Device and method for host-mediated arbuscular mycorrhizal fungus gene overexpression

Abstract

The invention discloses a device and a method for host-mediated arbuscular mycorrhizal fungus gene overexpression, the device comprises a mycorrhizal chamber and a hypha chamber which are arranged up and down, the bottom of the mycorrhizal chamber is provided with hypha holes, and a support net and a nylon net are padded above the hypha holes to allow hypha to pass but limit plant root systems to pass. Cultivating associated plants in a mycorrhizal room, inoculating AM fungus fungicide, and irrigating with a low-phosphorus nutrient solution; a high-phosphorus nutrient solution is injected into the hypha chamber, and due to the fact that only hyphae can absorb the high-phosphorus nutrient solution, mutualism between AM fungi and associated plants and high-speed exchange of symbiotic interface nutrients are enhanced. Then, a target host plant over-expressing AM fungal genes is introduced, so that the target host plant is added into the symbiotic network which is subjected to high-speed material exchange with AM fungal hyphae. Under the symbiotic relationship, a large amount of mRNA overexpressed in the root system of the target host plant enters the arbuscular mycorrhizal fungi through an arbuscular interface and is overexpressed. The method provides a new thought for overexpression of AM fungal genes.

Description

Technical Field

[0001] The 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 host plants absorb water, mineral nutrients, and improve host plants' tolerance to adversity. Since AM fungi cannot survive stably alone, they must establish a symbiotic relationship with the host to complete their own life history. Therefore, at present, the living symbiosis between AM fungi and various mycorrhizal plants is still used to obtain a large number of AM fungal propagules, which are then used to infect plants, so that plants can achieve better yield increases and improved quality.

[0003] In recent years, with the in-depth study of the symbiotic mechanism between AM fungi and plants, researchers have found that in the process of AM fungi improving the stress resistance of host plants, a series of fungal signaling pathways and the participation of some fungal genes themselves are activated, that is, the transcription factors and functional genes of these AM fungi play an indispensable role in the coordinated stress resistance of AM fungal symbionts. In order to further explore the specific role of these genes, researchers began to use the research ideas of plant gene functions to conduct heterologous functional analysis of these transcription factors or functional genes. At the same time, with the in-depth study of symbiotic interactions and the infection of host plants by pests and diseases, people have found that small RNAs can be exchanged at the symbiotic interface or the interface where pathogens interact with host plants. Then, the host-induced gene silencing (HIGS) technology came into being. HIGS has been proven to be able to achieve gene silencing 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. That is, RNA interference is achieved by using small RNAs that shuttle through the interaction interface, thereby specifically reducing or silencing gene expression in pathogens, pests, or AM fungi, and achieving gene silencing in these organisms. During the symbiotic interaction of AM fungi, only a small amount of small RNA shuttles through the interaction interface, and a small amount of small RNA with a hairpin structure based on the RNA interference silencing principle can efficiently achieve the silencing of 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 AM fungal symbionts.

[0004] Due to the fact that AM fungi cannot survive stably alone, AM fungal strains that overexpress AM fungal genes cannot be obtained through conventional technical means. At present, although HIGS technology can achieve silencing of AM fungal target genes, there is no application for overexpression of AM fungal target genes. This may be because only a small amount of RNA shuttles through the interaction interface between AM fungi and plants, making it difficult for conventional HIGS strategies to achieve overexpression of AM fungal genes in the fungus. That is, there is currently no technology that can achieve efficient shuttle of RNA molecules at the symbiotic interface and accumulation in large quantities in the fungus. Patent CN106190944A discloses a method for enriching arbuscular mycorrhizal fungal spores by layered culture, in which a seedling culture container is divided 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 does not have a continuous water and fertilizer supply setting, no water 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, thereby limiting the growth of hyphae and making it difficult for host plants carrying AM fungal genes to quickly join the AM fungal symbiotic network that exchanges nutrients with plants at a high speed, that is, 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, the gravitropism of the root system is utilized to prevent the plant 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 further 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 shortcomings 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 by using the above-mentioned device and realizing host-mediated overexpression of arbuscular mycorrhizal fungi 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 invention provides a device for host-mediated arbuscular mycorrhizal (AM) fungal gene overexpression. The device comprises an upper and lower layer, wherein the upper layer is a mycorrhizal chamber and the lower layer is a hyphae chamber. A plurality of hyphae holes are arranged at the bottom of the mycorrhizal chamber, through which AM fungal hyphae can reach the mycorrhizal chamber to absorb nutrients. A support net is provided above the mycorrhizal holes, and a nylon net is provided above the support net. The aperture of the nylon net allows hyphae to pass through but restricts plant roots from passing through.

[0009] The device described in the present invention can accelerate the material exchange between AM fungi and target host plants at the symbiotic interface and establish a stable symbiotic relationship. It can also achieve host-mediated overexpression of AM fungal genes in AM fungi based on the HIGS strategy, 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 of the present invention includes a mycorrhizal chamber and a hyphae chamber placed up and down, the mycorrhizal chamber is used for co-cultivation of the target host plant and AM fungi, and the hyphae chamber is used to provide a continuous water and fertilizer environment that is convenient for AM fungal hyphae to absorb; hyphae holes are provided at the bottom of the mycorrhizal chamber, and a support net and a nylon net are placed above the hyphae holes. The above design allows hyphae to pass through but restricts the passage of plant roots. The mycorrhizal chamber and hyphae chamber stacked up and down can provide stable water vapor pressure conditions, which is conducive to the spread of AM fungal hyphae from the mycorrhizal chamber to the hyphae chamber, and continuously absorb water and fertilizer from the hyphae chamber to exchange nutrients with the plant roots in the mycorrhizal chamber at a high speed, that is, a stable symbiotic relationship is formed, and at the same time, the material exchange between AM fungi and the target host plant at the symbiotic interface is accelerated, which is conducive to the large-scale shuttling of RNA molecules in the root system at the symbiotic interface. The device was further used to cultivate companion plants in the mycorrhizal chamber and inoculate AM fungal agents, and water low-phosphorus nutrient solution; high-phosphorus nutrient solution was injected into the mycelium chamber, and the mutualism between AM fungi and companion plants was strengthened by taking advantage of the characteristic that only mycelium absorbs high-phosphorus nutrient solution, thereby establishing a symbiotic network of high-speed nutrient exchange between AM fungi and companion plants. Subsequently, hairy roots that overexpress AM fungal genes were introduced to transform the target host plants. By reducing the growth potential of the original companion plants, the target host plants were joined to the symbiotic network of high-speed nutrient exchange established by AM fungi and plants, and formed a stable symbiotic relationship with the AM fungal mycelium network. Under this symbiotic relationship, the overexpressed mRNA in the root system of the target host plant entered the AM fungi in large quantities through the arbuscule interface and accumulated excessively, thereby achieving overexpression. This method provides a new idea for overexpression of AM fungal genes.

[0011] Furthermore, the pore size of the nylon mesh is 40-45 μm.

[0012] Furthermore, the pore size of the support mesh is 0.5-0.8 cm.

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

[0014] Furthermore, the diameter of the mycelium pores is 2 to 5 cm.

[0015] Furthermore, a support column for supporting the mycorrhizal chamber is provided in the mycelium chamber.

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

[0017] Preferably, the hard material is plastic.

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

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

[0020] S2. Planting the companion plants in the mycorrhizal chamber and inoculating the AM fungal agent, pouring nutrient solution for plant growth into the mycorrhizal chamber, and symbiotically cultivating, so that the AM fungi and the companion plants establish a stable symbiotic relationship, and achieve the purpose of accelerating nutrient exchange under the symbiotic relationship between the AM fungi and the plants;

[0021] S3. The transformed plants with hairy roots overexpressing AM fungal genes are used as target host plants, transferred into the mycorrhizal chamber, and 1 / 2 to 3 / 4 of the aboveground parts of the companion plants are cut off with scissors for symbiotic cultivation to establish a symbiotic relationship between the target host plants and the AM fungi, so that the mRNA molecules overexpressed in the root system of the target host can efficiently pass through the arbuscular interface and be over-accumulated and expressed in the AM fungi during the high-speed material exchange between the root cells and the arbuscular interface of the AM fungi.

[0022] Furthermore, the symbiotic cultivation time is 2 to 4 months.

[0023] Preferably, the symbiotic cultivation time is 3 months.

[0024] Furthermore, the symbiotic culture condition is to cultivate under a light condition of 24,000 to 26,000 lux, and in addition to watering with nutrient solution for plant growth, tap water is also required for daily watering of plant seedlings.

[0025] Furthermore, 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, in step S1, the liquid level of the nutrient solution for mycelium growth should be 1 to 2 cm lower than the bottom of the mycorrhizal chamber.

[0032] Furthermore, during the symbiotic culture, the concentration of the nutrient solution for mycelium growth in the mycelium chamber must be ensured and replaced in time.

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

[0034] The mycorrhizal chamber and hyphae chamber superimposed on each other in the device of the present invention can provide stable water vapor pressure conditions, which is conducive to the spread of AM fungal hyphae from the mycorrhizal chamber to the hyphae chamber, and continuously absorb water and fertilizer from the hyphae chamber to exchange nutrients with the plant roots in the mycorrhizal chamber at a high speed, that is, forming a stable symbiotic effect, while accelerating the material exchange between AM fungi and target host plants at the symbiotic interface, which is conducive to the large-scale shuttling of RNA molecules in the root system at the symbiotic interface. Further using the device of the present invention, first establish a symbiotic network system in which AM fungi and conventional mycorrhizal plants exchange nutrients at a high speed at the symbiotic interface; then introduce the target host plant, and through the strategy of reducing the growth potential of the original mycorrhizal plant, the target host plant is quickly added to the symbiotic network in which nutrients are exchanged at a high speed with the AM fungi at the symbiotic interface, so that the mRNA molecules of the AM fungal genes overexpressed in the root system of the target host plant can be shuttled into the AM fungi in large quantities, that is, the purpose of overexpression of AM fungal genes mediated by the target host plant in the AM fungi is achieved. The present invention overcomes the defects that a small amount of RNA in the AM symbiotic interface shuttles across the interaction interface and the conventional HIGS strategy is difficult to achieve overexpression of AM fungal genes in the fungus, and also lays a solid foundation for using AM fungal key genes in the future to enhance the tolerance of AM fungal symbionts under environmental stress. In addition, the device of the present invention has a simple structure, is easy to use, can be handmade, and has low cost. 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 arbuscular mycorrhizal fungal gene overexpression strategy.

[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 display diagram of 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 the arbuscular structure using fluorescent dyes (white arrows indicate the location of the arbuscular structure).

[0038] Figure 4 This is a 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 display diagram of 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 the arbuscular structure using fluorescent dyes (white arrows indicate the location of the arbuscular structure).

[0039] Figure caption: 1- mycorrhizal chamber, 2- mycelium chamber, 101- nylon net, 102- support net, 103- mycelium hole, 201- support column. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. 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 are 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", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", "third", "fourth", etc. are only used for descriptive purposes and cannot be understood 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 As shown, an application device based on the host-mediated arbuscular mycorrhizal fungal gene overexpression strategy is provided. The device is a two-layer space formed by stacking two square containers, the upper layer is a mycorrhizal chamber 1, and the lower layer is a mycorrhizal chamber 2; the mycorrhizal chamber 1 is provided with a plurality of equidistant small holes at the bottom to form mycorrhizal holes 103, through which mycelia can reach the mycorrhizal chamber 2 to absorb nutrients, a support net 102 is padded above the mycorrhizal holes 103, and a nylon net 101 is padded above the support net 102; a support column 201 for supporting the mycorrhizal chamber 1 is provided inside the mycorrhizal chamber 2. The radius of the mycorrhizal hole 103 is 2 cm, the mesh gap of the support net 102 is 0.5 cm, and the pore size of the nylon net is 45 μm.

[0045] The device comprises a mycorrhizal chamber 1 and a hyphae 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 hyphae chamber 2 is used for providing high-phosphorus water and fertilizer conditions that can be absorbed by hyphae. A hyphae hole 103 is provided at the bottom of the mycorrhizal chamber 1, and a support net 102 is padded above the hyphae hole 103. A nylon net 101 is padded on the support net 102. Since the material of the nylon net 101 is relatively soft, when a culture matrix 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 provided under the nylon net 101 for support. The aperture design of the device allows hyphae to pass through but restricts the passage of plant roots. The mycorrhizal chamber 1 and mycelium chamber 2 stacked up and down can provide stable water vapor pressure conditions, which is conducive to the AM fungal hyphae spreading from the mycorrhizal chamber 1 to the mycelium chamber 2, and continuously absorbing water and fertilizer from the mycelium chamber 2 to exchange nutrients with the plant roots in the mycorrhizal chamber 1 at a high speed, thus forming a stable symbiotic relationship, while accelerating 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 hosts at the symbiotic interface and achieving overexpression of arbuscular mycorrhizal fungal genes in fungi

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

[0048] S1. Take two clean, alcohol-sterilized white rectangular hard plastic containers with a length of 38 cm, a width of 30 cm, and a height of 12 cm. Open 21 small holes with a radius of 2 cm at an interval of 1.25 cm at the bottom of one of the containers, with three rows of 7 small holes each, as the mycorrhizal chamber. First, place a layer of plastic support net with a mesh gap of 0.5 cm at the bottom of the container with small holes, and then cover the bottom of the container with a 45μm nylon net to prevent plant roots from entering the mycelial chamber. Fill the mycorrhizal chamber with the substrate (sand and vermiculite, volume ratio of 1:1) treated by a high-pressure high-temperature sterilizer (121°C, 120min).

[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 overlap of the two is the mycelium chamber, and a black hard plastic cylindrical support column (bottom radius of 2 cm, height of 5 cm) is arranged inside the mycelium chamber.

[0050] S3. Plant the surface-sterilized Chrysopogon zizanioides seeds in the mycorrhizal chamber and inoculate the arbuscular mycorrhizal fungus, Rhizophagus irregularis, for 2 months under the conditions of 25,000 lux of light and 28 degrees Celsius of air temperature to ensure that the vetiver is in a good growth state, so that the AM fungi can establish a stable symbiotic relationship with it. During the 2-month cultivation period, pour low-phosphorus nutrient solution into the mycorrhizal chamber every 15 days, and inject high-phosphorus nutrient solution into the mycelium chamber 2 with a syringe. The nutrient solution level in the mycelium chamber 2 is 1 to 2 cm lower than the bottom of the mycorrhizal chamber container in step 2. The phosphate concentration in the low-phosphorus nutrient solution, i.e., the Hoagland nutrient solution, is 100 μM, and the pH is 5.6. The phosphate concentration in the high-phosphorus nutrient solution, i.e., the Hoagland nutrient solution, is 2 mM, and the pH is 5.2. After 2 months of cultivation, the symbiotic relationship between arbuscular mycorrhizal fungi (Heteromorpha) and vetiver was strengthened. That is, the vetiver root system could not absorb enough phosphorus nutrients in the mycorrhizal chamber, and the 45μM nylon mesh barrier existed. Therefore, the vetiver root system could not absorb nutrients from the mycelium chamber. It needed the hyphae of the symbiotic AM fungi (Heteromorpha) to absorb a large amount of phosphorus nutrients from the mycelium chamber and exchange nutrients with it, so as to achieve the purpose of accelerating nutrient exchange under the symbiotic relationship between AM fungi and plants.

[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 rhizosporon into the vector, and the constructed vector was transferred into the rhizogenes Agrobacterium (Agrobacterium rhizogenes) ARqua1 strain for transformation of M. truncatula hairy roots:

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

[0053] (2) Agrobacterium rhizogenes was cultured at 30°C and 200 rpm for 12 h, and then taken out and 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 a 30°C incubator for 48 h. A blade was used to smoothly cut the root of Medicago truncatula (3 mm near the embryo) to produce a wound in the clean bench, and then the wound surface was inoculated with Agrobacterium rhizogenes on the culture plate for infection;

[0054] (3) After the infection is complete, the Medicago truncatula is spread on a culture dish containing a plant callus sprouting medium (sprouting 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 (the alfalfa roots should be inserted into the medium) in a clean bench. 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 hairy root transformed plant with overexpression of AM fungus RiEFa and green fluorescent protein fusion gene) was transplanted into mycorrhizal chamber 1, and 1 / 2 to 3 / 4 of the aboveground part of vetiver grass was cut off with scissors to reduce the growth potential of vetiver grass, thereby reducing the competition between the companion plant vetiver grass and the target host plant Medicago truncatula, and then accelerating the establishment of AM symbiotic relationship between the target host plant Medicago truncatula and the existing AM fungal mycelial network, and culturing for 1 month. In this way, the host-mediated overexpression of AM fungus RiEFa and green fluorescent protein fusion gene in the fungus body can be achieved, that is, the target host plant carrying overexpressed AM fungus gene can achieve the target host plant by adding the network of symbiotic interaction between the AM fungal mycelial network and the plant with high-speed nutrient exchange established in the above steps, so that the mRNA molecules of the overexpressed RiEFa and green fluorescent protein fusion gene in the root system of the target host can efficiently pass through the arbuscular interface and accumulate excessively in the arbuscular interface during the high-speed material exchange process between the root cells and the AM fungal arbuscular interface.

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

[0058] The target host plant and its roots in step S4 of Example 2 were dug out together with the root soil, and the roots of the target host plant were soaked in an ice-water mixture to soften the root soil, and then the roots were cleaned with the ice-water mixture, and repeated 3 to 4 times. The plant was clamped with clean tweezers and collected in a glass culture dish with a diameter of 15 cm. Due to the design of the RiEFa and green fluorescent protein fusion gene, it was possible to confirm whether green fluorescent protein luminescence existed in the arbuscular symbiotic structure by fluorescence microscopy, that is, to verify the success of overexpression of the RiEFa and green fluorescent protein fusion gene in the arbuscular structure.

[0059] The results are as follows Figure 3 As shown, by fluorescence microscopy, Figure 3 a is a display diagram of the green fluorescent protein (GFP) signal in the living mycorrhizal symbiotic root system. Figure 3 b is the localization map of arbuscular structure determined by using AF488WGA fluorescent dye (Nanjing Wobo Biotechnology, Wheat Germ Agglutinin Alexa Fluor 488, Catalog No. DU-035). Figure 3 a and Figure 3 From the correspondence between the branch structure positions in b, it is further confirmed that there is a green fluorescent signal in the arbuscular symbiotic structure of the living mycorrhizal symbiotic root system, which proves that the green fluorescent protein is highlighted 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] Comparative Example 1: Using conventional flowerpots to plant target host plants to mediate arbuscular mycorrhizal fungal gene overexpression

[0061] Surface-sterilized Chrysopogon zizanioides seeds were planted in conventional flowerpots (square, 20 cm long and 20 cm wide, i.e., without a mycelium chamber provided with a high-phosphorus nutrient solution) and inoculated with the arbuscular mycorrhizal fungus, Rhizophagus irregularis. Other culture conditions were the same as those in Example 2.

[0062] The method for plant hairy root transformation in Example 2 was used to obtain positive hairy root transformed plants overexpressing AM fungal genes (Medicago truncatula having a fusion gene of overexpressing AM fungal RiEFa and green fluorescent protein).

[0063] The target host plant Medicago truncatula was transplanted into a conventional flower pot for growing vetiver according to the method in Example 2, and 1 / 2 to 3 / 4 of the aboveground part of the vetiver was cut off with scissors to reduce the growth potential of the vetiver, and then cultured for 1 month. The method in Example 3 was then used to verify the overexpression of the AM fungal gene in the fungus of the target host plant.

[0064] The results are as follows Figure 4 As shown, by fluorescence microscopy, Figure 4 a is a display diagram of green fluorescent protein (GFP) signal in the living mycorrhizal symbiotic root system. Figure 4 b The localization map of the arbuscular structure was determined by using AF488WGA fluorescent dye (Nanjing Wobo Biotechnology, Wheat Germ Agglutinin Alexa Fluor 488Alexa Fluor488, Catalog No. DU-035). Figure 4 a and Figure 4 In b, the correspondence between the branch structure positions further confirmed that there was no green fluorescence signal in the arbuscular symbiotic structure of the living mycorrhizal symbiotic root system, that is, the green fluorescent protein was not brightly expressed in the arbuscular structure.

[0065] Combining Example 3 and Comparative Example 1, the results show that the 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.

Claims

1. A device for host-mediated arbuscular mycorrhizal fungal gene overexpression, characterized in that: The invention comprises two layers, the upper layer is a mycorrhizal chamber (1), and the lower layer is a mycorrhizal chamber (2); a plurality of mycorrhizal holes (103) are arranged at the bottom of the mycorrhizal chamber (1), and mycorrhizal can reach the mycorrhizal chamber (2) through the mycorrhizal holes (103) to absorb nutrients; a support net (102) is provided above the mycorrhizal holes (103), and a nylon net (101) is provided above the support net (102); the aperture of the nylon net (101) allows mycorrhizal passage but restricts the passage of plant roots.

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

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

4. The device according to claim 1, characterized in that: The diameter of the mycelium pores (103) is 2 to 5 cm.

5. The device 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. A method for accelerating the material exchange between arbuscular mycorrhizal fungi and target host plants at the symbiotic interface using the device described in any one of claims 1 to 5 and realizing host-mediated overexpression of arbuscular mycorrhizal fungi 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 any one of claims 1 to 5, and adding a nutrient solution for mycelium growth to the mycelium 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), symbiotically cultivating, so that the arbuscular mycorrhizal fungi and the companion plant establish a stable symbiotic relationship, and achieving the purpose of accelerating nutrient exchange under the symbiotic relationship between the arbuscular mycorrhizal fungi and the plant; S3. The hairy root transformed plants with overexpressed arbuscular mycorrhizal fungi genes are used as target host plants and transferred into the mycorrhizal chamber (1). The aboveground parts of the companion plants are cut off by scissors by 1 / 2 to 3 / 4, and symbiotically cultivated to establish a symbiotic relationship between the target host plants and the arbuscular mycorrhizal fungi. The mRNA molecules overexpressed in the root system of the target host can efficiently pass through the arbuscular interface during the high-speed material exchange between the root cells and the arbuscular mycorrhizal fungi, and be over-accumulated and expressed in the arbuscular mycorrhizal fungi.

7. The method according to claim 6, 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.

8. The method according to claim 6, 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.

9. The method according to claim 6, characterized in that: The symbiotic culture condition is to culture under a light condition of 24000-26000 lux and ensure the water required for growth.

10. The method according to claim 6, characterized in that: In step S1, the liquid level of the nutrient solution for mycelium growth should be 1 to 2 cm lower than the bottom of the mycorrhizal chamber (1).

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