An apparatus and method for host-mediated overexpression of genes of arbuscular mycorrhizal fungi

Abstract

The application discloses a device and method for host-mediated overexpression of arbuscular mycorrhizal fungi genes, which comprises a mycorrhizal chamber and a mycelium chamber placed upside down, the bottom of the mycorrhizal chamber is provided with a mycelium hole, and a supporting net and a nylon net are laid above the mycelium hole to allow the mycelium to pass through but limit the plant roots to pass through. The mycorrhizal chamber is used for culturing a companion plant and inoculating an AM fungal inoculum, and low-phosphorus nutrient solution is used for irrigation; the mycelium chamber is used for injecting high-phosphorus nutrient solution, and only the mycelium can absorb the high-phosphorus nutrient solution, so that the mutualistic symbiosis between the AM fungi and the companion plant and the high-speed exchange of nutrients at the symbiotic interface are strengthened. Subsequently, a target host plant with overexpressed AM fungal genes is introduced, so that the target host plant is added into the symbiotic network for high-speed material exchange with the AM fungal mycelium. Under the symbiotic relationship, the overexpressed mRNA in the root system of the target host plant enters the interior of the arbuscular mycorrhizal fungi in a large amount through the arbuscular interface and realizes overexpression. The method provides a new idea for overexpression of AM fungal genes.

Description

Technical Field

[0001] The present invention relates to the field of fungal gene genetic improvement, and in particular to a device and method for host-mediated arbuscular mycorrhizal fungal gene overexpression. Background Art

[0002] Arbuscular mycorrhiza (AM) fungi can establish beneficial symbioses with most terrestrial vascular plants, helping the host plant absorb water and mineral nutrients and improving its tolerance to adverse conditions. Because AM fungi cannot survive independently, they must establish a symbiotic relationship with their host to complete their life cycle. Therefore, currently, AM fungi are still used to cultivate large numbers of AM fungal propagules through the living symbiosis between AM fungi and various mycorrhizal plants. These propagules are then used to infect plants, thereby increasing plant yields and improving plant quality.

[0003] In recent years, as research into the mechanisms of AM fungal symbiosis with plants has deepened, researchers have discovered that AM fungi activate a series of fungal signaling pathways and the involvement of some fungal genes in enhancing host plant stress resistance. Specifically, these AM fungal transcription factors and functional genes play an indispensable role in the coordinated stress resistance of AM fungal symbionts. To further explore the specific roles of these genes, researchers have begun to use the research approach of plant gene function to conduct heterologous functional analysis of these transcription factors or functional genes. At the same time, as researchers delve deeper into symbiotic interactions and the infection of host plants by pests and diseases, they have discovered that small RNAs can be exchanged at the symbiotic interface or at the interface between pathogens and host plants. Soon after, the host-induced gene silencing (HIGS) technology emerged. HIGS has been shown to silence genes in pathogens, pests, and AM fungi when pathogens parasitize host plants, pests (including insects and nematodes) infect hosts, and AM fungi coexist with host plants. This involves using small RNAs that shuttle through the interaction interface to achieve RNA interference, thereby specifically reducing or silencing gene expression in pathogens, pests, or AM fungi, achieving gene silencing in these organisms. During the AM fungal symbiotic interaction, only a small number of small RNAs shuttle through the interaction interface. Based on the RNA interference silencing principle, a small number of small RNAs with hairpin structures can efficiently silence target genes, thereby ensuring the success of HIGS technology in silencing target genes in the fungus during the symbiosis between AM fungi and host plants, and then studying the impact of target gene silencing on the AM fungal symbiosis.

[0004] Due to the inability of AM fungi to survive stably on their own, AM fungal strains that overexpress AM fungal genes cannot be obtained through conventional techniques. Currently, while HIGS technology can achieve silencing of AM fungal target genes, there are no applications for overexpressing AM fungal target genes. This may be because only a small amount of RNA shuttles across the interaction interface between AM fungi and plants, making it difficult for conventional HIGS strategies to achieve overexpression of AM fungal genes within the fungus. In other words, there is currently no technology that can achieve efficient shuttling of RNA molecules across the symbiotic interface and their accumulation in the fungus. Patent CN106190944A discloses a method for enriching arbuscular mycorrhizal fungal spores through layered culture. The method involves dividing a seedling culture container into a lower layer accessible to hyphae, a middle layer isolating water, fertilizer, and roots, and an upper layer for plant root growth. The method blocks the root system from absorbing nutrients, thereby enhancing the plant's dependence on AM fungi for nutrient transfer, thereby enhancing the colonization of AM fungi in the root system and the proliferation and spore production of hyphae in the lower layer. However, the lower layer lacks a continuous water and fertilizer supply system, no vapor pressure conditions conducive to hyphae growth, and no mechanism for efficient and high-speed nutrient exchange between host plants carrying AM fungal genes and the AM fungal hyphae network. This limits the growth of hyphae and makes it difficult for host plants carrying AM fungal genes to quickly join the AM fungal symbiotic network that exchanges nutrients with plants at high speed. In other words, it is difficult to achieve host-mediated overexpression of AM fungal genes in AM fungi. Patent CN110476713A discloses a device and method for continuously collecting arbuscular mycorrhizal fungal hyphae. The device utilizes mycorrhizal chambers and mycelial chambers placed on the left and right. By inverting the plant growth, the gravitropism of the roots is utilized to prevent them from directly absorbing high-phosphorus nutrients, thereby improving the symbiotic relationship between arbuscular mycorrhizal fungi and plants and facilitating the collection of arbuscular mycorrhizal fungal hyphae. However, its inverted growth mode is not conducive to plant photosynthesis, thereby limiting the growth of the host plant, and is in turn not conducive to the exchange between the photosynthetic products of the host plant and the nutrients supplied by the mycorrhizal fungi. It is impossible to achieve high-speed exchange of nutrients at the arbuscular symbiotic interface, and it is difficult to achieve host-mediated overexpression of AM fungal genes in AM fungi. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a device for host-mediated arbuscular mycorrhizal fungal gene overexpression.

[0006] The second object of the present invention is to provide a method for accelerating the material exchange between arbuscular mycorrhizal fungi and target host plants at the symbiotic interface using the above-mentioned device and achieving host-mediated overexpression of arbuscular mycorrhizal fungal genes in arbuscular mycorrhizal fungi based on the HIGS strategy.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] The application provides a device for host-mediated arbuscular mycorrhizal (AM) fungal gene overexpression, which comprises an upper layer and a lower layer, wherein the upper layer is a mycorrhizal chamber and the lower layer is a hypha chamber; a plurality of hypha holes are arranged at the bottom of the mycorrhizal chamber, and AM fungal hyphae can pass through the hypha holes to the hypha chamber to absorb nutrients; a support net is arranged above the hypha holes, and a nylon net is arranged above the support net, wherein the pore size of the nylon net allows the hyphae to pass through but limits the plant roots to pass through.

[0009] The device can accelerate the material exchange between the AM fungi and the target host plants at the symbiotic interface, establish a stable symbiotic relationship, and realize the host-mediated overexpression of AM fungal genes in AM fungi based on the HIGS strategy, thereby laying a solid foundation for the future use of AM fungal key genes to enhance the stress resistance of AM fungal symbionts.

[0010] The device comprises a mycorrhizal chamber and a hypha chamber arranged in a top-bottom manner, the mycorrhizal chamber is used for co-culturing target host plants and AM fungi, and the hypha chamber is used for providing a continuous water and fertilizer environment for the AM fungal hyphae to absorb; the bottom of the mycorrhizal chamber is provided with hypha holes, and a support net and a nylon net are arranged above the hypha holes, which allows the hyphae to pass through but limits the plant roots to pass through. The top-bottom stacked mycorrhizal chamber and hypha chamber can provide stable water vapor pressure conditions, thereby facilitating the spread of AM fungal hyphae from the mycorrhizal chamber to the hypha chamber, and the continuous absorption of water and fertilizer from the hypha chamber to the plant roots in the mycorrhizal chamber for high-speed nutrient exchange, that is, a stable symbiotic relationship is formed, and the material exchange between the AM fungi and the target host plants at the symbiotic interface is accelerated, which is beneficial to the large shuttling of RNA molecules in the root system at the symbiotic interface. Further, the device is used for co-culturing a symbiotic plant in the mycorrhizal chamber and inoculating AM fungal agents, and low-phosphorus nutrient solution is irrigated; high-phosphorus nutrient solution is injected into the hypha chamber, and the characteristics of only the hyphae absorbing the high-phosphorus nutrient solution are utilized to strengthen the mutual symbiosis between the AM fungi and the symbiotic plant, thereby establishing a symbiotic network of high-speed nutrient exchange between the AM fungi and the symbiotic plant. Subsequently, hairy roots overexpressing AM fungal genes are introduced to transform the target host plant, the growth of the original symbiotic plant is reduced, the target host plant is added to the symbiotic network of high-speed nutrient exchange established by the AM fungi and the plant, and a stable symbiotic relationship is formed with the AM fungal hypha network. Under this symbiotic relationship, the overexpressed mRNA in the root system of the target host plant enters the AM fungi in large quantities through the arbuscular interface and accumulates in excess, thereby realizing overexpression. This method provides a new idea for AM fungal gene overexpression.

[0011] Further, the pore size of the nylon net is 40-45 μm.

[0012] Further, the pore size of the support net is 0.5-0.8 cm.

[0013] Further, the support net is a hard material with a space gap of 0.5-0.8 cm.

[0014] Further, the pore diameter of the mycelium hole is 2-5 cm.

[0015] Further, the mycelium chamber is provided with support columns for supporting the mycorrhizal chamber.

[0016] Preferably, the mycorrhizal chamber and the mycelium chamber are formed by stacking two identical plastic hard white rectangular containers, and the mycelium chamber is provided with a plurality of support columns made of hard material.

[0017] Preferably, the hard material is plastic.

[0018] The application also provides a method for accelerating the exchange of substances between AM fungi and host plants at the symbiotic interface and overexpressing host-mediated AM fungal genes in AM fungi based on HIGS strategy using the above device, comprising the following steps:

[0019] S1. Add sterilized substrate to the mycorrhizal chamber of any of the above devices, and add nutrient solution for mycelium growth to the mycelium chamber;

[0020] S2. Plant the companion plant in the mycorrhizal chamber and inoculate the AM fungal inoculum, water the mycorrhizal chamber with nutrient solution for plant growth, and co-culture to establish a stable symbiotic relationship between AM fungi and companion plants, achieving the purpose of accelerating nutrient exchange under AM fungal and plant symbiotic relationship;

[0021] S3. Transfer the hairy root transformed plant with overexpressed AM fungal genes as the target host plant to the mycorrhizal chamber, and cut off 1 / 2-3 / 4 of the aboveground part of the companion plant with scissors, and co-culture to establish a symbiotic relationship between the target host plant and the AM fungi, and realize the efficient crossing of the overexpressed mRNA molecules in the target host root into the AM fungal arbuscule interface and the overexpression of the mRNA molecules in the AM fungi during the high-speed material exchange process between the mRNA molecules in the target host root and the AM fungal arbuscule interface.

[0022] Further, the co-culture time is 2-4 months.

[0023] Preferably, the co-culture time is 3 months.

[0024] Further, the co-culture conditions are to culture under the light intensity of 24000-26000 lux, and in addition to watering the plants with nutrient solution for plant growth, the plants seedlings also need to be watered with tap water on a daily basis.

[0025] Further, the nutrient solution for plant growth in step S2 is a low-phosphorus nutrient solution; the phosphate concentration in the low-phosphorus nutrient solution is 80-120 μM, and the pH is 5.5-5.7.

[0026] Preferably, the phosphate concentration in the low-phosphorus nutrient solution is 100 μM and the pH is 5.6.

[0027] Furthermore, the nutrient solution for plant growth in step S2 is watered once every 10 to 20 days.

[0028] Preferably, the nutrient solution for plant growth in step S2 is watered once every 15 days.

[0029] Furthermore, the nutrient solution for mycelium growth in step S1 is a high-phosphorus nutrient solution; the phosphate concentration in the high-phosphorus nutrient solution is 1-3 mM, and the pH is 5.1-5.3.

[0030] Preferably, the phosphate concentration in the high-phosphorus nutrient solution is 2 mM and the pH is 5.2.

[0031] Furthermore, the liquid level of the nutrient solution for mycelial growth in step S1 should be 1 to 2 cm lower than the bottom of the mycorrhizal chamber.

[0032] Furthermore, during the symbiotic culture period, the concentration of the nutrient solution for mycelial growth in the mycelial chamber must be ensured and replaced in a timely manner.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The stacked mycorrhizal and hyphae chambers of the present invention provide stable vapor pressure conditions, thereby facilitating the spread of AM fungal hyphae from one mycorrhizal chamber to another. The hyphae continuously absorb water and fertilizer from the hyphae chambers, rapidly exchanging nutrients with the plant roots within the chambers. This creates a stable symbiotic effect and accelerates the exchange of substances between the AM fungi and the target host plant at the symbiotic interface, facilitating the large-scale shuttling of RNA molecules within the root system across the symbiotic interface. Furthermore, utilizing the present invention, a symbiotic network system is first established in which AM fungi and conventional mycorrhizal plants exchange nutrients at a high rate at the symbiotic interface. The target host plant is then introduced, and by reducing the growth potential of the original mycorrhizal plant, the target host plant is rapidly incorporated into the symbiotic network in which nutrients are rapidly exchanged with the AM fungi at the symbiotic interface. This allows the mRNA molecules of AM fungal genes overexpressed in the roots of the target host plant to be shuttled in large quantities into the AM fungi, thereby achieving the target host plant-mediated overexpression of AM fungal genes within the AM fungi. This invention overcomes the limitations of conventional HIGS strategies, which hinder the overexpression of AM fungal genes within the AM symbiotic interface due to the limited RNA shuttle across the interaction interface. This also lays a solid foundation for future use of key AM fungal genes to enhance the tolerance of AM fungal symbionts to environmental stress. Furthermore, the device has a simple structure, is easy to use, can be handcrafted, and is relatively inexpensive. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 A front cross-sectional view of an application device for host-mediated arbuscular mycorrhizal fungal gene overexpression strategy.

[0036] Figure 2 Schematic diagram of the bottom of the mycorrhizal chamber of a device for applying the host-mediated gene overexpression strategy in arbuscular mycorrhizal fungi.

[0037] Figure 3 This is the green fluorescence result of the arbuscular structure in the root system of the target host plant. Figure 3 a is a diagram showing the green fluorescent protein (GFP) signal in the living mycorrhizal symbiotic root system (the yellow arrow indicates the GFP luminescence position); Figure 3 b is a diagram confirming the location of arbuscule structures using fluorescent dyes (white arrows indicate the location of arbuscules).

[0038] Figure 4 This is the green fluorescence result of the arbuscular structure in the root system of the target host plant when the target host plant is planted in a conventional flower pot and the arbuscular mycorrhizal fungus gene is overexpressed. Figure 4 a is a diagram showing the green fluorescent protein (GFP) signal in the living mycorrhizal symbiotic root system (the yellow arrow indicates the GFP luminescence position); Figure 4 b is a diagram confirming the location of arbuscule structures using fluorescent dyes (white arrows indicate the location of arbuscules).

[0039] Figure caption: 1-mycorrhizal chamber, 2-hyphae chamber, 101-nylon mesh, 102-support mesh, 103-hyphae pores, 201-support column. DETAILED DESCRIPTION

[0040] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0041] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0042] In the description of the present invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and therefore should not be construed as limiting the present invention. Furthermore, the terms "first," "second," "third," and "fourth," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0043] Example 1 An application device based on host-mediated arbuscular mycorrhizal fungal gene overexpression strategy

[0044] like Figure 1 and Figure 2 The figure shows an application device for a host-mediated arbuscular mycorrhizal fungal gene overexpression strategy. The device consists of two stacked square containers, creating a two-layer space: the upper layer is a mycorrhizal chamber 1, and the lower layer is a mycelial chamber 2. The bottom of the mycorrhizal chamber 1 is provided with multiple equidistant small holes forming mycelial pores 103, through which mycelia can reach the mycelial chamber 2 to absorb nutrients. A support net 102 is placed above the mycelial pores 103, and a nylon mesh 101 is placed above the support net 102. Inside the mycelial chamber 2, support columns 201 are provided to support the mycorrhizal chamber 1. The mycelial pores 103 have a radius of 2 cm, the mesh spacing of the support net 102 is 0.5 cm, and the pore size of the nylon mesh is 45 μm.

[0045] The device includes a mycorrhizal chamber 1 and a mycelium chamber 2 placed one above the other. The mycorrhizal chamber 1 is used for co-cultivation of target host plants and arbuscular mycorrhizal fungi, and the mycelium chamber 2 is used for providing high-phosphorus water and fertilizer conditions that can be absorbed by the mycelium. Mycelium holes 103 are provided at the bottom of the mycorrhizal chamber 1, and a support net 102 is placed above the mycelium holes 103. A nylon net 101 is placed on the support net 102. Since the nylon net 101 is made of a relatively soft material, when a culture medium is added to the nylon net 101, it is easy to cause the nylon net 101 to collapse. Therefore, a support net 102 needs to be set under the nylon net 101 for support. The above-mentioned aperture design of the device allows mycelium to pass through but restricts the passage of plant roots. The superimposed mycorrhizal chamber 1 and mycelial chamber 2 can provide stable water vapor pressure conditions, which is conducive to the spread of AM fungal hyphae from the mycorrhizal chamber 1 to the mycelial chamber 2, and continuously absorb water and fertilizer from the mycelial chamber 2 to exchange nutrients with the plant roots in the mycorrhizal chamber 1 at a high speed, thus forming a stable symbiotic relationship. At the same time, it accelerates the material exchange between the arbuscular mycorrhizal fungi and the target host plants at the symbiotic interface, which is conducive to the large-scale shuttling of RNA molecules in the roots at the symbiotic interface.

[0046] Example 2 A method for accelerating the exchange of substances between arbuscular mycorrhizal fungi and their hosts at the symbiotic interface and achieving overexpression of arbuscular mycorrhizal fungal genes in the fungus

[0047] The above-mentioned device of the present invention is implemented by the following specific method:

[0048] S1. Take two clean, alcohol-sterilized, white, rectangular, rigid plastic containers that are 38 cm long, 30 cm wide, and 12 cm high. Open 21 small holes with a radius of 2 cm at intervals of 1.25 cm at the bottom of one of the containers, with three rows of 7 holes each, to serve as the mycorrhizal chamber. First, place a layer of plastic support mesh with a mesh gap of 0.5 cm at the bottom of the container with the small holes. Then, cover the bottom of the container with a 45 μm nylon mesh to prevent plant roots from entering the mycelial chamber. Fully fill the mycorrhizal chamber with a substrate (sand and vermiculite, volume ratio of 1:1) that has been treated in a high-pressure, high-temperature sterilizer (121°C, 120 min).

[0049] S2. Stack the mycorrhizal chamber with another hard plastic container, with the mycorrhizal chamber on top and the other container on the bottom. The space created by the two stacking is the mycelial chamber. A black hard plastic cylindrical support column (bottom radius of 2 cm, height of 5 cm) is set inside the mycelial chamber.

[0050] S3. Surface-sterilized vetiver (Chrysopogon zizanioides) seeds were planted in a mycorrhizal chamber and inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis. The vetiver was incubated under 25,000 lux of light and 28°C for two months to ensure optimal growth of the vetiver, allowing the AM fungi to establish a stable symbiotic relationship with the vetiver. During the two-month incubation period, a low-phosphorus nutrient solution was poured into the mycorrhizal chamber every 15 days. Simultaneously, a high-phosphorus nutrient solution was injected into mycelial chamber 2 using a syringe, with the nutrient solution level in mycelial chamber 2 1-2 cm below the bottom of the mycorrhizal chamber container in step 2. The low-phosphorus nutrient solution (Hoagland nutrient solution) had a phosphate concentration of 100 μM and a pH of 5.6, while the high-phosphorus nutrient solution (Hoagland nutrient solution) had a phosphate concentration of 2 mM and a pH of 5.2. After two months of cultivation, the symbiotic relationship between the arbuscular mycorrhizal fungus (Heteromorpha) and vetiver was strengthened. Specifically, the vetiver roots were unable to absorb sufficient phosphorus nutrients within the mycorrhizal chamber, and due to the presence of a 45μM nylon mesh barrier, they were unable to absorb nutrients from the mycelial chamber. Consequently, the vetiver roots needed the hyphae of the symbiotic AM fungus (Heteromorpha) to absorb large amounts of phosphorus nutrients from the mycelial chamber in order to exchange nutrients with it, thus accelerating nutrient exchange within the symbiotic relationship between the AM fungus and the plant.

[0051] S4. Referring to the prior art "Boisson-Dernier et al. (2001) Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizalsymbiotic associations.", a dicotyledonous plant overexpression vector pC1300-35S+eGFP vector was used to clone the RiEFa gene CDS of heteromorphic rhizosporium into the vector. The constructed vector was then transferred into the Agrobacterium rhizogenes ARqua1 strain for transformation of M. truncatula hairy roots:

[0052] (1) Medicago truncatula seeds were sterilized with 5% sodium hypochlorite solution for 20 minutes, and then spread on a culture dish containing germination medium. The seeds were placed in a refrigerator at 4 degrees Celsius for 24 hours, and then placed in a 25-degree Celsius incubator under light-shading to inhibit their growth rate.

[0053] (2) Agrobacterium rhizogenes was cultured at 30°C and 200 rpm for 12 h. After being taken out, it was plated on a culture medium prepared with LB and supplemented with resistance hormones in a clean bench. After the plate was plated, the culture dish was placed upside down in an incubator at 30°C for 48 h. In the clean bench, a blade was used to smoothly cut the root of Medicago truncatula (3 mm near the embryo) to produce a wound. The wound surface was then inoculated with Agrobacterium rhizogenes on the culture plate for infection.

[0054] (3) After the infection is completed, the Medicago truncatula is spread on a culture dish containing a culture medium for promoting plant callus budding (budding medium), and cultured in an incubator at 18 degrees Celsius for one week.

[0055] (4) After one week, take out the culture medium and transfer it to a new rooting medium with added resistance hormone in the clean bench (the alfalfa root should be inserted into the culture medium), and culture it in an incubator at 25 degrees Celsius and 25,000 lux to obtain positive hairy root transformed plants.

[0056] The target host plant, Medicago truncatula (a transformed hairy root plant overexpressing the AM fungus RiEFa / green fluorescent protein fusion gene), was transplanted into mycorrhizal chamber 1. Using scissors, 1 / 2 to 3 / 4 of the aboveground portion of the vetiver was cut off to reduce the growth potential of the vetiver, thereby reducing competition between the companion plant vetiver and the target host plant, Medicago truncatula. This, in turn, accelerated the establishment of an AM symbiotic relationship between the target host plant and the existing AM fungal mycelial network. The chamber was then incubated for one month. This achieved host-mediated overexpression of the AM fungus RiEFa / green fluorescent protein fusion gene within the mycorrhizal chamber. Specifically, the target host plant carrying the overexpressed AM fungus gene, through the integration of the AM fungal mycelial network established in the aforementioned steps and the symbiotic interaction with the plant, enabled the mRNA molecules of the overexpressed RiEFa / green fluorescent protein fusion gene within the target host root system to efficiently cross the arbuscular interface and accumulate excessively in the arbuscules during the high-speed exchange of nutrients between the root cells and the AM fungal arbuscules.

[0057] Example 3 Verification of overexpression of AM fungal genes in target host plants

[0058] The target host plant and its roots, along with the soil surrounding them, were excavated from step S4 of Example 2. The roots were then soaked in an ice-water mixture to loosen the soil. The roots were then cleaned with the ice-water mixture three to four times. The plant was then picked up with clean tweezers and placed in a 15-cm-diameter glass Petri dish. Due to the design of the RiEFa / green fluorescent protein fusion gene, the presence of green fluorescent protein emission in the arbuscular symbiosis could be confirmed using a fluorescence microscope, verifying the successful overexpression of the RiEFa / green fluorescent protein fusion gene in the arbuscular symbiosis.

[0059] The results are as follows Figure 3 As shown, by fluorescence microscopy, Figure 3 a is a diagram showing the green fluorescent protein (GFP) signal in the living mycorrhizal symbiotic root system. Figure 3 b is the arbuscular structure localization map determined by using AF488WGA fluorescent dye (Nanjing Wobo Biotechnology, Wheat Germ Agglutinin Alexa Fluor 488, Product No. DU-035). Figure 3 a and Figure 3 From the correspondence between the branch structure positions in b, it is confirmed that a green fluorescent signal exists in the arbuscular symbiotic structure of the living mycorrhizal symbiotic root system, which proves that the green fluorescent protein is brightly expressed in the arbuscular structure, indicating that the above-mentioned device and method of the present invention can achieve host-mediated overexpression of the AM fungal gene RiEFa and the green fluorescent protein fusion gene in the fungus.

[0060] Example 1 Target host plant-mediated overexpression of AM fungal gene using conventional pots

[0061] Surface-sterilized Chrysopogon zizanioides seeds were planted in conventional pots (square, 20 cm long, 20 cm wide, i.e., no mycelium chamber with high phosphorus nutrient solution provided) and inoculated with Rhizophagus irregularis inoculum, and other culture conditions were performed as in Example 2.

[0062] Positive hairy root transformed plants overexpressing AM fungal genes (Medicago truncatula with overexpressed AM fungal RiEFa and green fluorescent protein fusion gene) were obtained using the plant hairy root transformation method in Example 2.

[0063] The target host plant Medicago truncatula was transplanted into conventional pots planted with Chrysopogon zizanioides according to the method in Example 2, and the aboveground part of Chrysopogon zizanioides was reduced by 1 / 2 to 3 / 4 using scissors to reduce the growth vigor of Chrysopogon zizanioides, and cultured for 1 month. The overexpression of AM fungal genes in the mycelium of the target host plant was verified using the method in Example 3.

[0064] The results are shown in Figure 4 Figures Figure 4 a is a green fluorescent protein (GFP) signal display image in the living mycorrhizal symbiotic root system, Figure 4 b is a figure showing the location of the arbuscular structure determined by AF488WGA fluorescent dye (Wheat Germ Agglutinin Alexa Fluor 488, Alexa Fluor 488, item number DU-035, Nanjing Wobio Biological Technology), according to Figure 4 a and Figure 4 b, and the correspondence of the arbuscular structure location, it was further confirmed that there was no green fluorescent signal in the arbuscular symbiotic structure of the living mycorrhizal symbiotic root system, i.e., the green fluorescent protein was not highly expressed in the arbuscular structure.

[0065] In summary, Examples 3 and Comparative Example 1 show that the overexpression of AM fungal gene RiEFa and green fluorescent protein fusion gene in the mycelium using the device and method of the present application can be achieved using host-mediated AM fungal gene RiEFa and green fluorescent protein fusion gene.

Claims

1. A method for accelerating the exchange of substances between arbuscular mycorrhizal fungi and target host plants at the symbiotic interface and achieving host-mediated overexpression of arbuscular mycorrhizal fungal genes in arbuscular mycorrhizal fungi based on the HIGS strategy, characterized in that: The steps include: S1. Adding a sterilized substrate to the mycorrhizal chamber (1) of the apparatus for host-mediated arbuscular mycorrhizal fungal gene overexpression, and adding a nutrient solution for mycelial growth to the mycelial chamber (2); S2. Planting the companion plant in the mycorrhizal chamber (1) and inoculating the arbuscular mycorrhizal fungi agent, pouring a nutrient solution for plant growth into the mycorrhizal chamber (1), and symbiotically cultivating the arbuscular mycorrhizal fungi and the companion plant to establish a stable symbiotic relationship, thereby achieving the purpose of accelerating nutrient exchange under the symbiotic relationship between the arbuscular mycorrhizal fungi and the plant; S3. The hairy root transformed plant with overexpressed AMF genes is used as the target host plant and transferred to the mycorrhizal chamber (1). 1 / 2 to 3 / 4 of the aboveground part of the companion plant is cut off with scissors and symbiotically cultivated to establish a symbiotic relationship between the target host plant and the AMF. The mRNA molecules overexpressed in the target host root system can efficiently pass through the AMF interface during the high-speed material exchange between the root cells and the AMF and be over-accumulated and expressed in the AMF. The device for host-mediated arbuscular mycorrhizal fungal gene overexpression comprises two layers, the upper layer being a mycorrhizal chamber (1) and the lower layer being a mycorrhizal chamber (2); a plurality of mycorrhizal pores (103) are provided at the bottom of the mycorrhizal chamber (1), through which mycelia can reach the mycorrhizal chamber (2) to absorb nutrients; a support net (102) is provided above the mycorrhizal pores (103), and a nylon net (101) is provided above the support net (102); the aperture of the nylon net (101) allows mycelia to pass through but restricts the passage of plant roots.

2. The method according to claim 1, characterized in that The pore size of the nylon mesh (101) is 40-45 μm.

3. The method according to claim 1, characterized in that The pore size of the support net (102) is 0.5-0.8 cm.

4. The method according to claim 1, characterized in that The mycelium pores (103) have a pore diameter of 2 to 5 cm.

5. The method according to claim 1, characterized in that: A support column (201) for supporting the mycorrhizal chamber (1) is provided in the mycelium chamber (2).

6. The method according to claim 1, characterized in that The nutrient solution for plant growth in step S2 is a low-phosphorus nutrient solution; the phosphate concentration in the low-phosphorus nutrient solution is 80-120 μM, and the pH is 5.5-5.

7.

7. The method according to claim 1, characterized in that The nutrient solution for mycelium growth in step S1 is a high-phosphorus nutrient solution; the phosphate concentration in the high-phosphorus nutrient solution is 1-3 mM and the pH is 5.1-5.

3.

8. The method according to claim 1, characterized in that: The symbiotic culture conditions are to culture under a light condition of 24,000 to 26,000 lux and ensure the water required for growth.

9. The method according to claim 1, characterized in that: The liquid level of the nutrient solution for mycelial growth in step S1 should be 1 to 2 cm lower than the bottom of the mycorrhizal chamber (1).

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