Application of the gene EgNPF8.1 encoding the dipeptide transporter protein of Eucalyptol cylindrica.

Abstract

This invention discloses the application of the EgNPF8.1 gene, which encodes a dipeptide transporter protein in Eucalyptus macrocarpa, belonging to the fields of plant genetic engineering and biotechnology. The EgNPF8.1 gene provided by this invention participates in the infection and development of arbuscular mycorrhizal fungi (AM) symbionts. Transcriptional analysis shows that EgNPF8.1 is significantly induced by infection with the AM fungus *Rhizopus heterophyllus*. Functional studies of the EgNPF8.1 gene in Eucalyptus macrocarpa indicate that it encodes a dipeptide transporter and plays an important role in the nitrogen and phosphorus uptake pathway of Eucalyptus macrocarpa arbuscular mycorrhizal fungi. Silencing this gene has a significant impact on the nitrogen and phosphorus uptake pathway of Eucalyptus macrocarpa arbuscular mycorrhizal fungi and can be used for the genetic improvement of mycorrhizal forests. Furthermore, by adding nutrient solutions with different nitrogen and phosphorus concentrations, the infection rate, infection intensity, and arbuscular abundance of AM fungi were observed. Low phosphorus and nitrogen conditions significantly promoted the formation and development of Eucalyptus macrocarpa arbuscular mycorrhizae.

Description

Technical Field

[0001] This invention belongs to the fields of plant genetic engineering and biotechnology, and relates to an arbuscular mycorrhizal induced Eucalyptus dipeptide transporter gene EgNPF8.1 and its application in improving the infection rate of Eucalyptus arbuscular mycorrhizal fungi and the efficient absorption of nitrogen and phosphorus. Background Technology

[0002] Nitrogen (N) and phosphorus (P) are two essential macronutrients for plant growth and development, and their available content in soil directly affects normal plant growth and productivity. However, terrestrial plants often face stress from various nutrient deficiencies simultaneously in agroforestry soils, such as low nitrogen and low phosphorus. In recent years, the impact of ecological factors such as nitrogen and phosphorus deficiencies on forest and tree production has become a major concern, threatening timber yield and quality. To ensure a sustainable timber supply to meet growing market demand, forestry worldwide relies heavily on the use of nitrogen and phosphorus fertilizers to improve timber yield and quality. However, long-term use of chemical fertilizers has serious negative impacts on the agroforestry ecosystem. Therefore, sustainable forestry development requires trees to have a more efficient capacity for absorbing and utilizing nitrogen and phosphorus from the soil to reduce fertilizer and cost inputs in forestry production.

[0003] Mycorrhizal symbionts first appeared approximately 400 million years ago and have undergone a long evolutionary process, continuing to exist widely and conservatively in various ecosystems in nature to this day (Genre et al., 2020). Scientists have been studying mycorrhizae for 150 years (Bonfante, 2018), and research on the nutrient absorption pathways formed by arbuscular mycorrhizal (AM) fungi and host plants is flourishing (Harrison et al., 2002; Breuillin-Sessoms et al., 2015; Wang et al., 2020; Shi et al., 2021; Xie et al., 2022). However, research on the molecular mechanisms of interactions among multiple nutrients in arbuscular mycorrhizae is rarely reported (Bonneau et al., 2013; Xie et al., 2019), which greatly limits the widespread application of mycorrhizal biology techniques in agricultural and forestry production practices. Eucalyptus trees are widely planted globally due to their significant ecological and economic value in areas such as ecological protection, carbon sequestration, timber production, bioenergy, and papermaking. However, low productivity remains the most critical issue in eucalyptus plantation production. Fortunately, the rhizosphere soil of eucalyptus trees is rich in mycorrhizal fungi, which can form a mutually beneficial symbiotic relationship with eucalyptus roots. These fungi promote the absorption and translocation of mineral nutrients such as phosphorus and nitrogen from the soil, regulate the nitrogen-phosphorus nutrient balance within the plant, improve eucalyptus growth and health, and thus enhance its productivity. Therefore, researching a method to improve the arbuscular mycorrhizal infection rate and efficient nitrogen and phosphorus absorption of Eucalyptus grandis has become an urgent problem to be solved. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a dipeptide transport protein encoding gene of Eucalyptus macrocarpa, as well as the protein encoded by the gene and the related applications of the gene in nitrogen and phosphorus absorption.

[0005] The second objective of this invention is to provide a nutrient solution formula that promotes mycorrhizalization, efficient absorption of nitrogen and phosphorus nutrients, and rapid growth in Eucalyptus seedlings.

[0006] The third objective of this invention is to provide a method for promoting arbuscular mycorrhizal fungal infection and efficient nitrogen and phosphorus absorption in Eucalyptus macrocarpa, in order to solve the key scientific problems raised in the background art.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] Applications of the EgNPF8.1 gene encoding the eucalyptol dipeptide transporter include at least one of the following applications:

[0009] A) Application in promoting the colonization of Eucalyptus macrocarpa roots by arbuscular mycorrhizal fungi;

[0010] B) Application in improving the efficient absorption of nitrogen and phosphorus from the external environment by mycorrhizal Eucalyptus plants;

[0011] C) Application in determining the utilization efficiency of organic nitrogen in mycorrhizal Eucalyptus plants.

[0012] Preferably, the organic nitrogen comprises at least one of small molecule peptides, amino acids, and peptones; the small molecule peptides comprise small molecule peptides containing 2 to 10 amino acid residues; specifically, the small molecule peptides are dipeptides; the dipeptides include, but are not limited to, at least one of L-leucine dipeptide (Leu-Leu), L-histidine-L-leucine dipeptide (His-Leu), and glutamic acid-glutamic acid dipeptide.

[0013] Furthermore, it includes at least one of the following applications:

[0014] A1) Application in promoting the infection rate of arbuscular mycorrhizal fungi in the roots of Eucalyptus macrocarpa; specifically, the infection rate in the roots of control plants was significantly increased compared with gene-silenced plants; more specifically, under phosphorus-deficient (-P+N) or nitrogen-deficient (+PN) conditions, the infection rate in the roots of control plants was significantly increased compared with gene-silenced plants.

[0015] A2) Application in promoting the abundance of arbuscular mycorrhizal fungi in the roots of Eucalyptus grandis; specifically, compared with gene-silenced plants, the abundance of arbuscular fungi in the roots of control plants was significantly increased; more specifically, under phosphorus-deficient (-P+N) or phosphorus- and nitrogen-exposed (+P+N) conditions, the abundance of arbuscular fungi in the roots of control plants was significantly increased compared with gene-silenced plants.

[0016] Preferably, the arbuscular mycorrhizal fungi include *Rhizophagus irregularis*, and more particularly, *R. irregularis* DAOM 197198.

[0017] B1) Application in improving the efficient absorption of nitrogen from the external environment by mycorrhizal Eucalyptus plants; preferably, the nitrogen includes at least one of nitrate nitrogen, amino acids, small molecule peptides, peptone, etc. The small molecule peptides include small molecule peptides containing 2 to 10 amino acid residues; specifically, the small molecule peptides are dipeptides; the dipeptides include, but are not limited to, at least one of L-leucine dipeptide (Leu-Leu), L-histidine-L-leucine dipeptide (His-Leu), and glutamic acid-glutamic acid dipeptide.

[0018] Specifically, the total nitrogen content in the aboveground and underground tissues of the control plants was significantly higher than that of the gene-silenced plants under the same nutritional conditions.

[0019] B2) Application in improving the efficient absorption of phosphorus from the external environment in mycorrhizal Eucalyptus plants; specifically, under nitrogen deficiency (+PN) or phosphorus and nitrogen application (+P+N) conditions, the total phosphorus content in the aboveground tissues of control plants was significantly higher than that of gene-silenced plants under the same nutritional conditions; under phosphorus and nitrogen application (+P+N) conditions, the total phosphorus content in the underground tissues of control plants was significantly higher than that of gene-silenced plants under the same nutritional conditions.

[0020] Preferably, the gene-silencing plant is obtained by using virus-induced gene silencing (VIGS) technology, which involves designing an RNAi silencing region at the 5' end of the EgNPF8.1 gene to target a specific region of the target gene, thus obtaining an EgNPF8.1-RNAi silencing plant.

[0021] Preferably, the RNAi silencing region is an RNAi silencing region relative to the translation start codon (ATG) from -60 to +110.

[0022] Preferably, the nucleotide sequence of the gene encoding the eucalyptol dipeptide transporter EgNPF8.1 is shown in SEQ ID NO.1.

[0023] The amino acid sequence of the eucalyptol dipeptide transporter protein EgNPF8.1 is shown in SEQ ID NO.2.

[0024] The present invention also provides a method for improving the nitrogen and phosphorus absorption of Eucalyptus macrocarpa by arbuscular mycorrhizal fungi, comprising the following steps:

[0025] Inoculate the roots of Eucalyptus grandis seedlings with Heteromorpha rhizocarpium, and then apply modified Long-Ashton nutrient solution. This will improve the colonization of Eucalyptus grandis roots by arbuscular mycorrhizal fungi and promote the synergistic and efficient absorption of nitrogen and phosphorus by Eucalyptus grandis.

[0026] Preferably, the inoculation time with Rhizoctonia heterophylla is 60 days or more; more preferably 60 to 150 days.

[0027] Preferably, the concentration of phosphorus (Pi) in the modified Long-Ashton nutrient solution is 30 μM to 300 μM; nitrogen (N-NO3) concentration is 30 μM to 300 μM. - The concentration of ) is 250μM~2500μM.

[0028] Furthermore, the modified Long-Ashton nutrient solution is formulated as follows: MgSO4·7H2O: 0.75mM, K2SO4: 1mM, CaCl2·2H2O: 2mM, NaH2PO4: 30μM~300μM, NaNO3: 250μM~2500μM, FeNa·EDTA: 0.025mM, MnSO4·H2O: 0.005mM, CuSO4·5H2O: 0.0005mM, ZnSO4·7H2O: 0.00025mM, H3BO3: 0.025mM, NaMoO4·2H2O: 0.0001mM, and 1g / L peptone or 2.5mM small molecule peptides.

[0029] The small molecule peptide includes small molecule peptides containing 2 to 10 amino acid residues; specifically, the small molecule peptide is a dipeptide; the dipeptide includes, but is not limited to, at least one of L-leucine dipeptide (Leu-Leu), L-histidine-L-leucine dipeptide (His-Leu), and glutamic acid-glutamic acid dipeptide.

[0030] First, peptone is prepared into a peptone solution with a concentration of 10 g / L, and then added to the modified Long-Ashton nutrient solution for further preparation. The peptone solution needs to be prepared separately and used immediately.

[0031] This invention uses *Eucalyptus grandis*, a native tree species of South China, as the host plant. After inoculating with the arbuscular mycorrhizal fungus *Rhizophora irregularis* DAOM 197198 and treating the plants under different nitrogen and phosphorus levels for 60 days, plant morphological indicators were recorded and physiological indicators of *Eucalyptus grandis* were measured. The optimal nitrogen-phosphorus ratio for *Eucalyptus grandis* growth was explored through analysis of plant height, root length, and biomass. Simultaneously, the total nitrogen and total phosphorus contents in the aboveground and underground tissues of *Eucalyptus grandis* were measured, and the infection rate of the roots inoculated with *Rhizophora irregularis* was determined.

[0032] The present invention has the following advantages and effects compared with the prior art:

[0033] (1) The EgNPF8.1 gene provided by this invention is involved in the infection and development of arbuscular mycorrhizal fungi and mycorrhizal symbionts. Gene transcription level analysis shows that EgNPF8.1 is significantly induced to express by infection with the AM fungus Heterocystis heterophylla. Figure 3 ).

[0034] (2) This invention, through functional studies of the dipeptide transporter protein encoding gene EgNPF8.1 of Eucalyptus macrocarpa, shows that this gene encodes a dipeptide transporter ( Figure 4This gene plays an important role in the nitrogen and phosphorus uptake pathway of Eucalyptus arbuscular mycorrhizal fungi, and silencing this gene has a significant impact on the nitrogen and phosphorus uptake pathway of Eucalyptus arbuscular mycorrhizal fungi. Figure 5 It can be used for the genetic improvement of mycorrhizal trees.

[0035] (3) Based on the function of the EgNPF8.1 gene in this invention, combined with the physiological and molecular mechanism of nitrogen-phosphorus synergistic effect in Eucalyptus arbuscular mycorrhizal, organic nitrogen (peptone, AOBOX) and inoculation with R. irregularis were applied in Eucalyptus pot experiment to confirm the effect of organic nitrogen addition on nitrogen and phosphorus nutrient absorption and growth of mycorrhizal Eucalyptus. At the same time, an effective and reliable nutrient solution formula for rapid seedling cultivation of Eucalyptus is provided (Table 2).

[0036] (4) This invention observed the infection rate, infection intensity, and arbuscular abundance of the AM fungus *Rhizoctonia irregularis* by adding nutrient solutions with different nitrogen and phosphorus concentrations. Under low phosphorus and nitrogen conditions, the formation and development of arbuscular mycorrhizae in *Eucalyptus macrocarpa* were significantly promoted. Using genomics and real-time quantitative PCR, a dipeptide transport protein encoding gene, EgNPF8.1, was identified in *Eucalyptus macrocarpa* that is induced to express by arbuscular mycorrhizae. The expression of this gene significantly promoted the absorption of organic nitrogen (such as leucine dipeptide) by *Eucalyptus macrocarpa* under *Rhizoctonia irregularis* infection conditions and maintained the normal development of *Eucalyptus macrocarpa* arbuscular mycorrhizae. Based on the research results, further improvement of the mycorrhizal plant nutrient solution formula and the addition of organic nitrogen (peptone) can more significantly promote the absorption of nitrogen and phosphorus by *Eucalyptus macrocarpa*, thereby accelerating the growth of *Eucalyptus macrocarpa* seedlings and increasing *Eucalyptus macrocarpa* biomass. Through creative exploration and experimentation, this invention modulates the nutrient ratio in the nutrient solution, improves the infection rate of Eucalyptus globulus mycorrhizal fungi and the efficient absorption of nitrogen and phosphorus, and provides a new biological technology for Eucalyptus globulus mycorrhizal seedling cultivation. Attached Figure Description

[0037] Figure 1 This diagram illustrates the effect of different nitrogen and phosphorus levels on the infection of Eucalyptus macrocarpa roots by the AM fungus *Rhizophora heterophylla*. A: Mycorrhizal infection level after WGA488 (wheat germ lectin 488) staining; F%: Total infection rate of AM fungi; M%: Mycorrhizal infection intensity of roots; A%: Root arbuscular abundance. According to Duncan's multiple comparison analysis, P < 0.05. B: Fluorescence microscopic images of Eucalyptus macrocarpa mycorrhizae grown under different nitrogen and phosphorus levels. Scale bar: 50 μm.

[0038] Figure 2 This is a graph showing the total nitrogen (A, B) and total phosphorus (C, D) content in Eucalyptus macrocarpa under different nitrogen and phosphorus levels according to the present invention; where -P: 30 μM Pi, +P: 300 μM Pi, -N: 250 μM N-NO3 - +N: 2500μMN-NO3- According to Duncan's multiple comparison test, the values ​​are mean ± SE (n=3), P<0.05; AM: arbuscular mycorrhizal fungi inoculation, NM: no inoculation.

[0039] Figure 3 This diagram shows the transcriptional levels of EgNPF8.1 induced by inoculation with *Rhizomycorrhizal fungi* and different nitrogen and phosphorus treatments in the aboveground (A) and underground (B) parts of *Eucalyptus macrocarpa*. The *Eucalyptus macrocarpa* ubiquitin 3 (EgUBI3) gene was used as a normalized internal reference gene. According to Duncan's multiple comparison test, the values ​​are mean ± SE (n=3), P<0.05; gray: inoculated with arbuscular mycorrhizal fungi, white: uninoculated.

[0040] Figure 4 This diagram illustrates the functional analysis of the EgNPF8.1 gene encoding the eucalyptol dipeptide transporter in *Saccharomyces cerevisiae*. A: Growth of overexpression sites of expression vectors pFL61-EgNPF8.1 and empty vector EV (i.e., empty vector pFL61) in wild-type yeast strain BY4741 on ammonium sulfate-free yeast uracil synthesis-deficient media containing different NaNO3 concentrations; B: Growth of overexpression sites of expression vectors pFL61-EgNPF8.1 and EV (i.e., empty vector pFL61) in wild-type yeast strain BY4741 on ammonium sulfate-free yeast uracil synthesis-deficient media containing different L-leucine dipeptide concentrations; C: Growth of overexpression sites of expression vectors pFL61-EgNPF8.1 and empty vector pFL61. D: Functional complementarity of FL61 in wild-type yeast strain BY4741 and dipeptide uptake-deficient yeast strain Δptr2; E: Functional complementarity of expression vector pYNR-EgNPF8.1 and empty vector pYNR-EX in wild-type Hansenula polymorpha strain CBS4732 and high-affinity nitrate transporter-deficient yeast mutant strain Δynt; E: L-leucine dipeptide content in wild-type yeast strain BY4741 and dipeptide uptake-deficient yeast strain Δptr2 ​​grown with expression vector pFL61-EgNPF8.1 and empty vector EV (i.e., empty vector pFL61) at a concentration of 0.25 mL L-leucine dipeptide; According to Duncan's multiple comparison test, the values ​​are mean ± SE (n = 3), P < 0.05.

[0041] Figure 5This diagram illustrates the effect of EgNPF8.1-RNAi silencing on AM fungal infection of Eucalyptus roots under different nitrogen and phosphorus levels. In the diagram, AC represents the mycorrhizal fungal infection level determined after WGA488 staining; F% represents AM fungal infection rate; M% represents mycorrhizal infection intensity; and A% represents root arbuscular abundance. D represents fluorescence microscopy images of the arbuscular structure of *Rhizopus heterophyllus* in control and EgNPF8.1-RNAi-silenced plants grown under different nitrogen and phosphorus levels; ma represents mature arbuscular structures; ca represents collapsed arbuscular structures; scale bar: 50 μm. EH represents the relative expression levels of genes related to arbuscular development in control and EgNPF8.1-RNAi-silenced plants. All values ​​are expressed as mean ± SE (n = 3), P < 0.05, based on Duncan's multiple comparison analysis.

[0042] Figure 6 The graph shows the total nitrogen (A, B) and total phosphorus (C, D) contents in the control and EgNFP8.1-RNAi-silenced plants of mycorrhizal Eucalyptus under different nitrogen and phosphorus concentrations according to the Duncan multiple comparison test. The values ​​are mean ± SE (n = 3), P < 0.05.

[0043] Figure 7 This diagram illustrates the growth phenotypes of *Eucalyptus macrocarpa* control and EgNPF8.1 gene-silenced plants treated with leucine dipeptide, as well as the silencing efficiency and leucine dipeptide content. A: Growth phenotypes of *Eucalyptus macrocarpa* control and silent plants treated with leucine dipeptide and AM fungi (scale bar: 10cm); BC: EgNPF8.1 expression levels in *Eucalyptus macrocarpa* control and silent plants treated with leucine dipeptide and AM fungi; D: Leucine dipeptide content determination in *Eucalyptus macrocarpa* control and silent plants treated with leucine dipeptide and AM fungi; AM: Arbuscular mycorrhizal fungi inoculation; NM: No inoculation; Control: Control plants; VIGS-EgNPF8.1-RNAi: EgNPF8.1 gene-silenced plants; -Leu-Leu: 0mM leucine dipeptide; +Leu-Leu: 2.5mM leucine dipeptide. Values ​​are expressed as mean ± SE (n=3), P<0.05, according to Duncan's multiple comparison test.

[0044] Figure 8This figure shows the effect of EgNPF8.1 silencing under leucine dipeptide treatment on arbuscular mycorrhizal formation and functional gene expression in *Eucalyptus macrocarpa*. In the figure, AC: mycorrhizal fungal infection level determined after WGA488 staining; F%: AM fungal infection rate; M%: mycorrhizal infection intensity; A%: root arbuscular abundance; D: fluorescence microscopy images of *Rhizophora heterophylla* arbuscular structure in control and gene-silenced plants of *Eucalyptus macrocarpa* with or without (+Leu-Leu) leucine dipeptide; ma: mature arbuscular; ca: collapsed arbuscular; scale bar: 50 μm; EH: relative expression levels of arbuscular development-related genes in control and gene-silenced plants. According to Duncan's multiple comparison analysis, P < 0.05.

[0045] Figure 9 This figure shows the effect of additional organic nitrogen application at different nitrogen and phosphorus levels on the growth phenotypes of non-mycorrhizal and mycorrhizal Eucalyptus macrocarpa. In Figure A: Eucalyptus macrocarpa treated with 3.6 mg / L sodium dihydrogen phosphate (i.e., 30 μM Pi) (-PN), 3.6 mg / L sodium dihydrogen phosphate and 1 g / L peptone (-P+N), 36 mg / L sodium dihydrogen phosphate (+PN), and 36 mg / L sodium dihydrogen phosphate and 1 g / L peptone (+P+N), with a baseline sodium nitrate addition of 21.25 mg / L (i.e., 250 μM N-NO3). - ), were cultured in quartz sand substrate pots for 90 days, n=3; BC: the effect of additional organic nitrogen application at different nitrogen and phosphorus levels on the plant height and crown width of non-mycorrhizal and mycorrhizal Eucalyptus species. According to Duncan's multiple comparison test, the values ​​are mean ± SE (n=3), P<0.05.

[0046] Figure 10 This figure shows the effect of additional organic nitrogen application at different nitrogen and phosphorus levels on the growth phenotype of non-mycorrhizal and mycorrhizal Eucalyptus grandis cultured in pots with quartz sand substrate for 150 days (n=3). The treatments included Eucalyptus grandis with 3.6 mg / L sodium dihydrogen phosphate (i.e., 30 μM Pi) (-PN), 3.6 mg / L sodium dihydrogen phosphate and 1 g / L peptone (-P+N), 36 mg / L sodium dihydrogen phosphate (+PN), and 36 mg / L sodium dihydrogen phosphate and 1 g / L peptone (+P+N), with a basal sodium nitrate addition of 21.25 mg / L (i.e., 250 μM N-NO3). - ). Detailed Implementation

[0047] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0048] The following examples further illustrate the outstanding features and significant advancements of the present invention. These examples are merely illustrative and do not limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0049] Unless otherwise specified, the test methods in the following examples are generally performed under standard experimental conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used are commercially available.

[0050] Example 1

[0051] (1) Inoculate the maize root system with AM fungus R. irregularis DAOM 197198 to promote the propagation of fungal agents. When the mycorrhizal maize plants have grown for 4 months, take the mycorrhizal root segments and the mixture of soil gravel and mix them thoroughly to make mycorrhizal fungal agents for later use.

[0052] (2) R. irregularis spores were isolated and collected by wet sieving and sucrose centrifugation and stored in a 4°C refrigerator. R. irregularis spores were placed in a 12-well cell culture plate containing sterile water and placed in a 25°C dark incubator to induce spore germination tubes. After 7 days of culture, the germinating spores were collected in centrifuge tubes and used for Eucalyptus root inoculation experiments.

[0053] (3) Eucalyptus seeds were surface sterilized with 1% NaClO for 15 min, rinsed three times with sterile water, and then germinated on 1 / 4 MS solid medium. Germination was carried out in the dark at 25℃ for 3 days to obtain a radicle of about 0.5 cm. When the hypocotyl was fully expanded and two cotyledons had just appeared, the seedlings were transplanted into nitrogen- and phosphorus-free quartz sand pots for a nitrogen- and phosphorus deficiency adaptation test. After 14 days, seedlings with uniform growth were transferred to individual pots. *Rhizopus heterophyllus* was inoculated at the base of the seedlings, with four biological replicates for each treatment. The microclimate conditions in the plant growth chamber were: photoperiod of 16 h daytime, temperature of 24℃, and light intensity of 100-200 Wm. -2 At 8 pm, the temperature was 19℃.

[0054] (4) During the first week, water the plant once with a low-phosphorus (30 μM NaH2PO4) modified Long-Ashton (mLA) nutrient solution (Hewitt, 1966). Afterward, water with modified mLA nutrient solutions of different nitrogen-phosphorus ratios every three days, using 50 mL of nutrient solution each time. The nitrogen and phosphorus concentrations were as follows: 30 μM Pi and 250 μM N-NO3. - (-PN), 30 μM Pi and 2500 μM N-NO3 -(-P+N), 300μM Pi and 250μM N-NO3 - (+PN), 300μM Pi and 2500μM N-NO3 - Eucalyptus seedlings were treated with (+P+N). The concentrations of phosphorus and nitrogen in the standard LA solution were 30 μM NaH2PO4 and 1 mM NaNO3, respectively. Sixty days after inoculation with the AM fungus Rhizophora irregularis, the abundance of extraroot hyphae in the root system of Eucalyptus seedlings was observed under a stereomicroscope to preliminarily determine the degree of mycorrhizalization of Eucalyptus seedlings, and mycorrhizal samples were collected.

[0055] (5) The aboveground tissues of each bioreplication of Eucalyptus grandis were randomly divided into 4 groups for nitrogen and phosphorus content determination. Similarly, the underground tissues were randomly divided into 4 groups, and the total nitrogen and total phosphorus content in each tissue was determined using a continuous flow analyzer (Series SA1100, SKALAR). At the same time, the infection rate of R. irregularis in the roots of Eucalyptus grandis was detected.

[0056] The results are as follows Figure 1 and Figure 2 As shown, root samples were stained with WGA488 (wheat germ lectin 488), and the effects of different nitrogen and phosphorus levels on the infection rate of *R. irregularis* in *Eucalyptus macrocarpa* roots were observed under a microscope. The results showed that under phosphorus-added low-nitrogen (+PN) or low-phosphorus-low-nitrogen (-PN) treatments, the infection rate of the AM fungus *R. irregularis* and the formation of arbuscular mycorrhizae were significantly inhibited, while low-phosphorus-added nitrogen (-P+N) treatment significantly promoted the establishment of arbuscular mycorrhizae and arbuscular development in *Eucalyptus macrocarpa*. The results also indicated that nitrate and phosphate synergistically regulate the formation of arbuscular mycorrhizae and the growth of *Eucalyptus macrocarpa*, and that appropriate nitrate addition can promote the formation of *Eucalyptus macrocarpa* mycorrhizal symbionts. Under nitrogen-deficient conditions, appropriate phosphorus supply significantly promoted the increase of total nitrogen content in the aboveground tissues of *Eucalyptus macrocarpa*. Simultaneously, under phosphorus-added conditions, nitrogen treatment significantly increased the total phosphorus content in the underground tissues of *Eucalyptus macrocarpa*. On the other hand, under nitrogen treatment, phosphorus addition led to a significant increase in total nitrogen content in both the aboveground and underground tissues of Eucalyptus grandis, indicating that appropriate phosphate addition promoted the absorption of nitrate nitrogen from the environment by Eucalyptus grandis. During the arbuscular mycorrhizal stage, under conditions of nitrogen and phosphorus deficiency, the total nitrogen and total phosphorus contents of the aboveground tissues of Eucalyptus grandis inoculated with Rhizopus irregularis were significantly higher than those of uninoculated Eucalyptus grandis tissues, indicating that the formation of arbuscular mycorrhizae enhanced the absorption of nitrogen and phosphorus by Eucalyptus grandis.

[0057] Example 2

[0058] The EgNPF8.1 of this invention contains 3 introns and 4 exons, encoding 571 amino acids. Phylogenetic analysis showed that it is closely related to dipeptide transporters such as Populus alba PaNPF8.1, Pistacia vera PvNPF8.1, and Arabidopsis thaliana AtNPF8.1 / 8.2, indicating that EgNPF8.1 and its homologous proteins may have similar dipeptide transport functions. qRT-PCR primers were designed using the ORF (open reading frame) of EgNPF8.1 (see Table 1), with the Ubiquitin 3 (EgUBI3) gene used as an internal reference gene for homogenization. Quantitative results showed that the expression level of EgNPF8.1 in the roots of Eucalyptus globulus tufts was approximately 48 times that in non-mycorrhizal Eucalyptus globulus roots. Figure 3 The results showed that EgNPF8.1 was strongly induced by arbuscular mycorrhizae, suggesting that it may play an important role in the nitrogen uptake pathway of Eucalyptus arbuscular mycorrhizae.

[0059] Table 1 Primers used in the qRT-PCR of this invention

[0060]

[0061] Example 3

[0062] To verify whether EgNPF8.1 encodes a nitrogen transporter, the full-length coding sequence of EgNPF8.1 (as shown in SEQ ID NO.1) was inserted into the yeast expression vector pFL61 (empty vector) through the NotI restriction site, obtaining the recombinant vector pFL61-EgNPF8.1. This pFL61-EgNPF8.1 recombinant vector was then transformed into *Saccharomyces cerevisiae* strain BY4741. The results showed that the BY4741 strain overexpressing EgNPF8.1 exhibited better cell growth on ammonium sulfate-free yeast uracil synthesis-deficient media supplemented with 0.5 mM, 2.5 mM, or 10 mM NaNO3 compared to the BY4741 strain transformed with the empty vector. Figure 4 A). This discovery reveals the potential role of EgNPF8.1 in nitrogen uptake in yeast cells.

[0063] Given that some plant NPF proteins have been shown to possess the ability to transport dipeptides and amino acids, as well as other substrates (organic nutrients) (Tsay et al., 2007; Ouyang et al., 2010), and combining phylogenetic tree relationships and protein structure-function predictions, the EgNPF8.1 transporter may transport nitrates and may also possess the ability to transport organic nitrogen (such as amino acids AAs and small peptides SPs). In this study, the BY4741 strain transformed with EgNPF8.1 and the empty vector was subjected to a spotting experiment on a sulfate-free yeast uracil synthesis-deficient selective medium containing 0.1 mM, 0.25 mM, and 0.5 mM L-leucine dipeptide (Leu-Leu). The results showed that the yeast overexpressing EgNPF8.1 exhibited significantly better growth than the yeast transformed with the empty vector. Figure 4 B). To determine whether EgNPF8.1 possesses dipeptide transport activity, the pFL61-EgNPF8.1 expression vector was further expressed in the *Saccharomyces cerevisiae* mutant Δptr2, which had its endogenous dipeptide transporter gene ScPTR2 knocked out using a CRISPR / Cas9 method. The EgNPF8.1-expressing yeast mutant Δptr2 ​​was inoculated into a sulfate-free yeast uracil synthesis-deficient selective medium containing 0.25 mM L-leucine dipeptide (Leu-Leu), L-histidine-L-leucine dipeptide (His-Leu), or glutamate-glutamate dipeptide (Glu-Glu) as the sole nitrogen source. The results showed that Δptr2 ​​cells expressing EgNPF8.1 grew well on Leu-Leu, His-Leu, or Glu-Glu dipeptide selective media, while the growth of Δptr2 ​​cells transformed with the empty vector was extremely slow, indicating that EgNPF8.1 can transport dipeptides in yeast. Figure 4 C). The content of L-leucine dipeptide in yeast grown under 0.25 mM Leu-Leu medium as the sole nitrogen source was determined by high-performance liquid chromatography (HPLC). The results showed that the wild-type yeast BY4741 transformed with an empty vector had good absorption capacity for the dipeptide, while the absorption of L-leucine dipeptide in the yeast mutant Δptr2 ​​transformed with an empty vector was inhibited. The EgNPF8.1 gene, however, could play a role in dipeptide absorption. Figure 4E). To determine whether EgNPF8.1 possesses nitrate transport activity, the pYNR-EgNPF8.1 expression vector was constructed and further expressed in a high-affinity nitrate transporter-deficient yeast mutant strain Δynt (wild-type *Hansenula polymorpha* strain NCYC495). The yeast mutant Δynt expressing EgNPF8.1 was inoculated onto a yeast uracil synthesis-deficient selective medium containing 0.5 mM NaNO3 as the sole nitrogen source, free of ammonium sulfate. The pYNR-EgNPF8.1 expression vector was constructed by inserting the full-length coding sequence of EgNPF8.1 (as shown in SEQ ID NO.1) between the SalI and SpeI restriction sites on the yeast shuttle vector pYNR-EX (empty vector). The *Hansenula polymorpha* mutant Δynt had its nitrate reductase gene (YNR1) knocked out using the CRISPR / Cas9 method. Driven by the nitrate reductase gene (YNR1) promoter, the constructed pYNR-EgNPF8.1 was used to express the EgNPF8.1 gene in the *Hansenula polymorpha* mutant Δynt. Linearization was performed at the BstEII site before yeast transformation. The *Hansenula polymorpha* mutant Δynt and wild-type NCYC495 strain were transformed and cultured according to the description by Martín et al. (2008), followed by a complementation experiment. The results showed that Δynt cells expressing EgNPF8.1 and Δynt cells transformed with the empty vector pYNR-EX grew extremely slowly, while wild-type *Hansenula polymorpha* transformed with the empty vector grew well, indicating that EgNPF8.1 cannot transport nitrate in yeast. Figure 4 D). In summary, these results indicate that the Eucalyptus EgNPF8.1 gene encodes a dipeptide transporter.

[0064] The yeast shuttle vector pYNR-EX was published in the literature “Huang Jiu, Shi Shuangfeng, Zhang Erte, et al. Cloning and functional study of GeNRT2.1 nitrate transport protein gene in dwarf pearl [J]. Progress in Biotechnology, 2022, 12(02):256-264.”

[0065] Example 4

[0066] By using virus-induced gene silencing (VIGS) technology, an RNAi silencing region relative to the translation start codon (ATG) -60 to +110 is designed at the 5' end of the EgNPF8.1 gene to target a specific region of the target gene, thereby avoiding off-target effects.

[0067] The specific DNA fragment from the silenced EgNPF8.1 was then cloned into the pTRV2 plasmid of tobacco brittle virus. At least 72 independent EgNPF8.1-RNAi silent plants were created using the VIGS method (Zhang and Liu, 2014; Buendia et al., 2016), and were used to determine nitrogen and phosphorus content in *Eucalyptus macrocarpa*, analyze its arbuscular mycorrhizal phenotype, and analyze the relative expression levels of arbuscular development-related genes (EgFatM, EgRAM2, EgPT4, RiMST2). The results showed that under low phosphorus and nitrogen (-P+N) conditions, the total mycorrhizal infection rate (F%) and arbuscular abundance (A%) of the EgNPF8.1-RNAi silent plants were significantly lower than those of the control plants. Figure 5 AC). Furthermore, compared to control plants, some arbuscular structures in the roots of silent plants were disrupted (AC). Figure 5 D). Meanwhile, compared to control plants, the relative expression levels of the gene EgFatM in the underground parts of silent plants were significantly reduced. Figure 5 E); Under -PN and +PN treatments, the relative expression level of the gene EgRAM2 in the underground part of the silent plant was significantly reduced. Figure 5 F); Under -P+N treatment, the relative expression level of the gene EgPT4 in the underground part of the silent plant was significantly reduced (F). Figure 5 G), the relative expression level of RiMST2 in the AM fungus *Rhizopus heterophyllus* was significantly reduced ( Figure 5 H).

[0068] Further analysis was conducted to determine the total nitrogen content in the aboveground and underground tissues of control and silent plants inoculated with *Rhizoctonia solani* (R. irregularis). Under four different nitrogen and phosphorus treatments, the nitrogen content in the aboveground and underground parts of silent *Eucalyptus macrocarpa* plants inoculated with *R. irregularis* was significantly lower than that in control plants grown under the same nutrient conditions. Figure 6 Specifically, under four different nitrogen and phosphorus levels—-PN, -P+N, +PN, and +P+N—the total nitrogen content in the aboveground tissues of arbuscular mycorrhizal EgNPF8.1-RNAi silenced plants was reduced by 21.95%, 19.05%, 30.97%, and 14.80%, respectively, compared to the control plants. Figure 6 A). On the other hand, compared with the control plants, the total nitrogen content in the arbuscular mycorrhizae (underground tissues) of EgNPF8.1-RNAi gene-silenced plants was reduced by 35.84%, 25.48%, 45.43%, and 26.83% under four different nitrogen and phosphorus treatments, respectively. Figure 6 B). This result indicates that EgNPF8.1 plays an important role in the nitrogen uptake pathway of Eucalyptus arbuscular mycorrhizal fungi.

[0069] Example 5

[0070] Functional studies of EgNPF8.1 in yeast revealed that it functions as a dipeptide transporter. A VIGS silencing assay was performed on Eucalyptus macrocarpa. After inoculation with AM fungi, the fungus was irrigated with a low-phosphorus (30 μM NaH2PO4) modified mA nutrient solution once during the first week, followed by irrigation with a phosphorus-added (300 μM NaH2PO4) modified mA nutrient solution. An additional 2.5 mM leucine dipeptide was added to the +Leu-Leu treatment, while no leucine dipeptide was added to the -Leu-Leu treatment. Nutrient solutions were irrigated every three days, with 50 mL applied each time. The inoculation period was 60 days. Figure 7 After A), by detecting the relative expression levels of the EgNPF8.1 gene under different treatments, it was shown that EgNPF8.1 was successfully silenced. Figure 7 BC). Simultaneous observation of Eucalyptus globulus growth revealed that, under conditions of leucine dipeptide addition and AM fungal inoculation, the uptake of leucine dipeptide by the control group Eucalyptus globulus was significantly higher than that by the EgNPF8.1-RNAi gene-silenced Eucalyptus globulus plants. Figure 7 D).

[0071] Mycorrhizal infection rate detection revealed that, compared with EgNPF8.1-RNAi silenced plants, the total infection rate, infection intensity, and arbuscular abundance of the control Eucalyptus arbuscular mycorrhizae treated with leucine dipeptide were significantly increased. Figure 8 AD), and the expression of marker genes closely related to arbuscular formation and development (EgFatM, EgRAM2, EgPT4, RiMST2) was detected. The results showed that the addition of leucine dipeptide significantly upregulated the expression of these genes. Figure 8 EH).

[0072] In summary, the expression of the EgNPF8.1 gene can positively regulate the organic nitrogen uptake pathway in Eucalyptus arbuscular mycorrhizal vines under conditions of organic nitrogen (such as leucine dipeptide) supply. This indicates that the EgNPF8.1 transporter participates in the organic nitrogen transport process in Eucalyptus arbuscular mycorrhizal vines, and that the addition of organic nitrogen can promote the development of Eucalyptus arbuscular mycorrhizal vines.

[0073] Example 6

[0074] Based on the above research on the nitrogen-phosphorus synergistic mechanism in the arbuscular mycorrhizae of Eucalyptus grandis, further experiments were conducted on the cultivation of mycorrhizal Eucalyptus grandis seedlings under nitrogen and phosphorus nutrient stress. Under the condition of added organic nitrogen (peptone), the growth of Eucalyptus grandis inoculated with *R. irregularis* for 90 days was significantly greater than that of uninoculated Eucalyptus grandis. Figure 9Under low phosphorus (-PN or -P+N) conditions, uninoculated *Eucalyptus irregularis* seedlings showed obvious phosphorus deficiency symptoms, manifested as purple leaves. However, *Eucalyptus irregularis* seedlings inoculated with *R. irregularis* under the same conditions showed significant relief from nutrient deficiency stress. Measurements of Eucalyptus globulus plant height and crown width showed that under nitrogen and phosphorus deficiency (-PN) conditions, Eucalyptus globulus inoculated with *R. irregularis* increased in height by 88.89% and crown width by 102.06% compared to uninoculated *R. irregularis*. Under phosphorus deficiency (-P+N) conditions, the inoculated *R. irregularis* plant height increased by 44.81% and crown width by 60.19% compared to uninoculated *R. irregularis*. Under nitrogen deficiency (+PN) conditions, the inoculated *R. irregularis* plant height increased by 19.36% and crown width by 18.85% compared to uninoculated *R. irregularis*. Under phosphorus and nitrogen application (+P+N) conditions, the increase in plant height of inoculated *R. irregularis* plant compared to uninoculated *R. irregularis* was not significant, but its crown width increased by 30.91%. Figure 9 ). 150 days after inoculation with *R. irregularis*, by comparing the growth phenotypes of *Eucalyptus grandis* under different treatments, seedlings inoculated with *R. irregularis* and supplemented with organic nitrogen showed greater tolerance to low nitrogen and low phosphorus nutrient stress and grew more vigorously than the control plants. Figure 10 The above results indicate that the addition of organic nitrogen and inoculation with AM fungi significantly promote the growth of Eucalyptus grandis seedlings.

[0075] The above examples demonstrate that the arbuscular mycorrhizal-related gene EgNPF8.1 cloned in this invention is the first arbuscular mycorrhizal-inducible dipeptide transport protein encoding gene isolated and identified in *Eucalyptus macrocarpa*. The function of such genes in plant mycorrhizal symbiosis has not been described to date. Examples 3, 4, and 5 show that this gene is closely related to dipeptide uptake in the mycorrhizal symbiotic pathway. Therefore, Example 6 further verifies that inoculation with *Rhizopus irregularis* and the addition of organic nitrogen (peptone) significantly improves the arbuscular mycorrhizal infection rate of *Eucalyptus macrocarpa* and its efficient uptake of nitrogen and phosphorus. Simultaneously, an effective and reliable nutrient solution formula for rapid seedling cultivation of *Eucalyptus macrocarpa* is provided (Table 2).

[0076] Table 2. Formula of mycorrhizal plant nutrient solution containing added organic nitrogen used in this invention.

[0077]

[0078] The embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

[0079] References:

[0080] Bonfante P.(2018)The future has roots in the past: the ideas and scientists that shaped mycorrhizal research[J].New Phytologist.220:982-995.

[0081] Bonneau L, Huguet S, Wipf D, Pauly N, Truong HN. (2013) Combined phosphate and nitrogen limitation generates a nutrient stress transcriptome favorable for arbuscular mycorrhizal symbiosis in Medicago truncatula[J]. NewPhytologist 199:188-202.

[0082] Breuillin-Sessoms F, Floss DS, Gomez SK, Pumplin N, Ding Y, Levesque-Tremblay V, Noar RD, Daniels DA, Bravo A, Eaglesham JB, Benedito VA, Udvardi MK, Harrison MJ. (2015) Suppression of arbusculedegeneration in Medicago truncatulaphosphate transporter4 mutants is dependent on the ammonium transporter2family protein AMT2;3[J].Plant Cell.27:1352-1366.

[0083] Buendia L,Wang T,Girardin A,Lefebvre B.(2016)The LysM receptor-likekinase SlLYK10regulates the arbuscular mycorrhizal symbiosis in tomato[J].NewPhytologist.210:184-195.

[0084] Genre A,Lanfranco L,Perotto S,Bonfante P.(2020)Unique and commontraits in mycorrhizal symbioses[J].Nature Reviews Microbiology.18:649-660.

[0085] Harrison MJ,Dewbre GR,Liu J.(2002)A phosphate transporter fromMedicago truncatula involved in the acquisition of phosphate released byarbuscular mycorrhizal fungi[J].Plant Cell.14:2413-2429.

[0086] Hewitt EJ.(1966)Sand and water culture methods used in the study ofplant nutrition[M].Farnham Royal:England,UK 22:315-709.

[0087] Martín Y,Navarro FJ,Siverio JM(2008)Functional characterization ofthe Arabidopsis thaliana nitrate transporter CHL1 in the yeast Hansenulapolymorpha[J].Plant Molecular Biology.68:215-224.

[0088] Ouyang J,Cai Z,Xia K,Wang Y,Duan J,Zhang M(2010)Identification andanalysis of eight peptide transporter homologs in rice[J].Plant Science 179:374-382.

[0089] Shi J,Zhao B,Zheng S,Zhang X,Wang X,Dong W,Xie Q,Wang G,Xiao Y,ChenF,Yu N,Wang E.(2021)Aphosphate starvation response-centered network regulatesmycorrhizal symbiosis[J].Cell.184:5527-5540.

[0090] Tsay YF,Chiu CC,Tsai CB,Ho CH,Hsu PK(2007)Nitrate transporters andpeptide transporters[J].FEBS Lett 581:2290-2300.

[0091] Wang S,Chen A,Xie K,Yang X,Luo Z,Chen J,Zeng D,Ren Y,Yang C,Wang L,Feng H,López-Arredondo DL,Herrera-Estrella LR,Xu G.(2020)Functional analysisof the OsNPF4.5 nitrate transporter reveals a conserved mycorrhizal pathwayof nitrogen acquisition in plants[J].Proc Natl Acad Sci USA.117:16649-16659.

[0092] Xie X, Hu W, Fan

[0093] Xie X,Lai W,Che

[0094] Zhang H,Liu Y.(2014)VIGS Assays[J].Bio-protocol.4:e1057.

[0095] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Gene encoding the eucalyptol dipeptide transporter protein EgNPF8.1 The application, characterized in that, The application is at least one of the following: the gene encoding the macroeucalyptol dipeptide transporter protein. EgNPF8.1 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2; A) Application in promoting the colonization of Eucalyptus macrocarpa roots by arbuscular mycorrhizal fungi; B) Application in improving the efficient absorption of external organic nitrogen by mycorrhizal Eucalyptus plants; C) Application in determining the utilization efficiency of organic nitrogen in mycorrhizal Eucalyptus plants; The arbuscular mycorrhizal fungus mentioned is *Rhizocarpium heterotropoides* (… Rhizophagus irregularis ); The fungus in the mycorrhizalized Eucalyptus plant is *Heteromorpha rhizocarp* ( Rhizophagus irregularis ).

2. The application according to claim 1, characterized in that: The application is specifically at least one of the following applications: A1) Application in promoting the infection rate of arbuscular mycorrhizal fungi on the roots of Eucalyptus macrocarpa; A2) Application in promoting the arbuscular abundance of arbuscular mycorrhizal fungi on Eucalyptus roots; B1) Application in improving the efficient absorption of external organic nitrogen by mycorrhizal Eucalyptus plants; The arbuscular mycorrhizal fungus mentioned is *Rhizocarpium heterotropoides* (… Rhizophagus irregularis ); The fungus in the mycorrhizalized Eucalyptus plant is *Heteromorpha rhizocarp* ( Rhizophagus irregularis ).

3. The application according to claim 2, characterized in that: In A1), with EgNPF8.1 Compared with the control plants, the infection rate in the roots of gene-silenced plants was significantly increased; In A2), with EgNPF8.1 Compared with the control plants, the abundance of tufts in the roots of gene-silenced plants was significantly increased; In B1), the total nitrogen content in both the aboveground and underground tissues of the control plants was significantly higher than that under the same nutritional conditions. EgNPF8.1 Gene-silenced plants.

4. The application according to claim 2, characterized in that: In A1), under phosphorus-deficient or nitrogen-deficient conditions, with EgNPF8.1 Compared with the control plants, the infection rate in the roots of gene-silenced plants was significantly increased; In A2), under phosphorus deficiency or phosphorus and nitrogen application conditions, compared with... EgNPF8.1 Compared with gene-silenced plants, control plants showed a significant increase in the abundance of tufts in their roots.

5. The application according to claim 1, characterized in that: In B) or C), the organic nitrogen comprises at least one of small molecule peptides, amino acids, and peptones; The small molecule peptides include small molecule peptides containing 2 to 10 amino acid residues.

6. The application according to claim 5, characterized in that: The small molecule peptide is a dipeptide; the dipeptide includes at least one of L-leucine dipeptide, L-histidine-L-leucine dipeptide, and glutamic acid-glutamic acid dipeptide.

7. The application according to claim 1 or 2, characterized in that: The gene encoding the macroeucalyptol dipeptide transport protein EgNPF8.1 The nucleotide sequence is shown in SEQ ID NO.

1.

8. The application according to claim 3 or 4, characterized in that: The EgNPF8.1 Gene-silenced plants are produced using virus-induced gene silencing technology, through... EgNPF8.1 An RNAi silencing region is designed at the 5' end of the gene to target a specific region of the target gene, resulting in... EgNPF8.1-RNAi Silent plant.

9. The application according to claim 8, characterized in that: The RNAi silencing region is the RNAi silencing region relative to the translation start codon ATG -60 to +110.

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