Role of flavonoid glycosides in weed resistance in rice

Abstract

The present invention discloses the effect of flavonoid glycosides in weed resistance in rice. The present invention provides the use of isoorientin in any of the following: suppressing weed growth, controlling plant allelopathy, and controlling plant resistance to weeds. The results of the present invention show that isoorientin can control plant allelopathy and can be used to suppress weed growth. The present invention is of great significance to the development of environmentally friendly green pesticides and provides theoretical guidance for weed control in paddy fields.

Description

[Technical field]

[0001] The present invention relates to the field of biotechnology, specifically to the effect of flavonoid glycosides in weed resistance in rice. [Background technology]

[0002] Weed growth in paddy fields has a serious impact on rice yield and quality (Amb and Ahluwalia, 2016; Chung et al., 2018). Currently, the application of herbicides is the main method of weed control, but the large-scale application of herbicides entails many risks, such as causing environmental pollution (Annett et al., 2014), endangering human health, and increasing resistant weeds (Jabran et al., 2015). Therefore, the development of environmentally friendly weed management techniques, especially weed control strategies based on allelopathy, has attracted increasing attention (Cheng and Cheng, 2015). Plant allelopathy refers to the interaction between organisms, i.e., the direct or indirect effect of one plant (or microorganism) on the growth of another plant (or microorganism) by releasing one or more chemicals (allelopathic substances) into the environment (Rice, 1984).

[0003] Currently, the allelopathic substances discovered in rice are mainly divided into indoles, phenolic acids, terpenoids, flavonoids, fatty acids, alkaloids, etc. (Khanh et al., 2007; Kato-Noguchi et al., 2011; Chung et al., 2018). The main flavonoid substance with allelopathic activity is tricin (5,7,4'-trihydroxy-3',5'-dimethoxyflavone). It has not been clarified whether other flavonoid substances have allelopathic activity, and the genetic mechanism and weed suppression mechanism of allelopathy in rice. Summary of the Invention

[0004] The present invention seeks to address the role of flavonoid glycosides in weed resistance in rice.

[0005] In a first aspect, the present invention seeks protection for the use of isoorientin in any of the following: P1: Weed growth suppression, P2: Regulation of plant allelopathy, P3: Control of plant resistance to weeds.

[0006] The control of plant allelopathy is achieved by increasing the content of isoorientin in the plant body, thereby enhancing the plant's allelopathy.

[0007] The control of the resistance of a plant to weeds is achieved by increasing the content of isoorientin in the plant body, thereby enhancing the resistance of the plant to weeds.

[0008] In a second aspect, the present invention seeks protection for a method for controlling the growth of weeds.

[0009] A method for suppressing the growth of the weeds against which the present invention seeks to protect may comprise the step of applying isoorientin to the weeds.

[0010] Here, the effective concentration of isoorientin may be 500 mg / L or more.

[0011] In a specific embodiment of the present invention, the working concentration of isoorientin is 500 mg / L.

[0012] In a specific embodiment of the present invention, inhibiting the growth of weeds specifically refers to inhibiting the growth of the roots or radicles of weeds.

[0013] In a third aspect, the present invention seeks protection for any of the following methods: Method I: A method for breeding a plant variety with enhanced allelopathy, comprising a step of obtaining a plant variety with enhanced allelopathy by improving the content of isoorientin in a recipient plant. Method II: A method for breeding a plant variety with reduced allelopathy, comprising a step of reducing the content of isoorientin in a recipient plant, thereby obtaining a plant variety with reduced allelopathy. Method III: A method for breeding a plant variety with enhanced resistance to weeds, comprising the step of obtaining a plant variety with enhanced resistance to weeds by improving the content of isoorientin in a recipient plant. Method IV: A method for breeding a plant variety with reduced resistance to weeds, comprising a step of reducing the content of isoorientin in a recipient plant to obtain a plant variety with reduced resistance to weeds.

[0014] In the above method, improving the content of isoorientin in the recipient plant can be achieved as follows. (A1) reducing the activity and / or expression level of a protein capable of catalyzing the conversion of isoorientin to other substances in a recipient plant, and / or (A2) Improving the activity and / or expression level of a protein capable of catalyzing the conversion of other substances to isoorientin in a recipient plant.

[0015] In the above method, reducing the content of isoorientin in the recipient plant can be achieved as follows. (B1) improving the activity and / or expression level of a protein capable of catalyzing the conversion of isoorientin to other substances in a recipient plant, and / or (B2) The activity and / or expression level of a protein capable of catalyzing the conversion of other substances to isoorientin in a recipient plant is reduced.

[0016] Furthermore, (A1) can be achieved by the following (a1), and (A2) can be achieved by the following (a2). (a1) suppressing the expression of a gene encoding a protein capable of catalyzing the conversion of isoorientin to another substance in a recipient plant; (a2) A gene encoding a protein capable of catalyzing the conversion of other substances to isoorientin is introduced into a recipient plant.

[0017] Furthermore, (B1) can be achieved by the following (b1), and (B2) can be achieved by the following (b2). (b1) introducing into a recipient plant a gene encoding a protein capable of catalyzing the conversion of isoorientin to another substance; (b2) suppressing the expression in a recipient plant of a gene encoding a protein capable of catalyzing the conversion of other substances to isoorientin;

[0018] Here, the other substance is a substance that cannot enhance the allelopathy of the recipient plant, or a substance that enhances the allelopathy of the recipient plant to a lesser extent than isoorientin, or a substance that cannot enhance the resistance of the recipient plant to weeds, or a substance that enhances the resistance of the recipient plant to weeds to a lesser extent than isoorientin.

[0019] In a specific embodiment of the present invention, the other substance in (A1), (a1), (B1) and (b1) is specifically isoorientin-2″-O-glucopyranoside.

[0020] In each of the above embodiments, the plant may be rice and the weed may be barnyard grass.

[0021] In a fourth aspect, the present invention seeks protection for the use of Os02g0589400 protein or related biological material in any of the following: Q1: Catalyzing the glycosylation reaction of isoorientin, Q2: Preparation of isoorientin-2''-O-glucopyranoside, Q3: Control of isoorientin content in plants.

[0022] Relevant biological material is a nucleic acid molecule capable of expressing an Os02g0589400 protein or an expression cassette comprising the nucleic acid molecule, a recombinant vector, a recombinant bacterium or a transgenic cell line.

[0023] The Os02g0589400 protein may be any of the proteins shown below. (D1) a protein having an amino acid sequence of SEQ ID No. 1; (D2) a protein having one or more amino acid residues substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No. 1 and having the same function; (D3) A protein having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with an amino acid sequence defined in any one of (D1) to (D2) and having the same function. (D4) A fusion protein obtained by binding a tag to the N-terminus and / or C-terminus of a protein specified in any one of (D1) to (D3).

[0024] In the above proteins, the tag refers to a polypeptide or protein that is expressed by fusing with a target protein using in vitro recombinant DNA technology to facilitate expression, detection, tracking and / or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and / or SUMO tag, etc.

[0025] In the above proteins, homology refers to the identity of amino acid sequences. The identity of amino acid sequences can be measured using a homology search site on the Internet, such as the BLAST page on the NCBI homepage. For example, in the advanced BLAST2.1, the program blastp is used, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as the matrix, and the Gap existence cost, Per residue gap cost, and Lambda ratio are set to 11, 1, and 0.85 (default values), respectively, to search and calculate the identity of a pair of amino acid sequences, and the identity value (%) can be obtained.

[0026] In the above proteins, 99% or more identity may be at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identity. 95% or more identity may be at least 96%, 97%, 98% identity. 90% or more identity may be at least 91%, 92%, 93%, 94% identity. 85% or more identity may be at least 86%, 87%, 88%, 89% identity. 80% or more identity may be at least 81%, 82%, 83%, 84% identity.

[0027] The nucleic acid molecule capable of expressing the Os02g0589400 protein may be a DNA molecule as set forth below: (E1) a DNA molecule represented by SEQ ID No. 2; (E2) a DNA molecule that hybridizes under stringent conditions with the DNA molecule defined in (E1) and encodes the Os02g0589400 protein; (E3) A DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to a DNA molecule defined by (E1) or (E2) and encoding an Os02g0589400 protein.

[0028] In the above nucleic acid molecule, stringent conditions are as follows: hybridization may be performed in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5 M Na3PO4, and 1 mM EDTA at 50° C., followed by washing with 2×SSC and 0.1% SDS at 50° C.; alternatively, hybridization may be performed in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA at 50° C., followed by washing with 1×SSC and 0.1% SDS at 50° C.; alternatively, hybridization may be performed in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA at 50° C., followed by washing with 0.5×SSC and 0.1% SDS at 50° C. Alternatively, hybridization may be performed in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA at 50° C., followed by washing with 0.1×SSC, 0.1% SDS at 50° C. Alternatively, hybridization may be performed in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA at 50° C., followed by washing with 0.1×SSC, 0.1% SDS at 65° C. Alternatively, hybridization may be performed in a 6×SSC, 0.5% SDS solution at 65° C., followed by washing once each with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.

[0029] In the above nucleic acid molecules, homology refers to the identity of nucleotide sequences. The identity of nucleotide sequences can be measured using a homology search site on the Internet, such as the BLAST page on the NCBI homepage. For example, in the advanced BLAST2.1, the program blastp is used, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as the matrix, and the Gap existence cost, Per residue gap cost, and Lambda ratio are set to 11, 1, and 0.85 (default values), respectively, to search and calculate the identity of a pair of nucleotide sequences, thereby obtaining an identity value (%).

[0030] In the above nucleic acid molecules, 95% or more homology may be at least 96%, 97%, or 98% identity. 90% or more homology may be at least 91%, 92%, 93%, or 94% identity. 85% or more homology may be at least 86%, 87%, 88%, or 89% identity. 80% or more homology may be at least 81%, 82%, 83%, or 84% identity.

[0031] The recombinant expression vector can be constructed using an existing plant expression vector. The plant expression vector includes binary Agrobacterium vectors and vectors that can be used for microprojectile bombardment, such as pCAMBIA-1300-221, pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN, or plant expression vectors derived from these vectors. The plant expression vector can further include the 3'-end untranslated region of the foreign gene, i.e., a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can direct the addition of polyadenylate to the 3' end of the mRNA precursor. When constructing a recombinant expression vector using the above gene, any of a strong, constitutive, tissue-specific, or inducible promoter can be added before the transcription initiation nucleotide. Examples include the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin gene Ubiquitin promoter (pUbi), and the stress-inducible promoter rd29A, which can be used alone or in combination with other plant promoters. Furthermore, when constructing a recombinant expression vector using the gene of the present invention, enhancers such as translation enhancers and transcription enhancers can also be used. These enhancer regions can be the ATG start codon or adjacent region start codons, but must be in line with the reading frame of the coding sequence to ensure translation of the entire sequence. The translation control signals and start codons can be from a variety of sources and can be natural or synthetic. The translation initiation region can be derived from the transcription initiation region or from a structural gene. The recombinant expression vector used can be engineered to facilitate identification and screening of transgenic plant cells or plants. For example, genes encoding color-changing enzymes or luminescent compounds that can be expressed in plants, antibiotic markers with resistance, or chemical drug resistance marker genes, etc. can be added.Alternatively, plants can be transformed directly by stress screening without adding a selective marker gene.

[0032] In a fifth aspect, the present invention further seeks protection for a plant variety obtained by breeding using the method according to the third aspect of the preamble.

[0033] Plant varieties include, but are not limited to, whole plants, seeds, reproductive tissues or organs, and the like.

[0034] Depending on the breeding method, the plant variety may be a plant variety with enhanced allelopathic activity, a plant variety with reduced allelopathic activity, a plant variety with enhanced resistance to weeds, or a plant variety with reduced resistance to weeds.

[0035] In a sixth aspect, the present invention further seeks to protect against weed growth inhibiting compounds.

[0036] The weed growth-inhibiting compound against which the present invention seeks to protect is isoorientin.

[0037] Here, the weed may be barnyard grass.

[0038] In each of the above embodiments, the structural formula of isoorientin is shown in Figure 1. The structural formula of isoorientin-2"-O-glucopyranoside is shown in Figure 2. [Brief description of the drawings]

[0039] [Figure 1] This is the structural formula of isoorientin. [Diagram 2] This is the structural formula of isoorientin-2''-O-glucopyranoside. [Diagram 3]Purification of Os02g0589400 protein. The arrow indicates the size of the recombinant protein after protein purification. [Figure 4] Chromatogram of in vitro enzyme activity assay of Os02g0589400. [Diagram 5] A comparison of the weed suppression effects of the compounds isoorientin and isoorientin-2''-O-glucopyranoside is shown. A shows the effects of isoorientin and isoorientin-2''-O-glucopyranoside on the growth of barnyard grass roots, and B shows the effects of isoorientin and isoorientin-2''-O-glucopyranoside on the growth of barnyard grass shoots. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] The following examples are provided to facilitate understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are all performed in triplicate and the results are averaged.

[0041] Example 1, Purification of Compounds (1) Fresh leaves of Indica rice with high content of the target compound were harvested about 55 days after sowing, dried in a freeze-dryer, and crushed into powder. Eight times the volume of 75% ethanol was added and the mixture was extracted with ultrasound three times for 20 minutes each time.

[0042] (2) The extract is filtered and concentrated using a rotary evaporator until the odor of alcohol disappears, and then an equal volume of petroleum ether is added and extracted three times. The petroleum ether phase extract is collected, and then an equal volume of dichloromethane is added to the remaining aqueous phase and extracted three times. The dichloromethane phase extract is collected, and then an equal volume of n-butanol is added to the remaining aqueous phase and extracted three times. The n-butanol phase extract is collected.

[0043] (3) Take 1 mL of the extract from each phase in (2), dry it, dissolve it in methanol, centrifuge it, aspirate the supernatant and transfer it to an Agilent vial, and measure the compounds in each phase using UPLC-QTOF / MS. Column: RRHD SB-C18 (Agilent), packing particle size: 1.8 μm, specifications: 2.1 mm (inner diameter) × 100 mm (length). Column temperature: 40 °C, injection volume: 5 μL. Mobile phase A: H2O (containing 0.1% formic acid, % represents volume percentage), mobile phase B: acetonitrile. Elution gradient: 0-2 min: 5% B-10% B, 2-12 min: 10% B-25% B, 12-18 min: 25% B-70% B, 18-23 min: 70% B-90% B, 23-25 ​​min: 90% B-100% B, 25-30 min: 100% B, post-run 5 min. Flow rate: 0.3 mL / Min. The mass spectrometry system used an electrospray ionization source (ESI) in positive ion measurement mode, with high purity nitrogen as the carrier gas, pressure: 40 psi, and temperature: 325 °C. The measurement results showed that the target compound was mainly present in the n-butanol phase.

[0044] (4) The n-butanol phase was concentrated using a rotary evaporator and then purified using a low- and medium-pressure preparative high-performance liquid chromatograph (Biotage Isolera TM Coarse fractionation was performed by wet loading using a column chromatography column (Prime). The packing material of the silica gel column was YMC*GEL C18 spherical packing material with a particle size of 50 μm and a pore size of 12 nm. The mobile phase was water (containing 0.1% formic acid, % indicates volume percentage) and methanol. The mobile phase ratio was 10 column volumes of 10%-90% methanol and 2 column volumes of 90%-100% methanol, % indicates volume percentage. Flow rate: 50 mL / min.

[0045] (5) The content of the target compound in each fraction is measured using UPLC-QTOF / MS in the same manner as in (3). Then, the fractions containing the target compound are combined and concentrated using a rotary evaporator.

[0046] (6) The concentrate is pre-separated on an ODS column and eluted sequentially with methanol:H2O solutions of 18:82, 24:76, 30:70, 35:65, 40:60, 50:50, 60:40, 70:30, 80:20, and 100:00 (volume ratio). After obtaining a total of eight fractions, the content of the target compound in each fraction is measured using UPLC-QTOF / MS. As a result of the measurement, it was found that fraction 2 (Fr2) and fraction 3 (Fr3) contained the target compound.

[0047] (7) Concentrate and weigh Fr2. Then, separate 203 mg of Fr2 using a reversed-phase semi-preparative high-performance liquid chromatograph. Mobile phase: acetonitrile-H2O (15:85, v / v), flow rate: 2.5 mL / min. A total of 13 fractions (Fr2.1 to Fr2.13) were obtained. Measurements showed that Fr2.10 contained the target compound. This was concentrated to obtain 43 mg of the concentrate, which was then further separated using a reversed-phase semi-preparative high-performance liquid chromatograph. Mobile phase: methanol-H2O (40:60, v / v), flow rate: 2.5 mL / min, and isoorientin-2''-O-glucopyranoside (13.0 mg, RT = 18.3 min) was obtained.

[0048] (8) Fr3 was concentrated to obtain 108 mg of concentrate. It was then pre-separated on a Sephadex LH-20 column, and the eluent was methanol. A total of 8 fractions were obtained, of which fraction 2 (Fr3.2) contained the target compound. After concentration, Fr3.2 (54 mg) was purified by reversed-phase semi-preparative high-performance liquid chromatography. Mobile phase: acetonitrile-H2O (20:80, v / v), flow rate: 2.5 mL / min, isoorientin (16.9 mg, RT = 16.3 min) was obtained.

[0049] (9) The structure of the obtained compound was identified using NMR (Bruker AVIII 400 spectrometer). The structure of isoorientin is shown in Figure 1, and the specific NMR results are as follows: 1 H-NMR(400MHz,Methanol-d4)δ:6.53(1H,s,H-3),6.47(1H,s,H-8),7.35(1H ,s,H-2′),6.89(1H,brsH-5′),7.35(1H,s,H-6′),4.89(1H,d,J=8.0Hz,H-1′′ ),4.18(1H,m,H-2′′),3.48(1H,m,H-3′′),3.50(1H,m,H-4′′),3.43(1H,m,H -5′′),3.91(1H,d,J=12.4Hz,H-6a′′),3.75(1H,dd,J=12.4,5.2Hz,H-6b′′); 13 C-NMR(100MHz,Methanol-d4)δ:166.2(C-2),103.9(C-3),184.0(C-4),162.0(C- 5),109.1(C-6),164.8(C-7),95.2(C-8),158.7(C-9),105.2(C-10),123.5(C-1' ),114.1(C-2′),147.0(C-3′),151.0(C-4′),116.8(C-5′),120.3(C-6′),75.3(C -1′′), 72.6(C-2′′), 80.1(C-3′′), 71.8(C-4′′), 82.6(C-5′′), 62.9(C-6′′). The structure of isoorientin-2''-O-glucopyranoside is shown in Figure 2, and the specific NMR results are as follows: 1H-NMR(400MHz,DMSO-d6)δ:13.65(1H,s,5-OH),7.35(1H,s,H-6′),7.35(1H,s,H-2′),6.84(1H,brsH-5 ′),6.54(1H,s,H-3),6.40(1H,s,H-8),4.65(1H,d,J=8.0Hz,H-1′′),4.52(1H,m,H-2′′),3.41(1H,m,H -3′′),3.18(1H,m,H-4′′),3.18(1H,m,H-5′′),3.19(1H,m,H-6′′),4.22(1H,d,J=8.5Hz,H-1′′′),2.8 5(1H,m,H-2′′′),3.04(1H,m,H-3′′′),3.04(1H,m,H-4′′′),3.04(1H,m,H-5′′′),3.66(1H,m,H-6′′′); 13 C-NMR(100MHz,DMSO-d6)δ:163.3.2(C-2),102.3(C-3),181.6(C-4),160.5(C-5),108.2(C-6),161.5(C -7),93.2(C-8),156.9(C-9),104.7(C-10),121.1(C-1′),112.8(C-2′),146.0(C-3′),150.6(C-4′),116 .11(C-5′), 118.9(C-6′), 71.6(C-1′′), 81.3(C-2′′), 78.6(C-3′′), 70.5(C-4′′), 81.6(C-5′′), 60.7(C-6′′), 105.6(C-1′′′), 74.6(C-2′′′), 76.6(C-3′′′), 69.8(C-4′′′), 76.6(C-5′′′), 61.7(C-6′′′).

[0050] Example 2. Use of the Os02g0589400 gene (1) Gene cloning The Os02g0589400 gene is cloned using genomic DNA from the leaf blade of Japonica rice (Oryza sativa L. ssp. japonica cv. Zhonghua11, ZH11) seedling stage as a template. The target gene is ligated to the pEASY-T3 vector (TransGen Biotech, pEASY (registered trademark)-T3 Cloning Kit) by enzyme cleavage and ligation method. The ligation product is then transformed into DH5α competent cells, and positive clones are screened by blue-white spots, and the resulting recombinant plasmid is named pEASY-T3-Os02g0589400. The structure of the recombinant vector pEASY-T3-Os02g0589400 is described as a recombinant plasmid in which a small fragment between the enzyme cleavage sites BamHI and HindIII in the pEASY-T3 vector is replaced with the DNA fragment shown in SEQ ID No. 2. SEQ ID No. 2 is the CDS sequence of the Os02g0589400 gene, which encodes the protein shown in SEQ ID No. 1.

[0051] (2) Prokaryotic expression of Os02g0589400 The recombinant vector pEASY-T3-Os02g0589400 is completely digested with the restriction enzymes BamHI and HindIII, and the expression vector pMAL-c2X (Hua Yueyang, VECT-570) is simultaneously digested. After agarose electrophoresis, a fragment of about 1.3 Kb in size containing Os02g0589400 and a pMAL-c2X vector fragment of about 6.6 Kb are collected and ligated, and the resulting recombinant plasmid is confirmed to be correct by sequencing, and then named pMAL-Os02g0589400. The structure of pMAL-Os02g0589400 is described as a vector in which the sequence between the two enzyme cleavage sites BamHI and HindIII in the pMAL-c2X vector is replaced with the Os02g0589400 gene (SEQ ID No. 2).

[0052] The obtained recombinant prokaryotic expression vector pMAL-Os02g0589400 is transformed into E. coli NovaBlue (Hua Yueyang, WR4478) competent cells. At the same time, pMAL-c2X empty vector is transformed as a control. The positive clones are screened on LB plates containing ampicillin antibiotics, and the positive clones are identified by PCR amplification, the primers are F1 / R1, and the size of the amplified fragment is 414bp.

[0053] The positive clones were inoculated into 20 mL of LB liquid medium containing ampicillin resistance and cultured at 37°C and 220 rpm for 12 to 14 hours with shaking. Then, the bacterial suspension was transferred to 400 mL of LB liquid medium (containing 100 μg / mL ampicillin) at a ratio of 1:1000 (volume ratio) and cultured at 37°C and 220 rpm to measure the OD. 600 Culture until the pH reaches 0.6-0.8. Remove the bacterial solution, place on ice to cool, and add 160 μL of 500 mM IPTG to make the final IPTG concentration 0.2 mM. To induce protein expression, culture at 16°C and 100 rpm for 24 hours with shaking. Centrifuge at 4°C and 10,000 rpm for 10 minutes to recover the bacteria. Then, resuspend the bacteria in column buffer and freeze at -20°C overnight. The column buffer (1 L) is formulated as follows: NaCl 11.7 g; DTT 154 mg; 0.5 M EDTA 2 mL; 1 M Tris-HCl (pH = 7.4) 40 mL; the remainder is water. The next day, after the sample has melted, disrupt the cells with an ultrasonic disrupter and centrifuge at 10,000 rpm for 10 minutes. Purify the target protein using a linear starch column.

[0054] The specific purification method is as follows. a. Activate the affinity column packing material using column buffer (see above for formulation) (flow rate 1 mL / min). b. The resulting crude protein supernatant is slowly loaded onto the activated linear starch column, and the flow rate is adjusted to approximately 0.5 mL / min so that the target protein is sufficiently bound to the affinity column packing. c, After all the sample has flowed through the packing, column buffer is added and washed several times to remove unbound contaminating proteins. d. Add 15 mL of column buffer containing maltose (1 L of column buffer contains 3.6 g of maltose) to elute the target protein, and repeat this process once. e) Add the eluate to the ultrafiltration tube in several portions and centrifuge at 4°C, 5000 rpm for 15 minutes each. f. Add 1 mL of 100 mM Tris-HCl, centrifuge at 5000 rpm for 15 min at 4°C, and pour off the waste liquid. Transfer the target protein solution to a 1.5 mL Eppendorf centrifuge tube. g. 2 μL of the obtained target protein solution was sucked up and used in a Bradford (Quick Start TM Add 1 mL of Bradford 1× Dye Reagent (BioRAD, CAS67-56-1) and measure the OD at 595 nm (OD ) of the protein to be measured using a spectrophotometer. 595 The absorption peak at 1000 nm was measured. The absorption value of the target protein was converted to protein concentration, and the concentration of the purified Os02g0589400 recombinant protein was found to be 2.03 μg / μL. h. After SDS-PAGE electrophoresis, the size of the recombinant protein Os02g0589400 was confirmed to be approximately 90 KD by Coomassie brilliant blue staining (Figure 3). Primer F1: 5'-GGCGACACAGCTCTCATGTCCTCGT-3'; Primer R1: 5'-CCCTCTCGCCCCTTCCAAGTAGCTC-3'.

[0055] (3) In vitro enzyme activation of Os02g0589400 The enzyme activation reaction system is 38 μL of Tris-HCl (pH 7.0, 50 mM, containing 10 mM DTT), 1 μL of 10 mM glycosyl acceptor (the glycosyl acceptor of Os02g0589400 is isoorientin), 1 μL of 100 mM glycosyl donor (UDPG), and 10 μL of Os02g0589400 recombinant protein obtained in (2) (protein concentration of Os02g0589400 is 2.03 μg / μL). After reacting for 1 hour in a water bath at 30 °C, the reaction is stopped with an equal volume of methanol. The pMAL-c2X empty vector is added to the transformed E. coli, and the protein sample obtained according to the above step (2) is used as the negative control. After centrifugation at 12,000 rpm at 4° C. for 10 minutes, 30 μL of the supernatant was taken and measured by UPLC-QTOF / MS using the same method as that used in Example 1.

[0056] The results showed that, compared with the empty vector control, Os02g0589400 recombinant protein could catalyze the production of isoorientin-2''-O-glucopyranoside from isoorientin in the reaction system to which it was added. This indicated that Os02g0589400 plays an important role in the process of catalyzing the production of isoorientin-2''-O-glucopyranoside from isoorientin (see Figure 4).

[0057] Example 3. Application of Compounds to Lettuce and Barnyardgrass 1. Experimental procedure for application of compounds to lettuce and barnyard grass Test compounds: isoorientin and isoorientin-2''-O-glucopyranoside. (1) The test compound is dried to a powder form using a centrifugal concentrator, weighed out, and an appropriate amount of sterile water is added to make the final concentration 10 mg / mL. (2) Prepare 500 mL of 0.8% agar and sterilize in an autoclave. (3) After cooling the sterilized agar to room temperature on a clean bench (note: do not allow the medium to solidify), accurately aspirate 4.75 mL of the medium into a sterilized 10 mL centrifuge tube. Add 250 μL of 10 mg / mL test compound solution to make the final concentration of the compound 500 mg / L. Quickly pour the medium containing the test compound into a 10 cm x 10 cm square petri dish and quickly shake the dish to spread the medium evenly and flatly on the square dish. Use the medium to which an equal amount of sterilized water has been added as a control. (4) Wash barnyard grass seeds several times with distilled water and place them on sterile filter paper to dry. (5) After the medium has solidified, place barnyard grass seeds with sterile tweezers, placing 26 seeds in each petri dish (2 rows of 13 seeds each). Each treatment contains three biological replicates. (6) Place the dish in a 28°C incubator and culture in the dark for 3 days. (7) Phenotypic statistics: After 3 days, the lengths of the root and shoot of each treatment were counted.

[0058] II. Phenotype of compounds applied to barnyardgrass and lettuce As shown in FIG. 5, when isoorientin and isoorientin-2''-O-glucopyranoside were applied to barnyard grass at a concentration of 500 mg / L, respectively, the root length of barnyard grass treated with isoorientin was significantly shorter than that of the control (p=1.539×10 -3), but the root length of barnyardgrass treated with isoorientin-2''-O-glucopyranoside was not significantly different from that of the control (p=0.321). The shoot length of barnyardgrass treated with isoorientin and isoorientin-2''-O-glucopyranoside was not significantly different from that of the control (p=0.297, p=0.902). This indicated that application of the compound isoorientin mainly affected the growth of barnyardgrass roots, and that isoorientin had a more significant inhibitory effect on the growth of barnyardgrass roots than the product isoorientin-2''-O-glucopyranoside.

[0059] The present invention has been described in detail above. Those skilled in the art can carry out the present invention in a broader scope under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although the present invention has been described in specific embodiments, it is understood that the present invention can be further improved. In short, based on the principles of the present invention, the present application is intended to include any modification, use, or improvement to the present invention, including modifications made using conventional techniques known in the art that depart from the scope disclosed in the present application. Some basic features may be applied within the scope of the following attached claims. [Industrial Applicability]

[0060] The flavonoid glycoside substance related to allelopathy provided by the present invention is isoorientin. Compound application experiments showed that isoorientin has a significant inhibitory effect on the root growth of barnyardgrass. The gene involved in the formation of the isoorientin compound provided by the present invention is Os02g0589400. In vitro enzyme activation experiments showed that Os02g0589400 can catalyze the production of the glycosylation product isoorientin-2''-O-glucopyranoside from isoorientin. When the compound was applied at a concentration of 500 mg / L, isoorientin had a more significant inhibitory effect on the growth of barnyardgrass roots than the product isoorientin-2''-O-glucopyranoside. This indicates that isoorientin can control the allelopathic effects of plants and can be used to suppress the growth of weeds. The present invention is of great significance for the development of environmentally friendly green pesticides and provides a theoretical guide for the control of weeds in paddy fields.

Claims

1. Use of isoorientin or isoorientin-2″-O-glucopyranoside in any of the following: (1) Suppression of weed growth in paddy fields, (2) Regulation of plant allelopathy, (3) Controlling plant resistance to weeds, (4) Breeding of plant varieties with enhanced allelopathic properties; (5) Breeding plant varieties with enhanced resistance to weeds; (6) Control of isoorientin content in plants.

2. The method for suppressing the growth of weeds in paddy fields comprises increasing the content of isoorientin in a recipient plant, the recipient plant is rice, The use according to claim 1, wherein preferably, improving the content of isoorientin in a recipient plant comprises reducing the glycosylation reaction of isoorientin.

3. Improving the content of isoorientin in the recipient plant comprises reducing the activity and / or expression level of a protein capable of catalyzing the conversion of isoorientin to another substance in the recipient plant; Preferably, the step of reducing the activity and / or expression level of a protein capable of catalyzing the conversion of isoorientin to other substances in the recipient plant comprises suppressing the expression of a gene encoding a protein capable of catalyzing the conversion of isoorientin to other substances in the recipient plant. The use according to claim 2.

4. The protein that catalyzes the conversion of isoorientin to another substance is Os02g0589400 protein or a biological material related thereto, The use according to claim 3, wherein the relevant biological material is preferably a nucleic acid molecule capable of expressing the Os02g0589400 protein or an expression cassette, recombinant vector, recombinant bacterium or transgenic cell line comprising said nucleic acid molecule.

5. The Os02g0589400 protein is a protein represented by any one of the following: (D1) a protein having the amino acid sequence of SEQ ID No. 1; (D2) A protein having one or more amino acid residues substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No. 1 and having the same function; (D3) A protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with an amino acid sequence defined in any one of (D1) to (D2) and having the same function; (D4) The use according to claim 4, which is a fusion protein obtained by binding a tag to the N-terminus and / or C-terminus of a protein defined by any one of (D1) to (D3).

6. In the above (2), the control of the allelopathic effect of the plant is to enhance the allelopathic effect of the plant by increasing the content of isoorientin in the plant body, The use according to claim 1, wherein in (3), the control of the weed resistance of the plant is to enhance the weed resistance of the plant by increasing the content of isoorientin in the plant body.

7. 2. The use according to claim 1, wherein the plant is rice and the weed is barnyard grass or lettuce.

8. The use according to claim 1, wherein the growth inhibition of weeds is to inhibit the growth of roots or rootlets of weeds.

9. The use according to claim 1, wherein (4) and (5) include a step of obtaining a plant variety with enhanced allelopathy by increasing the content of isoorientin in a recipient plant, or a step of obtaining a plant variety with enhanced resistance to weeds.

10. In the above (6), controlling the isoorientin content in a plant includes improving the isoorientin content in the recipient plant; Preferably, this is accomplished as follows: (A1) reducing the activity and / or expression level of a protein capable of catalyzing the conversion of isoorientin to another substance in the recipient plant, and / or (A2) improving the activity and / or expression level of a protein capable of catalyzing the conversion of other substances to isoorientin in the recipient plant; More preferably, the above-mentioned (A1) is achieved by the following (a1), and the above-mentioned (A2) is achieved by the following (a2): (a1) suppressing the expression of a gene encoding a protein capable of catalyzing the conversion of isoorientin to another substance in the recipient plant; (a2) introducing into the recipient plant a gene encoding a protein capable of catalyzing the conversion of other substances to isoorientin; 2. The use according to claim 1.

11. the other substance is a substance that cannot enhance the allelopathy of the recipient plant or enhances the allelopathy of the recipient plant to a lesser extent than isoorientin, Alternatively, the other substance is a substance that cannot enhance the weed resistance of the recipient plant, or is a substance that enhances the weed resistance of the recipient plant to a lesser extent than isoorientin.

12. The use according to claim 10, wherein in (A1) or (a1), the other substance is isoorientin-2''-O-glucopyranoside.

13. A plant variety obtained by breeding using the method according to claim 1 (4) or (5).

14. A compound for use in suppressing the growth of weeds in paddy fields, comprising: The compound is isoorientin and the weed is barnyard grass or lettuce.

15. The compound described in claim 14, wherein the isoorientin is used at a concentration of 500 mg / L or more.

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