Application of the soybean isoflavone synthesis gene GmCHI4a in improving plant resistance to Phytophthora soybeanis

Abstract

This invention relates to the field of bioengineering technology, and in particular to a gene for synthesizing soybean isoflavones. GmCHI4a Applications in improving plant resistance to Phytophthora soybeanis. Soybean isoflavone synthesis gene. GmCHI4a The CDS sequence is shown in SEQ ID NO.2 of the sequence listing; the application includes utilizing the gene. GmCHI4a Construct a plant expression vector and transfer it into the target plant to express the gene. GmCHI4a Overexpression in plants. The plant expression vector is modified with a RUBY visual selection marker. Advantages include: GmCHI4a Overexpression can significantly increase the isoflavone content in hairy roots and enhance the defense against Phytophthora sacchariformis, and can be applied to broad resistance to different physiological races of Phytophthora sacchariformis.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more particularly to a gene for synthesizing soybean isoflavones. GmCHI4a Applications to improve plant resistance to Phytophthora in soybean. Background Technology

[0002] Soybeans Glycine max As a globally important food and economic crop, soybean is one of the main sources of plant protein and oil. However, it is susceptible to damage from the oomycete pathogen—Phytophthora soybean (…). Phytophthora sojae Phytophthora root rot (PRR), caused by *Phytophthora infestans*, has become a devastating soil-borne disease severely restricting soybean production. This pathogen interferes with the host's immune system by secreting effector proteins, and its infection process exhibits significant spatiotemporal specificity: zoospores preferentially colonize the root epidermis, inducing root browning necrosis and vascular bundle dysfunction through molecular mechanisms such as cell wall polysaccharide degradation and inhibition of the MAPK signaling pathway, ultimately leading to systemic wilting of the plant. Although traditional chemical control and the breeding of resistant varieties have achieved some success, the rapid mutation of the pathogen and the problem of drug resistance are becoming increasingly prominent, necessitating an analysis of the soybean disease resistance mechanism at the molecular level to develop novel control strategies.

[0003] Isoflavones are an important class of secondary metabolites in plants, playing a crucial role in plant-microbe interactions. Studies have shown that isoflavones can enhance plant disease resistance by directly inhibiting pathogen growth, activating systemic resistance (SAR) in plants, or acting as signaling molecules to regulate the expression of defense-related genes. Based on the spatiotemporal specificity of *Phytophthora sojae* infection in soybeans, targeted utilization of key genes in the isoflavone pathway can increase the isoflavone content in soybean root tissues, which has significant application value for soybean defense against *Phytophthora sojae*. Summary of the Invention

[0004] To address the above problems, this invention provides a gene for the synthesis of soybean isoflavones. GmCHI4a Applications to improve plant resistance to Phytophthora in soybean.

[0005] The primary objective of this invention is to provide a gene for the synthesis of soybean isoflavones. GmCHI4a In applications to improve plant resistance to Phytophthora soybeanis, the gene... GmCHI4a The CDS sequence is shown in SEQ ID NO.2 of the sequence listing; the application includes utilizing the gene. GmCHI4a Construct a plant expression vector and transfer it into the target plant to express the gene. GmCHI4a Overexpression in plants.

[0006] Preferably, the plant expression vector carries a RUBY visual screening marker.

[0007] Preferably, the plant expression vector is the overexpression vector pCAMBIA1300-GmCHI4a with RUBY visual screening markers.

[0008] Preferably, in the plant expression vector, the RUBY visual selection marker replaces the original Hyg selection marker, and expression is driven by a 35s promoter and terminated by a polyA terminator.

[0009] The preferred target plant is soybean.

[0010] The second objective of this invention is to provide a method for improving plant resistance to Phytophthora soybeanis, comprising the following steps: constructing a plant expression vector; transforming the expression vector into Agrobacterium tumefaciens; and transmitting the soybean isoflavone synthesis gene shown in SEQ ID NO. 2 via Agrobacterium-mediated transformation. GmCHI4a Transferred into the target plant and screened to obtain GmCHI4a Transgenic plants with overexpressed genes; resistance was identified by inoculation with Phytophthora soybeanis, and strains with enhanced resistance were screened out.

[0011] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0012] Soy isoflavone synthesis GmCHI4a Genes and their encoded proteins were transformed into the target gene using Agrobacterium rhizogenes-mediated soybean genetic transformation. GmCHI4a After being transferred into the hairy roots of soybean, the identification results showed that... GmCHI4a Overexpression significantly increases isoflavone content in hairy roots and enhances defense against Phytophthora sacchariformis. This invention further demonstrates that naringenin, a chalcone isomerase catalyzed product, can inhibit multiple physiological races of Phytophthora sacchariformis and can be used to improve defense against Phytophthora sacchariformis, providing a molecular target for the development of resistance-modified varieties of different physiological races of Phytophthora sacchariformis. Attached Figure Description

[0013] Picture 1 Soybeans provided according to embodiments of the present invention GmCHI4a A schematic diagram of the gene and protein structures.

[0014] Picture 2 It is provided according to the embodiments of the present invention. GmCHI4a Identification of gene overexpression results in hairy-rooted species; Figure A shows the results of gene overexpression in hairy-rooted species. GmCHI4a A schematic diagram of the gene and RUBY visual tag overexpression vector pCAMBIA1300-GmCHI4a; BC diagram is... GmCHI4a Results of gene expression level identification in control group (EV) and overexpression hairy roots.

[0015] Picture 3This is a schematic diagram (Figure A) of the CRISPR / Cas vector backbone PGES201 element provided according to an embodiment of the present invention. GmCHI4a Gene structure, corresponding location of knockout target site, and sgRNA sequence (Figure B).

[0016] Picture 4 The results of the antibacterial experiment on multiple races of Phytophthora soybean provided by the embodiments of the present invention are shown in Figure A, which shows the difference in growth status of Phytophthora soybean in the culture dish containing naringenin compared with the control; Figure B shows the statistical results of the growth area of ​​Phytophthora soybean in Figure A.

[0017] Picture 5 It is provided according to the embodiments of the present invention. GmCHI4a Gene knockout materials ( GmCHI4a KO ) and overexpression materials ( GmCHI4a OE The results of resistance identification against Phytophthora soybeanis; Figures AB show the phenotypic images of Phytophthora zoospores treated for 7 days; Figures C and E are respectively... GmCHI4a KO and GmCHI4a OE Before and after processing GmCHI4a Gene expression levels; D and F are GmCHI4a KO and GmCHI4a OE Biomass of Phytophthora soybean after treatment; EV represents the non-GMO control, and Zs represents the treatment with Phytophthora soybean zoospores.

[0018] Picture 6 It is provided according to the embodiments of the present invention. GmCHI4a KO and GmCHI4a OE Results of naringenin content determination before and after infection with Phytophthora in soybean; Figure A shows... GmCHI4a The result of material removal is shown in Figure B. GmCHI4a The results of overexpressing materials. Detailed Implementation

[0019] In the following description, embodiments of the invention will be described with reference to the accompanying drawings. In the description below, the same modules are denoted by the same reference numerals. Where the same reference numerals are used, their names and functions are also the same. Therefore, their detailed description will not be repeated.

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not constitute a limitation thereof.

[0021] This invention provides a gene for the synthesis of soybean isoflavones. GmCHI4a , GmCHI4a The genome sequence is shown in SEQ ID NO.1, the CDS sequence is shown in the sequence listing SEQ ID NO.2, and the protein it encodes has the amino acid sequence shown in the sequence listing SEQ ID NO.3, or the nucleotide sequence shown in SEQ ID NO.1-2 with one or more substituted, deleted, or added nucleotides having the same or similar function, or the amino acid sequence shown in SEQ ID NO.3 with one or more substituted, deleted, or added amino acids having the same or similar function.

[0022] Provided with GmCHI4a Biological materials for genes, wherein the biological material is an expression vector, expression cassette, or host.

[0023] Provides genes for the synthesis of soy isoflavones GmCHI4a The application of its encoded protein chalcone isomerase in regulating soybean's defense against Phytophthora sojae.

[0024] This study provides the application of naringenin in inhibiting different physiological races of Phytophthora soybeanis, including but not limited to R1, R2, R4, R6, R17, and R19.

[0025] Provides an sgRNA that reduces GmCHI4a Target sites for gene expression levels include: TATTTGGAGCCAGAAGTAGT.

[0026] A soybean GmCHI4a Inhibitors of genes or their encoded proteins can reduce GmCHI4a Decreasing gene expression levels, or reducing the level and / or activity of endogenous chalcone isomerase in plants, can reduce soybean isoflavone content and decrease resistance to Phytophthora soybeanis. Inhibitors can be selected from any one or more of the following:

[0027] i) Knockout or knockdown GmCHI4a Gene expression vectors;

[0028] ii) A recombinant host containing i);

[0029] iii) Inhibition GmCHI4a Gene expression formulations (including nucleic acid molecular formulations);

[0030] v) Agents that inhibit chalcone isomerase activity;

[0031] vi) make GmCHI4a Preparations that are inactivated by gene mutation.

[0032] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0033] The encoding chalcone isomerase GmCHI4a Genes include genomic DNA, cDNA, recombinant DNA or mRNA, hnRNA encoding the GmCHI4a protein; or nucleic acid molecules that are inversely complementary to the aforementioned DNA, cDNA, recombinant DNA or mRNA.

[0034] The above GmCHI4a Genes can be modified or optimized according to actual needs, thereby making gene expression more efficient; for example: (1) according to the codons preferred by the recipient plant, while maintaining the gene expression efficiency described in this invention. GmCHI4a (1) Simultaneously altering the amino acid sequence of a gene to conform to the preferences of the recipient plant; (2) Modifying the gene sequence adjacent to the initiation methionine to enable efficient translation initiation; for example, using sequences known to be effective in plants for modification; (3) Linking to promoters of various plant expression to facilitate their expression in plants; the promoters may include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-selective, and tissue-specific promoters; the choice of promoter will vary with the time and space requirements of expression and also depends on the target species; (4) Introducing enhancer sequences, such as intron sequences (e.g., derived from Adhl and Bronzel) and viral leader sequences (e.g., derived from TMV, MCMV, and AMV).

[0035] In this invention, the vector may be a plasmid, granule, bacteriophage, or viral vector. The host may be a fungus, bacteria, algae, or cell.

[0036] The soybean variety Williams 82 involved in this invention is a resistant variety of soybean Phytophthora infestans, while Williams is a susceptible variety. They are not the same material; the strain is knocked out in Williams 82. GmCHI4a The gene can reduce the resistance of Williams82 to Phytophthora soybeanis to study gene function and molecular mechanism; overexpression in Williams GmCHI4a Genes can enhance Willams' resistance to Phytophthora soysarum, and the above methods can be applied to other varieties susceptible to Phytophthora soysarum.

[0037] The present invention will be further illustrated below with reference to the embodiments.

[0038] Example 1

[0039] in soybeans GmCHI4a Gene structure analysis: DNA was extracted from young leaves of soybean variety Williams 82, and amplified using this genomic DNA as a template. GmCHI4a Genome fragments, GmCHI4aThe genome sequence is shown in SEQ ID NO.1, with a total length of 2011 bp, containing 4 exons and 3 introns. GmCHI4a The CDS sequence of the gene is shown in SEQ ID NO.2. GmCHI4a The full-length CDS sequence of the gene is 630 bp; the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO.3, consisting of 210 amino acids. Picture 1 ).

[0040] Example 2

[0041] soybeans GmCHI4a The construction of gene overexpression vectors and hairy root transformation are detailed below:

[0042] (1) GmCHI4a Construction of gene overexpression vectors

[0043] Total RNA was extracted from leaves of soybean variety Williams 82. Using the RNA as a template, it was reverse transcribed into cDNA using a reverse transcription kit. Primers were designed using the cDNA as a template.

[0044] Forward primer:

[0045] 3'-GGACCTCGAGAATTCGAGCTCATGGCTACTGAAGAGGTT-5';

[0046] Reverse primer:

[0047] 3'-CACCATGGTGGCGACCGGTGGCTTGGACAACTCCTGCGA-5';

[0048] Amplification using high-fidelity enzymes GmCHI4a The gene CDS sequence was cloned into a modified pCAMBIA1300 overexpression vector with a RUBY visual selection marker (e.g., Picture 2 (A) In the picture: GmCHI4a The gene is inserted between two restriction enzyme sites, EcoRI and SalI, where EcoRI(0) indicates... GmCHI4a The start position of the gene insertion fragment, represented by SalI (1496). GmCHI4a The termination position of the gene insertion fragment; the RUBY visual selection marker replaces the original Hyg selection marker, with expression driven by a 35s promoter and terminated by a polyA terminator.

[0049] (2) GmCHI4a Gene overexpression hairy root transformation

[0050] Select plump soybean seeds and sow them in vermiculite. Five days later, for seedlings whose cotyledons have not yet separated, make a 45° incision at the hypocotyl with a blade. Simultaneously, collect Agrobacterium K599 carrying the pCAMBIA1300-GmCHI4a vector and apply it to the incision. Lay sterile filter paper flat in a glass dish, add 10 mL of sterile water to moisten the filter paper, and place the seedlings with the incision side up in the glass dish for co-culturing for 24 hours. Then, transfer the soybean plants to transparent pots containing vermiculite to induce hairy roots. Hairy root formation is clearly visible after 14 days (e.g., ...). Picture 2 (B)

[0051] (3) GmCHI4a Gene overexpression plant expression level detection

[0052] extract GmCHI4a Gene overexpression of total RNA from hairy roots was used to reverse transcriptase the RNA into cDNA. Primers were designed using the cDNA as a template:

[0053] Forward primer: 3'-ACCAAGCCCCTATCTTTGCT-5';

[0054] Reverse primer: 3'-ATAAACTTCTCCACCGGGGC-5';

[0055] Detection by quantitative PCR GmCHI4a The expression levels of the gene in overexpressing plants were analyzed. GmCHI4a The expression level in overexpressing plants was significantly higher than that in control plants (e.g., Picture 2 C).

[0056] Example 3

[0057] soybeans GmCHI4a The construction of gene knockout vectors and Agrobacterium-mediated transformation of leguminous plants are detailed below:

[0058] Through analysis GmCHI4a The CDS sequence of the gene was obtained to acquire highly specific sgRNAs (forward primer: 3'-ggattgTATTTGGAGCCAGAAGTAGT-5'; reverse primer: 3'-AAACACTACTTCTGGCTCCAAATACA-5'). The sgRNA primers were annealed and cloned into the PGEs201 knockout vector.

[0059] In this embodiment, soybean cotyledonary nodes were transformed using the Agrobacterium-mediated transformation method to obtain... GmCHI4a Genetically modified materials with heritable editing capabilities.

[0060] (1) Obtaining soybean explants

[0061] Mature soybean seeds with smooth surfaces, no damage, no disease spots, and no cracks were selected and sterilized with chlorine for 14 hours. The sterilized seeds were then ventilated on a clean bench to allow the chlorine to completely evaporate, and germinated on germination medium for 6 hours. Half of the hypocotyl was removed from the soybean, and the soybean was longitudinally cut along the hypocotyl. The remaining hypocotyl was used as the recipient material for Agrobacterium-mediated transformation.

[0062] (2) Soybean conversion

[0063] The Agrobacterium-mediated method involved two Agrobacterium infections, followed by 5 days of dark culture at 22°C on a co-culture medium; then 7 days of culture under strong light on SI-I medium; explant buds were removed, and the plants were cultured under strong light on SI-II medium for 14 days; cotyledons and hypocotyls of the explants were removed, and the plants were subcultured every 14 days on SE medium; clustered buds of approximately 3 cm were cut and placed in rooting medium to root; plants with well-developed root growth on RM rooting medium were transferred to soil for planting. Thirty resistant plants were screened using Bar resistance testing. After 5 months of cultivation in a greenhouse, the fruit pods began to mature, and seed harvesting was completed after 6 months.

[0064] (3) Detection of heritable editing methods in transgenic plants

[0065] The harvested T1 generation seeds were planted in the field, and genomic DNA was extracted from the leaves of the transgenic plants. Primers were designed using the DNA as a template.

[0066] Forward primer: 3'-CTTAAGGCAGCTAGGGATCT-5';

[0067] Reverse primer: 3'-GATGGGGGCATGCCGATTAA-5';

[0068] Fragments containing the edited sites were amplified by PCR and then subjected to first-generation sequencing. The sequencing results were compared with a reference sequence to obtain... GmCHI4a Gene-edited transgenic plants (such as Picture 3 ).

[0069] Example 4

[0070] soybeans GmCHI4a The effect of genetically synthesized naringenin on the growth of Phytophthora soybeanis: due to GmCHI4a The metabolite synthesized by the gene is naringenin. To verify the effect of naringenin on root rot of *Phytophthora sojae*, a concentration of 100 μg / ml of naringenin was added to 10% V8 medium (methanol was used as a control). *Phytophthora sojae* races 1, 2, 4, 6, 17, and 19 were cultured in the naringenin-containing medium for 7 days at 25°C in the dark, and the area of ​​*Phytophthora sojae* growth was counted. Results are as follows: Picture 4As shown in the figure, naringenin has a significant inhibitory effect on the growth of different physiological races of Phytophthora soybean.

[0071] Example 5

[0072] soybeans GmCHI4a The gene knockout and overexpression experiments for identifying resistance to Phytophthora in soybean are detailed below:

[0073] (1) Soybeans GmCHI4a Effects of gene knockout on resistance to Phytophthora in soybean

[0074] To verify GmCHI4a The function of the gene in resisting soybean Phytophthora infestation was compared between wild-type Williams 82 and knockout genes. GmCHI4a Transgenic plants GmCHI4a KO Plants were inoculated with zoospores of Phytophthora soybeanis for 7 days, and the root phenotype was observed. Root tissue was also collected 4 hours after inoculation for comparison. GmCHI4a Gene expression level detection and soybean Phytophthora biomass detection.

[0075] The results showed that knockout GmCHI4a Seven days after inoculating transgenic plants with Phytophthora soybean zoospores, significant lesions appeared on the roots. Picture 5 (A) and 4 hours after vaccination. GmCHI4a KO In the root system GmCHI4a Gene expression levels were significantly lower than in control plants ( Picture 5 (C) and GmCHI4a KO The biomass of Phytophthora soybean in the root system was significantly higher than that in the control (e.g., Picture 5 (D). This illustrates the knockout. GmCHI4a The gene reduced the soybean's resistance to Phytophthora soybeanis.

[0076] (2) Soybeans GmCHI4a Gene overexpression for resistance to Phytophthora sojae

[0077] Williams, the disease-sensitive material, and overexpression GmCHI4a Transgenic plants GmCHI4a OE Plants were inoculated with zoospores of Phytophthora soybeanis for 7 days, and plant phenotypes were observed. Root tissue was also collected 4 hours after inoculation for comparison. GmCHI4a Gene expression levels and biomass of *Phytophthora soybeanae* were detected. Results showed that the susceptible material *Williams*, inoculated with *Phytophthora soybeanae* zoospores, exhibited a death phenotype 7 days after inoculation. GmCHI4a OE Overexpression material inoculated with zoospores of Phytophthora soybean can continue to survive. Picture 5 (B) and 4 hours after vaccination. GmCHI4aOE In the root system GmCHI4a Gene expression levels were significantly higher in plants than in control plants. Picture 5 (E); and GmCHI4a OE The biomass of Phytophthora in soybean roots was significantly lower than that in the control (e.g., Picture 5 (F). This illustrates GmCHI4a Gene overexpression enhances soybean resistance to Phytophthora sojae.

[0078] Example 6

[0079] soybeans GmCHI4a The experiment on the changes in naringenin content in gene knockout and overexpression materials is as follows:

[0080] To verify GmCHI4a Changes in naringenin content in genetically modified materials will GmCHI4a Gene knockout GmCHI4a Gene overexpression and control materials were inoculated with zoospores of *Phytophthora sojae*; root materials were collected after inoculation to detect the content of naringenin. Results are as follows: Picture 6 As shown in the figure, after inoculation with Phytophthora soybeanii... GmCHI4a The naringenin content in the roots of gene knockout was significantly lower than that in the control Williams 82. Picture 6 (A) GmCHI4a The naringenin content in roots of overexpressing the gene was significantly higher than that in the control group Williams. Picture 6 (B)

[0081] The key technical points of this invention include: a method related to the synthesis of soybean isoflavones. GmCHI4a Genes and their encoded proteins were transformed into the target gene using Agrobacterium rhizogenes-mediated soybean genetic transformation. GmCHI4a After being transferred into the hairy roots of soybean, the identification results showed that... GmCHI4a Overexpression significantly increased isoflavone content in hairy roots and enhanced defense against Phytophthora soybeanii; using CRISPR / Cas9 technology and genetic transformation at the cotyledonary node, isoflavones were expressed in the whole soybean plant. GmCHI4a Knockout reduces the isoflavone content in the roots of the material and also reduces its defense against Phytophthora soybean.

[0082] Furthermore, chalcone isomerase catalyzes the synthesis of naringenin, an important flavonoid compound. This invention utilizes plate inhibition experiments to demonstrate that naringenin exhibits inhibitory effects against different physiological races (R1, R2, R4, R6, R17, R19) of *Phytophthora sacchariformis*. Therefore, the invention provides… GmCHI4a The gene and its encoded protein can be applied to broad resistance to different physiological races of Phytophthora soybean.

[0083] Example 7

[0084] This embodiment describes a method for improving plant resistance to Phytophthora soybeanis, comprising the following steps:

[0085] S1. Construct a plant expression vector; in the plant expression vector, the RUBY visual tag is located downstream of the promoter and the soybean isoflavone synthesis gene. GmCHI4a Upstream;

[0086] S2. The expression vector was transformed into Agrobacterium; the soybean isoflavone synthesis gene shown in SEQ ID NO. 2 was expressed via Agrobacterium-mediated transformation. GmCHI4a Transferred into the target plant, soybean;

[0087] S3. Use Ruby's visual filter markers to perform visual filtering and obtain... GmCHI4a Transgenic plants with overexpressed genes;

[0088] S4. Resistance was identified by inoculating with Phytophthora soybeanis and strains with enhanced resistance were selected.

[0089] It should be understood that the various forms of processes shown above can be used to reorder, add, or delete steps. For example, the steps described in this invention disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution disclosed in this invention can be achieved, and this is not limited herein.

[0090] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. Soy isoflavone synthesis gene GmCHI4a In the application of improving plant resistance to Phytophthora soybeanis, the characteristics are as follows: The gene GmCHI4a The CDS sequence is shown in SEQ ID NO.2 of the sequence listing; the application includes utilizing the gene. GmCHI4a Construct a plant expression vector and transfer it into soybean to express the gene. GmCHI4a Overexpression in plants; The plant expression vector carries a RUBY visual selection marker; the RUBY visual selection marker replaces the original Hyg selection marker, and expression is driven by a 35s promoter and terminated by a polyA terminator.

2. The soybean isoflavone synthesis gene according to claim 1 GmCHI4a In the application of improving plant resistance to Phytophthora soybeanis, the characteristics are as follows: The plant expression vector is an overexpression vector pCAMBIA1300-GmCHI4a with a RUBY visual screening marker.

3. A method for improving plant resistance to Phytophthora soybeanis, characterized in that: Includes the following steps: A plant expression vector was constructed; the expression vector was transformed into Agrobacterium; and the soybean isoflavone synthesis gene shown in SEQ ID NO.2 was expressed via Agrobacterium-mediated transformation. GmCHI4a Transferred into the target plant and screened to obtain GmCHI4a Transgenic plants with overexpressed genes; resistance was assessed by inoculation with Phytophthora soybeanis, and strains with enhanced resistance were screened out; the target plant was soybean.

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