Peach MADS BOX transcription factor PpAGL24 gene and application thereof

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a peach tree MADS BOX transcription factor PpAGL24 gene and application thereof. Specifically, an MADS BOX transcription factor PpAGL24 gene is cloned in a peach tree root system, and the nucleotide sequence of the gene is shown as SEQ ID NO.1. Molecular biology experiments and genetic engineering means prove that the gene actively responds to low-nitrogen signals, peach lateral root generation and nitrogen absorption and utilization can be promoted, the nitrogen utilization efficiency of transgenic plants can be effectively improved, and the transgenic plants can be applied to the field of plant growth. Gene resources are provided for peach tree breeding with high nitrogen utilization efficiency, the current situation that nitrogen fertilizer is excessively used due to low nitrogen utilization efficiency in peach tree production can be solved, and the application prospect is very high.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering, and in particular relates to a peach MADS BOX transcription factor PpAGL24 gene and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Peach is one of the most popular fruits. In recent years, due to the one-sided pursuit of yield, producers often apply a large amount of nitrogen fertilizer during the cultivation process. Excessive use of nitrogen fertilizer will lead to reduced peach fruit quality and environmental pollution. In the growth and development of peach, the root system is an important nutritional organ. A well-developed root system can improve the efficiency of nitrogen absorption and utilization. Discovering the key genes for peach nitrogen efficiency is crucial to improving nitrogen utilization efficiency and reducing nitrogen use.

[0004] MADS BOX transcription factors are a class of proteins with MADS-box domains. Nowadays, people have found that MADS BOX family genes in different plants have functions such as regulating plant growth and development, coping with adverse stress, flower development, and regulating fruit ripening, but the function of MADS BOX genes in peaches has not yet been studied. Therefore, studying genes related to root development and nitrogen metabolism in peaches and exploring their related transcription factors is of great significance for reducing nitrogen input in peach orchards and improving nitrogen absorption and utilization efficiency in peach trees. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention aims to provide a peach MADS BOX transcription factor PpAGL24 gene and its application. Specifically, the present invention cloned a MADS BOX transcription factor PpAGL24 gene in peach trees, and proved through molecular biology experiments and genetic engineering methods that the gene actively responds to low nitrogen signals, can effectively promote the root development and nitrogen absorption and utilization of transgenic plants, and provides gene resources for peach tree high nitrogen utilization efficiency (NUE) breeding, which can effectively solve the current situation of low NUE in peach production. Based on the above research results, the present invention is completed.

[0006] In order to achieve the above technical objectives, the technical solution provided by the present invention is as follows:

[0007] In a first aspect of the present invention, a gene PpAGL24 is provided, wherein the gene PpAGL24 is selected from:

[0008] (a1) the nucleotide sequence shown in SEQ ID NO.1;

[0009] (a2) a degenerate sequence that differs from the sequence in (a1) due to redundancy of genetic codes but encodes the same amino acid sequence;

[0010] (a3) a nucleotide sequence that has ≥90% identity with the nucleotide sequence shown in (a1) or (a2) and encodes a nucleotide sequence having the same or similar functional protein;

[0011] (a4) A nucleotide sequence that is complementary to any one of (a1) to (a3).

[0012] The second aspect of the present invention provides a protein, which is encoded by the above-mentioned gene PpAGL24.

[0013] Specifically, the protein includes:

[0014] (b1) the amino acid sequence shown in SEQ ID NO.2;

[0015] (b2) a functionally equivalent protein obtained by (conservative) amino acid residue substitution and / or deletion and / or addition to the amino acid sequence shown in SEQ ID NO.2;

[0016] (b3) A protein having a sequence identity of ≥90% with SEQ ID NO.2 and having equivalent biological activity.

[0017] The third aspect of the present invention provides a recombinant expression vector, an engineered host cell or a transgenic plant containing the above gene.

[0018] The fourth aspect of the present invention provides the use of the gene PpAGL24, protein, recombinant expression vector containing the gene PpAGL24, engineered host cell in any one or more of the following:

[0019] (c1) Regulation of plant traits;

[0020] (c2) Screening of plant varieties with high nitrogen utilization efficiency;

[0021] (c3) Improvement and cultivation of plant varieties with high nitrogen utilization efficiency.

[0022] In the present invention, the plant may be a seed plant, further a dicotyledonous plant, and further a plant of the Rosaceae family, and peach is most preferred.

[0023] A fifth aspect of the present invention provides a method for improving and cultivating peach trees, the method comprising: increasing the expression level and / or activity of the PpAGL24 gene in the peach trees, thereby obtaining peach trees with more lateral roots and higher nitrogen utilization efficiency.

[0024] Beneficial technical effects of one or more of the above technical solutions:

[0025] The above technical scheme screened out the peach MADS BOX transcription factor PpAGL24 gene, which can respond to low nitrogen signals and regulate the occurrence of peach lateral roots and nitrogen absorption and utilization. Overexpression of the PpAGL24 gene in plants can regulate root development, improve peach nitrogen utilization efficiency, reduce nitrogen input in production, improve fruit quality, and protect the environment. Therefore, it has broad application prospects and extremely high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings required for use in the description of the embodiment will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0027] Figure 1 0.1 mM KNO in the embodiment of the present invention 3 Relative expression level of PpAGL24 in peach roots after treatment with 0.1 mM KCL for 30 min.

[0028] Figure 2 The results of GUS staining and qRT-PCR of relative expression of GUS in tobacco leaves transiently transformed after treatment with different nitrogen concentrations in the present invention are shown in Figure 1. A is a staining result of tobacco leaves, and B is an analysis of relative expression of GUS in tobacco leaves.

[0029] Figure 3 Figure 1 is the phenotype of the regenerated root system after PpAGL24 infects the peach root system and the detection of related physiological indicators in the embodiment of the present invention. A is a photo of the regenerated root system of transgenic peach. B is the relative expression of PpAGL24 in the transgenic root system. CE is the number of lateral roots, root dry weight and root nitrogen content of the transgenic root system.

[0030] Figure 4 is the relative expression level of related genes in the peach root system of PpAGL24 overexpression in the embodiment of the present invention

[0031] Figure 5 : phenotypes of Arabidopsis root system of different genotypes in the present invention. A is a photo of the root system of Arabidopsis thaliana of different genotypes after vertical cultivation. B is the number of lateral roots. C is the length of the main root.

[0032] Figure 6 It is the relative expression level of genes related to root development and nitrogen metabolism in the root systems of Arabidopsis thaliana of different genotypes in the examples of the present invention. DETAILED DESCRIPTION

[0033] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0034] It should be noted that the terms used herein are only to describe specific embodiments, and are not intended to limit the exemplary embodiments according to the application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should also be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates that there are features, steps, operations, devices, components and / or combinations thereof. The present invention uses conventional techniques and methods in the field of genetic engineering and molecular biology. Those skilled in the art can adopt other conventional techniques, methods and reagents in this area on the basis of the embodiments provided by the present invention, without being limited to the limitation of the specific embodiments of the present invention.

[0035] In the present invention, the term "sequence identity" or "homology" specifically refers to the degree of similarity between biological macromolecular sequences. The similarity can be evaluated by manual comparison or using bioinformatics tools. When a computer program is used for sequence comparison, the sequence to be tested and the reference sequence need to be input into the analysis system, and after setting the comparison parameters and subsequence coordinates, the program will automatically calculate and output the sequence similarity percentage based on a preset algorithm to quantitatively characterize the degree of homology between sequences.

[0036] To perform sequence comparison, generally, one sequence is compared to a test sequence as a reference sequence. When a sequence comparison algorithm is used, the test and reference sequences are input into a computer, the coordinates of the subsequences are specified if necessary, and the parameters of the sequence algorithm program are specified. Then, based on the selected program parameters, the sequence comparison algorithm will calculate the percent sequence identity (consistency) of the test sequence relative to the reference sequence.

[0037] In the scope of gene sequence, the nucleic acid molecules of the present invention include DNA and its derivative forms, including but not limited to complementary DNA (cDNA), chromosomal DNA, recombinant DNA, etc. In the field of plant genetic transformation technology, the nucleic acid molecules of the present invention can be used in conjunction with a variety of conventional plant transformation vectors. The selection of specific vectors needs to be combined with the characteristics of transformation methodology (such as Agrobacterium-mediated method, gene gun method, etc.) and target plant species for adaptive design.

[0038] In the present invention, conservative amino acids refer to amino acids that are highly conserved during evolution due to their functional or structural importance, and usually remain unchanged or undergo only substitutions of similar properties in similar proteins of different species, such as serine, histidine, aspartic acid, glutamic acid, arginine, lysine, etc., which are not specifically limited here.

[0039] As mentioned above, studying genes related to root development and nitrogen metabolism in peach and exploring its related transcription factor PpAGL24 are of great significance for reducing nitrogen input in peach orchards and improving nitrogen absorption and utilization efficiency of peach trees.

[0040] In view of this, in a typical embodiment of the present invention, a gene PpAGL24 is provided, wherein the gene PpAGL24 is selected from:

[0041] (a1) the nucleotide sequence shown in SEQ ID NO.1;

[0042] (a2) a degenerate sequence that differs from the sequence in (a1) due to redundancy of genetic codes but encodes the same amino acid sequence;

[0043] (a3) a nucleotide sequence that has ≥90% identity with the nucleotide sequence shown in (a1) or (a2) and encodes a nucleotide sequence having the same or similar functional protein;

[0044] (a4) A nucleotide sequence that is complementary to any one of (a1) to (a3).

[0045] In another specific embodiment of the present invention, a protein is provided, wherein the protein is encoded by the above gene PpAGL24. The protein is selected from:

[0046] (b1) the amino acid sequence shown in SEQ ID NO.2;

[0047] (b2) a functionally equivalent protein obtained by (conservative) amino acid residue substitution and / or deletion and / or addition to the amino acid sequence shown in SEQ ID NO.2;

[0048] (b3) A protein having a sequence identity of ≥90% with SEQ ID NO.2 and having equivalent biological activity.

[0049] In another specific embodiment of the present invention, a recombinant expression vector, an engineered host cell or a transgenic plant containing the above gene is provided.

[0050] In a specific embodiment of the present invention, the recombinant expression vector is obtained by connecting the above-mentioned gene to a vector through conventional molecular cloning technology. The vector can be a cloning vector or an expression vector. The vector can be a genetic element such as a viral vector, a plasmid vector, a phage vector or an artificial chromosome.

[0051] In another specific embodiment of the present invention, the engineered host cell refers to a host cell whose function has been changed by operating and modifying the genes of the target cell, such as an engineered host cell obtained by introducing an exogenous target gene or a recombinant expression vector into the target cell, or an engineered host cell obtained by directly editing the endogenous gene of the target cell.

[0052] The target cell may be a eukaryotic cell or a prokaryotic cell; further, the target cell may be a microbial cell or a plant cell.

[0053] Furthermore, the target cells may be bacteria, fungi, actinomycetes and the like, including bacteria such as Escherichia, Agrobacterium, Bacillus, fungi such as yeast, and actinomycetes such as Streptomyces, Nocardia, and Micromonospora.

[0054] In the present invention, the plant cell can be isolated, in vitro, cultured, or preferably a part of a plant; wherein the plant cell can be a seed plant cell, and the seed plant is specifically a dicotyledonous plant, further a Rosaceae plant, and further a peach.

[0055] In another specific embodiment of the present invention, the use of the gene PpAGL24, protein, recombinant expression vector containing the gene PpAGL24, and engineered host cell in any one or more of the following is provided:

[0056] (c1) Regulation of plant traits;

[0057] (c2) Screening of plant varieties with high nitrogen utilization efficiency;

[0058] (c3) Improvement and cultivation of plant varieties with high nitrogen utilization efficiency.

[0059] In the present invention, the plant may be a seed plant, further a dicotyledonous plant, and further a plant of the Rosaceae family, and peach is most preferred.

[0060] Wherein, the (c1) regulating plant traits is specifically manifested in regulating the root development and nitrogen absorption and utilization efficiency of plants.

[0061] In the above (c2), the screening of plant varieties with high nitrogen utilization efficiency is specifically carried out by analyzing the expression of the gene PpAGL24 to screen plant varieties (peach varieties) with high expression of PpAGL24, thereby obtaining peach varieties with higher nitrogen utilization efficiency.

[0062] In (c3), the improvement and cultivation of plant varieties with high nitrogen utilization efficiency are specifically manifested in the improvement and cultivation of peach trees with more lateral roots and higher nitrogen utilization efficiency, thereby obtaining peach varieties with higher peach fruit quality. Specifically, the present invention proves through experiments that compared with the control group, the hairy peaches overexpressing the gene PpAGL24 show an increase in the number of lateral roots, an increase in root biomass, a stronger root system than the control group plants, and an increase in root nitrogen content. And from the perspective of gene expression levels, compared with the control group roots, the expression levels of genes such as PpNRT2.1, PpNPF1.2, PpNiR1, PpWRKY75, and PpTAA1 in the PpAGL24 overexpression root system have changed significantly, indicating that the PpAGL24 gene regulates the ability of peach plants to absorb nitrogen metabolism by affecting the expression of genes related to nitrogen metabolism and root development in the peach root system, thereby regulating the occurrence of peach lateral roots.

[0063] In another specific embodiment of the present invention, a method for improving and cultivating peach trees is provided, the method comprising: increasing the expression level and / or activity of the PpAGL24 gene in the peach trees, thereby obtaining peach trees with more lateral roots and higher nitrogen utilization efficiency.

[0064] In the method, increasing the expression level and / or activity of the PpAGL24 gene in peach trees includes introducing a plasmid containing the PpAGL24 gene, connecting a strong promoter to the PpAGL24 gene, enhancing the promoter activity of the endogenous PpAGL24 gene, and introducing an enhancer, etc., which are not specifically limited here.

[0065] In the following examples, the materials, reagents, strains, plasmids, enzymes, kits, etc. used were obtained from commercial sources unless otherwise specified. Plant materials: The peach seeds used for germination were Peach oleraceus, the variety was Qingzhou peach, the wild Arabidopsis thaliana was Columbia type, and the tobacco was Nicotiana benthamiana.

[0066] Example

[0067] 1. Experimental Methods

[0068] (1) CTAB method to extract RNA and synthesize cDNA

[0069] First, preheat the CTAB extract in a 65°C water bath; take a small amount of leaves or roots and place them in a 2.0mL RNase-free EP tube, add steel balls, freeze them in liquid nitrogen, and then place them in a sample grinder for vibration grinding; add 500μL of preheated CTAB extract to the EP tube and shake to mix; then place the premixed solution in a 65°C metal bath and keep it warm for 20 minutes, turning it upside down 4-5 times during this period; add an equal volume of extract (chloroform: isoamyl alcohol = 24:1) to the EP tube, and shake to mix. Mix well; centrifuge at 12000rpm for 10min at 4℃, take the supernatant and put it in a new 1.5mL RNase-free EP tube, add an equal volume of extraction solution (chloroform: isoamyl alcohol = 24:1), extract again, transfer the supernatant to a new EP tube, add 2.5 times the volume of anhydrous ethanol to precipitate, mix well by inversion, put it in a -20℃ refrigerator for 30min, centrifuge at 12000rpm for 10min at 4℃, and discard the supernatant; add 500μL 75% anhydrous ethanol to the EP tube to wash the precipitate, invert it, centrifuge at 12000rpm for 10min at 4℃, and discard the supernatant; centrifuge briefly to remove the excess supernatant; put the EP tube in a clean bench to blow dry, and add 50μL DEPC water to dissolve the precipitate. Remove DNA with DNaseⅠ, and then reverse transcribe it into cDNA.

[0070] (2) Cloning of peach PpAGL24 gene fragment

[0071] The peach PpAGL24 gene sequence was obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov), multiple primers were designed based on the sequence, and high-fidelity enzyme 2×Phanta Flash Master Mix was used for gene cloning. The reaction system was as follows:

[0072]

[0073] The reaction conditions of PCR were as follows: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 58℃ annealing for 5s, 72℃ extension for 20s, 35 cycles, and finally 72℃ extension for 1min, paused at 4℃. After the reaction was completed, agarose gel electrophoresis was performed to detect the size and specificity of the PCR product bands. The bands corresponding to the correct fragment length were recovered by gel electrophoresis.

[0074] (3) Vector construction

[0075] The recovered PpAGL24 fragment was connected to the 35s-driven pCAMBIA1300 vector, and then transformed into DH5α E. coli competent cells, and a single spot on the screening medium was selected for sequencing. The sequencing results were confirmed to be correct using SnapGene software.

[0076] (4) Plasmid extraction and Agrobacterium transformation

[0077] The correct monoclone in the sequencing result was inoculated into 5 mL of LB liquid culture medium containing Kan, placed in a shaker at 37°C and 200 rpm for overnight culture, and centrifuged at 10,000 rpm for 5 min at room temperature to collect the bacteria; after removing the culture medium, the plasmid was extracted using a plasmid extraction kit (Nanjing Novozymes Company), and the concentration of the plasmid was detected using a NanoDrob 2000 microspectrophotometer; 2 μL of the extracted plasmid was added to 8 μL of the freshly melted GV3101 and K599 Agrobacterium competent cells, left to stand on ice for 5 min, frozen in liquid nitrogen for 5 min, heat shocked at 37°C for 5 min, and then placed on ice for another 5 min, and finally 700 μL of YEP liquid culture medium was added, placed in a shaker at 28°C and 200 rpm, shaken for 2-3 h, and finally applied to YEP solid culture medium containing rifampicin and Kan, and cultured in a 28°C incubator for 2 d.

[0078] (5) Gene expression detection

[0079] The cDNA obtained by reverse transcription was used as a template to detect the relative expression of PpAGL24 and other genes using a 2×UltraSYBR Mixture fluorescent quantitative PCR kit (Kangwei Century Company). The reaction system and reaction procedure are as follows:

[0080]

[0081] The above reaction was carried out in an ABI7500 PCR amplifier. The reaction conditions were 95°C pre-denaturation for 10 min, 95°C denaturation for 15 s, 60°C annealing for 30 s, 40 cycles, and finally a melting curve analysis was performed: 95°C for 15 s, 60°C for 1 min, 95°C for 15 s, and 60°C for 15 s.

[0082] (6) GUS staining of tobacco leaves

[0083] The PpAGL24 promoter sequence was recombined to obtain the proPpAGL24::GUS plasmid, and the correctly sequenced vector was transformed into Agrobacterium GV3101. The Agrobacterium containing proPpAGL24::GUS and the Agrobacterium containing the empty vector were injected into 4-week-old tobacco leaves, respectively. After 24 hours of darkness after injection and normal cultivation under 16h / 8h light / darkness for 28 hours, 1 cm leaf discs were taken with a cork puncher, stained at 28℃ for 24 hours, and decolorized with 70% ethanol until the green color of the control group disappeared completely.

[0084] (7) Phenotypic identification of root morphology

[0085] Root morphology was examined on 0.7% (w / v) agar-solidified MS medium. Seeds were cultured horizontally on MS medium and, after germination for 3 days, seedlings with similar growth conditions were selected and transferred to 13×13 cm 2 MS medium was used for vertical growth. From the day of transplanting, the number of visible lateral roots and the length of the main root were counted and photographed 6 days later, and the root length of the plants was manually measured using digital images of ImageJ software (NIH).

[0086] The peach root phenotype was analyzed 25 days after K599 infection (PpAGL24-GFP overexpression line), and the transformation status of each transgenic line was detected by qRT-PCR. Then, the root system was scanned and data analyzed using a root scanner.

[0087] (8) Total nitrogen content detection

[0088] The harvested plant materials were sterilized at 115°C for 15 minutes, dried at 70°C to constant weight, fully ground in a grinder, sieved, and weighed 3 portions of 0.2g each for digestion. The digested samples were fixed to 50mL with distilled water. The nitrogen content was determined using a fully automatic Kjeldahl nitrogen analyzer.

[0089] 2. Experimental results

[0090] First, we analyzed the nitrogen treatment (0.1 mM KNO) using qRT-PCR. 3 )30min later, the relative expression level of PpAGL24 in peach roots was found to increase after low nitrogen treatment ( Figure 1 ), and at the same time, the proPpAGL24::GUS reporter line was constructed and injected into 4-week-old Nicotiana benthamiana plants under the mediation of Agrobacterium tumefaciens (GV3101). The injected tobacco plants were then treated with different concentrations of nitrogen (0mM, 0.5mM, 5mM, 15mM) by root irrigation and foliar spraying. The results of qRT-PCR and GUS staining showed that ( Figure 2 ), PpAGL24 is able to respond to low nitrogen signals.

[0091] The PpAGL24 gene was transferred into the plant by genetic transformation technology to construct peach and Arabidopsis plants with stable and transient overexpression of the PpAGL24 gene.

[0092] PpAGL24 was overexpressed in the peach root system through the root transformation system mediated by Agrobacterium rhizogenes (K599). The results showed that the peach root system overexpressing PpAGL24 showed an increase in the number of lateral roots and root biomass, and had a stronger root system than the control plant, and the nitrogen content in the root system increased ( Figure 3). qRT-PCR was used to detect the expression levels of genes such as PpNRT2.1, PpNPF1.2, PpNiR1, PpWRKY75, and PpTAA1 in the peach roots overexpressing PpAGL24. The results showed that compared with the control roots, the expression levels of these genes in the PpAGL24 overexpressing roots changed significantly ( Figure 4 ). This indicates that the PpAGL24 gene regulates the ability of peach plants to absorb nitrogen and metabolize nitrogen by affecting the nitrogen metabolism of peach roots and the expression of genes related to root development, thereby regulating the development of peach lateral roots.

[0093] The recombinant plasmid containing the PpAGL24 gene was transformed into wild-type Arabidopsis by the floral dipping method to obtain PpAGL24 transgenic Arabidopsis. PpAGL24-overexpressing Arabidopsis (OE1, OE2), AGL24 mutant Arabidopsis (AGL24-1, AGL24-2) and Col-0 plants were grown vertically on Murashige and Skoog (MS) medium to observe the root phenotype. The results showed that heterologous overexpression of PpAGL24 could promote the development of Arabidopsis root system and increase the number of lateral roots ( Figure 5 ). qRT-PCR results showed that the expression of AtLBD29, AtNR1 and AtNRT2.1, genes related to root development and nitrogen absorption and utilization, were significantly induced in the roots of the transgenic lines ( Figure 6 ). These results indicate that overexpression of PpAGL24 in Arabidopsis can regulate the expression of AtLBD29, AtNR1, and AtNRT2.1, increase the number of lateral roots, and improve the ability to absorb and utilize nitrogen.

[0094] In summary, the peach PpAGL24 gene actively responds to low nitrogen signals, can promote the occurrence of peach lateral roots and nitrogen absorption and utilization, can effectively improve the nitrogen utilization efficiency of transgenic plants, and provides genetic resources for the breeding of peach trees with high nitrogen utilization efficiency. It can solve the current situation of excessive use of nitrogen fertilizer in peach production due to low nitrogen utilization efficiency, and has high application prospects.

[0095] Nucleotide and amino acid sequence information used in the present invention

[0096] PpAGL24 gene

[0097] atgacgaggaggaaaatccagatcaagaagattgacaacacaacggcgaggcaggtgacgttttcgaagaggaggagagggctttt

[0098] caagaaagcccaggagctctctactctctgtgatgctgagattgctcttgtagtcttctcagctactgggaagctctttgaatacaccagct

[0099] ccagcgtgcaacaagtaattgaaaggcatggcttgctttcttccaattatgaccagttgaatcaaccatctcttgagctgcagtcctttggt

[0100] atgtctcagcttgagagcagtacttccgccgcattgagcaaggaaattgcggagagtacacatgagctaaggaagctaatgggagaa

[0101] gagctccaagaactaaacatgaaagagttgcaggaactagagaaactgctcggatcaggattgaggcgtgttagagatgcaaagggt

[0102] gaattttttctgaaggagatcacctctcttaagtggaagggatcccaaatgatgcaagaaaacaagcgattgaagcagatggcaaaccg

[0103] acaggtccaaacacttgaacttgaacaaggccaatcctccgagccaataggcgatttcatccattcatatccttctcaagaccacgacagctctgacacttctctcaagttggggcaagcttttcctaacgggatatga(SEQ ID NO.1)

[0104] PpAGL24 protein

[0105] MTRRKIQIKKIDNTTARQVTFSKRRRGLFKKAQELSTLCDAEIALVVFSATGKLFEYTS

[0106] SSVQQVIERHGLLSSNYDQLNQPSLELQSFGMSQLESSTSAALSKEIAESTHELRKLM

[0107] GEELQELNMKELQELEKLLGSGLRRVRDAKGEFFLKEITSLKWKGSQMMQENKRLKQMANRQVQTLELEQGQSSEPIGDFIHSYPSQDHDSSDTSLKLGQAFPNGI(SEQ ID NO.2)

[0108] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A gene PpAGL24, characterized in that: The PpAGL24 gene is selected from: (a1) the nucleotide sequence shown in SEQ ID NO.1; (a2) a degenerate sequence that differs from the sequence in (a1) due to redundancy of genetic codes but encodes the same amino acid sequence; (a3) a nucleotide sequence that has ≥90% identity with the nucleotide sequence shown in (a1) or (a2) and encodes a nucleotide sequence having the same or similar functional protein; (a4) A nucleotide sequence that is complementary to any one of (a1) to (a3).

2. A protein, characterized in that The protein is encoded by the above gene PpAGL24.

3. The protein according to claim 2, characterized in that The protein is selected from: (b1) the amino acid sequence shown in SEQ ID NO.2; (b2) a functionally equivalent protein obtained by (conservative) amino acid residue substitution and / or deletion and / or addition to the amino acid sequence shown in SEQ ID NO.2; (b3) A protein having a sequence identity of ≥90% with SEQ ID NO.2 and having equivalent biological activity.

4. A recombinant expression vector, an engineered host cell or a transgenic plant containing the gene PpAGL24 according to claim 1.

5. The recombinant expression vector, engineered host cell or transgenic plant according to claim 4, characterized in that: The recombinant expression vector is obtained by connecting the gene PpAGL24 to a vector, and the vector is a cloning vector or an expression vector. Further, the vector is any one or more of a viral vector, a plasmid, a phagemid or an artificial chromosome.

6. Use of the gene PpAGL24 according to claim 1, the protein according to any one of claims 2-3, the recombinant expression vector containing the gene PpAGL24 according to any one of claims 4-6, and the engineered host cell in any one or more of the following: (c1) Regulation of plant traits; (c2) Screening of plant varieties with high nitrogen utilization efficiency; (c3) Improvement and cultivation of plant varieties with high nitrogen utilization efficiency.

7. The use according to claim 6, characterized in that The plant is a seed plant, further a dicotyledonous plant, further a plant of the Rosaceae family, and most preferably a peach.

8. The use according to claim 6, characterized in that The (c1) regulating plant traits is specifically manifested in regulating the root development and nitrogen absorption and utilization efficiency of plants; In the above (c2), the screening of plant varieties with high nitrogen utilization efficiency is specifically carried out by analyzing the expression of the gene PpAGL24 and screening plant varieties (peach varieties) with high expression of the gene PpAGL24, so as to obtain peach varieties with higher nitrogen utilization efficiency. In the above (c3), the improvement and cultivation of plant varieties with high nitrogen utilization efficiency is specifically manifested in the improvement and cultivation of peach trees with more lateral roots and higher nitrogen utilization efficiency, thereby obtaining peach tree varieties with higher peach fruit quality.

9. A method for improving and cultivating peach trees, characterized in that: The method comprises: increasing the expression amount and / or activity of the gene PpAGL24 in peach trees, thereby obtaining peach trees with more lateral roots and higher nitrogen utilization efficiency.

10. The method according to claim 9, characterized in that The method for increasing the expression level and / or activity of the PpAGL24 gene in peach trees comprises introducing a plasmid containing the PpAGL24 gene, connecting a strong promoter to the PpAGL24 gene, enhancing the promoter activity of the endogenous PpAGL24 gene and introducing an enhancer.

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