Application of soybean BSP family Glyma.20G130000.1 gene in soybean resistance to phytophthora root rot

Abstract

This invention belongs to the field of genetic engineering technology, and specifically discloses the soybean BSP family. Glyma.20G130000.1 Application of a gene in soybean resistance to soybean Phytophthora root rot. This invention clones a gene from soybean. Glyma.20G130000.1 The expression was efficiently achieved in soybean plants through transient transformation, resulting in overexpressed soybean plants. Functional identification results showed that, compared with control plants, the overexpression... Glyma.20G130000.1 Soybean plants containing this gene exhibit significantly enhanced resistance to Phytophthora soybeanii. Furthermore, this invention provides the application of biomaterials related to this gene, and methods for screening or breeding disease-resistant soybean plants based on this gene. Therefore, Glyma.20G130000.1 Genes not only provide an effective tool for revealing the basic resistance mechanism of soybean to Phytophthora root rot, but also provide valuable genetic resources for soybean disease resistance breeding.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically involving the application of the soybean BSP family Glyma.20G130000.1 gene in soybean resistance to soybean Phytophthora root rot. Background Technology

[0002] Soybeans Glycine max L. Soybean seeds are rich in high-quality oils and proteins, and there is a huge market demand for them in the food industry, feed processing, and chemical production. For a long time, through breeding high-yield and high-quality varieties, optimizing cultivation management measures, and expanding planting areas, soybean production has been able to basically meet the continuously growing market demand. However, soybean stem and root rot (PSRR), caused by Phytophthora sojae, is a highly destructive soybean disease worldwide, causing economic losses of $1-2 billion annually and posing a serious threat to global soybean production.

[0003] In rainy and humid environments, *Phytophthora sojae* produces a large number of chemotactic zoospores. These zoospores actively attract and accumulate at the soybean roots, infecting the root and stem tissues and causing plant rot and death. When the external environment is unsuitable for infection, the pathogen can form oospores to resist adverse conditions; once environmental conditions become suitable, the oospores germinate again, completing the infection cycle. These infection and survival characteristics make the control of *Phytophthora sojae* extremely difficult. Therefore, fully utilizing soybean's own disease-resistant genetic resources and breeding soybean varieties resistant to *Phytophthora sojae* has become an important means to reduce yield losses and ensure safe soybean production.

[0004] Since the first soybean antifungal gene, Rps1a, was identified in 1964, researchers have successively obtained several other soybean antifungal genes, including Rps1c, Rps1k, and Rps3a. However, related studies have shown that the resistance mediated by Rps1a lost its effectiveness in 1972. Research confirms that the effective utilization period of a single Rps resistance gene is typically only 6-15 years, after which it needs to be replaced by a new resistance gene.

[0005] Based on the above situation, discovering new, efficient, and durable soybean Phytophthora blight gene resources is of great significance for continuously controlling soybean Phytophthora blight and ensuring the stable development of the soybean industry. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a soybean BSP family. Glyma.20G130000.1 Application of genes in soybean resistance to soybean Phytophthora root rot.

[0007] In a first aspect, the present invention provides overexpression of the soybean BSP family. Glyma.20G130000.1 The application of genes in improving soybean resistance to soybean Phytophthora root rot, the aforementioned Glyma.20G130000.1 The amino acid sequence of the protein BSP06 encoded by the gene is any of the following: A1) The amino acid sequence shown in SEQ ID NO.2; A2) A protein that has more than 90% identity with and has the same function as the protein shown in SEQ ID NO.2, obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO.2; A3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

[0008] Furthermore, the aforementioned Glyma.20G130000.1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0009] In this invention, the soybean BSP family was identified through screening. Glyma.20G130000.1 The gene, whose nucleotide sequence is shown in SEQ ID NO.1, and whose encoded protein BSP06 has the amino acid sequence shown in SEQ ID NO.2, have the GeneBank Sequence ID NP_001238289.2. Experiments showed successful overexpression. Glyma.20G130000.1 Compared with the control material, the soybean plants with the gene showed stronger resistance to Phytophthora soybeanis and were better able to resist Phytophthora soybeanis root rot caused by Phytophthora soybeanis.

[0010] In a second aspect, the present invention provides the soybean BSP family as described in the first aspect. Glyma.20G130000.1 The application of gene-related biomaterials, specifically to enhance the resistance of soybeans to soybean Phytophthora root rot. The biomaterial is at least one of the following (a1)-(a4): (a1) A nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.1; (a2) An expression cassette containing the nucleic acid molecule described in (a1); (a3) A recombinant vector containing the nucleic acid molecule described in (a1), or a recombinant vector containing the expression cassette described in (a2); (a4) A recombinant microorganism containing the nucleic acid molecule described in (a1), or a recombinant microorganism containing the expression cassette described in (a2), or a recombinant microorganism containing the recombinant vector described in (a3).

[0011] Thirdly, the present invention also provides a method for improving the resistance of soybean to soybean root rot, the method comprising overexpressing the soybean BSP family. Glyma.20G130000.1Gene screening was used to select soybean plants resistant to soybean Phytophthora root rot.

[0012] Furthermore, the overexpression of the soybean BSP family Glyma.20G130000.1 Genes are introduced into soybean plants by transferring the biological material associated with them.

[0013] Furthermore, the biomaterial is at least one of the following (a1)-(a4): (a1) A nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.1; (a2) An expression cassette containing the nucleic acid molecule described in (a1); (a3) A recombinant vector containing the nucleic acid molecule described in (a1), or a recombinant vector containing the expression cassette described in (a2); (a4) A recombinant microorganism containing the nucleic acid molecule described in (a1), or a recombinant microorganism containing the expression cassette described in (a2), or a recombinant microorganism containing the recombinant vector described in (a3).

[0014] Fourthly, the present invention also provides a method for screening soybeans with resistance to Phytophthora root rot, the method comprising detecting in soybean plants to be tested... Glyma.20G130000.1 The steps for determining the expression level of a gene or the content of its encoded protein BSP06.

[0015] Furthermore, the aforementioned Glyma.20G130000.1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein BSP06 is shown in SEQ ID NO.2.

[0016] Fifthly, the present invention also provides a method for cultivating soybeans with resistance to Phytophthora root rot, the method comprising detecting in soybean plants to be tested... Glyma.20G130000.1 By analyzing the expression level of the gene or the content of its encoded protein BSP06, soybean plants with resistance to soybean root rot were screened and used as parents for hybridization or backcrossing with other soybean plants.

[0017] Furthermore, the aforementioned Glyma.20G130000.1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein BSP06 is shown in SEQ ID NO.2.

[0018] Compared with the prior art, this invention discovers the soybean BSP family in soybeans. Glyma.20G130000.1 Gene. This invention successfully achieved overexpression of [gene name] in soybean plants using transient expression technology. Glyma.20G130000.1Soybean plants with the gene showed stronger resistance to Phytophthora soybeanae compared to the control group. Simultaneously, using stable transgenic soybean technology, the gene was stably knocked out in the soybean variety Williams 82. Glyma.20G130000.1 The gene was successfully knocked out. Glyma.20G130000.1 Gene knockout transformants, experiments have shown that, Glyma.20G130000.1 Gene knockout transformants showed significantly reduced resistance to soybean mold. Therefore, Glyma.20G130000.1 Genes not only provide an effective tool for revealing the basic resistance mechanism of soybean to Phytophthora root rot, but also provide valuable genetic resources for soybean disease resistance breeding. Attached Figure Description

[0019] Figure 1 For transient overexpression Glyma.20G130000.1 Western blot results of protein BSP06 in soybean plants.

[0020] Figure 2 For transient overexpression Glyma.20G130000.1 Statistical results of soybean plant leaf lesions and biomass measurements. Figure A shows photographs of soybean plant leaf lesions; Figure B shows the statistical results of soybean plant leaf lesion diameter; Figure C shows the statistical results of soybean plant leaf biomass.

[0021] Figure 3 For genes Glyma.20G130000.1 Schematic diagram of the knockout vector 。

[0022] Figure 4 For genes Glyma.20G130000.1 Statistical results of leaf lesions and biomass measurements of soybean plants with knockout mutants. Figure A shows photographs of leaf lesions on soybean plants; Figure B shows the statistical results of leaf lesion diameter; Figure C shows the statistical results of leaf biomass on soybean plants. Detailed Implementation

[0023] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0024] Example 1 DNA was extracted from soybean Williams 82, and primer pairs were used as a template. Glyma.20G130000.1 -F: 5'-ATGATCAATCATGCTCTTTG-3' (SEQ ID NO.3) and Glyma.20G130000.1-R: 5'-GGCAATATTGCCATACTTGG-3' (SEQ ID NO.4) was amplified by PCR to obtain the amplified product gene. Glyma.20G130000.1 .

[0025] The PCR reaction volume was 50 μL, including 25 μL of PCR Mix enzyme, 1 μL of DNA template, 1 μL of each primer, and 22 μL of ddH2O. The reaction conditions were: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 1 min, for 33 cycles, and a final extension at 72℃ for 5 min.

[0026] Using infusion method to extract genes Glyma.20G130000.1 The sequence was constructed into the plant expression vector Pbin-HA. 3 μL of Pbin-HA was added... Glyma.20G130000.1 The HA vector was transformed into Agrobacterium strain GV3101 using heat shock transformation. After culturing at 28°C for 2 days, single colonies were picked and PCR amplified using primers Pbin-HA-F: 5'-CGAATCTCAAGCAATCAAGCATTC-3' (SEQ ID NO. 5) and Pbin-HA-R: 5'-GCGTAATCTGGAACATCGTATG-3' (SEQ ID NO. 6) to verify the DNA band of the positive strain. The positive strain Pbin- was finally obtained. Glyma.20G130000.1 -HA.

[0027] Example 2

[0028] Prepare 200 mL OD 600 =1 Pbin- Glyma.20G130000.1 -HA positive strain, with Pbin-GUS-HA as negative control, leaves of 14-year-old soybean plants were placed in the bacterial solution and vacuum filtered for 10 min. The filtered plants were then moistened for 24 h before being subjected to Phytophthora infestation (…). Ps JS2 strain) in vitro inoculation.

[0029] To ensure the successful expression of the target protein BSP06 using the transient expression technology, soybean leaves were sampled two days after filtration, ground in liquid nitrogen, and the protein was extracted. Western blot was then used to verify the expression of the target protein BSP06.

[0030] like Figure 1 As shown, compared to the negative control, in soybean plants Glyma.20G130000.1 FLAG -OE#1 and Glyma.20G130000.1 FLAG Transient overexpression of protein BSP06 was successfully achieved in OE#2.

[0031] Example 3

[0032] Statistical analysis was conducted on soybean plants (Glyma.20, G130000.1) transiently expressing the target protein BSP06. FLAG -OE#1 and Glyma.20G130000.1 FLAG -Diseases were observed 2 days after inoculation with OE#2, and the bioexpression level of Phytophthora soybean was determined. Leaves measuring 1.5 cm x 1.5 cm were fixed at the inoculation site, with three leaves per sample. DNA was extracted from the obtained samples for later use.

[0033] qPCR was performed using quantitative primers actin-F: 5'-ACTGCACCTTCCAGACCATC-3' (SEQ ID NO.7) and actin-R: 5'-CCACCACCTTGATCTTCATG-3' (SEQ ID NO.8), GmCYP2-F: 5'-CGGGACCAGTGTGCTTCTTCA-3' (SEQ ID NO.9) and GmCYP2-R: 5'-CCCCTCCACTACAAAGGCTCG-3' (SEQ ID NO.10) to identify the infection status of the pathogen.

[0034] Statistical results of lesions and biomass measurement results are as follows: Figure 2 As shown, it can be seen that soybean plants Glyma.20G130000.1 transiently express the target protein BSP06. FLAG -OE#1 and Glyma.20G130000.1 FLAG - The lesions on the leaves of OE#2 were significantly smaller than those on the negative control plants. Figure 2 (Figure A) Furthermore, statistical analysis of lesion diameter showed that the lesion diameter of soybean plants transiently expressing the target protein BSP06 was significantly smaller than that of the negative control plants. Figure 2 (See Figure B). Simultaneously, biomass measurements showed a significant decrease in the biomass of *Phytophthora soybeani* in the leaves of soybean plants transiently expressing the target protein BSP06. Figure 2 (Figure C in the middle)

[0035] Example 4

[0036] Reference gene Glyma.20G130000.1 The nucleotide sequence (SEQ ID NO.1) was used to predict the gene using the CCTop-CRISPR / Cas9 target online predictor (uni-heidelberg.de). Glyma.20G130000.1The target sequences were ultimately identified, with 5'-GGCGGCGTGGCTTTCCGTGA-3' (SEQ ID NO.11) and 5'-TCTCAGTGCAAGATACGTTG-3' (SEQ ID NO.12) being the most effective targets. Glyma.20G130000.1 Gene knockout target sequence.

[0037] The target sequence was synthesized into the pFGC5941(-)-GmU6-DR-8sgRNA-DR-GmUBQ-hcpf1 vector (e.g., Figure 3 In this study, the obtained knockout vector was heat-shocked and transformed into Agrobacterium strain AGL1. Gene knockout in soybean was then achieved through stable transformation experiments. Glyma.20G130000.1 The DNA sequence of the obtained transformant material was extracted and sequenced using primer pairs. Glyma.20G130000.1 -F: 5'-ATGATCAATCATGCTCT TTG-3' (SEQ ID NO.3) and Glyma.20G130000.1 -R: 5'-GGCAATATTGCCATACTTGG-3' (SEQ ID NO.4) After PCR amplification of the target sequence, sequencing was performed to verify the knockout results of the positive material, and knockout mutants Glyma.20G130000.1-KO#1 and Glyma.20G130000.1-KO#2 were obtained.

[0038] Example 5

[0039] To verify the gene Glyma.20G130000.1 To assess the resistance of knockout materials to Phytophthora soybeanis, wild-type soybean Williams 82 was used as a control. Phytophthora soybeanis was in vitro inoculated onto 14-year-old knockout mutant plants Glyma.20G130000.1-KO#1 and Glyma.20G130000.1-KO#2. Ps Two days after inoculation, lesion counts and biomass measurements were performed using the JS2 strain (methods as in Example 3) to obtain quantitative results for identifying the infection status of the pathogen.

[0040] The results are as follows Figure 4 As shown, in genes Glyma.20G130000.1 The lesions on the leaves of the knocked-out soybean plants Glyma.20G130000.1-KO#1 and Glyma.20G130000.1-KO#2 were significantly larger than those on the wild type. Figure 4 (See Figure A in the image). Furthermore, statistical analysis of lesion diameter showed that the lesion diameter of plants with knockout mutants Glyma.20G130000.1-KO#1 and Glyma.20G130000.1-KO#2 was significantly larger than that of the wild type. Figure 4(See Figure B). Simultaneously, biomass measurements showed that the biomass of *Phytophthora soybeani* in the leaves of plants from the knockout mutants *Glyma.20G130000.1-KO#1* and *Glyma.20G130000.1-KO#2* was significantly increased. Figure 4 (Figure C in the middle)

[0041] In summary, successful overexpression Glyma.20G130000.1 Soybean plants with the gene knocked out showed stronger resistance to Phytophthora soybean compared to control plants, while those with the gene knocked out... Glyma.20G130000.1 The genetically modified soybean plants showed significantly lower resistance to Phytophthora soybean compared to wild-type materials.

[0042] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. Overexpression of the soybean BSP family Glyma.20G130000.1 The application of genes in improving soybean resistance to soybean Phytophthora root rot is characterized by, The Glyma.20G130000.1 The amino acid sequence of the protein BSP06 encoded by the gene is any of the following: A1) The amino acid sequence shown in SEQ ID NO.2; A2) A protein that has more than 95% identity with and has the same function as the protein shown in SEQ ID NO.2, obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO.2; A3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

2. The application according to claim 1, characterized in that, The Glyma.20G130000.1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. The soybean BSP family as described in claim 1 or 2 Glyma.20G130000.1 The application of gene-related biomaterials is characterized by, The application is to improve the resistance of soybeans to soybean Phytophthora root rot. The biomaterial is at least one of the following (a1)-(a4): (a1) A nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.1; (a2) An expression cassette containing the nucleic acid molecule described in (a1); (a3) A recombinant vector containing the nucleic acid molecule described in (a1), or a recombinant vector containing the expression cassette described in (a2); (a4) A recombinant microorganism containing the nucleic acid molecule described in (a1), or a recombinant microorganism containing the expression cassette described in (a2), or a recombinant microorganism containing the recombinant vector described in (a3).

4. A method for improving soybean resistance to soybean Phytophthora root rot, characterized in that, The method includes overexpression of the soybean BSP family. Glyma.20G130000.1 Gene screening was used to select soybean plants resistant to soybean Phytophthora root rot.

5. The method according to claim 4, characterized in that, The overexpression of soybean BSP family Glyma.20G130000.1 Genes are introduced into soybean plants by transferring the biological material associated with them.

6. The method according to claim 5, characterized in that, The biomaterial is at least one of the following (a1)-(a4): (a1) A nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.1; (a2) An expression cassette containing the nucleic acid molecule described in (a1); (a3) A recombinant vector containing the nucleic acid molecule described in (a1), or a recombinant vector containing the expression cassette described in (a2); (a4) A recombinant microorganism containing the nucleic acid molecule described in (a1), or a recombinant microorganism containing the expression cassette described in (a2), or a recombinant microorganism containing the recombinant vector described in (a3).

7. A method for screening soybeans with resistance to Phytophthora root rot, characterized in that, The method includes detecting [something] in the soybean plant to be tested. Glyma.20G130000.1 The steps for determining the expression level of a gene or the content of its encoded protein BSP06.

8. The method according to claim 7, characterized in that, The Glyma.20G130000.1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein BSP06 is shown in SEQ ID NO.

2.

9. A method for cultivating soybeans with resistance to Phytophthora root rot, characterized in that, The method includes detecting in the soybean plant to be tested... Glyma.20G130000.1 By analyzing the expression level of the gene or the content of its encoded protein BSP06, soybean plants with resistance to soybean root rot were screened and used as parents for hybridization or backcrossing with other soybean plants.

10. The method according to claim 9, characterized in that, The Glyma.20G130000.1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein BSP06 is shown in SEQ ID NO.2.

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