Application of GmTINY gene in enhancing soybean cyst nematode resistance of plant

Abstract

The invention belongs to the technical field of biology, and particularly provides application of a GmTINY gene in enhancing nematode resistance of plants, and a CDS region coding sequence of the GmTINY gene is as shown in SEQ ID NO: 2. The invention provides the application of the GmTINY gene in improving the soybean cyst nematode resistance of the plant, and particularly, the nematode resistance of the plant is enhanced by up-regulating the expression of the GmTINY gene in the plant, so that the soybean cyst nematode resistance effect of the plant is effectively improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the use of the GmTINY gene in enhancing plant disease resistance. Background Art

[0002] Soybean Cyst Nematode (SCN) is one of the main pathogens in soybean production globally and has a profound impact on the soybean industry. As a parasitic nematode, SCN invades the soybean roots, disrupts their water and nutrient absorption functions, and thus affects the normal growth of the plants. Its specific harms are manifested as stunted growth of soybean plants, yellowing of leaves, dwarfing of plants, and even whole-plant death in severe cases. Globally, soybean cyst nematode disease is one of the most serious diseases causing soybean yield losses. According to research, in areas severely infected with SCN, the yield loss rate can be as high as 30%. In addition, SCN indirectly affects the stability of the global soybean supply chain by reducing the market value of soybeans and increasing production costs, posing a long-term threat to the global soybean market.

[0003] With global climate change and the transformation of agricultural planting methods, the spread and harm of SCN are becoming increasingly serious, threatening not only agricultural production but also bringing non-negligible challenges to the global soybean supply chain.

[0004] Currently, the control strategies for soybean cyst nematode mainly rely on the breeding of disease-resistant varieties and crop rotation in farmland. However, with the increasing diversity and adaptability of the nematode population, the resistance of disease-resistant varieties gradually weakens. Crop rotation, as a traditional control method, also faces problems such as the limitation of crop planting cycles and the unreasonable allocation of land resources, making it difficult to maintain stable control effects in the long term. The continuous pressure on food and oilseed supplies globally further highlights the urgency and necessity of developing more efficient and durable nematode-resistant breeding strategies. Ensuring the sustainable development of soybean production and controlling the outbreak of major diseases such as SCN have become one of the urgent problems to be solved in the global agricultural science and technology field.

[0005] In the research and development field of soybean cyst nematode control, scientists are constantly exploring and innovating nematode-resistant breeding technologies, aiming to cultivate new soybean varieties with stronger disease resistance through genetic engineering, molecular breeding and other means. These new varieties can not only effectively cope with the invasion of SCN but also adapt to different growth environments, improving the yield and quality of soybeans. In recent years, with the rapid development of biotechnology, nematode-resistant breeding strategies based on gene editing and transgenic technologies have gradually become the focus of research. By regulating the gene expression related to disease resistance in plants, it is expected to significantly enhance the resistance of soybeans to SCN, thereby reducing the economic losses caused by diseases.

[0006] In addition, the use of modern molecular biology techniques to explore and functionally validate soybean disease-resistant genes is also a hot topic in the current agricultural research field. Through in-depth analysis of the soybean genome, researchers can identify key genes related to SCN resistance and use gene editing techniques to improve the functions of these genes, enabling them to play a stronger disease-resistant role in soybean plants.

[0007] In summary, against the backdrop of numerous challenges faced by the global soybean industry, the development of innovative nematode-resistant technologies and the cultivation of new varieties with strong disease resistance have become an urgent need in the current agricultural science and technology field. Through effective nematode-resistant breeding strategies, not only can the yield and quality of soybeans be improved, losses caused by diseases be reduced, but also the cost of international soybean trade can be lowered, enhancing China's competitiveness in the global soybean market and promoting the sustainable development of the soybean industry. In the future, through in-depth research on soybean disease-resistant genes and the wide application of modern breeding techniques, it is expected to provide more efficient and lasting solutions for the global soybean industry, ensuring the long-term healthy development of this important crop. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a method for effectively enhancing the ability of soybeans to resist cyst nematodes, so as to solve the problem of insufficient effectiveness in the prior art.

[0009] To solve the above technical problem, the present invention provides the use of the GmTINY gene in enhancing plant nematode resistance, and the CDS region coding sequence of the GmTINY gene is as described in SEQ ID NO:2.

[0010] Note: The sequence of the GmTINY gene is from:

[0011] https: / / phytozome-next.jgi.doe.gov / report / gene / Gmax_Wm82_a6_v1 / Glyma.01G147600.

[0012] As an improvement to the use of the present invention: enhancing the nematode resistance of the plant by overexpressing the GmTINY gene in the plant, or reducing the nematode resistance of the plant by silencing the GmTINY gene in the plant.

[0013] As a further improvement to the use of the present invention: the plant is soybean.

[0014] The present invention also simultaneously provides a method for enhancing plant nematode resistance, by overexpressing the GmTINY gene to regulate the expression of resistance genes GmAAT Rhg1 , GmSNAP18 and other resistance-related genes, thereby enhancing the nematode resistance of the plant.

[0015] As an improvement to the method of the present invention: The GmTINY gene functions by binding to the ERELEE4 cis-acting element in the promoter region of the soybean cyst nematode resistance-related gene Rhg1.

[0016] Note: The promoter region of the Rhg1 major resistance gene is the promoter region of the soybean cyst nematode resistance-related gene.

[0017] As a further improvement to the method of the present invention: The expression of the GmTINY gene is regulated by the soybean hairy root transformation method to enhance plant nematode resistance.

[0018] As a further improvement to the method of the present invention: The GmTINY gene is overexpressed or silenced through the following steps: constructing a transient overexpression vector of the GmTINY gene using the pAGM4673 vector, or constructing an RNA interference (RNAi) vector using the pGRNAi2 vector, and transforming the above vectors into the cotyledons of Williams 82 (Wm 82) and Forrest soybeans through the Agrobacterium rhizogenes ARqua1 strain to induce the generation of transgenic hairy roots, so as to achieve overexpression or silencing of the GmTINY gene in soybeans.

[0019] As a further improvement to the method of the present invention: The expression level of the GmTINY gene is verified by real-time quantitative PCR (RT-qPCR);

[0020] Soybean hairy roots with successfully overexpressed or silenced GmTINY gene are screened by fluorescence;

[0021] The transformed soybean hairy roots are inoculated with the J2 stage larvae of soybean cyst nematode (SCN) for resistance testing;

[0022] The number of nematodes infecting the soybean roots and their developmental stages are observed and recorded by the fuchsin staining method;

[0023] By analyzing the data of the number of nematode infections and developmental stages, the effect of overexpression or silencing of the GmTINY gene on soybean nematode resistance is evaluated.

[0024] Based on in-depth research and experiments, the present invention has made the following important discoveries and achievements:

[0025] First of all, through the study of the soybean cyst nematode (SCN) infection process, the present invention found that when SCN infects soybeans, the expression of the Rhg1 major resistance gene will be significantly up-regulated. To further explore the related mechanism, the inventor constructed a yeast one-hybrid library and used the Rhg1 major resistance gene GmAAT Rhg1Using the promoter regions of GmSNAP18 as bait to screen for specific host proteins, the inventors finally discovered that the soybean TINY gene is a potential interacting transcription factor. Through in vivo and in vitro experiments, the inventors further demonstrated that the TINY gene can bind to the ERELEE4 cis-acting element within the promoter regions of GmAAT Rhg1 and GmSNAP18 genes, activate the expression of these resistance genes, and thereby enhance the plant's resistance to SCN.

[0026] In the present invention, the inventors adopted the Agrobacterium rhizogenes-mediated hairy root transformation technique and successfully obtained soybean hairy roots overexpressing GmTINY. Through fluorescence screening and RT-qPCR verification, the inventors found that the overexpression of the GmTINY gene significantly inhibited the development of nematodes in the roots of Wm82 soybeans, and in the resistant cultivar Forrest, it further enhanced its ability to resist SCN. Secondly, the inventors verified the continuous expression of the GmTINY gene in soybeans by constructing stably transformed soybean plants and obtained T1 generation seeds. The overexpression level of the GmTINY gene was confirmed by RT-qPCR analysis. Subsequently, the results of the cyst inoculation experiment showed that the transgenic soybean plants overexpressing the GmTINY gene exhibited stronger resistance to SCN compared to the control group. To further study the function of the GmTINY gene, the inventors also conducted a silencing experiment on the GmTINY gene through the soybean hairy root transformation technique. The experimental results showed that after silencing the GmTINY gene, the susceptibility of the Wm82 soybean roots to SCN increased significantly. This result indicates that overexpressing the GmTINY gene can effectively enhance the nematode resistance of soybeans.

[0027] Based on the above research results, the present invention for the first time reveals the important role of the GmTINY gene in soybean resistance to SCN. Specifically, the GmTINY gene binds to the promoter region of the Rhg1 major resistance locus, activates the expression of resistance genes, and thereby enhances the resistance of soybeans to SCN. In addition, the inventors further verified the significant effect of GmTINY gene overexpression in enhancing soybean SCN resistance through stable transformation and hairy root transformation experiments. The technical method of the present invention provides a new approach for soybean disease-resistant breeding, has broad application prospects, and is suitable for popularization and use in agricultural production.

[0028] The present invention is not limited to soybeans and is applicable to a variety of plant species. Applicable plants include but are not limited to soybeans, tobacco, tomatoes, peppers, potatoes, rice, wheat, corn, cotton, etc. By regulating the expression of the TINY gene or its homologous family genes in these plants, the disease resistance of plants can be effectively enhanced, especially showing significant effects in improving the nematode resistance of crops such as soybeans.

[0029] In addition, the method of the present invention has great flexibility in enhancing the expression of target genes. Any known gene expression regulation method can be used to achieve an increase in the expression level of specific genes in plants, such as virus-mediated overexpression, transient overexpression mediated by hairy roots or Agrobacterium tumefaciens, or long-term overexpression by constructing stable transgenic plants. As long as the selected method can precisely and specifically upregulate the expression of the GmTINY gene or its homologous genes in plants, the effect of enhancing plant disease resistance can be achieved.

[0030] The additional features and advantages of the present invention will be further described in detail in the following specific implementation process and will be more clearly presented during the implementation process.

[0031] The beneficial effects of the present invention are mainly reflected in:

[0032] The present invention provides the application of the GmTINY gene in improving the ability of plants to resist soybean cyst nematodes. Specifically, by upregulating the expression of the GmTINY gene in plants, the nematode resistance of plants is enhanced, thereby effectively improving the effect of plants in resisting soybean cyst nematodes.

[0033] It should be emphasized that: as an important member of the DREB subfamily in the AP2 / ERF transcription factor gene family, this gene family plays an important role in promoting plant growth and improving plant immunity, but its role in the interaction between plants and plant parasitic nematodes has not been revealed. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The above and / or other aspects and advantages of the present invention will become more obvious and easier to understand by combining the following drawings to describe the embodiments, where:

[0035] Figure 1 shows the results of a yeast one-hybrid experiment, indicating that GmTINY can interact with the promoter region of the Rhg1 resistance gene. According to Figure 1 , it can be known that: both the GmTINY protein and the GmERF7 protein can interact with the promoter regions of the Rhg1 resistance genes GmAAT and GmSNAP18, but if the ERELEE4 element in the promoter region mutates, this interaction disappears.

[0036] Figure 2 shows the results of an electrophoretic mobility shift assay (EMSA) experiment:

[0037] A represents the binding of biotin-labeled ERE4 probe to GmTINY and GmERF7 proteins respectively; B represents the results of a cold probe competition experiment. Unlabeled ERE4 probes (50x, 100x, 200x) were used to compete with the biotin-labeled ERE4 probe for binding to the GmTINY protein.

[0038] According to Figure 2 , it can be known that the GmTINY protein interacts with the ERE4 probe, the GmERF7 protein does not interact with the ERE4 probe, and the interaction between the GmTINY protein and ERE4 can be weakened by cold probe competition.

[0039] Figure 3 It shows the significant expression of the GmTINY gene in soybeans on the 3rd day (3 dpi) after soybean cyst nematode infection.

[0040] Figure 4 It shows the effect of overexpression (OE) of GmTINY on the resistance of soybean hairy roots to SCN;

[0041] Figure 4 Among them: A represents a representative picture of fuchsin staining after SCN infects soybean hairy roots; B represents the statistical results of the proportion of J3, J4 stages and cyst numbers to the total number of infected nematodes.

[0042] Figure 5 It shows the effect of stable overexpression (OE) of GmTINY transgenic soybeans on SCN;

[0043] Figure 5 Among them: A represents a representative picture of root cysts after SCN infects transgenic soybeans; B represents the statistical results of the number of cysts per gram of roots.

[0044] Figure 6 It shows the effect of GmTINY gene silencing (RNAi) on the resistance of soybean hairy roots to SCN;

[0045] Figure 6 Among them: A represents a representative picture of fuchsin staining after SCN infects soybean hairy roots; B represents the statistical results of the proportion of J3, J4 stages and cyst numbers to the total number of infected nematodes.

[0046] Figure 7 It shows the RT-qPCR verification results, demonstrating significant changes in the transcriptional levels of GmTINY in GmTINY overexpression (OE), GmTINY gene silencing (RNAi) and control groups;

[0047] Figure 7 Among them: A represents the change in the transcriptional level of GmTINY in GmTINY overexpression (OE) hairy roots; B represents the change in the transcriptional level of GmTINY in GmTINY gene silencing (RNAi) hairy roots; C represents the change in the transcriptional level of GmTINY in 3 lines (#36, #185, #197) of GmTINY overexpression (OE) transgenic soybeans. Specific implementation manners

[0048] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0049] For the technologies or experimental conditions not specifically described in the embodiments, reference can be made to relevant technical literature in the field (such as "Molecular Cloning: A Laboratory Manual", Third Edition, edited by J. Sambrook et al. and translated by Huang Peitang et al., Science Press) or operations can be carried out according to the product specifications. Reagents or equipment without indicating the manufacturer are all conventional products and can be purchased from the market, for example, from Nanjing Novoprotein Co., Ltd.

[0050] Example 1

[0051] In this example, the inventors studied the interaction between the GmTINY gene and the promoter region of the Rhg1 resistance gene using the yeast one-hybrid experiment, and verified the role of GmTINY in regulating soybean resistance to soybean cyst nematode (SCN).

[0052] First, the inventors constructed a yeast one-hybrid system to detect the interaction between GmTINY and the promoter region of the Rhg1 major resistance gene. Rhg1 is a known major soybean SCN resistance locus, and the resistance genes it regulates include GmAAT Rhg1 and GmSNAP18. The promoter regions of these genes contain multiple cis-acting elements that may bind to transcription factors. In this experiment, the inventors selected the ERELEE4 element in the promoter region of the Rhg1 major resistance gene as the bait region for yeast one-hybrid screening to explore the ability of GmTINY to bind to it ( Figure 1 ).

[0053] The specific operation steps are as follows:

[0054] 1.1. Construction of the yeast one-hybrid bait plasmid

[0055] Clone the promoter region of the Rhg1 major resistance gene (SEQ ID NO:1) from the soybean genome, including the ERELEE4 element. Insert the promoter region fragment into the yeast one-hybrid vector pHIS2 ( Figure 1 as shown in pHIS2-pGmAAT Rhg1 and pHIS2-pGmSNAP18) to construct a bait plasmid containing the Rhg1 promoter region.

[0056] 1.2. Construction and screening of the yeast one-hybrid library

[0057] The inventors constructed an activation domain fusion plasmid containing the soybean GmTINY gene, and cloned the coding region of the GmTINY gene (SEQ ID NO:2) into the yeast expression vector pGADT7 ( Figure 1pGADT7-GmTINY shown below) as the library plasmid. Meanwhile, other activation domain plasmids (such as Figure 1 pGADT7-GmERF7 in the figure) were constructed for further verifying the binding of different transcription factors to the Rhg1 promoter region. The bait plasmid and the library plasmid were co-transformed into yeast strain Y187, and positive clones were screened on selective media in the following steps.

[0058] 1.3. Yeast transformation and interaction verification

[0059] The transformed yeast grew in the defective medium SD-Trp-Leu-His, and X-α-gal staining was used to screen yeast clones expressing blue markers to determine whether GmTINY interacted with the ERELEE4 element in the Rhg1 promoter region. The experimental results showed that yeast clones transfected with the plasmid containing GmTINY could grow normally on the selective medium and showed blue staining, indicating that the GmTINY gene could specifically bind to the ERELEE4 element in the Rhg1 promoter region and activate the expression of downstream resistance genes.

[0060] Note: The transformed yeast grew on SD-Trp-Leu medium (OD values of 1, 0.1, 0.01) and SD-Trp-Leu-His medium with 50 mM 3-AT (OD values of 1, 0.1, 0.01). If the yeast could grow normally on both media, it was considered that there was an interaction between the target protein and DNA.

[0061] 1.4. Analysis of interaction results

[0062] As Figure 1 shown, the yeast one-hybrid experiment results showed that yeast clones transfected with pGADT7-GmTINY and pHis2-pGmAAT _Rhg1 and pHis2-pGmSNAP18 plasmids grew normally on the selective medium (SD-Trp-Leu-His) and showed blue markers, indicating that the GmTINY protein could specifically bind to the ERELEE4 element in the promoter region of the Rhg1 major resistance gene. This binding activated the expression of the reporter gene, further verifying the function of GmTINY as a transcription factor. In summary, the experimental results showed that as a transcription factor, GmTINY could regulate GmAAT_ by specifically binding to the ERELEE4 element in the promoter region of the Rhg1 major resistance gene. Rhg1The expression of resistance genes such as GmSNAP18. This function may enhance the resistance of soybeans to SCN by increasing the expression level of resistance genes. This discovery reveals the potential role of GmTINY in the mechanism of soybean resistance to SCN, providing a new idea for improving soybean disease resistance by regulating the expression of the GmTINY gene.

[0063] 1.5. Control experiment

[0064] To ensure the accuracy of the experiment, the inventors set up a negative control experiment ( Figure 1 ), in which yeast clones without the plasmid containing the GmTINY gene failed to grow on the selective medium and no blue label appeared, further proving the specific binding of GmTINY to the Rhg1 promoter region.

[0065] Example 2

[0066] In this example, the inventors used electrophoretic mobility shift assay (EMSA) to further verify that the GmTINY transcription factor can specifically bind to the ERELEE4 cis-acting element in the promoter region of the Rhg1 resistance gene in vitro. Through this experiment, the direct physical interaction between GmTINY and the Rhg1 promoter region was clarified, providing evidence for revealing the molecular mechanism of GmTINY regulating the expression of genes related to soybean resistance to soybean cyst nematode (SCN) ( Figure 2 ).

[0067] 2.1. Cloning and labeling of the target fragment

[0068] According to the promoter sequence of the Rhg1 major resistance gene in the soybean genome (SEQ ID NO: 1), the region containing the ERELEE4 cis-acting element was selected, and the target fragment was designed and synthesized based on the sequence information of this region. The selected promoter fragment was biotin-labeled by Zhejiang Youkang Biotechnology Co., Ltd. for DNA-protein binding detection in EMSA. The biotin-labeled ERELEE4 fragment will be used as a probe for the binding experiment with the GmTINY protein.

[0069] 2.2. Expression and purification of GmTINY protein

[0070] Next, the present invention expresses the GmTINY protein through a prokaryotic expression system. The coding region of GmTINY (SEQ ID NO: 2) was cloned into the prokaryotic expression vector pET-28a and expressed in Escherichia coli. The GmTINY protein with a His tag was purified by nickel column affinity chromatography to obtain a high-purity recombinant GmTINY protein for subsequent in vitro binding experiments.

[0071] 2.3. Process of EMSA experiment

[0072] The purified GmTINY protein was incubated with a biotin-labeled ERELEE4 probe in a binding buffer (11 μL of ultrapure water, 2 μL of 10× binding buffer, 1 μL of 50% Glycerol, 1 μL of 1% NP-40, 1 μL of Poly(dI.dC), 2 μL of the probe, 2 μL of the protein) (the incubation conditions were 28 °C for 30 min), and then non-denaturing polyacrylamide gel electrophoresis was performed. Whether the GmTINY protein could bind to the ERELEE4 element in the Rhg1 promoter region was detected by the change in electrophoretic mobility. The binding reaction was monitored by the change in the mobility of the protein complex bound to the probe.

[0073] 2.4. Detection of binding results

[0074] After the electrophoresis was completed, the DNA-protein complex was detected by a membrane transfer and biotin probe imaging system. Figure 2 Result A showed that the GmTINY protein could form a specific DNA-protein complex with the biotin-labeled ERELEE4 probe, indicating that the GmTINY protein directly bound to the ERELEE4 element in the promoter region of the Rhg1 resistance gene in vitro. Compared with the control group without the addition of the GmTINY protein, an obvious change in mobility occurred in the reaction system with the GmTINY protein, showing a slower migration rate, further verifying the binding ability of the GmTINY protein to the ERELEE4 element.

[0075] 2.5. Competition experiment

[0076] To further verify the specificity of the binding, the inventors conducted a competition experiment. An excess of unlabeled ERELEE4 fragment was added to the reaction system as a competing probe. Figure 2 Result B showed that the unlabeled ERELEE4 probe could competitively inhibit the binding of the biotin-labeled probe to the GmTINY protein, resulting in the disappearance of the mobility shift complex, which further proved the specificity of the binding of the GmTINY protein to the ERELEE4 element.

[0077] 2.6. Control experiment

[0078] To ensure the accuracy of the results, a negative control experiment was set up. Figure 2 A In the reaction system without the addition of the GmTINY protein, the formation of the DNA-protein complex was not observed, proving that the binding of the ERELEE4 element to the GmTINY protein was specific. This result was consistent with the results of the positive experiment, further confirming the specific binding of GmTINY to the ERELEE4 element in the Rhg1 promoter region.

[0079] Example 3

[0080] In this example, the expression changes of the GmTINY gene in the roots of soybean Wm82 after being infected by soybean cyst nematode (SCN) were analyzed by RT-qPCR experiments. The experimental results showed that the expression level of the GmTINY gene was significantly up-regulated 3 days after SCN infection (3 dpi), demonstrating the important role of this gene in the soybean's anti-SCN response( Figure 3 ).

[0081] 3.1. Experimental materials and treatments

[0082] The wild-type soybean variety Wm82 was selected as the experimental material, and two groups of treatments were set: one group was the SCN infection group, and the other group was the Mock (non-inoculated with SCN) control group. The roots of the two groups of soybeans were sampled at 3 days (3 dpi) and 10 days (10 dpi) after soybean cyst nematode infection for subsequent gene expression analysis.

[0083] 3.2. RNA extraction and RT-qPCR detection

[0084] Total RNA was extracted from the root samples of each treatment group, and cDNA was synthesized using a reverse transcription system. Subsequently, the expression level changes of the GmTINY gene after SCN infection were analyzed by real-time quantitative PCR (RT-qPCR). The expression level of GmTINY was normalized relative to the control gene to ensure the accuracy and comparability of the data.

[0085] 3.3. Result analysis

[0086] As Figure 4 shown, on the 3rd day after SCN infection (3 dpi), the GmTINY gene showed a significant up-regulated expression in the infected soybean roots. Compared with the non-infected Mock group, the expression level of GmTINY in the SCN infection group increased significantly, increasing by approximately 20-fold, indicating that the expression of this gene was significantly enhanced at the initial stage of SCN infection (p < 0.01, indicating significant difference). However, at 10 days (10 dpi), the expression level of GmTINY returned to a level close to that of the Mock control group, showing no significant difference (ns, not significant). These results suggest that the GmTINY gene may enhance the soybean's defense ability against nematodes by regulating the expression of resistance-related genes at the initial stage of SCN infection, but its expression gradually returns to normal in the later stage of infection.

[0087] 3.4. Conclusion

[0088] The experimental results of this example clearly show that the GmTINY gene is significantly upregulated at the early stage of SCN infection (3 dpi), suggesting that this gene may play a key role in the initial defense response of soybeans against SCN infection. This provides an experimental basis for further studying the role of the GmTINY gene in the soybean nematode resistance mechanism and theoretical support for applying this gene to soybean disease-resistant breeding.

[0089] Example 4

[0090] In this example, the inventors evaluated the effect of overexpression (OE) of the GmTINY gene on the resistance of soybean cyst nematode (SCN) through soybean hairy root transformation experiments. By detecting the infection of SCN at different developmental stages in soybean roots, the inhibitory effect of GmTINY gene overexpression on SCN development was revealed ( Figure 4 ).

[0091] 4.1. Materials and Treatments

[0092] The wild-type soybean variety Wm82 and the resistant variety Forrest were selected as experimental materials. Using the Agrobacterium-mediated transformation technique, the GmTINY overexpression vector was introduced into soybean hairy roots to obtain transgenic hairy roots with overexpression of GmTINY (GmTINY-OE). The Mock group was the control group without overexpression of GmTINY. After inoculating SCN cysts, soybean root samples were collected at 14 dpi (days) for subsequent experimental analysis.

[0093] Referring to the article "Enhancing Agrobacterium-mediated soybean transformation efficiency with an auxiliary solution. Crop Health 2024", a transient overexpression vector of the GmTINY gene was constructed using the homologous recombination method. The specific construction method was as follows: The open reading frame (ORF) of soybean GmTINY was PCR amplified using the cDNA of wild-type soybean Williams 82 (Wm82), and the reagent was KOD one PCR Master Mix from Toyobo Company. The PCR product was detected by agarose gel electrophoresis, and the target fragment was recovered by cutting the gel.

[0094] The PCR reaction system was:

[0095]

[0096] The amplified GmTINY gene fragment was integrated into the binary vector pAGM4673 by homologous recombination method, together with the cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (NOS) terminator. This backbone vector also carried the RFP fluorescent marker gene for subsequent screening of transgenic plants.

[0097] The reaction system was as follows:

[0098]

[0099] Reaction conditions: 50 °C, 15 min.

[0100] Further screening was carried out by means of Escherichia coli transformation and colony PCR. Samples with the target band were further verified by PCR, and finally the successfully ligated vector OE-GmTINY was obtained.

[0101] (1) Escherichia coli transformation:

[0102]

[0103] Let it stand on ice for 30 min; water bath at 42 °C for 45 s; stand on ice for 2 min; add 1 ml of antibiotic-free LB liquid medium, place it at 37 °C, and shake it on a shaker at 200 rpm for 1 h; after taking it out, centrifuge it at 6000 rpm for 2 min, remove the supernatant, and spread the remaining on Kan LB solid medium and culture it overnight at 37 °C.

[0104] (2) Colony PCR:

[0105]

[0106] Use a pipette tip to pick the colonies obtained in step (1) and add them to the prepared system.

[0107] The PCR reaction conditions were as follows:

[0108] (1) Pre-denaturation at 95 °C for 5 min; (2) Denaturation at 95 °C for 30 sec, annealing at 55 °C for 30 sec, extension at 72 °C for 45 sec, for a total of 35 cycles; (3) Store at 4 °C.

[0109] 4.2. Microscopic observation

[0110] The infection of different developmental stages of SCN (J2 to J4 and cysts) in soybean hairy roots was microscopically observed by acid fuchsin staining method. Figure 4A shows the SCN infection of the root systems of Wm82 and Forrest varieties after GmTINY - OE and Mock treatments. The results show that in the GmTINY overexpression treatment group, for both the Wm82 and Forrest varieties, the development and infection of SCN were significantly reduced. In particular, the number of nematodes entering the J3 and J4 stages was significantly decreased, indicating that GmTINY overexpression significantly inhibited the development of SCN.

[0111] 4.3. Data analysis

[0112] Figure 4 B shows the proportions of J3, J4 stages and cyst numbers among the total infected nematodes. The results show that in the Wm82 variety, the SCN development proportion in the GmTINY overexpression group was significantly lower than that in the Mock group (p < 0.05). In addition, in the resistant variety Forrest, GmTINY overexpression further enhanced its resistance to SCN, and the proportions of J3 and J4 stage nematodes were significantly lower than those in the Mock group and the Wm82 group. Statistical results show that GmTINY - OE treatment significantly inhibited the proportion of nematodes developing to the late stages (J3, J4 and cysts), especially more obvious in the Forrest variety.

[0113] 4.4. Discussion of results

[0114] The experimental results show that overexpression of the GmTINY gene can significantly enhance the resistance of soybean root systems to SCN and inhibit the development of nematodes in the root systems. Compared with the control group, the infection number and development stage proportion of SCN in the hairy roots of soybeans overexpressing GmTINY were significantly reduced. Especially in the resistant variety Forrest, overexpression of GmTINY further enhanced the resistance of soybeans. This finding indicates that GmTINY, as a key gene against SCN, can significantly inhibit the development process of SCN by regulating the resistance mechanism of the root system.

[0115] Example 5

[0116] In this example, by constructing stable GmTINY overexpression (OE) soybean plants, the effect of GmTINY gene overexpression on the resistance of soybean cyst nematode (SCN) was further studied. The results show that soybean varieties stably overexpressing GmTINY can significantly inhibit the development of SCN and reduce the number of cyst formations ( Figure 5 ).

[0117] 5.1. Construction of stable transgenic plants

[0118] Using the Agrobacterium-mediated transformation technique, multiple soybean transgenic lines stably overexpressing the GmTINY gene (GmTINY-OE#36, GmTINY-OE#185, and GmTINY-OE#197) were constructed. These lines were further constructed and screened after the function of GmTINY was preliminarily verified through the transgenic hairy root system in Example 4, for in-depth study of the role of the GmTINY gene in soybean resistance to SCN. The Mock group was the control group without GmTINY transformation. Soybean root samples were collected at 21 dpi for observation and statistics of the SCN development stage.

[0119] 5.2. Microscopic Observation

[0120] Figure 6 A shows the comparison of SCN development in transgenic soybean lines stably overexpressing GmTINY and the Mock control group. Through the observation of the roots, it can be seen that the SCN infection in the Mock group is more severe and the number of cysts is larger, while in the soybean roots overexpressing GmTINY, the SCN infection is significantly reduced, especially the number of nematodes at the J4 stage is significantly decreased, indicating that overexpression of GmTINY can effectively inhibit the development and reproduction of nematodes.

[0121] 5.3. Data Analysis

[0122] Figure 6 B shows the statistical results of the number of J4 and cysts per gram of fresh roots. The results show that in the Mock group, the number of J4 and cysts is significantly higher than that in each GmTINY-OE transgenic line (p<0.05). Among them, the development of SCN in the GmTINY-OE#36 line is significantly inhibited, and the number of J4 and cysts is significantly reduced, while the GmTINY-OE#185 and GmTINY-OE#197 lines also show strong resistance, and the number of cysts is significantly lower than that in the Mock group. This result indicates that stable overexpression of the GmTINY gene can effectively reduce the development and reproduction of SCN in soybean roots.

[0123] 5.4. Result Discussion

[0124] The experimental results clearly show that stable overexpression of the GmTINY gene can significantly enhance soybean resistance to SCN and reduce cyst formation. Compared with the Mock control group, the number of nematodes at the J4 stage in the soybean roots overexpressing GmTINY is significantly reduced, indicating that GmTINY inhibits the development process of SCN by regulating the root defense response.

[0125] Example 6

[0126] In this example, the inventors studied the role of the GmTINY gene in soybean resistance to soybean cyst nematode (SCN) through the GmTINY gene silencing (RNAi) technique. The experimental results showed that the silencing of the GmTINY gene significantly reduced the resistance of soybeans to SCN, indicating that this gene plays an important positive regulatory role in the process of soybean resistance to nematodes. Figure 6 )

[0127] 6.1. Materials and Treatments

[0128] Two soybean varieties were used in the experiment: Wm82 and the resistant variety Forrest. The GmTINY gene in soybeans was silenced using RNA interference technology (RNAi). Specifically, the TINY gene fragment (SEQ ID NO: 3) was constructed into the pGRNAi2 vector and used for hairy root transformation. Hairy roots transformed with the empty vector were used as the control. After inoculating with SCN cysts, soybean root samples were collected at 14 dpi (days) for subsequent experimental analysis.

[0129] 6.2. Fuchsin Staining Observation

[0130] Figure 6 A shows the SCN infection in the Wm82 and Forrest varieties after GmTINY-RNAi and Mock treatments. The development of nematodes in soybean hairy roots was observed through acid fuchsin staining. The results showed that in the GmTINY-RNAi treatment group, the development of SCN in the Wm82 and Forrest varieties was significantly accelerated, and the number of nematodes entering the J3 and J4 stages increased significantly. Especially in the resistant variety Forrest, the infection of SCN was significantly enhanced, indicating that the ability of soybeans to resist SCN decreased significantly after the silencing of the GmTINY gene.

[0131] 6.3. Data Analysis

[0132] Figure 6 B shows the proportion of the number of nematodes in the J3 and J4 stages and cysts in the total number of infected nematodes. The results showed that in the Wm82 variety, the development proportion of SCN in the GmTINY-RNAi group was significantly higher than that in the Mock group, indicating that the silencing of the GmTINY gene led to the accelerated development of SCN. Similarly, in the Forrest variety, the silencing of the GmTINY gene significantly reduced the resistance of this variety, and the proportion of nematodes in the J3 and J4 stages was significantly higher than that in the control group (p < 0.05). These results indicate that the expression of GmTINY plays a key role in inhibiting the development of SCN to the late stages (J3, J4, and cysts).

[0133] 6.4. Result Discussion

[0134] The experimental results showed that the silencing of the GmTINY gene led to a significant acceleration in the development of SCN in soybean roots. Especially in the resistant cultivar Forrest, the GmTINY-RNAi treatment significantly reduced the SCN resistance of soybeans. Compared with the Mock control group, the number of nematodes at the J3 and J4 stages in the GmTINY gene-silenced group increased significantly, indicating that the GmTINY gene plays a positive regulatory role in the process of soybean resistance to SCN.

[0135] Example 7

[0136] In this example, the changes in the expression levels of the GmTINY gene in overexpressing (OE), silenced (RNAi), and control group soybeans were verified by real-time quantitative PCR (RT-qPCR), and the relationship between the expression of the GmTINY gene and soybean resistance to soybean cyst nematode (SCN) was further determined ( Figure 7 ).

[0137] 7.1. Experimental design

[0138] This experiment consisted of three parts, which respectively detected the relative expression levels of the GmTINY gene in GmTINY overexpression, GmTINY gene silencing, and different GmTINY overexpression lines. Using the Mock group as a control, transgenic soybean lines with GmTINY overexpression (OE) and gene silencing (RNAi) were constructed respectively, and RT-qPCR detection was carried out.

[0139] 7.2. RT-qPCR analysis

[0140] The experiment used RT-qPCR technology to detect the expression levels of the GmTINY gene under different treatment conditions, and was normalized using a reference gene.

[0141] Figure 7 A shows the expression of the GmTINY gene in the GmTINY overexpressing soybean lines. Compared with the Mock control group, the expression of the GmTINY gene in the overexpressing lines was significantly upregulated (p < 0.01), proving that the GmTINY gene was effectively expressed in the overexpressing lines.

[0142] Figure 7 B shows the expression level of the GmTINY gene in the RNAi treatment group. The results showed that the expression of the GmTINY gene in the RNAi group was significantly downregulated (p < 0.01), indicating that the silencing effect of the GmTINY gene was obvious, verifying the effectiveness of the GmTINY gene silencing strategy.

[0143] Figure 7C shows the expression of three different GmTINY overexpression lines (OE-GmTINY#36, OE-GmTINY#185, OE-GmTINY#197). The results show that the expression level of the GmTINY gene in each overexpression line is significantly higher than that in the Mock control group, and the expression level of the OE-GmTINY#36 line is the highest, followed by OE-GmTINY#185 and OE-GmTINY#197, and there are significant differences among the three (p<0.05). This indicates that there are differences in the expression levels of the GmTINY gene in different overexpression lines, and the highest expression level is reached in the OE-GmTINY#36 line.

[0144] 7.3. Results and Discussion

[0145] The experimental results clearly show that the GmTINY gene is significantly up-regulated in the overexpression lines and significantly down-regulated in the RNAi silencing lines. These data further verify the regulatory role of the GmTINY gene in the process of resistance to SCN, and clarify the differences in the expression levels of GmTINY in different transgenic lines through quantitative results. In addition, these RT-qPCR results are highly consistent with the SCN resistance performance in other experiments, further supporting the importance of the GmTINY gene in improving the disease resistance of soybeans.

[0146] It should be emphasized that the present invention first discovers the up-regulated expression of GmAAT Rhg1 and GmSNAP18 resistance genes in the GmTINY overexpression transgenic roots, and proves through experiments that overexpression of the GmTINY gene can enhance resistance to SCN. In addition, using the method for enhancing the nematode resistance of soybeans in the present invention can effectively enhance the nematode resistance of soybeans and has no effect on the growth and development of soybeans, and can be popularized and applied.

[0147] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention.

[0148] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0149] Although embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. Use of the GmTINY gene in enhancing plant resistance to nematodes, characterized in that: The CDS region coding sequence of the GmTINY gene is as shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that: The nematode resistance of the plant is enhanced by overexpressing the GmTINY gene in the plant, or the nematode resistance of the plant is reduced by silencing the GmTINY gene in the plant.

3. The use according to claim 2, characterized in that: The plant is soybean.

4. A method for enhancing plant resistance to nematodes, characterized in that: Regulation of the resistance gene GmAAT by overexpressing the GmTINY gene Rhg1 , GmSNAP18 and other resistance-related genes, thereby enhancing the plant's resistance to nematodes.

5. The method according to claim 4, characterized in that: The GmTINY gene exerts its function by binding to the ERELEE4 cis-acting element in the promoter region of the soybean cyst nematode resistance-related gene Rhg1.

6. The method according to claim 5, characterized in that: The expression of GmTINY gene was regulated by soybean hairy root transformation to enhance plant resistance to nematodes.

7. The method according to claim 6, characterized in that: The GmTINY gene was overexpressed or silenced by the following steps: a transient overexpression vector of the GmTINY gene was constructed using the pAGM4673 vector, or an RNA interference (RNAi) vector was constructed using the pGRNAi2 vector, and the above vectors were transformed into Williams 82 (Wm 82) and Forrest soybean cotyledons using the Agrobacterium rhizogenes ARqua1 strain to induce the generation of transgenic hairy roots, so as to achieve overexpression or silencing of the GmTINY gene in soybean.

8. The method according to claim 7, characterized in that: The expression level of GmTINY gene was verified by real-time quantitative PCR (RT-qPCR); The soybean hairy roots that successfully overexpressed or silenced the GmTINY gene were screened by fluorescence. Successfully transformed soybean hairy roots were inoculated with J2 stage larvae of soybean cyst nematode (SCN) for resistance testing; The number of nematodes infecting soybean roots and their developmental stages were observed and recorded by fuchsin staining. The effects of overexpression or silencing of the GmTINY gene on soybean nematode resistance were evaluated by analyzing the data on the number of nematodes infected and the developmental stages.

Images