearization The plant expression vector pRI101 was digested with restriction endonucleases BamHI and SalI using standard methods. The modified pRI101 RNAi vector was digested with XbaI and SalI. The digestion products were separated by 1.0% agarose gel electrophoresis, and the linearized vector fragments were recovered, dissolved in sterile ddH2O, and stored at -20℃ for later use.

[0035] The construction method of the modified pRI101 RNAi vector (pRI101-RNAi) is as follows: (1) Using pKANNBAL plasmid DNA as a template, PCR amplification was carried out using primers Intron-F (Sma1) / Intron-R; the 5′ ends of the primers were introduced with the SmaⅠ recognition sequence CCCGGG and the KpnⅠ recognition sequence GGTACC, respectively. The amplified target intron sequences are as follows: ccaattggtaaggaaataattattttcttttttccttttagtataaaaatagttaagtgatgttaattagtatgattataataatatagttgttataattgtgaaaaaataatttataaatatattgtttacataaacaacatagtaatgtaaaaaaatatgacaagtgatgtgtaagacgaagaagataaaagttgagag taagtatattatttttaatgaatttgatcgaacatgtaagatgatatactagcattaatatttgttttaatcataatagtaattctagctggtttgatgaattaaatatcaatgataaaatactatagtaaaaataagaataaataaattaaaataatatttttttatgattaatagtttattatataattaaatatcta taccattactaaatattttagtttaaaagttaataaatattttgttagaaattccaatctgcttgtaatttatcaataaacaaaatattaaataacaagctaaagtaacaaataatatcaaactaatagaaacagtaatctaatgtaacaaaacataatctaatgctaatataacaaagcgcaagatctatcaattttat atagtattatttttcaatcaacattcttattaatttctaaataatacttgtagttttattaacttctaaatggattgactattaattaaatgaattagtcgaacatgaataaacaaggtaacatgatagatcatgtcattgtgttatcattgatcttacatttggattgattacagttgggaagctgggttcgaaatcgat The PCR products were purified by agarose gel electrophoresis and gel recovery to obtain intron DNA fragments.

[0036] (2) The pRI101 backbone vector and the purified intron PCR fragment were double-digested with SmaⅠ and KpnⅠ restriction endonucleases, respectively. After the digestion system was incubated, the linearized pRI101 vector backbone fragment and the target intron fragment were recovered.

[0037] (3) Ligation, transformation and positive clone verification The linearized vector fragment and intron fragment were mixed at an appropriate molar ratio, and T4 DNA ligase was added for ligation. The ligation product was transformed into competent *E. coli* cells, and the transformed colonies were screened on kanamycin-containing solid medium. Single colonies were picked and cultured by shaking, and plasmids were extracted. Restriction enzyme digestion was performed for preliminary identification, followed by full-length DNA sequencing for verification. The sequencing results showed that the aforementioned intron sequence was directionally inserted between the SmaⅠ and KpnⅠ sites of the pRI101 vector multiple cloning site, with no base sequence mutations, successfully constructing the modified pRI101-RNAi vector.

[0038] (3) The linearized vector and the intron fragment were ligated with T4 DNA ligase. The ligation product was transformed into Escherichia coli, and positive colonies were screened with kanamycin. The plasmid was extracted and digested with enzymes and sequenced to confirm that the intron sequence was oriented between the multiple cloning sites SmaⅠ and KpnⅠ of the pRI101 vector, thus obtaining the modified pRI101-RNAi vector.

[0039] 2. Homologous recombination linkage The connection was made using the ClonExpress MultiS One Step Cloning Kit (Nanjing Nuoweizan).

[0040] Construction of overexpression vector: Take 50 ng of linearized pRI101 vector and... MdMYB73L The full-length PCR product of the coding region (including the homologous sequence at the end of the vector) was mixed at a molar ratio of 1:3, and 4 μL of 5× CE MultiS Buffer and 2 μL of Exnase MultiS were added. ddH2O was added to bring the total volume to 20 μL. The mixture was incubated at 37°C for 30 minutes and then cooled on ice.

[0041] RNAi vector construction: MdMYB73L The sense fragment (SEQ.ID.NO.3) was inserted into the modified pRI101-RNAi vector (XbaI / SalI site). The resulting recombinant vector was then double-digested with KpnI and SacI, and the antisense fragment (a 315 bp sequence complementary to SEQ.ID.NO.3) was inserted into the linearized vector fragment. The ligation system was the same as above.

[0042] 3. Transformation and identification of positive clones The ligation product was heat-shocked and transformed into *E. coli* DH5α chemocompetent cells, plated on LB agar plates containing 50 mg / L kanamycin, and incubated upside down at 37°C for 14 hours. Single colonies were picked for preliminary identification by colony PCR. Plasmids were extracted from positive clones by shaking and verified again by double digestion with BamHI and SalI. Finally, sequencing confirmed that the sequences were completely correct and free of base mutations. The correctly sequenced recombinant plasmids were named pRI101- MdMYB73L -OE (overexpression) and pRI101- MdMYB73L -RNAi (interference expression), plasmids are stored at -20℃ for later use.

[0043] Example 3: MdMYB73L Obtaining genetically modified apples (1) Preparation of the inoculation solution Transformation: The plasmid containing the target fragment was transformed into Agrobacterium strain EHA105 and incubated upside down at 28°C for 36-48 h. Positive clones were screened for later use.

[0044] Shaking: Add the positive strain to 10 mL LB liquid medium (containing 50 mg / L kanamycin) and incubate at 28℃ and 200 rpm for 16-24 h with shaking.

[0045] Large shaking: Take 1 mL to 5 mL of the bacterial culture from the small shaking and add it to 100 mL of new LB resistant medium. Incubate at 28 °C with shaking for 16-24 h.

[0046] Preparation of infection solution: Aliquot the bacterial suspension into two 50 mL centrifuge tubes, centrifuge at 5000 rpm for 5 min, and collect the bacterial cells; resuspend in M20 solution with AS&Bet, centrifuge at 5000 rpm for 5 min, collect the bacterial cells, resuspend once more, zero the M20, and measure the OD. 600 =0.5-1.0. The preparation of the inoculum is complete.

[0047] (2) Preparation of explants Place healthy 'GL-3' leaves that have grown for about 20 days in M20 liquid, with the underside of the leaves facing up, and cut open the main veins with a sterile scalpel; then place the underside of the leaves in the prepared infection solution and incubate at room temperature for 8 minutes, followed by 3 days of dark culture in co-culture medium.

[0048] (3) Screening of positive plants After co-culture, the leaves were washed 2-3 times with a solution of 500 mg / L cephalosporin (1L sterile water + 2 mL cephalosporin), each time for 5-10 minutes. They were then transferred to an extension selection medium and cultured in the dark for 2 days, followed by another transfer to the selection medium and cultured in the dark for 3-4 weeks. Afterward, they were exposed to light to grow until resistant shoots appeared. Once the resistant shoots had grown, they were transferred to a subculture medium containing antibiotics for further selection. Positive lines were detected using DNA and RNA assays, propagated extensively, and then transplanted to a greenhouse.

[0049] (4) Molecular identification of transgenic positive lines ( Figure 1 ) (1) DNA level detection: Young leaves of transplanted plants were taken and genomic DNA was extracted using the CTAB method. Electrophoresis was performed to screen positive plants.

[0050] (2) RNA expression analysis: Total RNA was extracted from leaves of PCR-positive plants and wild-type plants, reverse transcribed into cDNA, and detected by qRT-PCR. MdMYB73L The relative expression level. The results are as follows: Figure 1 As shown, compared to the wild type, the overexpression line (OE) MdMYB73L The expression level was significantly upregulated (P<0.01), while the expression level in the RNAi interference line was significantly downregulated (P<0.01), indicating successful acquisition. MdMYB73L Transgenic apple plants with overexpression and suppressed expression.

[0051] Example 4: Phenotypic and nitrogen use efficiency evaluation of transgenic apple plants under different nitrogen conditions 1. Experimental material processing and design Wild-type (WT), overexpression lines, and RNAi interference lines with uniform growth, approximately 2 months of age, and no significant differences in root length and plant height were selected. After root washing, the plants were transferred to a nitrogen-free nutrient solution for 7 days of nitrogen starvation pretreatment. After pretreatment, the plants were transferred to nutrient solutions containing different concentrations (0.5 mM KNO3 for low nitrogen treatment and 10 mM KNO3 for high nitrogen treatment) for further treatment. The nutrient solution was changed every 3 days, and the treatment lasted for 40 days.

[0052] 2. Plant phenotypic observation and biomass statistics After treatment, the phenotypes of plants in each treatment group were photographed and recorded (see...). Figure 2 Carefully remove the plant, wash the root surface with deionized water to remove any adhering substances, and blot dry with absorbent paper. Measure the following parameters: Plant height: The vertical height from the base of the roots to the tip of the growing point; Fresh weight: Whole plant fresh weight (accurate to 0.01 g); The results are as follows Figure 3As shown, under low nitrogen (0.5 mM) conditions, MdMYB73L The plant height and fresh weight of RNAi interference lines were significantly higher than those of wild type (P<0.05), while those of OE overexpression lines were significantly lower than those of wild type (P<0.05). There were no significant differences among genotypes under high nitrogen conditions.

[0053] 3. Measurement of physiological indicators related to nitrogen utilization Take a mixed sample of treated plant leaves and roots, flash-freeze and grind them into powder using liquid nitrogen, and use it for the detection of the following indicators: Nitrate nitrogen (NO3) ⁻ -N) content determination: Weigh 0.5 g of sample, add 10 mL of deionized water, extract in a boiling water bath for 60 minutes, cool, and centrifuge at 12,000 rpm for 10 minutes. Take the supernatant and determine the absorbance at 410 nm using the salicylic acid-concentrated sulfuric acid colorimetric method. Calculate the content using a standard curve prepared with KNO3.

[0054] Total nitrogen content determination: Weigh 0.2 g of dried sample (blanch at 105℃ for 30 minutes, dry at 65℃ to constant weight), and determine the total nitrogen content using the H2SO4-H2O2 digestion method and a Kjeldahl nitrogen analyzer (FOSS Kjeltec 8400).

[0055] Nitrate reductase (NR) activity assay: An in vitro method was used. 0.5 g of fresh leaves were chopped and placed in a reaction solution containing 0.1 mM KH₂PO₄ buffer (pH 7.5, containing 10 mM KNO₃ and 5 mM propanol). The solution was incubated under vacuum for 30 minutes at 25°C, followed by a dark reaction for 30 minutes. The NO₂ generated in the reaction solution was then measured. ⁻ Content, expressed as NO2 generated per unit time per unit fresh weight ⁻ The quantity represents NR activity.

[0056] Nitrite reductase (NiR) activity assay: Take 1.0 g of fresh leaves, add 4 mL of extraction buffer (containing 0.1 M Tris-HCl pH 7.4, 10 mM MgCl2, 5 mM DTT, 1 mM EDTA, 5% PVP), grind in an ice bath, centrifuge at 12,000 rpm for 15 minutes, and collect the supernatant. The reaction system contains 0.1 M phosphate buffer (pH 6.8), 0.1 mM NaNO2, 0.5 mM MV (methyl viologen), and 0.2 mM Na2S2O4. Add enzyme solution to start the reaction, react at 30℃ for 15 minutes, and then measure the remaining NO2. ⁻ Content, expressed as NO2 consumed per unit time ⁻ The quantity represents the activity of NiR.

[0057] The results are as follows Figure 4 As shown: Under low nitrogen (0.5 mM KNO3) conditions,MdMYB73L Nitrogen use-related physiological indicators of overexpressing plants were significantly lower than those of wild type, while nitrogen use-related physiological indicators of RNAi lines were significantly higher than those of wild type (P<0.05).

[0058] Based on the above phenotypic and physiological data, under low nitrogen conditions, interference MdMYB73L The expression significantly promotes nitrogen use-related physiological indicators in apple plants, thereby increasing plant biomass (plant height, fresh weight). This result indicates that... MdMYB73L It is a negative regulator of nitrogen use efficiency in apples. Silencing this gene using RNAi technology can effectively improve the adaptability of apple plants to low-nitrogen environments and their ability to efficiently utilize nitrogen nutrients.

[0059] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. An apple gene MdMYB73L Use in (1) or (2) below: (1) Improve the nitrogen use efficiency of apples under low nitrogen conditions; (2) To cultivate transgenic apple varieties that utilize nitrogen nutrients efficiently under low-nitrogen conditions; The low-nitrogen condition refers to a condition containing a low concentration of nitrate nitrogen. The apple gene MdMYB73L is a DNA molecule according to any one of I) to III) below: I) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; II) DNA molecules encoding the amino acid sequence shown in SEQ ID NO.2, excluding those in I); (III) DNA molecules that have 80% or more identity with the DNA fragment defined in (I) and whose encoded protein is functionally equivalent to the protein shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The method for improving the nitrogen utilization efficiency of apples under low nitrogen conditions by inhibiting the expression of the apple gene MdMYB73L to improve the nitrogen utilization efficiency of apples under low nitrogen conditions.

3. The application according to claim 1, characterized in that, The improvement in nitrogen use efficiency of apples under low nitrogen conditions is manifested in the following ways: the growth of apple plants under low nitrogen conditions and the improvement of physiological and biochemical indicators related to nitrogen absorption, translocation and assimilation capacity. The growth includes plant height and / or fresh weight; The physiological and biochemical indicators related to nitrogen absorption, transport, and assimilation capacity include nitrate content, total nitrogen content, nitrate reductase activity, and nitrite reductase activity.

4. For interfering with the apple gene of claim 1 MdMYB73L The recombinant vector for expression is used in the following (1) or (2): (1) Improve the nitrogen use efficiency of apples under low nitrogen conditions; (2) Develop transgenic apple varieties that utilize nitrogen nutrients efficiently under low nitrogen conditions.

5. The application according to claim 4, characterized in that, The recombinant vector contains the apple gene shown in SEQ ID NO.

3. MdMYB73L Antonyms.

6. Apple genes MdMYB73L The application of the encoded protein in improving nitrogen use efficiency in apples under low-nitrogen conditions is characterized by, The apple gene MdMYB73L The encoded protein is shown in either (A1) or (A2) below: (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2 of the sequence listing; (A2) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

7. A method for improving nitrogen use efficiency in apples, characterized in that, By reducing the target apple plants MdMYB73L Gene expression levels may decrease. MdMYB73L The activity of gene-encoded proteins can improve the nitrogen utilization efficiency of apples.

8. The method according to claim 7, characterized in that, The reduction of target apple plants MdMYB73L Gene expression levels are achieved through gene silencing, gene editing, RNA interference, promoter mutation, or transcriptional repression.

9. The method according to claim 7, characterized in that, The method is to improve the nitrogen use efficiency of apples under low nitrogen conditions, wherein the low nitrogen conditions are 0.5 mM KNO3.

10. A method for cultivating a transgenic apple variety with high nitrogen nutrient utilization under low-nitrogen conditions, characterized in that, Includes the following steps: (1) Construct an apple gene containing the one shown in SEQ ID NO.3 MdMYB73L Interference carriers for antonymous fragments; (2) The interference expression vector was transformed into Agrobacterium, and the transformation of apple explants was mediated by Agrobacterium. (3) Screening and identifying positive transgenic plants to obtain MdMYB73L Transgenic apple varieties with interference expression and high nitrogen nutrient utilization efficiency.

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