Use of wild soybean zinc finger protein gszfp4 or encoding material thereof

Abstract

The use of a wild soybean zinc finger protein GsZFP4 or an encoding material thereof. The nucleotide sequence of a gene GsZFP4 which encodes the wild soybean zinc finger protein GsZFP4 is shown as SEQ ID NO. 1. A constructed plant over-expression vector pBA002-GsZFP4 is transformed into soybeans. After treatment with the SMV strain SC7 for 21 days, it is found that soybeans that over-express GsZFP4 have obviously relieved disease symptoms and remarkably reduced SMV content, and show resistance against SMVs. In general, GsZFP4 may positively regulate the resistance of soybeans against SMVs.

Description

Application of wild soybean zinc finger protein GsZFP4 or coding material thereof

[0001] The present application claims priority to the Chinese patent application No. CN202410405467.8, filed on April 7, 2024, and entitled "Application of wild soybean zinc finger protein transcription factor coding gene GsZFP4", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application belongs to the field of genetic engineering, and relates to the application of wild soybean zinc finger protein GsZFP4 or coding material thereof. BACKGROUND

[0003] Soybean is susceptible to various diseases and environmental stresses during growth. Among the diseases, soybean mosaic virus (SMV) can cause serious damage to the yield and quality of soybean. The yield loss caused by SMV is generally about 10%, and in severe years, it can reach 35%-50% or even zero yield (Li et al., 2013). Since SMV is widely distributed, causes serious damage, and is difficult to control by chemical agents, breeding and planting disease-resistant varieties are the most economical and effective control method for this disease.

[0004] Wild soybean (Glycine soja Sieb. et Zucc.) is a wild relative of cultivated soybean. China is the origin center of soybean, and there are rich wild soybean resources. At present, there are more than 6000 wild soybean resources preserved in China, accounting for more than 90% of the total number in the world (Ma Xiaoping et al., 2009). Molecular marker analysis shows that the genetic diversity of wild soybean is higher than that of cultivated soybean. By comparing the genetic diversity of wild soybean and local varieties, it is found that only 51% of the alleles in wild soybean are retained in local soybean varieties (Wen et al., 2009). Therefore, using wild soybean resources to explore new SMV disease resistance genes plays a key role in reducing SMV damage and improving SMV breeding effectiveness.

[0005] SUMMARY

[0006] The purpose of the present application is to disclose the disease resistance gene engineering application of wild soybean zinc finger protein transcription factor coding gene GsZFP4. The gene is rapidly up-regulated in leaves after SMV induction. In addition, subcellular localization analysis shows that GsZFP4 is a nuclear localization protein. GsZFP4 gene can be introduced into soybean as a target gene to positively regulate the resistance of soybean to SMV.

[0007] The purpose of the present application can be achieved by the following technical solutions:

[0008]

[0009] The application also provides a recombinant expression vector containing the wild soybean zinc finger protein transcription factor encoding gene GsZFP4.

[0010] When the plant expression vector is constructed using the gene GsZFP4, any enhanced promoter or inducible promoter can be added before the transcription initiation nucleotide. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding a selective marker gene (GUS gene, luciferase gene, etc.) to the plant expression vector. From the safety point of view of transgenic plants, no selective marker gene can be added, and the transformed plants can be screened by stress.

[0011] The plant expression vector carrying the gene GsZFP4 described in the application can be transformed into plant cells or tissues by using Ti plasmid, Ri plasmid, plant virus vector, DNA direct transformation, microinjection, electroporation, Agrobacterium-mediated, etc. Conventional biological methods, and the transformed plant tissues are cultivated into plants. The plant host to be transformed can be a monocotyledonous plant such as sorghum, rice, wheat, corn, etc., or a dicotyledonous plant such as peanut, soybean, rape, tomato, poplar, lawn grass, alfalfa, etc.

[0012] The wild soybean zinc finger protein transcription factor encoding gene GsZFP4 described in the application is found to positively regulate the resistance of soybean to SMV after being transformed into soybean by genetic engineering. Advantages

[0013] The wild soybean GsZFP4 is a zinc finger protein transcription factor, and the gene positively regulates the resistance of transgenic soybean to SMV strain SC7. The gene is rapidly up-regulated in the leaf after SMV induction, and the subcellular localization analysis shows that GsZFP4 is a nuclear localization protein. At the same time, it is found through function verification that GsZFP4 positively regulates the resistance of transgenic soybean to SMV. Therefore, GsZFP4 can be used as a target for regulating the resistance of soybean to SMV, and used for the modification of soybean resistance to SMV. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0015] Figure 1 is the PCR amplification result of the GsZFP4 gene; wherein, Marker: 5000bp, GsZFP4 gene target band 1275bp;

[0016] Figure 2 is the subcellular localization of GsZFP4; wherein, GFP: green fluorescent protein; M-cherry: red fluorescent protein; BF: bright field; Merge: fusion protein; 35S:GFP: empty control; 35S:GsZFP4-GFP: GsZFP4 protein with GFP tag; scale bar: 50 μm;

[0017] Figure 3 is the expression amount of GsZFP4 in resistant material NJAU_W052 and susceptible material HAAS_075 after induction by SMV-SC7 for 0, 2, 4, 8, 12, 48 h; wherein, the values are three biological average values ± standard error (SE), and * and ** are significant at the 0.05 and 0.01 probability levels, respectively;

[0018] Figure 4 is the relative expression amount of GsZFP4 gene in overexpression material; wherein, OE3 and OE14 represent two GsZFP4 overexpression lines; the values are three biological average values ± standard error (SE), and * and ** are significant at the 0.05 and 0.01 probability levels, respectively;

[0019] Figure 5 is the detection result of GsZFP4 gene overexpression soybean test strip; wherein, the negative is a control wild type, non-transgenic plant one band;

[0020] Figure 6 is the resistance phenotype of GsZFP4 overexpression line material to SMV-SC7 21 days after inoculation;

[0021] Figure 7 is the expression amount of coat protein (CP) encoding gene in GsZFP4 overexpression line material detected by qRT-PCR; wherein, the values are three biological average values ± standard error (SE), and * is significant at the 0.05 probability level. DETAILED DESCRIPTION

[0022] The present application is further described below in conjunction with the accompanying drawings and examples.

[0023] The methods used in the following examples are conventional methods unless otherwise specified.

[0024] Example 1

[0025] Cloning and expression characteristic analysis of wild soybean GsZFP4 gene

[0026] 1) Cloning of wild soybean zinc finger protein transcription factor encoding gene GsZFP4

[0027] The base sequence corresponding to the gene was found in the Soybase database according to the GsZFP4 gene number Glysoja.04G010500, and specific primers F1: 5'-CTCGTCGTACAATCCAT CTC-3'(SEQ ID NO. 3) and F2: 5'-CATTTCCACTCAAACATTTATC-3'(SEQ ID NO. 4) were designed according to the sequence.

[0028] The wild soybean material NJAU_W052 was taken as the material, and its leaves were taken and ground with a mortar. The lysate was added to a 1.5 mL EP tube, shaken well, and then transferred to a 1.5 mL EP tube. Total RNA was extracted (Tiangen, Beijing, China). The total RNA quality was identified by formaldehyde denaturing gel electrophoresis, and the RNA content was determined by spectrophotometer.

[0029] The obtained total RNA was used as a template, and reverse transcription was performed according to the instructions of the reverse transcription kit provided by Takara company. After obtaining the first strand of cDNA, PCR amplification was performed. The PCR program was as follows: 95°C pre-denaturation for 3 minutes, 95°C denaturation for 15 seconds, 60°C annealing for 15 seconds, 72°C extension for 1 minute and 30 seconds, a total of 35 cycles, finally 72°C for 5 minutes, followed by 12°C constant temperature. Then, PCR product gel purification, ligation and transformation were performed, and positive single clones were selected for sequencing. After sequencing, the CDS sequence of the wild soybean GsZFP4 gene with a length of 1275 bp was obtained (SEQ ID NO. 1), and the PCR amplification results are shown in Figure 1. The gene sequence was synthesized by Beijing Qikexin Biotechnology Co., Ltd. for subsequent vector construction.

[0030] 2) Subcellular localization of GsZFP4

[0031] Primers (not containing stop codon) F3: 5'-ACAAATCTATCTCTCTCGAGATGTCCTCCGAAGACATCAC-3'(SEQ ID NO. 5) and F4: 5'-GCTCACCATGGATCCAAGCCAATTAGGTGGCATCT-3'(SEQ ID NO. 6) containing the complete ORF of GsZFP4 gene were designed, and the specific PCR process and steps were the same as step 1).

[0032] Then the complete ORF of GsZFP4 gene without stop codon was homologously recombined into the expression vector pFGC5941 by double digestion with Xhol and BamHI, so that the complete ORF of GsZFP4 gene was fused with the 3' end of the reporter gene GFP on the expression vector pFGC5941 to form a 35S-GsZFP4-GFP chimeric gene, and a subcellular localization vector pFGC5941-GsZFP4 was constructed. The target gene GsZFP4 was transformed into tobacco leaf cells by Agrobacterium transformation method with the empty vector, respectively. The results showed that the GsZFP4 protein was located in the nucleus (Figure 2).

[0033] 3) Expression analysis of GsZFP4 after induction by SMV

[0034] The resistant material NJAU_W052 and the susceptible material HAAS_075 were inoculated with SMV-SC7, respectively, to detect the difference in the expression amount of GsZFP4 between the resistant and susceptible materials. The treated leaves were collected at 0h, 2h, 4h, 8h, 12h and 48h after treatment, and were quickly frozen in liquid nitrogen and stored at -80°C.

[0035] The total RNA extraction step 1).

[0036] Soybean constitutively expressed Tubulin (accession number AY907703) was used as an internal reference, and the primers were F5: 5'-GGAGTTCACAGAGGCAGAG-3' (SEQ ID NO. 7) and F6: 5'-CACTTAC GCATCACATAGCA-3' (SEQ ID NO. 8). The total RNA from the leaves of the wild soybean resistant material NJAU_W052 and the susceptible material HAAS_075 under different treatment conditions was used as a template, and after reverse transcription to cDNA, real-time fluorescent quantitative PCR reaction was carried out, and the primer sequences were F7: 5'-CTATTCGCA CTATCTTCACG-3' (SEQ ID NO. 9) and F8: 5'-ATCCTTCCTCCCACTAAACC-3' (SEQ ID NO. 10), to detect the expression change of GsZFP4 after induction by SMV.

[0037] It was found in the present application that the expression of GsZFP4 in the resistant material was strongly induced by SMV, and reached a peak at 8h after inoculation, about 18 times of that at 0h (Figure 3). This result shows that GsZFP4 in the resistant material can actively respond to the induction of SMV to increase the expression amount.

[0038] Example 2

[0039] Genetic engineering application of GsZFP4 gene

[0040] 1) Construction of plant overexpression vector

[0041] The CDS sequence of GsZFP4 gene was obtained by PCR amplification from PUC19-T Vector containing the CDS sequence of wild soybean GsZFP4 gene shown in SEQ ID NO. 1 synthesized by Bio Company, using primers F9: 5'-CGCGCCGGGCCCAGGCCTACGCGTATGTCCTCCGAAGACATCACTCT-3' (SEQ ID NO. 11) and F10: 5'-ATCGGGGAAATTCGAGCTCCTAAAGCCAATTAGGTGGCATCT-3' (SEQ ID NO. 12). The GsZFP4 was ligated into pBA002 vector by recombination reaction to obtain pBA002-GsZFP4 plant overexpression vector. The plant transformation vector pBA002 contains a strong 35S promoter that can strongly induce the expression of the target gene GsZFP4 in the recipient. Then the pBA002-GsZFP4 plant overexpression vector was transformed into Agrobacterium tumefaciens strain EHA105 by freeze-thaw method, and soybean was transformed by soybean cotyledon node transformation method.

[0042] 2) Relative expression amount of GsZFP4 gene in overexpression materials

[0043] Two stable transformed overexpression positive lines, named OE3 and OE14, were obtained by tissue culture method. The GsZFP4 overexpression soybean materials were planted in a constant temperature basement, and the leaves were taken after 21 days, frozen in liquid nitrogen and stored at -80°C. The total RNA extraction step 1). Using soybean constitutive expression Tubulin as internal reference, the primer sequences were F5 and F6, and the total RNA of GsZFP4 overexpression soybean material was used as template, and after reverse transcription to cDNA, real-time fluorescent quantitative PCR reaction was carried out, the primer sequences were F7 and F8, and the expression amount of GsZFP4 gene in different materials was detected. It can be seen from Figure 4 that the expression amount of GsZFP4 overexpression line is significantly higher than that of wild type.

[0044] 3) Phenotype identification

[0045] Two overexpression lines OE3 and OE14 were obtained by tissue culture, and the overexpression positive seedlings were detected by bar test strip (Figure 5). After the true leaves fully expanded, the wild type WT and overexpression lines were inoculated with SMV-SC7 virus or 0.01 mol / L phosphate buffer, and the disease was observed 21 days after inoculation. The leaves of the control plants WT showed obvious shrinkage, and the leaves of the overexpression lines did not show obvious mosaic symptoms (Figure 6). The expression level of the virus CP gene was analyzed, and it was found that the expression level of SMV-CP gene in the two overexpression lines was significantly lower than that of the wild type (Figure 7). The primer sequences for detecting the expression level of SMV-CP gene are F11: 5'-CAGATGGGTGTGGTTATG-3'(SEQ ID NO. 13) and F12: 5'-ACAATGGGTTTCAGCGGATA-3'(SEQ ID NO. 14). The above results show that overexpression of GsZFP4 gene reduces the accumulation of virus and improves the resistance of soybean to SMV-SC7.

[0046] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

[0047] References

[0048] Ma X P, Yang G Y, Yang Z Y, et al. Application of wild soybean in soybean breeding [J]. Crop Research, 2009, 23(01): 11-12.

[0049] Wu W X, Yang X F, Liu Y, et al. Prokaryotic expression and functional study of tobacco C3HC4-type zinc finger protein in transgenic plants [J]. Biotechnology Bulletin, 2014(07): 100-105.

[0050] Guo H., Bi X., Wang Z., Jiang D., Cai M., An M., et al. (2022). Reactive oxygen species-related genes participate in resistance to cucumber green mottle mosaic virus infection regulated by boron in Nicotiana benthamiana and watermelon. Front. Plant Sci. 13. doi: 10.3389 / fpls.2022.1027404.

[0051] Hernandez, J. A., Gullner, G., Clemente-Moreno, M. J., Kunstler, A., Juhasz, C, Diaz-Vivancos, P. et al. (2016) Oxidative stress and antioxidative responses in plant-virus interactions. Physiological and Molecular Plant Pathology, 94, 134-148.

[0052] Li K, Liu ZT, Li HC, Zhang K, Wang CK, Ren R, Lu WG, Zhi HJ (2013) Resistance to soybean mosaic virus and soybean cyst nematode of soybean cultivars from China national soybean uniform trials. Soybean Science 32(5):670-675.

[0053] Li, W., Zhu, Z., Chern, M., Yin, J., Yang, C, Ran, L. et al. (2017) A natural allele of a transcription factor in rice confers broad-spectrum blast resistance. Cell, 170, 114-126.

[0054] Wen Z, Ding Y, Zhao T. et al. (2009) Genetic diversity and peculiarity of annual wild soybean (G. soja Sieb. et Zucc.) from various eco-regions in China. Theoretical and Applied Genetics, 119:371-381.

Claims

1. Application of wild soybean zinc finger protein GsZFP4 or biological materials encoding said wild soybean zinc finger protein GsZFP4 in regulating soybean resistance to soybean mosaic virus; The amino acid sequence of the wild soybean zinc finger protein GsZFP4 is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The biological material includes wild soybean zinc finger protein transcription factor encoding gene GsZFP4, plant expression vector or recombinant bacteria; The nucleotide sequence of the GsZFP4 gene is shown in SEQ ID NO.1; The plant expression vector comprises a basic vector and the GsZFP4 gene inserted into the basic vector; The recombinant bacteria include basic bacteria and the GsZFP4 gene or the plant expression vector introduced into the basic bacteria.

3. The use according to claim 2, characterized in that The basic vector includes pBA002 vector or PUC19-T vector; The basic bacteria include Agrobacterium.

4. The use according to any one of claims 1 to 3, characterized in that The regulation includes overexpressing the wild soybean zinc finger protein GsZFP4, reducing the content of soybean mosaic virus in soybean, and enhancing the resistance of soybean to soybean mosaic virus.

5. A plant expression vector, characterized in that The plant expression vector comprises a basic vector and a GsZFP4 gene inserted into the basic vector; the nucleotide sequence of the GsZFP4 gene is shown in SEQ ID NO.

1.

6. The plant expression vector according to claim 5, characterized in that The basic vector includes pBA002 vector or PUC19-T vector.

7. A recombinant bacterium, characterized in that The recombinant bacteria include a basic bacteria and a GsZFP4 gene introduced into the basic bacteria or the plant expression vector according to claim 5 or 6; the nucleotide sequence of the GsZFP4 gene is shown in SEQ ID NO.

1.

8. The recombinant bacterium according to claim 7, characterized in that The basic bacteria include Agrobacterium.

9. The recombinant bacterium according to claim 8, characterized in that The Agrobacterium is Agrobacterium tumefaciens strain EHA105.

10. Use of the plant expression vector according to claim 5 or 6 or the recombinant bacterium according to any one of claims 7 to 9 in improving the resistance of soybean to soybean mosaic virus.

11. Use of the plant expression vector according to claim 5 or 6 or the recombinant bacterium according to any one of claims 7 to 9 in cultivating transgenic plants resistant to soybean mosaic virus.

12. The use according to claim 11, characterized in that The transgenic plants include transgenic monocotyledonous plants or transgenic dicotyledonous plants.

13. The use according to claim 12, characterized in that The monocot transgenic plants include sorghum, rice, wheat and corn; The dicotyledonous plants include peanut, soybean, rapeseed, tomato, poplar, turf grass and alfalfa.

14. A method for improving soybean resistance to soybean mosaic virus, characterized in that: include: The biological material encoding the wild soybean zinc finger protein GsZFP4 is introduced into soybeans to overexpress the wild soybean zinc finger protein GsZFP4 in soybeans; the amino acid sequence of the wild soybean zinc finger protein GsZFP4 is shown in SEQ ID NO.

2.

15. The method according to claim 14, characterized in that The introduction method is the soybean cotyledonary node transformation method.

16. A method for cultivating transgenic plants resistant to soybean mosaic virus, characterized in that: The host plant cells or tissues are transformed with biological materials encoding wild soybean zinc finger protein GsZFP4 and then cultivated.

17. The method according to claim 16, characterized in that The host plant includes a monocotyledonous plant or a dicotyledonous plant; The monocot transgenic plants include sorghum, rice, wheat and corn; The dicotyledonous plants include peanut, soybean, rapeseed, tomato, poplar, turf grass and alfalfa.

18. The method according to claim 16, characterized in that The transformation methods include: Ti plasmid transformation, Ri plasmid transformation, plant virus vector transformation, DNA direct transformation, microinjection, electroporation or Agrobacterium-mediated transformation.

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