Application of soybean isoflavone synthetic gene GmCHI4a in improving resistance of plant to phytophthora sojae

Abstract

The invention relates to the technical field of bioengineering, in particular to application of a soybean isoflavone synthetic gene GmCHI4a in improving resistance of plants to phytophthora sojae. The CDS sequence of the soybean isoflavone synthetic gene GmCHI4a is as shown in SEQ ID NO. 2 in a sequence table; the application comprises the following steps: constructing a plant expression vector by using the gene GmCHI4a, and transferring the plant expression vector into a target plant, so that the gene GmCHI4a is over-expressed in the plant. After being modified, the plant expression vector has an RUBY visual selection marker. The method has the advantages that overexpression of the GmCHI4a can obviously improve the content of isoflavone in hairy roots and enhance the defense effect on phytophthora sojae, and the method can be applied to wide resistance of different physiological races of the phytophthora sojae.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a soybean isoflavone synthesis gene. GmCHI4a Application in improving plant resistance to soybean phytophthora. Background Art

[0002] Soybean ( Glycine max ) is an important food and economic crop in the world and one of the main sources of plant protein and oil. However, the Oomycete pathogen, Phytophthora sojae ( Phytophthora sojae Phytophthora root rot (PRR), caused by Phytophthora root rot (PRR), has become a devastating soilborne disease that severely restricts soybean production. This pathogen interferes with the host immune system by secreting effector proteins, and its infection process exhibits remarkable spatiotemporal specificity: zoospores preferentially colonize the root epidermis and, through molecular mechanisms such as degradation of cell wall polysaccharides and inhibition of the MAPK signaling pathway, induce root browning and necrosis, vascular dysfunction, and ultimately systemic wilting. Although traditional chemical control and breeding of disease-resistant varieties have achieved some success, the rapid mutation and drug resistance of the pathogen are becoming increasingly prominent, necessitating a molecular understanding of soybean disease resistance mechanisms to develop novel control strategies.

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

[0004] In order to solve the above problems, the present invention provides a soybean isoflavone synthesis gene GmCHI4a Application in improving plant resistance to soybean phytophthora.

[0005] The first purpose of the present invention is to provide a soybean isoflavone synthesis gene GmCHI4a In the application of improving plant resistance to soybean phytophthora, the gene GmCHI4a The CDS sequence of the gene is shown in SEQ ID NO.2; the application includes utilizing the gene GmCHI4a Construct a plant expression vector and transfer it into the target plant to make 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 a RUBY visual screening marker.

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

[0009] Preferably, the target plant is soybean.

[0010] The second object of the present invention is to provide a method for improving plant resistance to Phytophthora sojae, comprising the following steps: constructing a plant expression vector; transferring the expression vector into Agrobacterium; and translating the soybean isoflavone synthesis gene shown in SEQ ID NO. 2 into the plant by Agrobacterium-mediated method. GmCHI4a Transfer into target plants and screen to obtain GmCHI4a Transgenic plants with gene overexpression; resistance is identified by inoculation with soybean phytophthora, and strains with enhanced resistance are screened out.

[0011] Compared with the prior art, the present invention can achieve the following beneficial effects: Soy isoflavone synthesis related GmCHI4a The target gene and its coding protein were transformed into soybean by Agrobacterium rhizogenes-mediated genetic transformation. GmCHI4a The results showed that GmCHI4a Overexpression significantly increases isoflavone content in hairy roots and enhances defense against Phytophthora sojae. This study further demonstrates that naringenin, a product of chalcone isomerase catalysis, can inhibit multiple races of Phytophthora sojae. This finding suggests that naringenin can be used to enhance defense against Phytophthora sojae and provide a molecular target for the development of improved varieties resistant to different races of Phytophthora sojae. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Picture 1 The soybean provided by the embodiment of the present invention GmCHI4a Schematic diagram of the gene structure and protein structure.

[0013] Picture 2 According to an embodiment of the present invention, GmCHI4a Identification of the results after gene overexpression in hairy root species; Figure A shows the results of gene overexpression in hairy root species. GmCHI4a Schematic diagram of the overexpression vector pCAMBIA1300-GmCHI4a of the gene and RUBY visual tag; BC is GmCHI4a Identification results of gene expression levels in control group (EV) and overexpression hairy roots.

[0014] Picture 3Schematic diagram of the CRISPR / Cas vector backbone PGES201 element according to an embodiment of the present invention (Figure A) and GmCHI4a Gene structure, corresponding positions of knockout target sites, and sgRNA sequences (Figure B).

[0015] Picture 4 These are the results of an antibacterial experiment using naringenin according to an embodiment of the present invention against multiple species of Phytophthora sojae. Figure A shows the difference in growth of Phytophthora in a culture dish containing naringenin compared to a control. Figure B shows the statistical results of the growth area of ​​Phytophthora sojae corresponding to Figure A.

[0016] Picture 5 According to an embodiment of the present invention, GmCHI4a Gene knockout materials ( GmCHI4a KO ) and overexpression materials ( GmCHI4a OE ) are the results of resistance identification to Phytophthora sojae; Figures AB are phenotypic photos 7 days after Phytophthora zoospore treatment; Figures C and E are GmCHI4a KO and GmCHI4a OE Before and after treatment GmCHI4a The expression levels of genes; D and F are GmCHI4a KO and GmCHI4a OE Biomass of P. sojae after treatment; EV indicates non-transgenic control, and Zs indicates P. sojae zoospore treatment.

[0017] Picture 6 According to an embodiment of the present invention, GmCHI4a KO and GmCHI4a OE The results of naringenin content determination before and after infection with Phytophthora sojae; Figure A is GmCHI4a The result of knocking out the material, Figure B is GmCHI4a Results of overexpressed materials. DETAILED DESCRIPTION

[0018] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the following description, identical modules are denoted by identical reference numerals. In the case of identical reference numerals, their names and functions are also identical. Therefore, their detailed description will not be repeated.

[0019] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not constitute a limitation of the present invention.

[0020] The present invention provides a soybean isoflavone synthesis gene GmCHI4a , GmCHI4a The genomic sequence is shown in SEQ ID NO.1, the CDS sequence is shown in SEQ ID NO.2 in the sequence listing, and the encoded protein, the amino acid sequence is shown in SEQ ID NO.3 in the sequence listing, or a nucleotide sequence having the same or similar function as the nucleotide sequence shown in SEQ ID NO.1-2 by substitution, deletion or addition of one or several nucleotides, or an amino acid sequence having the same or similar function as the amino acid sequence shown in SEQ ID NO.3 by substitution, deletion or addition of one or several amino acids.

[0021] Provided with GmCHI4a The biological material of the gene is an expression vector, an expression cassette or a host.

[0022] Provides soybean isoflavone synthesis genes GmCHI4a and its encoded protein chalcone isomerase in regulating soybean defense against Phytophthora sojae.

[0023] Provided is the use of naringenin in inhibiting different physiological races of Phytophthora sojae, including but not limited to R1, R2, R4, R6, R17, and R19.

[0024] Provide an sgRNA to reduce GmCHI4a Target sites for gene expression levels include: TATTTGGAGCCAGAAGTAGT.

[0025] Provided is a soybean GmCHI4a Inhibitors of genes or their encoded proteins can reduce GmCHI4a Inhibitors can reduce the expression level of the gene, or reduce the level and / or activity of endogenous chalcone isomerase in plants, thereby reducing soybean isoflavone content and resistance to Phytophthora sojae. The inhibitor can be selected from any one or more of the following: i) Knockout or knockdown GmCHI4a Gene expression vector; ii) a recombinant host comprising i); iii) Inhibition GmCHI4a Gene expression preparations (including nucleic acid molecule preparations); v) Preparations that inhibit chalcone isomerase activity; vi) Make GmCHI4a Agents that inactivate gene mutations.

[0026] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0027] The chalcone isomerase encoding GmCHI4aThe gene includes genomic DNA, cDNA, recombinant DNA, mRNA, or hnRNA encoding the GmCHI4a protein; or a nucleic acid molecule that is reverse complementary to the above DNA, cDNA, recombinant DNA, or mRNA.

[0028] above GmCHI4a The gene can be modified or optimized according to actual needs to make the gene expression more efficient; for example: (1) according to the codons preferred by the recipient plant, while maintaining the GmCHI4a (1) modifying the amino acid sequence of the gene while changing its codons to match the preferences of the recipient plant; (2) modifying the gene sequence adjacent to the initiator methionine to enable efficient translation initiation; for example, using sequences known to be effective in plants. (3) connecting to various plant-expressed promoters to facilitate their expression in plants; the promoters may include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the temporal and spatial requirements of expression and also depends on the target species; (4) introducing enhancer sequences, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV).

[0029] In the present invention, the vector may be a plasmid, cosmid, phage or viral vector. The host may be a fungus, bacteria, algae or cell.

[0030] The soybean variety Williams 82 involved in the present invention is a soybean Phytophthora resistant variety, and Williams is a soybean Phytophthora susceptible variety. The two are not the same material. GmCHI4a Gene can reduce the resistance of Williams82 to soybean phytophthora to study gene function and molecular mechanism; overexpression in Williams GmCHI4a The gene can improve Williams' resistance to soybean phytophthora, and the above method can be applied to other soybean varieties susceptible to soybean phytophthora.

[0031] The present invention will be further described below with reference to the embodiments.

[0032] Example 1 in soybeans GmCHI4a Gene structure analysis: DNA was extracted from young leaves of soybean variety Williams 82, and the genomic DNA was used as a template to amplify the GmCHI4a Genome fragments, GmCHI4a The genome sequence is shown in SEQ ID NO.1, with a total length of 2011 bp, including 4 exons and 3 introns. GmCHI4a The CDS sequence of the gene is shown in SEQ ID NO.2. GmCHI4a The full length of the gene CDS sequence is 630 bp; the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO.3, which contains 210 amino acids ( Picture 1 ).

[0033] Example 2 soybeans GmCHI4a The construction of gene overexpression vector and hairy root transformation are as follows: (1) GmCHI4a Gene overexpression vector construction Total RNA was extracted from the leaves of soybean variety Williams 82 and converted to cDNA using a reverse transcription kit. Using cDNA as a template, primers were designed: Forward primer: 3'-GGACCTCGAGAATTCGAGCTCATGGCTACTGAAGAGGTT-5'; Reverse primer: 3'-CACCATGGTGGCGACCGGTGGCTTGGACAACTCCTGCGA-5'; Amplification using high-fidelity enzymes GmCHI4a Gene CDS sequence was cloned into the modified pCAMBIA1300 overexpression vector with RUBY visual screening marker (such as Picture 2 (A in the middle). In the figure: GmCHI4a The gene is inserted between the EcoRI and SalI restriction sites, EcoRI (0) indicates GmCHI4a The starting position of the gene insertion fragment, indicated by SalI (1496) GmCHI4a The termination position of the gene insertion fragment; the RUBY visual screening marker replaces the original Hyg screening marker, and the expression is driven by the 35s promoter and terminated by the polyA terminator.

[0034] (2) GmCHI4a Gene overexpression hairy root transformation Select plump soybean seeds and sow them in vermiculite. After 5 days, use a blade to create a wound at a 45° angle on the hypocotyl of the seedlings whose cotyledons have not yet separated. At the same time, collect K599 Agrobacterium carrying the pCAMBIA1300-GmCHI4a vector and apply it to the wound. Spread sterilized filter paper on a glass dish and add 10 mL of sterile water to soak the filter paper. Place the seedlings with the wound facing up in the glass dish and co-cultivate for 24 hours. Then transfer the soybean plants to a transparent pot containing vermiculite to induce hairy roots. After 14 days of growth, hairy roots are clearly visible (such as Picture 2 Middle B).

[0035] (3) GmCHI4a Detection of gene expression levels in overexpressed plants extract GmCHI4a Gene overexpression hairy root total RNA was reversed into cDNA using a reverse transcription kit. Using cDNA as a template, primers were designed: Forward primer: 3′-ACCAAGCCCCTATCTTTGCT-5′; Reverse primer: 3′-ATAAACTTCTCCACCGGGGC-5′; Detection by quantitative PCR GmCHI4a The expression level of gene overexpression plants showed GmCHI4a The expression of gene overexpression plants was significantly higher than that of control plants (e.g. Picture 2 C).

[0036] Example 3 soybeans GmCHI4a The construction of gene knockout vector and Agrobacterium-mediated transformation of legumes are as follows: Through analysis GmCHI4a The CDS sequence of the gene was used to generate 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.

[0037] In this embodiment, the soybean cotyledonary node was transformed by Agrobacterium-mediated method to obtain GmCHI4a Genetically edited transgenic material that is heritable.

[0038] (1) Obtaining soybean explants Select mature soybean seeds with smooth surfaces, no damage, lesions, or cracks. Sterilize them with chlorine for 14 hours. Ventilate the sterilized seeds on a clean bench to completely evaporate the chlorine. Germinate them on germination medium for 6 hours. Remove half of the hypocotyl and cut the soybeans longitudinally along the hypocotyl. The remaining hypocotyl will serve as the recipient material for Agrobacterium-mediated transformation.

[0039] (2) Soybean transformation The Agrobacterium-mediated method involves secondary Agrobacterium infection. Plants are cultured in co-culture medium at 22°C in the dark for 5 days, then in SI-I medium under strong light for 7 days. Large explanted buds are removed and cultured in SI-II medium under strong light for 14 days. Cotyledons and hypocotyls are removed and subcultured every 14 days in SE medium. Approximately 3 cm long buds are excised and placed in rooting medium for rooting. Plants with well-developed roots in RM rooting medium are transferred to soil for planting. Thirty resistant plants were screened for Bar resistance. After five months of greenhouse cultivation, pods begin to mature, and harvest is completed after six months.

[0040] (3) Detection of heritable transgenic plant editing methods 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: Forward primer: 3′-CTTAAGGCAGCTAGGGATCT-5′; Reverse primer: 3′-GATGGGGGCATGCCGATTAA-5′; The fragment containing the editing site was amplified by PCR and sequenced for the first generation. The sequencing results were compared with the reference sequence to obtain GmCHI4a Gene-edited transgenic plants (e.g. Picture 3 ).

[0041] Example 4 soybeans GmCHI4a Effects of genetically synthesized naringenin on the growth of Phytophthora sojae: GmCHI4a The metabolite synthesized by the gene is naringenin. In order to verify the effect of naringenin on soybean Phytophthora root rot, naringenin was added to 10% V8 medium (methanol was used as a control) at a concentration of 100ug / ml. Soybean Phytophthora Race1, Race2, Race4, Race6, Race17, and Race19 were cultured in the naringenin-containing medium. The culture was carried out at 25°C in the dark for 7 days, and the area of ​​soybean Phytophthora growth was counted. The results are as follows: Picture 4 As shown in the figure, it was found that naringenin had a significant inhibitory effect on the growth of different physiological races of soybean Phytophthora.

[0042] Example 5 soybeans GmCHI4a The gene knockout and overexpression resistance identification experiments to soybean Phytophthora are as follows: (1) Soybeans GmCHI4a Effects of gene knockout on resistance to Phytophthora spp. in soybean To verify GmCHI4a The function of the gene for resistance to Phytophthora sojae was determined by comparing the wild-type Williams 82 and knockout GmCHI4atransgenic plants GmCHI4a KO The plants were inoculated with zoospores of Phytophthora sojae for 7 days, and the root phenotypes were observed. Root tissues were collected 4 hours after inoculation. GmCHI4a Gene expression detection and soybean Phytophthora biomass detection.

[0043] The results showed that knockout GmCHI4a Seven days after the transgenic plants were inoculated with zoospores of Phytophthora sojae, significant lesions appeared on their roots ( Picture 5 Middle A); At the same time, 4 hours after inoculation GmCHI4a KO In the root system GmCHI4a The gene expression level was significantly lower than that in the control plants ( Picture 5 C); and GmCHI4a KO The number of soybean Phytophthora organisms in the root system was significantly higher than that in the control (e.g. Picture 5 This indicates that the knockout GmCHI4a Genes reduce soybean resistance to Phytophthora sojae.

[0044] (2) Soybeans GmCHI4a Gene overexpression for resistance to Phytophthora sojae The susceptible material Williams and overexpression GmCHI4a transgenic plants GmCHI4a OE Plants were inoculated with zoospores of Phytophthora sojae for 7 days, and the phenotypes of the plants were observed. Root tissues were collected 4 hours after inoculation. GmCHI4a Gene expression detection and soybean phytophthora biomass detection. The results showed that the susceptible material Williams inoculated with soybean phytophthora zoospores showed a death phenotype 7 days later, while GmCHI4a OE Zoospores of overexpressing material inoculated with Phytophthora sojae can continue to survive ( Picture 5 Middle B); At the same time, 4 hours after inoculation GmCHI4a OE In the root system GmCHI4a The gene expression level was significantly higher than that in the control plants ( Picture 5 E); and GmCHI4a OE The number of soybean phytophthora organisms in the root system was significantly lower than that in the control (e.g. Picture 5 F). This shows GmCHI4a Gene overexpression improves soybean resistance to Phytophthora sojae.

[0045] Example 6 soybeans GmCHI4a The experiment on the changes in naringenin content in gene knockout and overexpression materials is as follows: To verify GmCHI4a The changes in naringenin content in genetically modified materials will GmCHI4a Gene knockout, GmCHI4a The gene overexpression and control materials were inoculated with zoospores of Phytophthora sojae; after inoculation, the root materials were collected to detect the content of naringenin. Picture 6 As shown in the figure, it can be seen that after inoculation of soybean phytophthora GmCHI4a The naringenin content in the knockout roots was significantly lower than that in the control Williams 82 ( Picture 6 Middle A), GmCHI4a The naringenin content in the roots of the gene overexpression was significantly higher than that in the control Williams ( Picture 6 Middle B).

[0046] The key technical points of the present invention include: a method for synthesizing soybean isoflavones GmCHI4a The target gene and its coding protein were transformed into soybean by Agrobacterium rhizogenes-mediated genetic transformation. GmCHI4a The results showed that GmCHI4a Overexpression can significantly increase the isoflavone content in hairy roots and enhance the defense against soybean phytophthora; through CRISPR / Cas9 technology, soybean cotyledonary nodes were genetically transformed and the isoflavone content in the whole soybean plant was significantly increased. GmCHI4a Knockout: The isoflavone content in the roots of the knockout material is reduced, and the defense effect against soybean phytophthora is also reduced.

[0047] In addition, chalcone isomerase catalyzes the synthesis of naringenin, an important flavonoid compound. The present invention uses plate antibacterial experiments to prove that naringenin has an inhibitory effect on different physiological subspecies (R1, R2, R4, R6, R17, R19) of soybean phytophthora. Therefore, the present invention provides GmCHI4a The gene and its encoded protein can be used to provide broad resistance to different physiological races of soybean Phytophthora.

[0048] Example 7 This embodiment provides a method for improving plant resistance to Phytophthora sojae, comprising the following steps: S1. Construction of 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; S2. The expression vector was transformed into Agrobacterium; the soybean isoflavone synthesis gene shown in SEQ ID NO.2 was expressed by Agrobacterium-mediated method. GmCHI4a Transferred into the target plant soybean; S3. Use RUBY visual screening markers for visual screening to obtain GmCHI4a Transgenic plants with gene overexpression; S4. Identify resistance by inoculating soybean phytophthora and screen out strains with enhanced resistance.

[0049] It should be understood that the various forms of the processes shown above can be used to reorder, add, or delete steps. For example, the steps described in the present disclosure can be performed in parallel, sequentially, or in a different order, as long as the desired results of the technical solutions disclosed in the present disclosure can be achieved. This is not limited herein.

[0050] The above specific embodiments do not limit the scope of protection of the present invention. Those skilled in the art will appreciate that various modifications, combinations, sub-combinations, and substitutions may be made based on design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. Soy isoflavone synthesis gene GmCHI4a Application in improving plant resistance to Phytophthora sojae, characterized by: The gene GmCHI4a The CDS sequence of the gene is shown in SEQ ID NO.2; the application includes utilizing the gene GmCHI4a Construct a plant expression vector and transfer it into the target plant to make the gene GmCHI4a Overexpression in plants.

2. The soybean isoflavone synthesis gene according to claim 1 GmCHI4a Application in improving plant resistance to Phytophthora sojae, characterized by: The plant expression vector carries a RUBY visual screening marker.

3. The soybean isoflavone synthesis gene according to claim 2 GmCHI4a Application in improving plant resistance to Phytophthora sojae, characterized by: The plant expression vector is an overexpression vector pCAMBIA1300-GmCHI4a with a RUBY visual screening marker.

4. The soybean isoflavone synthesis gene according to claim 3 GmCHI4a Application in improving plant resistance to Phytophthora sojae, characterized by: In the plant expression vector, the RUBY visual screening marker replaces the original Hyg screening marker, and the expression is driven by a 35s promoter and terminated by a polyA terminator.

5. The soybean isoflavone synthesis gene according to claim 1 GmCHI4a Application in improving plant resistance to Phytophthora sojae, characterized by: The target plant is soybean.

6. A method for improving plant resistance to Phytophthora sojae, characterized by: The following steps are involved: Construct a plant expression vector; transform the expression vector into Agrobacterium; and use Agrobacterium-mediated method to express the soybean isoflavone synthesis gene shown in SEQ ID NO.2 in the sequence table. GmCHI4a Transfer into target plants and screen to obtain GmCHI4a Transgenic plants with gene overexpression; resistance is identified by inoculation with soybean phytophthora, and strains with enhanced resistance are screened out.

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