Application of a zinc finger protein in resisting soybean sudden death syndrome virus

Abstract

The present invention belongs to the field of biotechnology and discloses an application of a zinc finger protein in resisting soybean sudden death syndrome virus. The present invention discovers that the zinc finger protein ZNF1 in soybeans can inhibit virus infection during the infection of soybean sudden death syndrome virus (SoSGV). Overexpression of this gene can significantly inhibit the infection of SoSGV, and transgenic overexpression of ZNF1 can endow plants with significant resistance to SoSGV. Therefore, the zinc finger protein ZNF1 can be used to prepare transgenic plants with enhanced resistance to soybean sudden death syndrome virus, and has great application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of a zinc finger protein in resisting soybean sudden death syndrome virus. Background Art

[0002] Soybean ( Glycine max (L.) Merr ) is a high-quality and high-protein food crop, known as the "golden crop" and "plant meat", providing 65% of plant protein and 33% of edible vegetable oil for humans, and is one of the important crops in the world (Brar, G. S., Thomas, J. R. (1993). Soybean: Glycine max (L) Merrill-Science Direct. Genetic Improvement of Vegetable Crops. 427-463.). In addition to being used as food, soybeans have a wide range of uses. Soybean oil is one of the four major vegetable oils in the world and is widely used in the fields of food, medicine, cosmetics, etc.; defatted soybean meal is a high-quality feed and can be used for livestock and poultry breeding; soybeans can also be used as raw materials for green manure, desertification control, biofuels, etc. (Masuda, T., Goldsmith, P. D. (2009). Worldsoybean production: area harvested, yield, and long-term projections. IntFood Agribus Manag Rev. 12(1030-2016-82753), 1-20). The global demand for soybeans is increasing year by year.

[0003] Soybean yield can be affected by various factors, such as climate, variety, planting techniques, pests and diseases, etc. Among them, diseases are important factors affecting soybean yield. For example, in recent years, the large-scale prevalence and outbreak of soybean "stay-green" in the Huang-Huai-Hai region have posed a great threat to the healthy development of the soybean industry. Soybean "stay-green", commonly known as "greedy green" and "inverted green" (Wang, J., Yang, M., Deng, D. (1982). Causes and preventive measures of soybean greedy green and empty pods. Henan Agricultural Sciences. (9), 20), the main field symptom is that when soybeans are normally mature, the soybean plants still show green branches and leaves, and there are pods but the pods are empty (Li, K., Zhang, X., Guo, J., Penn, H., Wu, T., Li, L., Jiang, H., Chang, L., Wu, C., Han, T. (2019). Feeding of Riptortus pedestris on soybean plants, the primary cause of soybean staygreen syndrome in the Huang-Huai-Hai river basin. Crop J. 7(3), 360-367). Due to the complexity of the cause of stay-green, the pathogenic pathogen of soybean stay-green disease has not been known for a long time. In 2022, Cheng et al. in this research group identified a recombinant geminivirus - soybean stay-green associated virus (SoSGV) in soybean stay-green samples through small RNA sequencing, and confirmed through Koch's postulates that this virus can cause the stay-green phenotype in soybeans. After this virus infects soybeans, phenotypes such as leaves staying green, empty pods and abnormal seed development can appear, which is also consistent with most of the stay-green phenotypes observed in the field; further research found that in addition to infecting soybeans, SoSGV can also infect Nicotiana benthamiana, causing delayed senescence and empty pods in Nicotiana benthamiana (Cheng, R., Mei, R., Yan, R., Chen, H., Miao, D., Cai, L., Fan, J., Li, G., Xu, R., Lu, W., Gao, Y., Ye, W., Su, S., Han, T., Gai, J., Wang, Y., Tao, X., Xu, Y. (2022). A new distinct geminivirus causes soybean stay-green disease. Molecular Plant. 15(6), 927-930).A total of 368 symptomatic soybean samples collected from 17 regions in 8 provinces were further tested. The results showed that 228 of them tested positive (61.96%), and typical symptomatic soybean symptoms were observed in 96.93% of the positive samples. This indicates that symptomatic soybean caused by SoSGV has become the mainstream in the field and shows a trend of spreading from the Huang-Huai-Hai region to the surrounding areas (Cheng, R., Yan, R., Mei, R., Wang, Y., Niu, W., Ai, H., Qiao, S., Xu, M., Yu, W., Ye, W., Wang, Y., Tao, X., Zhou, X., Xu, Y. (2023). Epidemiological evaluation and identification of the insect vector of soybean stay-green associated virus. Phytopathology Research. 5(1), 20). Meanwhile, Cheng et al. in this research group identified the natural transmission vector of this virus as *Paranephotettix asiaticus* (.). Orosius orientalis This virus is a newly identified novel species of the Geminiviridae family. Currently, its pathogenic mechanism is unclear and no effective disease-resistant genes have been identified. Therefore, there is still a lack of effective means for the prevention and control of this disease. At present, no related genes and technologies have been reported that can be used to resist soybean symptomatic virus disease.

[0004] To defend against pathogen invasion, host plants have evolved a two-layered innate immune system. One is PTI (Pattern-triggered immunity) immunity triggered by the recognition of conserved pathogen-associated molecular patterns (PAMPs) produced by pathogenic microorganisms by pattern recognition receptors (PRRs) on the plant cell surface. The other is ETI (Effector-triggered immunity) immunity triggered by the recognition of pathogen effectors by intracellular immune proteins (Jones, J. D and Dangl, J. L. (2006). The plant immune system. Nature. 444(7117), 323-329). After plant immunity is activated, it involves a series of immune signal transduction processes. Among them, transcriptional reprogramming is an important feature of plant immunity, and this process is regulated by various transcription factors and proteins related to transcript complexes (Tsuda, K and Somssich, I. E. (2015). Transcriptional networks in plant immunity. New Phytologist. 206(3), 932-947). The zinc finger protein family is a large class of proteins in plants responsible for regulating gene transcription and expression. Proteins in this family are divided into various types, such as C2H2, C2HC, C2HC5, C3HC4, CCCH, C4, C4HC3, C6, and C8 types (Li, W., He, M., Wang, J., Wang, Y. (2013). Zinc finger protein (ZFP) in plants - A review. Plant Omics. 6(6)). Multiple members of this type of protein can regulate plant immunity. For example, the C2H2-type zinc finger protein TaZFP8-5B can negatively regulate the host's resistance to pathogens (Huang, L., Xie, R., Hu, Y., Du, L., Wang, F., Zhao, X., Huang, Y., Chen, X., Hao, M., Xu, Q., Feng, L., Wu, B., Wei, Z., Zhang, L., Liu, D. (2024). A C2H2-type zinc finger protein TaZFP8-5B negatively regulates disease resistance. BMC Plant Biology. 24(1), 1116.); in soybean GmZFP03Regulating the expression of two superoxide dismutase genes can promote the resistance of soybeans to Phytophthora sojae (Li, W., Zheng, X., Cheng, R., Zhong, C., Zhao, J., Liu, T. H., Yi, T., Zhu, Z., Xu, J., Meksem, K., Dai, L., Liu, S. (2023). Soybean ZINC FINGERPROTEIN03 targets two SUPEROXIDE DISMUTASE1s and confers resistanceto Phytophthora sojae . Plant Physiology. 192(1), 633-647). Therefore, using such genes to improve crop resistance is a very practical and effective way to improve disease resistance. SUMMARY OF THE INVENTION

[0005] Based on the research of the inventors, a gene capable of expressing the zinc finger protein ZNF1 in soybeans was first discovered. Overexpression of ZNF1 can significantly inhibit the infection and replication of the soybean chlorotic stunt virus; by transgenic overexpression in Nicotiana benthamiana or using virus-mediated overexpression technology in soybeans, it was found that overexpression of ZNF1 can significantly inhibit the chlorotic stunt phenotype induced by the soybean chlorotic stunt virus. Thus, the present invention was completed.

[0006] The present invention first provides a zinc finger protein, the amino acid sequence of which is shown in SEQ ID No. 2.

[0007] Furthermore, the present invention provides the coding gene of the above-mentioned zinc finger protein.

[0008] An expression element containing the above-mentioned coding gene is also provided.

[0009] A recombinant vector containing the above-mentioned coding gene is also provided.

[0010] The present invention further provides a recombinant host bacterium containing the above-mentioned coding gene.

[0011] The present invention also provides the use of the above-mentioned zinc finger protein or its coding gene in the preparation of transgenic plants with improved disease resistance. Specifically, the plant is a dicotyledonous plant.

[0012] Preferably, the plant is soybean or Nicotiana benthamiana, and the disease resistance refers to the soybean chlorotic stunt disease caused by the soybean chlorotic stunt virus.

[0013] The present invention particularly provides a method for preparing transgenic plants with enhanced resistance to the soybean chlorotic stunt virus, which includes the steps of overexpressing the above-mentioned coding gene in transgenic plants by transgenic methods and screening to obtain transgenic plants with enhanced resistance to the soybean chlorotic stunt disease.

[0014] Specifically, the plant is a dicotyledon. Preferably, the plant is soybean; the disease resistance refers to the soybean zhengqing virus disease caused by the soybean zhengqing virus.

[0015] The inventors of the present invention have found through research that overexpression of soybean ZNF1 in plants can significantly enhance the resistance to soybean zhengqing virus and is expected to be popularized for practical applications. Description of the Drawings

[0016] Figure 1 For transient overexpression of soybean ZNF1 to inhibit the infection and replication of SoSGV. Among them, Figure A shows the inhibition of SoSGV replication by overexpressing soybean ZNF1 through fluorescence indication. Figure B shows the detection results by western blot.

[0017] Figure 2 For using PVX to overexpress soybean ZNF1 to inhibit the systemic infection of SoSGV. Among them, Figure A is a representative image of PVX overexpressing GUS control and soybean ZNF1 inoculated with SoSGV for 10 days. Figure B is the accumulation level of SoSGV virus protein CP and DNA after PVX overexpressing GUS control and soybean ZNF1 were inoculated with SoSGV for 10 days.

[0018] Figure 3 Transgenic overexpressing ZNF1 plants can inhibit the systemic infection of SoSGV. Among them, Figure A is a representative image of inoculating SoSGV in wild-type and overexpressing ZNF1 plants for 10 days. Figure B is the virus DNA accumulation level in wild-type and overexpressing ZNF1 plants after inoculating SoSGV for 10 days. Figure 3 In C is the detection result of the virus DNA accumulation level.

[0019] Figure 4 For using ALSV (apple latent spherical virus) to overexpress ZNF1 in soybean to inhibit the systemic infection of SoSGV. Among them, Figure A is a representative image of ALSV overexpressing GUS control and ZNF1 inoculated with SoSGV for 12 days. Figure B is the virus DNA accumulation level of SoSGV after ALSV overexpressing GUS control and ZNF1 were inoculated with SoSGV for 12 days. Detailed Embodiments

[0020] The present invention will be described below through specific examples for better understanding of the present invention, but it does not constitute a limitation to the present invention.

[0021] Example 1: Transient overexpression of soybean ZNF1 inhibits the infection and replication of SoSGV

[0022] Previously, by analyzing the transcriptome data of soybean and Nicotiana benthamiana infected with SoSGV, a zinc finger protein ZNF1 that was significantly up-regulated in response to virus infection was identified. Its amino acid sequence is shown in SEQ ID NO: 2, and the full-length nucleotide sequence of the CDS is shown in SEQ ID NO: 1.

[0023] To further verify whether this protein functions in virus infection, ZNF1 was constructed into the plant transient expression vector pCOMBIA, and then through the transient overexpression technology mediated by Agrobacterium tumefaciens, soybean ZNF1 was highly expressed in Nicotiana benthamiana leaves to verify its effect on the infection and replication of SoSGV.

[0024] The results showed that transient overexpression of soybean ZNF1 protein could inhibit the replication of SoSGV. When co-expressed with the SoSGV replicon (SoSGV-GFP) through fluorescence observation, the fluorescence intensity was significantly lower than that of the control group ( Figure 1 in A), and the fluorescence result was also confirmed by western blot ( Figure 1 in B). This indicates that ZNF1 is a host factor that negatively regulates the infection of SoSGV.

[0025] Example 2: Using PVX to overexpress soybean ZNF1 to inhibit the systemic infection of SoSGV and the symptom of "symptomless greening"

[0026] To further explore the effect of soybean ZNF1 on the systemic infection of SoSGV, the full-length CDS of soybean ZNF1 was inserted into the genome of PVX (pGR106 vector). The pGR106 vector is an overexpression tool for foreign proteins modified based on potato virus X, which can infect solanaceous plants such as Nicotiana benthamiana. Inserting the target gene into the virus genome can achieve the high expression of this gene and produce a large amount of corresponding proteins. Soybean ZNF1 was highly expressed in Nicotiana benthamiana using PVX, and then SoSGV was inoculated to verify its effect on systemic infection.

[0027] Ten days after inoculating PVX, SoSGV was inoculated again. Ten days later, it was observed that compared with the control group, the symptoms of the group inoculated with SoSGV after PVX overexpressed soybean ZNF1 were significantly weaker than those of the control group overexpressing GUS with PVX ( Figure 2 in A). In the control group overexpressing GUS, obvious leaf curling was observed in the plants, while no systemic symptoms of SoSGV infection were observed in the plants overexpressing ZNF1.

[0028] At the same time, the CP protein and DNA accumulation levels of SoSGV were detected by western blot and qPCR respectively, showing that the accumulation levels of SoSGV CP and DNA in the treatment group inoculated with SoSGV after PVX overexpressed soybean ZNF1 were significantly lower than those of the control group.Figure 2 In B), this indicates that overexpression of PVX - soybean ZNF1 significantly inhibits the systemic infection of SoSGV.

[0029] Example 3: Transgenic overexpression of GmZNF1 plants inhibits the systemic infection of SoSGV

[0030] For the construction of the overexpression vector in Nicotiana benthamiana, the full - length CDS fragment of ZNF1 was inserted into the p2300 vector with a 35s promoter by recombinant cloning method, and transgenic materials were created in the laboratory ( Figure 3 In A). The overexpression of ZNF1 in transgenic T0 plants was confirmed by western blot experiment.

[0031] After collecting the seeds of T0 positive plants, T1 plants were used to conduct resistance tests, that is, whether overexpression of GmZNF1 confers the ability of plants to resist SoSGV infection.

[0032] At the 4 - 5 leaf stage of T1 tobacco plants overexpressing ZNF1, SoSGV virus was inoculated. After 10 days, the symptoms caused by the virus and the accumulation level of viral DNA were detected. The results of the resistance test showed that the viral symptoms produced by the two transgenic lines overexpressing ZNF1 were significantly weaker than those of the wild - type 10 days after inoculation with SoSGV. In the wild - type plants, slight leaf curling under was shown, while no obvious viral symptoms were observed in the two transgenic lines ( Figure 3 In A). The overexpression of ZNF1 in T1 tobacco plants overexpressing ZNF1 was confirmed by western blot ( Figure 3 In B). Subsequently, the detection results of the viral DNA accumulation level showed that the viral DNA accumulation levels in the two transgenic lines overexpressing ZNF1 were significantly lower than those of the wild - type control group ( Figure 3 In C).

[0033] Therefore, based on the above results, it is shown that plants with transgenic overexpression of soybean ZNF1 can significantly inhibit the systemic infection of SoSGV.

[0034] Example 4: Overexpression of GmZNF1 in soybean using ALSV inhibits the systemic infection of SoSGV

[0035] To further clarify whether ZNF1 plays a role in resisting SoSGV infection in soybeans and whether it has application potential in soybeans, an ALSV (apple latent spherical virus) virus overexpression vector was used. This vector is similar to the PVX vector but can overexpress the target protein in soybeans. By this method, ZNF1 (ALSV-ZNF1) was overexpressed in soybeans, and SoSGV was transmitted to soybeans overexpressing ZNF1 as the treatment group and soybeans overexpressing GUS as the control group through the oriental greenhouse leafhopper as a vector insect to verify the effect of overexpressing ZNF1 on SoSGV infection.

[0036] Twelve days after inoculation with SoSGV, it was observed that the plant height of plants overexpressing ZNF1 with ALSV was higher than that of the control group and there was no significant difference in plant height from the treatment group without SoSGV inoculation ( Figure 4 in A). Further detection of the accumulation level of SoSGV viral DNA showed that the accumulation level of viral DNA in soybeans overexpressing ZNF1 with ALSV was significantly lower than that of the control group ( Figure 4 in B), indicating that overexpression of ZNF1 with ALSV significantly inhibited the systemic infection of SoSGV in soybeans.

Claims

1. Use of a zinc finger protein or its encoding gene in the preparation of a transgenic plant with improved disease resistance, characterized in that, The zinc finger protein is a protein with the amino acid sequence shown in SEQ ID No.2; The plant is Nicotiana benthamiana or soybean, and it is achieved by overexpressing the zinc finger protein coding gene in the plant; The disease resistance refers to resistance to soybean sudden death syndrome caused by soybean sudden death syndrome virus.

2. The application according to claim 1, wherein The nucleotide sequence of the coding gene is shown in SEQ ID No.

1.

3. A method for preparing a transgenic plant with enhanced resistance to soybean symptomless virus, characterized in that, It includes the steps of overexpressing the coding gene of the zinc finger protein with the amino acid sequence shown in SEQ ID No.2 in transgenic plants by transgenic methods, and screening and obtaining transgenic plants with enhanced resistance to soybean sudden death syndrome; The plant is soybean or Nicotiana benthamiana.

4. The method according to claim 3, characterized in that, The nucleotide sequence of the coding gene is shown in SEQ ID No.1.

Images