Application of zinc finger protein in resisting soybean disease blue virus

Abstract

The invention belongs to the technical field of biology, and discloses application of zinc finger protein in resisting soybean disease green virus. It is found that the zinc finger protein ZNF1 in soybeans can inhibit virus infection in SoSGV infection, SoSGV infection can be remarkably inhibited by overexpressing the gene, and plants can be endowed with remarkable resistance to SoSGV through transgenic overexpression of the ZNF1. Therefore, the zinc finger protein ZNF1 can be used for preparing transgenic plants for enhancing soybean disease green virus resistance, and has great application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to application of zinc finger protein in resistance to soybean symptomatic virus. Background Art

[0002] Soybean ( Glycine max(L.) Merr ) is a high-quality, high-protein food crop, known as the "golden crop" and "plant meat". It provides 65% of plant protein and 33% of edible vegetable oil for humans and is one of the most important crops in the world (Brar, GS, Thomas, JR (1993). Soybean: Glycine max (L) Merrill-Science Direct. Genetic Improvement of Vegetable Crops. 427-463.). In addition to being a 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 food, medicine, cosmetics and other fields; soybean defatted meal is a high-quality feed that 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, PD (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 many factors, such as climate, variety, planting technology, pests and diseases, etc. Diseases are an important factor affecting soybean yield. For example, in recent years, the large-scale outbreak of soybean staygreen syndrome in the Huang-Huai-Hai region has posed a great threat to the healthy development of the soybean industry. Soybean staygreen syndrome is commonly known as greedy green or inverted green (Wang Jiaxiang, Yang Mingxing, Deng Daosheng. (1982). Causes and preventive measures of soybean greedy green pods. Henan Agricultural Science. (9), 20). The main field symptoms are that when soybeans mature normally, the soybean plants still have 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. CropJ. 7(3), 360-367). Due to the complexity of the causes of soybean greening disease, the pathogenic pathogen of soybean greening disease has remained unclear for a long time. In 2022, Cheng et al. from our research group identified a recombinant geminivirus, soybean stay-green associated virus (SoSGV), in soybean stay-green disease samples through small RNA sequencing, and confirmed through Koch's postulates that the virus can cause soybean stay-green phenotypes. After the virus infects soybeans, the phenotypes of green leaves, shrunken leaves and abnormal grain development can appear, which is consistent with most stay-green phenotypes observed in the field. Further studies found that in addition to infecting soybeans, SoSGV can also infect Nicotiana benthamiana, causing delayed senescence and shrunken 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 newdistinct geminivirus causes soybean stay-green disease. Molecular Plant. 15(6), 927-930).Further testing of 368 samples of soybean stay-green associated virus collected from 17 regions in 8 provinces showed that 228 samples were positive (61.96%), and 96.93% of the positive samples showed typical symptoms of soybean stay-green. This indicates that the soybean stay-green caused by SoSGV has become the mainstream in the field and is spreading from the Huanghuaihai 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). Epidemiologicalevaluation and identification of the insect vector of soybean stay-green associated virus. Phytopathology Research. 5(1), 20). At the same time, Cheng et al. of this research group identified the virus as the Japanese net-house leafhopper (. Orosius orientalis ). This virus is a newly identified new species of Geminiviridae. The pathogenic mechanism of the virus is still unclear and no effective disease-resistant genes have been identified. Therefore, there is still a lack of effective means to prevent and control the disease. At present, no related genes and technologies have been reported to be able to be used to resist soybean symptomatic green virus.

[0004] To resist pathogen invasion, host plants have evolved a two-layer innate immune system. One is PTI (Pattern-triggered immunity) immunity, which is triggered by the recognition of conserved pathogen-associated molecular patterns (PAMPs) produced by pathogenic microorganisms by pattern recognition receptors (PRRs) on the surface of plant cells. The other is ETI (Effector-triggered immunity) immunity, which is triggered by the recognition of pathogen effectors by intracellular immune proteins (Jones, J. D and Dangl, JL (2006). The plant immune system. Nature. 444(7117), 323-329). Plant immune activation involves a series of immune signal transductions, among which transcriptional reprogramming is an important feature of plant immunity, which is regulated by a variety of transcription factors and transcript complex-related proteins (Tsuda, K and Somssich, IE (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 that are responsible for regulating gene transcription and expression. This family of proteins is divided into many 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).). Many members of this class of proteins can regulate plant immunity, such as C2H2 type zinc finger protein TaZFP8-5B Can negatively regulate host 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.); GmZFP03Soybean ZINC FINGERPROTEIN03 targets two SUPEROXIDE DISMUTASE1s and confers resistance to Phytophthora sojae (Li, W., Zheng, X., Cheng, R., Zhong, C., Zhao, J., Liu, TH, Yi, T., Zhu, Z., Xu, J., Meksem, K., Dai, L., Liu, S. (2023). Soybean ZINC FINGERPROTEIN03 targets two SUPEROXIDE DISMUTASE1s and confers resistance to Phytophthora sojae (Li, W., Zheng, X., Cheng, R., Zhong, C., Zhao, J., Liu, TH, Yi, T., Zhu, Z., Xu, J., Meksem, K., Dai, L., Liu, S. (2023). Phytophthora sojae . Plant Physiology. 192(1), 633-647). Therefore, using this type of gene to improve crop resistance is a very effective way to improve disease resistance. Summary of the invention

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

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

[0007] Furthermore, the present invention provides a gene encoding the zinc finger protein.

[0008] An expression element containing the encoding gene is also provided.

[0009] Also provided is a recombinant vector containing the encoding gene.

[0010] The present invention further provides a recombinant host bacterium containing the encoding gene.

[0011] The present invention also provides the use of the zinc finger protein or its encoding gene in preparing a transgenic plant 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 soybean cyanosis virus disease caused by soybean cyanosis virus.

[0013] The present invention particularly provides a method for preparing transgenic plants with enhanced resistance to soybean cyanosis virus, which comprises the steps of overexpressing the coding gene in transgenic plants by transgenic methods, and screening transgenic plants with enhanced resistance to soybean cyanosis virus.

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

[0015] The inventors have found that overexpression of soybean ZNF1 in plants can significantly enhance resistance to soybean chlorosis virus and is expected to be promoted for practical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Transient overexpression of soybean ZNF1 inhibits the infection and replication of SoSGV. Figure A shows the inhibition of SoSGV replication by overexpression of soybean ZNF1 through fluorescence indication. Figure B shows the results of western blot detection.

[0017] Figure 2 To inhibit SoSGV systemic infection by overexpressing soybean ZNF1 using PVX. Figure A shows representative images of PVX overexpressing GUS control and soybean ZNF1 10 days after SoSGV inoculation. Figure B shows the accumulation levels of SoSGV viral protein CP and DNA 10 days after PVX overexpressing GUS control and soybean ZNF1 inoculation.

[0018] Figure 3 Transgenic plants overexpressing ZNF1 can inhibit SoSGV systemic infection. Figure A shows representative images of wild-type and ZNF1-overexpressing plants 10 days after inoculation with SoSGV. Figure B shows the level of viral DNA accumulation in wild-type and ZNF1-overexpressing plants 10 days after inoculation with SoSGV. Figure 3 Middle C is the test result of viral DNA accumulation level.

[0019] Figure 4 To inhibit SoSGV systemic infection by overexpressing ZNF1 in soybean using ALSV (apple latent spherical virus). Figure A shows representative images of ALSV overexpressing GUS control and ZNF1 inoculated with SoSGV 12 days later. Figure B shows the viral DNA accumulation level of SoSGV 12 days after ALSV overexpressing GUS control and ZNF1 inoculated with SoSGV. DETAILED DESCRIPTION

[0020] The present invention is described below by means of specific embodiments to facilitate a better understanding of the present invention, but the present invention is not limited thereto.

[0021] Example 1: Transient overexpression of soybean ZNF1 inhibits SoSGV infection and replication In the early stage, by analyzing the transcriptome data of soybean and Nicotiana benthamiana infected with SoSGV, a zinc finger protein ZNF1 was identified whose expression was significantly upregulated in response to virus infection. Its amino acid sequence is shown in SEQ ID NO: 2, and the full-length CDS nucleotide sequence is shown in SEQ ID NO: 1.

[0022] In order to further verify whether this protein plays a role in viral infection, ZNF1 was constructed into the plant transient expression vector pCOMBIA vector, and then soybean ZNF1 was expressed in large quantities in Nicotiana benthamiana leaves through Agrobacterium tumefaciens-mediated transient overexpression technology to verify its effect on SoSGV infection and replication.

[0023] The results showed that transient overexpression of soybean ZNF1 protein could inhibit SoSGV replication. The fluorescence intensity of ZNF1 co-expressed with SoSGV replicon (SoSGV-GFP) was significantly lower than that of the control group ( Figure 1 The fluorescence results were also confirmed by western blot ( Figure 1 This suggests that ZNF1 is a host factor that negatively regulates SoSGV infection.

[0024] Example 2: Using PVX to overexpress soybean ZNF1 to inhibit SoSGV system infection and symptom-greening phenotype To further explore the effect of soybean ZNF1 on SoSGV systemic infection, the full-length CDS of soybean ZNF1 was inserted into the genome of PVX (pGR106 vector). The pGR106 vector is a tool for overexpressing foreign proteins based on potato virus X virus, which can infect Solanaceae plants such as Nicotiana benthamiana. Inserting the target gene into the viral genome can achieve efficient expression of the gene and produce a large amount of corresponding protein. PVX was used to express soybean ZNF1 in large quantities in Nicotiana benthamiana, and then SoSGV was inoculated to verify the effect on its systemic infection.

[0025] Ten days after PVX inoculation, SoSGV was inoculated again. Ten days later, it was observed that the symptoms of PVX overexpressing soybean ZNF1 and then inoculated with SoSGV were significantly weaker than those of the PVX overexpressing GUS control group ( Figure 2 (A) Obvious leaf curling was observed in the plants of the control group overexpressing GUS, while no systemic symptoms of SoSGV infection were observed in the plants overexpressing ZNF1.

[0026] At the same time, western blot and qPCR were used to detect the accumulation levels of SoSGV CP protein and DNA, respectively. The results showed that the accumulation levels of SoSGV CP and DNA in the PVX overexpression soybean ZNF1 group and then inoculated with SoSGV were significantly lower than those in the control group ( Figure 2(B), indicating that PVX overexpression of soybean ZNF1 significantly inhibited the systemic infection of SoSGV.

[0027] Example 3: Transgenic plants overexpressing GmZNF1 inhibit SoSGV system infection For the construction of Nicotiana benthamiana overexpression vector, the full-length CDS fragment of ZNF1 was inserted into the p2300 vector with 35S promoter by recombination cloning, and transgenic materials were created in the laboratory ( Figure 3 (A) Transgenic T0 plants were confirmed by western blot experiments to have overexpressed ZNF1 in transgenic plants.

[0028] After collecting seeds of T0 generation positive plants, resistance tests were carried out using T1 generation plants to determine whether overexpression of GmZNF1 confers the ability to resist SoSGV infection on plants.

[0029] The T1 tobacco plants overexpressing ZNF1 were inoculated with SoSGV at the 4-5 leaf stage, and the virus symptoms and viral DNA accumulation levels were detected 10 days later. The resistance test results showed that the virus symptoms produced by the two transgenic lines overexpressing ZNF1 were significantly weaker than those of the wild type 10 days after inoculation with SoSGV. The wild type plants showed slight leaf curling, while no obvious virus symptoms were observed in the two transgenic lines ( Figure 3 Middle A). The overexpression of ZNF1 in T1 generation tobacco plants overexpressing ZNF1 was confirmed by western blot ( Figure 3 The results of subsequent detection of viral DNA accumulation levels showed that the viral DNA accumulation levels in the two transgenic lines overexpressing ZNF1 were significantly lower than those in the wild-type control group ( Figure 3 Middle C).

[0030] Therefore, based on the above results, it was shown that transgenic soybean plants overexpressing ZNF1 could significantly inhibit the systemic infection of SoSGV.

[0031] Example 4: Overexpression of GmZNF1 in soybean using ALSV to inhibit SoSGV system infection To further clarify whether ZNF1 plays a role in resisting SoSGV infection in soybeans and whether it has application potential in soybeans, we used the ALSV (apple latent spherical virus) virus overexpression vector, which is similar to the PVX vector but can overexpress the target protein in soybeans. ZNF1 (ALSV-ZNF1) was overexpressed in soybeans by this method, and SoSGV was transmitted to soybeans with ZNF1 overexpression as the treatment group and GUS overexpression as the control group using the Oriental net house leafhopper as the vector insect, respectively, to verify the effect of overexpression of ZNF1 on SoSGV infection.

[0032] Twelve days after inoculation with SoSGV, it was observed that the height of the plants overexpressing ZNF1 in ALSV was higher than that in the control group, and there was no significant difference in plant height between the plants inoculated with ALSV and those not inoculated with SoSGV ( Figure 4 Further testing of the SoSGV viral DNA accumulation level showed that the viral DNA accumulation level in soybeans with ALSV overexpressing ZNF1 was significantly lower than that in the control group ( Figure 4 (B) showed that ALSV overexpression of ZNF1 significantly inhibited the systemic infection of SoSGV in soybean.

Claims

1. An isolated zinc finger protein, characterized in that It is a protein having the amino acid sequence shown in SEQ ID No.

2.

2. The isolated gene encoding the zinc finger protein according to claim 1, characterized in that It is a nucleic acid molecule with a nucleotide sequence shown in SEQ ID No.

1.

3. An expression element containing the coding gene as claimed in claim 2.

4. A recombinant vector containing the coding gene as claimed in claim 2.

5. A recombinant host bacteria containing the coding gene as claimed in claim 2.

6. Use of the zinc finger protein according to claim 1, or a gene encoding the zinc finger protein, in preparing a transgenic plant with improved disease resistance.

7. The use according to claim 6, characterized in that The plant is a dicotyledonous plant.

8. The use according to claim 7, characterized in that The plant is soybean or Nicotiana benthamiana, and the disease resistance refers to soybean cyanosis virus disease caused by soybean cyanosis virus.

9. A method for preparing a transgenic plant with enhanced resistance to soybean scurvy virus, characterized in that: The method comprises the steps of over-expressing the coding gene as claimed in claim 2 in transgenic plants by a transgenic method, and screening transgenic plants with enhanced resistance to soybean scurvy virus disease.

10. The method according to claim 9, characterized in that The plant is soybean; the disease resistance refers to soybean greening virus disease caused by soybean greening virus.

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