Use of gm tiny gene in enhancing plant resistance to soybean cyst nematode

Abstract

The application belongs to the technical field of biology, and specifically provides a use of a GmTINY gene in enhancing plant resistance to nematodes, wherein a CDS region coding sequence of the GmTINY gene is as shown in SEQ ID NO: 2. The application provides an application of the GmTINY gene in improving the ability of plants to resist soybean cyst nematodes, specifically by up-regulating the expression of the GmTINY gene in plants, enhancing the resistance of the plants to nematodes, and thus effectively improving the effect of the plants on resisting soybean cyst nematodes.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the use of the GmTINY gene in enhancing plant disease resistance. Background Technology

[0002] Soybean cyst nematode (SCN) is one of the major pathogens in soybean production worldwide, with a profound impact on the soybean industry. As a parasitic nematode, SCN invades the soybean root system, disrupting its water and nutrient absorption functions, thereby affecting the normal growth of the plant. Specific damage manifests as stunted growth, yellowing leaves, dwarfing, and in severe cases, even death of the entire plant. Globally, soybean cyst nematode disease is one of the most devastating diseases causing soybean yield losses. Studies have shown that in areas severely infected with SCN, yield losses can reach as high as 30%. Furthermore, 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 changes in agricultural practices, the spread and harm of SCNs are becoming increasingly serious, threatening not only agricultural production but also posing significant challenges to the global soybean supply chain.

[0004] Currently, control strategies against soybean cyst nematodes mainly rely on the breeding of resistant varieties and crop rotation. However, as nematode populations become more diverse and adaptable, the resistance of resistant varieties gradually weakens. Crop rotation, as a traditional control method, also faces challenges due to crop planting cycle limitations and irrational land resource allocation, making it difficult to maintain stable control effects in the long term. The ongoing pressure on global food and oilseed supplies further highlights the urgency and necessity of developing more efficient and sustainable nematode-resistant breeding strategies. Ensuring the sustainable development of soybean production and controlling the outbreak of major diseases like SCN has become one of the most pressing issues to be addressed in global agricultural science and technology.

[0005] In the research and development 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 methods. These new varieties can not only effectively cope with SCN infestation but also adapt to different growth environments, improving soybean yield and quality. In recent years, with the rapid development of biotechnology, nematode-resistant breeding strategies based on gene editing and transgenic technologies have gradually become a research focus. By regulating the expression of genes related to disease resistance in plants, it is hoped that soybean resistance to SCN can be significantly enhanced, thereby reducing economic losses caused by the disease.

[0006] Furthermore, the discovery and functional verification of soybean disease resistance genes using modern molecular biology techniques is a current hot topic in agricultural research. Through in-depth analysis of the soybean genome, researchers can identify key genes related to SCN resistance and use gene editing technology to functionally modify these genes, enabling them to exert stronger disease resistance in soybean plants.

[0007] In conclusion, given the numerous challenges facing the global soybean industry, developing innovative nematode-resistant technologies and breeding new disease-resistant varieties have become urgent needs in the field of agricultural science and technology. Effective nematode-resistant breeding strategies can not only improve soybean yield and quality and reduce losses caused by diseases, but also lower international soybean trade costs, enhance my country's competitiveness in the global soybean market, and promote the sustainable development of the soybean industry. In the future, in-depth research into soybean disease-resistant genes and the widespread application of modern breeding technologies are expected to provide more efficient and sustainable 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 an effective method for enhancing the resistance of soybeans to cyst nematodes, so as to solve the problem of insufficient effect in the prior art.

[0009] To address the aforementioned technical problems, this invention provides the use of the GmTINY gene in enhancing plant resistance to nematodes, wherein 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 comes 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: the plant’s resistance to nematodes is enhanced by overexpressing the GmTINY gene in the plant, or the plant’s resistance to nematodes is reduced 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] This invention also provides a method for enhancing plant resistance to nematodes by overexpressing the GmTINY gene to regulate the resistance gene GmAAT. Rhg1 The expression of GmSNAP18 and other resistance-related genes enhances the plant's resistance to nematodes.

[0015] As an improvement to the method of the present invention: 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.

[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 soybean hairy root transformation to enhance plant resistance to nematodes.

[0018] As a further improvement to the method of the present invention, the GmTINY gene is overexpressed or silenced by means of the following steps: a transient overexpression vector for the GmTINY gene is constructed using the pAGM4673 vector, or an RNA interference (RNAi) vector is constructed using the pGRNAi2 vector, and the above vector is transformed into the cotyledons of Williams 82 (Wm 82) and Forrest soybeans by Agrobacterium rhizogenes ARqua1 strain, inducing 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 was verified by real-time quantitative PCR (RT-qPCR);

[0020] Soybean hairy roots that were successfully overexpressed or silenced by fluorescent screening;

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

[0022] The number of nematodes infecting soybean roots and their developmental stages were observed and recorded using the fuchsin staining method.

[0023] By analyzing data on nematode infection numbers and developmental stages, the effects of GmTINY gene overexpression or silencing on soybean nematode resistance were assessed.

[0024] Based on in-depth research and experimentation, this invention has yielded the following important findings and results:

[0025] First, this invention, through research on the infection process of soybean cyst nematode (SCN), found that the expression of the Rhg1 major resistance gene is significantly upregulated when SCN infects soybeans. To further explore the relevant mechanism, the inventors constructed a yeast one-hybrid library and used the Rhg1 major resistance gene GmAAT... Rhg1Using the promoter region of GmSNAP18 as bait, specific host proteins were screened, ultimately revealing the soybean TINY gene as a potential interacting transcription factor. Through in vivo and in vitro experiments, the inventors further demonstrated that the TINY gene can interact with GmAAT... Rhg1 It binds to the ERELEE4 cis-acting element in the promoter region of the GmSNAP18 gene, activating the expression of these resistance genes and thereby enhancing the plant's resistance to SCN.

[0026] In this invention, the inventors successfully obtained soybean hairy roots overexpressing GmTINY using Agrobacterium rhizogenes-mediated hairy root transformation technology. Through fluorescence screening and RT-qPCR verification, the inventors found that GmTINY gene overexpression significantly inhibited nematode development in Wm82 soybean roots and further enhanced SCN resistance in the resistant variety Forrest. Secondly, the inventors verified the sustained expression of the GmTINY gene in soybean by constructing stable transformed soybean plants, obtaining T1 generation seeds. RT-qPCR analysis confirmed the overexpression level of the GmTINY gene, and subsequent sporangia inoculation experiments showed that the transgenic soybean plants overexpressing the GmTINY gene exhibited stronger SCN resistance compared to the control group. To further investigate the function of the GmTINY gene, the inventors also conducted GmTINY gene silencing experiments using soybean hairy root transformation technology. The experimental results showed that silencing the GmTINY gene significantly increased the susceptibility of Wm82 soybean roots to SCN, indicating that overexpression of the GmTINY gene can effectively enhance soybean's resistance to nematodes.

[0027] Based on the above research findings, this invention reveals for the first time 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 site, activating the expression of resistance genes and thus enhancing soybean resistance to SCN. Furthermore, 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 this invention provides a new approach for soybean disease resistance breeding, has broad application prospects, and is suitable for widespread use in agricultural production.

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

[0029] Furthermore, the method of this invention offers great flexibility in enhancing the expression of target genes. Any known gene expression regulation method can be employed to increase the expression level of specific genes in plants, such as virus-mediated overexpression, transient overexpression mediated by Agrobacterium tumefaciens or Agrobacterium root tumefaciens, or long-term overexpression achieved by constructing stable transgenic plants. As long as the chosen method can accurately and specifically upregulate the expression of the GmTINY gene or its homologs in plants, it can enhance plant disease resistance.

[0030] The additional features and advantages of the present invention will be further detailed in the following specific implementation process and will become clearer during implementation.

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

[0032] This invention provides the application of the GmTINY gene in improving the plant's resistance to soybean cyst nematode. Specifically, by upregulating the expression of the GmTINY gene in plants, the plant's resistance to nematodes is enhanced, thereby effectively improving the plant's resistance to soybean cyst nematode.

[0033] It is important to emphasize that the GmTINY gene is an important member of the DREB subfamily of the AP2 / ERF transcription factor gene family. This gene family plays an important role in promoting plant growth and enhancing plant immunity, but its role in plant-plant parasitic nematode interactions has not yet been revealed. Attached Figure Description

[0034] The above and / or other aspects and advantages of the present invention will become more apparent and readily understood through the description of the embodiments in conjunction with the following drawings, wherein:

[0035] Figure 1 The results of a yeast one-hybrid assay are shown, indicating that GmTINY can interact with the promoter region of the Rhg1 resistance gene. According to... Figure 1 It can be seen that both GmTINY and GmERF7 proteins can interact with the promoter regions of the Rhg1 resistance genes GmAAT and GmSNAP18, but this interaction disappears if the ERELEE4 element in the promoter region is mutated.

[0036] Figure 2 The results of the Electrophoretic Mobility Variation (EMSA) experiment are shown:

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

[0038] according to Figure 2 It can be concluded that the GmTINY protein interacts with the ERE4 probe, while the GmERF7 protein does not interact with the ERE4 probe, and the interaction between the GmTINY protein and ERE4 can be weakened by competition with cold probes.

[0039] Figure 3 The study showed significant expression of the GmTINY gene in soybean on day 3 (3 dpi) after infection with soybean cyst nematode.

[0040] Figure 4 This study demonstrated the effect of GmTINY overexpression (OE) on the resistance of soybean hairy roots to SCN.

[0041] Figure 4 In the image: A represents a representative image of soybean hairy roots infected with SCN and stained with magenta; B represents the statistical results of the J3 and J4 stages and the proportion of sporangia to the total number of infected nematodes.

[0042] Figure 5 This demonstrates the effect of stably GmTINY overexpression (OE) transgenic soybean on SCN;

[0043] Figure 5 In the image: A represents a representative image of root sporangia in transgenic soybeans infected with SCN; B represents the statistical result of the number of root sporangia per gram.

[0044] Figure 6 The study demonstrated the effect of GmTINY gene silencing (RNAi) on SCN resistance in soybean hairy roots.

[0045] Figure 6 In the image: A represents a representative image of soybean hairy roots infected with SCN and stained with magenta; B represents the statistical results of the J3 and J4 stages and the proportion of sporangia to the total number of infected nematodes.

[0046] Figure 7 The results of RT-qPCR validation show significant changes in the transcriptional level of GmTINY in GmTINY overexpression (OE), GmTINY gene silencing (RNAi), and the control group.

[0047] Figure 7 In the diagram: A represents the change in GmTINY transcription level in hairy roots with GmTINY overexpression (OE); B represents the change in GmTINY transcription level in hairy roots with GmTINY silencing (RNAi); C represents the change in GmTINY transcription level in three lines of transgenic soybean (#36, #185, and #197) with GmTINY overexpression (OE). Detailed Implementation

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

[0049] For any techniques or experimental conditions not specifically described in the examples, please refer to relevant technical literature in this field (e.g., J. Sambrook et al., 3rd edition of *Molecular Cloning: A Laboratory Manual*, translated by Huang Peitang et al., Science Press) or follow the product instructions. Reagents or equipment not specified by manufacturer are all conventional products and can be purchased commercially, for example, from Nanjing Novizan Pharmaceutical Co., Ltd.

[0050] Example 1

[0051] In this embodiment, the inventors used a yeast one-hybrid experiment to study the interaction between the GmTINY gene and the promoter region of the Rhg1 resistance gene, verifying 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 site for SCN resistance in soybeans, and the resistance genes it regulates include GmAAT. Rhg1 Genes like GmSNAP18 contain multiple cis-acting elements in their promoter regions that can potentially bind to transcription factors. In this experiment, the inventors selected the ERELEE4 element from 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 operating steps are as follows:

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

[0055] The promoter region (SEQ ID NO:1) of the Rhg1 major resistance gene, including the ERELEE4 element, was cloned from the soybean genome. This promoter region fragment was inserted into the yeast one-hybrid vector pHIS2. Figure 1 pHIS2-pGmAAT Rhg1 As shown in pHIS2-pGmSNAP18, a bait plasmid containing the Rhg1 promoter region was constructed.

[0056] 1.2. Construction and Screening of Yeast One-Hybrid Libraries

[0057] The invention constructed a fusion plasmid containing the activation domain of the soybean GmTINY gene, and cloned the coding region (SEQ ID NO:2) of the GmTINY gene into the yeast expression vector pGADT7. Figure 1pGADT7-GmTINY (as shown in the image) was used as the library plasmid. Simultaneously, other activation domain plasmids (such as pGADT7-GmTINY) were constructed. Figure 1 The pGADT7-GmERF7 plasmid was used to further verify the binding of different transcription factors to the Rhg1 promoter region. The bait plasmid and library plasmid were co-transformed into yeast cell line Y187, and positive clones were screened on selective media following the steps described below.

[0058] 1.3. Yeast Transformation and Interaction Verification

[0059] Transformed yeast was grown in the deficient medium SD-Trp-Leu-His. X-α-gal staining was used to screen for yeast clones expressing the blue marker to determine whether GmTINY interacts with the ERELEE4 element in the Rhg1 promoter region. The results showed that yeast clones transformed with the plasmid containing GmTINY could grow normally on the selective medium and exhibit blue staining, indicating that the GmTINY gene can specifically bind to the ERELEE4 element in the Rhg1 promoter region, activating the expression of downstream resistance genes.

[0060] Note: The transformed yeast was grown on SD-Trp-Leu medium (OD values ​​of 1, 0.1, and 0.01) and SD-Trp-Leu-His medium containing 50 mM 3-AT (OD values ​​of 1, 0.1, and 0.01), respectively. 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. Interaction Result Analysis

[0062] like Figure 1 As shown, the results of the yeast one-hybrid experiment indicate that the transformation of pGADT7-GmTINY and pHis2-pGmAAT... _Rhg1 Yeast clones containing the pHis2-pGmSNAP18 plasmid grew normally on selective medium (SD-Trp-Leu-His) and showed a blue marker, indicating that the GmTINY protein can 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 validating the function of GmTINY as a transcription factor. In summary, the experimental results show that GmTINY, as a transcription factor, can 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 was also enhanced. This function may improve soybean resistance to SCN by increasing the expression level of resistance genes. This discovery reveals the potential role of GmTINY in the soybean SCN resistance mechanism and provides a new approach to improving soybean disease resistance by regulating GmTINY gene expression.

[0063] 1.5. Controlled Experiment

[0064] To ensure the accuracy of the experiment, the inventors set up a negative control experiment. Figure 1 The yeast clones that were not transformed with the plasmid containing the GmTINY gene failed to grow on selective medium and did not show blue markings, further demonstrating the specific binding of GmTINY to the Rhg1 promoter region.

[0065] Example 2

[0066] In this embodiment, the inventors further verified using electrophoretic mobility shift assay (EMSA) 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. This experiment clarified the direct physical interaction between GmTINY and the Rhg1 promoter region, providing evidence for revealing the molecular mechanism by which GmTINY regulates the expression of the soybean cyst nematode (SCN) resistance gene. Figure 2 ).

[0067] 2.1. Cloning and Labeling of Target Fragments

[0068] Based on the promoter sequence of the Rhg1 major resistance gene in the soybean genome (SEQ ID NO:1), a region containing the ERELEEE4 cis-acting element was selected, and a 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 use in DNA-protein binding detection in EMSA. The biotin-labeled ERELEEE4 fragment will be used as a probe for binding experiments with GmTINY protein.

[0069] 2.2. Expression and purification of GmTINY protein

[0070] Next, the present invention expresses the GmTINY protein using 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 E. coli. The His-tagged GmTINY protein was purified by nickel column affinity chromatography to obtain high-purity recombinant GmTINY protein for subsequent in vitro binding experiments.

[0071] 2.3. EMSA Experimental Procedure

[0072] Purified GmTINY protein was incubated with a biotin-labeled ERELEE4 probe in binding buffer (11 μL ultrapure water, 2 μL 10x binding buffer, 1 μL 50% Glycerol, 1 μL 1% NP-40, 1 μL Poly(dI.dC), 2 μL probe, and 2 μL protein) at 28 °C for 30 min, followed by non-denaturing polyacrylamide gel electrophoresis. Changes in electrophoretic mobility were used to detect whether GmTINY protein could bind to the ERELEE4 element in the Rhg1 promoter region. The binding reaction was monitored by changes in the migration rate of the protein complex bound to the probe.

[0073] 2.4. Detection based on results

[0074] After electrophoresis, the DNA-protein complex was detected by membrane transfer and biotin probe imaging system. Figure 2 Results showed that GmTINY protein could form a specific DNA-protein complex with the biotin-labeled ERELEEE4 probe, indicating that GmTINY protein directly binds to the ERELEEE4 element in the promoter region of the Rhg1 resistance gene in vitro. Compared with the control group without GmTINY protein, the reaction system containing GmTINY protein showed a significant change in migration rate, exhibiting a slower migration rate, further verifying the binding ability of GmTINY protein to the ERELEEE4 element.

[0075] 2.5. Competition Experiment

[0076] To further verify the specificity of the binding, the inventors conducted a competition experiment. An excess of the unlabeled ERELEE4 fragment was added to the reaction system as a competing probe. Figure 2 The results showed that the unlabeled ERELEE4 probe competitively inhibited the binding of the biotin-labeled probe to the GmTINY protein, leading to the disappearance of the migration-variable complex, which further demonstrated the specificity of the binding of the GmTINY protein to the ERELEE4 element.

[0077] 2.6. Controlled Experiment

[0078] To ensure the accuracy of the results, a negative control experiment was set up. Figure 2 In reaction system without the addition of GmTINY protein, no DNA-protein complex formation was observed, demonstrating that the binding of the ERELEE4 element to GmTINY protein is specific. This result is consistent with the positive results, further confirming the specific binding of GmTINY to the ERELEE4 element in the Rhg1 promoter region.

[0079] Example 3

[0080] In this embodiment, the expression changes of the GmTINY gene in the roots of soybean Wm82 after infection with soybean cyst nematode (SCN) were analyzed by RT-qPCR. The results showed that the expression level of the GmTINY gene was significantly upregulated 3 days after SCN infection (3 dpi), demonstrating the important role of this gene in the soybean's resistance to SCN. Figure 3 ).

[0081] 3.1. Experimental Materials and Treatment

[0082] Wild-type soybean variety Wm82 was selected as the experimental material, and two treatment groups were set up: one group was infected with SCN, and the other group was a mock (uninoculated with SCN) control group. The roots of soybeans in both groups were sampled at 3 days (3 dpi) and 10 days (10 dpi) after infection with soybean cyst nematodes for subsequent gene expression analysis.

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

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

[0085] 3.3. Results Analysis

[0086] like Figure 4 As shown, on day 3 (3 dpi) after SCN infection, the GmTINY gene showed significant upregulation in infected soybean roots. Compared to the uninfected Mock group, the GmTINY expression level in the SCN-infected group increased dramatically, by approximately 20-fold, indicating a significant enhancement in gene expression during the early stages of SCN infection (p < 0.01, indicating a significant difference). However, at day 10 (10 dpi), the GmTINY expression level returned to levels close to those of the Mock control group, with no significant difference (ns, not significant). These results suggest that the GmTINY gene may enhance soybean's defense against nematodes in the early stages of SCN infection by regulating the expression of resistance-related genes, but its expression gradually returns to normal in the later stages of infection.

[0087] 3.4. Conclusion

[0088] The experimental results of this embodiment clearly demonstrate that the GmTINY gene is significantly upregulated in the early stages of SCN infection (3 dpi), suggesting that this gene may play a key role in the initial defense response of soybean against SCN infection. This provides experimental evidence for further research on the role of the GmTINY gene in soybean nematode resistance mechanisms and provides theoretical support for applying this gene to soybean disease resistance breeding.

[0089] Example 4

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

[0091] 4.1. Materials and Processing

[0092] Wild-type soybean variety Wm82 and resistant variety Forrest were selected as experimental materials. Using Agrobacterium-mediated transformation, the GmTINY overexpression vector was introduced into soybean hairy roots to obtain GmTINY-overexpressing transgenic hairy roots (GmTINY-OE). The Mock group served as the control group without GmTINY overexpression. Soybean root samples were collected at 14 days post-inoculation (dpi) 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 pAGM4673 vector via homologous recombination. Specifically, the open reading frame (ORF) of soybean GmTINY was amplified by PCR using cDNA from wild-type soybean Williams 82 (Wm82) with Toyobo's KOD one PCR Master Mix. The PCR products were detected by agarose gel electrophoresis, and the target fragment was recovered by gel excision.

[0094] The PCR reaction system is as follows:

[0095]

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

[0097] The reaction system is as follows:

[0098]

[0099] Reaction conditions: 50℃, 15min.

[0100] Further screening was conducted using E. coli transformation and colony PCR. Samples with the target band were then validated by PCR, ultimately yielding the successfully ligated vector OE-GmTINY.

[0101] (1) Escherichia coli transformation:

[0102]

[0103] Incubate on ice for 30 min; incubate in a 42°C water bath for 45 s; incubate on ice for 2 min; add 1 ml of antibiotic-free LB liquid medium, place in a 37°C, 200 rpm shaker for 1 h; remove and centrifuge at 6000 rpm for 2 min, remove the supernatant, spread the remainder onto Kan LB solid medium, and incubate overnight at 37°C.

[0104] (2) Colony PCR:

[0105]

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

[0107] The PCR reaction conditions are as follows:

[0108] (1) Pre-denaturation at 95℃ for 5 min; (2) Denaturation at 95℃ for 30 sec, 55℃ for 30 sec, and 72℃ for 45 sec for a total of 35 cycles; (3) Storage at 4℃.

[0109] 4.2. Microscopic observation

[0110] The infection status of SCN at different developmental stages (J2 to J4 and sporangia) in soybean hairy roots was observed under a microscope using acid fuchsin staining. Figure 4A shows the SCN infection status of the roots of Wm82 and Forrest varieties after GmTINY-OE and Mock treatments. The results indicate that in the GmTINY overexpression treatment group, both Wm82 and Forrest varieties showed significantly reduced SCN development and infection, especially a significant reduction in the number of nematodes entering the J3 and J4 stages, indicating that GmTINY overexpression significantly inhibited SCN development.

[0111] 4.3. Data Analysis

[0112] Figure 4 B shows the proportions of J3, J4 stages and cysts in the total number of infected nematodes. The results indicated that in the Wm82 variety, the proportion of SCN development in the GmTINY overexpression group was significantly lower than in the Mock group (p<0.05). Furthermore, in the resistant variety Forrest, GmTINY overexpression further enhanced its resistance to SCN, with the proportions of J3 and J4 stage nematodes significantly lower than in the Mock and Wm82 groups. Statistical results indicate that GmTINY-OE treatment significantly inhibited the proportion of nematodes developing to late stages (J3, J4, and cysts), especially in the Forrest variety.

[0113] 4.4. Discussion of Results

[0114] Experimental results showed that overexpression of the GmTINY gene significantly enhanced soybean root resistance to SCN and inhibited nematode development in the roots. Compared with the control group, the number of SCN infections and the proportion of developmental stages in soybean hairy roots overexpressing GmTINY were significantly reduced. In particular, in Forrest-resistant varieties, GmTINY overexpression further enhanced soybean resistance. This finding indicates that GmTINY, as a key gene for SCN resistance, can significantly inhibit SCN development by regulating root resistance mechanisms.

[0115] Example 5

[0116] In this embodiment, the effect of GmTINY gene overexpression on soybean cyst nematode (SCN) resistance was further investigated by constructing stable GmTINY overexpression (OE) soybean plants. The results showed that soybean varieties stably overexpressing GmTINY significantly inhibited SCN development and reduced the number of cysts formed. Figure 5 ).

[0117] 5.1. Constructing stable transgenic plants

[0118] Several transgenic soybean lines stably overexpressing the GmTINY gene (GmTINY-OE#36, GmTINY-OE#185, and GmTINY-OE#197) were constructed using Agrobacterium-mediated transformation. These lines, after the initial verification of GmTINY function via the transgenic hairy root system in Example 4, were further constructed and screened for in-depth research on the role of the GmTINY gene in soybean resistance to SCN. The Mock group served as a control group without GmTINY transformation. Soybean root samples were collected at 21 days post-conversion (dpi) for observation and statistical analysis of SCN development stages.

[0119] 5.2. Microscopic observation

[0120] Figure 6 A shows a comparison of SCN development in transgenic soybean lines stably overexpressing GmTINY and the Mock control group. Root observation revealed that SCN infection was more severe and the number of sporangia was higher in the Mock group, while SCN infection was significantly reduced in the GmTINY-overexpressing soybean roots, particularly the number of nematodes at the J4 stage, indicating that GmTINY overexpression effectively inhibits nematode development and reproduction.

[0121] 5.3. Data Analysis

[0122] Figure 6 B presents the statistical results of the number of J4 cells and sporangia per gram of fresh roots. The results showed that in the Mock group, the number of J4 cells and sporangia was significantly higher than in each GmTINY-OE transgenic line (p<0.05). Specifically, SCN development in the GmTINY-OE#36 line was significantly inhibited, with a significant reduction in the number of J4 cells and sporangia. The GmTINY-OE#185 and GmTINY-OE#197 lines also exhibited strong resistance, with significantly lower sporangia numbers compared to the Mock group. These results indicate that stable overexpression of the GmTINY gene can effectively reduce SCN development and reproduction in soybean roots.

[0123] 5.4. Discussion of Results

[0124] The experimental results clearly demonstrate that stable overexpression of the GmTINY gene significantly enhances soybean resistance to SCN and reduces cyst formation. Compared with the Mock control group, the number of J4 stage nematodes in the roots of soybeans overexpressing GmTINY was significantly reduced, indicating that GmTINY inhibits the development of SCN by regulating the root defense response.

[0125] Example 6

[0126] In this embodiment, the inventors investigated the effect of the GmTINY gene on soybean resistance to soybean cyst nematode (SCN) using GmTINY gene silencing (RNAi) technology. The experimental results showed that GmTINY gene silencing significantly reduced soybean resistance to SCN, indicating that this gene plays an important positive regulatory role in soybean nematode resistance. Figure 6 ).

[0127] 6.1. Materials and Processing

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

[0129] 6.2. Observation of fuchsin staining

[0130] Figure 6 A shows the SCN infection status of Wm82 and Forrest varieties after GmTINY-RNAi and Mock treatments. The development of nematodes in soybean hairy roots was observed using acidic forsythoside staining. The results showed that in the GmTINY-RNAi treatment group, SCN development in Wm82 and Forrest varieties was significantly accelerated, and the number of nematodes entering the J3 and J4 stages increased significantly. Particularly in the resistant variety Forrest, SCN infection was significantly enhanced, indicating that GmTINY gene silencing significantly reduced soybean's resistance to SCN.

[0131] 6.3. Data Analysis

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

[0133] 6.4. Discussion of Results

[0134] Experimental results showed that silencing the GmTINY gene significantly accelerated SCN development in soybean roots, especially in the resistant variety Forrest, where GmTINY-RNAi treatment significantly reduced soybean's resistance to SCN. Compared with the Mock control group, the number of nematodes at stages J3 and J4 was significantly increased in the GmTINY gene silencing group, indicating that the GmTINY gene plays a positive regulatory role in soybean's resistance to SCN.

[0135] Example 7

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

[0137] 7.1. Experimental Design

[0138] This experiment consisted of three parts, which 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, GmTINY overexpression (OE) and gene silencing (RNAi) soybean transgenic lines were constructed and detected by RT-qPCR.

[0139] 7.2. RT-qPCR Analysis

[0140] The expression level of the GmTINY gene under different treatment conditions was detected by RT-qPCR, and the control gene was used for standardization.

[0141] Figure 7 A shows the expression of the GmTINY gene in GmTINY-overexpressing soybean lines. Compared with the Mock control group, the expression of the GmTINY gene was significantly upregulated in the overexpressing lines (p<0.01), demonstrating 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-treated group. The results indicate that the expression of the GmTINY gene was significantly downregulated in the RNAi group (p<0.01), indicating a significant silencing effect of the GmTINY gene and verifying the effectiveness of the GmTINY gene silencing strategy.

[0143] Figure 7C shows the expression levels of three different GmTINY overexpression lines (OE-GmTINY#36, OE-GmTINY#185, and OE-GmTINY#197). The results showed that the expression level of the GmTINY gene in each overexpression line was significantly higher than that in the Mock control group, with the highest expression level observed in the OE-GmTINY#36 line, followed by OE-GmTINY#185 and OE-GmTINY#197. Significant differences were found among the three lines (p<0.05). This indicates that the expression level of the GmTINY gene varies among the different overexpression lines, and that the highest expression level was achieved in the OE-GmTINY#36 line.

[0144] 7.3. Discussion of Results

[0145] The experimental results clearly show that the GmTINY gene was significantly upregulated in overexpression lines and significantly downregulated in RNAi-silenced lines. These data further validate the regulatory role of the GmTINY gene in SCN resistance and clarify the differences in GmTINY expression levels among different transgenic lines through quantitative results. Furthermore, these RT-qPCR results are highly consistent with SCN resistance results from other experiments, further supporting the importance of the GmTINY gene in enhancing soybean disease resistance.

[0146] It is important to emphasize that this invention is the first to discover GmAAT in GmTINY overexpressing transgenic roots. Rhg1 The expression of the GmSNAP18 resistance gene was upregulated, and experiments demonstrated that overexpression of the GmTINY gene enhances resistance to SCN. Furthermore, the method for enhancing soybean nematode resistance using this invention effectively improves soybean nematode resistance without affecting soybean growth and development, and can be widely applied.

[0147] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

[0148] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

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

Claims

1. GmTINY The use of genes in enhancing plant resistance to nematodes is characterized by: The GmTINY The CDS region coding sequence of the gene is as described in SEQ ID NO:2; the plant is soybean.

2. The use according to claim 1, characterized in that: Through overexpression of plant GmTINY Genes are used to enhance the plant's resistance to nematodes, or by silencing genes in the plant. GmTINY Genes are used to reduce the plant's resistance to nematodes.

3. A method for enhancing plant resistance to nematodes, characterized in that: Through overexpression GmTINY Genes regulate resistance genes GmAAT Rhg1 , GmSNAP18 The expression of [the substance] enhances the plant's resistance to nematodes; GmTINY The CDS region coding sequence of the gene is as described in SEQ ID NO:

2.

4. The method according to claim 3, characterized in that: GmTINY Genes related to resistance to soybean cyst nematode Rhg1 The ERELEE4 cis-acting element in the promoter region works together to function.

5. The method according to claim 4, characterized in that: Regulation through soybean hairy root transformation method GmTINY Gene expression to enhance plant resistance to nematodes.

6. The method according to claim 5, characterized in that: The following steps help to express or remain silent. GmTINY Gene: Constructed using pAGM4673 vector GmTINY The gene was transiently overexpressed using a vector, or an RNA interference (RNAi) vector was constructed using the pGRNAi2 vector. These vectors were then transformed into the cotyledons of Williams 82 (Wm 82) and Forrest soybeans using the Agrobacterium rhizogenes ARqua1 strain, inducing the generation of transgenic hairy roots to achieve the desired effect in soybeans. GmTINY Gene overexpression or silencing.

7. The method according to claim 6, characterized in that: GmTINY Gene expression levels were verified using real-time quantitative PCR; Successful overexpression or silencing was screened using fluorescence. GmTINY Soybean hairy roots (genes); The transformed soybean hairy roots were inoculated with J2 stage larvae of soybean cyst nematode for resistance testing. The number of nematodes infecting soybean roots and their developmental stages were observed and recorded using the fuchsin staining method. By analyzing data on the number of infected nematodes and their developmental stages, an assessment was made. GmTINY The effects of gene overexpression or silencing on soybean nematode resistance.

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