Application of RanGAP in regulating symbiotic nodulation
By regulating the expression or activity of RanGAP, the infection and nodulation phenotype of rhizobia in leguminous plants can be modulated, which solves the problem of unclear molecular mechanism of nitrogen fixation in symbiosis between leguminous plants and rhizobia. This achieves effective regulation of nodule formation and improvement of nitrogen fertilizer utilization, thus promoting sustainable agricultural development.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
- Filing Date
- 2024-12-06
- Publication Date
- 2026-06-09
AI Technical Summary
In the existing technology, the molecular mechanism of nitrogen fixation in symbiosis between leguminous plants and rhizobia is not clear, which makes it difficult to effectively regulate root nodule formation and nitrogen fertilizer utilization, thus affecting the sustainable development of agriculture.
By regulating the expression or activity of RanGAP, using RanGAP inhibitors or promoters, the infection and nodulation phenotype of rhizobia in leguminous plants can be modulated, including silencing, knocking out or overexpressing the RanGAP gene, and gene editing using the CRISPR/Cas9 system to regulate the expression and activity of RanGAP.
It significantly regulates rhizobium infection and nodule formation, enhances nitrogen fixation capacity, reduces nitrogen fertilizer demand, promotes nitrogen utilization by leguminous plants, and improves soil quality and agricultural production efficiency.
Smart Images
Figure HDA0005178411030000011 
Figure HDA0005178411030000012 
Figure HDA0005178411030000021
Abstract
Claims
1. The application of a RanGAP or its regulatory agent for regulating rhizobium infection and nodulation phenotype in leguminous plants; wherein the RanGAP includes RanGAP1 or RanGAP2.
2. The application as described in claim 1, characterized in that, The regulatory agent is a RanGAP inhibitor, which inhibits rhizobium infection and reduces the number of nodules; the inhibitor includes: agents that silence or knock out RanGAP, agents that inhibit RanGAP activity; preferably, it includes: interfering molecules that specifically interfere with RanGAP gene expression, CRISPR gene editing agents, homologous recombination agents, or site-directed mutagenesis agents targeting RanGAP, wherein the agents induce loss-of-function mutations in RanGAP; preferably, the inhibitor includes: interfering molecules targeting the genomic sequence (or its complementary sequence) corresponding to positions 17-37 of SEQ ID NO:9 and the genomic sequence (or its complementary sequence) corresponding to positions 17-38 of SEQ ID NO:11; more preferably, the inhibitor includes: interfering material prepared using primers of the sequences shown in SEQ ID NO:9 and SEQ ID NO:10, or interfering material prepared using primers of the sequences shown in SEQ ID NO:11 and SEQ ID NO:12; or The regulatory agent is a RanGAP promoter, which promotes rhizobium infection and increases the number of nodules; the promoter includes: an exogenous RanGAP encoding gene or an expression construct or vector containing the encoding gene.
3. Methods for regulating rhizobium infection and nodulation phenotype in leguminous plants include: Regulate the expression or activity of RanGAP in legumes; said RanGAP includes RanGAP1 or RanGAP2.
4. The method as described in claim 3, characterized in that, The method involves reducing the expression or activity of RanGAP in plants to inhibit rhizobium infection and reduce the number of nodules. The method includes silencing or knocking out RanGAP in plants, or inhibiting the activity of RanGAP. Preferably, RanGAP is silenced using an interfering molecule that specifically interferes with the expression of the RanGAP gene. Silencing or knocking out RanGAP in plants includes: knocking out the RanGAP gene by gene editing using a CRISPR system, knocking out the RanGAP gene by homologous recombination, or performing a loss-of-function mutation on RanGAP in plants containing RanGAP. Preferably, the silencing is performed with the genomic sequence (or its complementary sequence) corresponding to positions 17-37 in SEQ ID NO:9 as the target and the genomic sequence (or its complementary sequence) corresponding to positions 17-38 in SEQ ID NO:11 as the target; More preferably, the silencing is performed using interfering material prepared with primers showing the sequences of SEQ ID NO:9 and SEQ ID NO:10, or using interfering material prepared with primers showing the sequences of SEQ ID NO:11 and SEQ ID NO:
12.
5. The method as described in claim 1, characterized in that, The method aims to enhance the expression or activity of RanGAP in plants, preferably in plants with low RanGAP expression, thereby promoting rhizobium infection and increasing nodule formation. Preferably, the method includes: introducing the RanGAP gene or an expression construct or vector containing the gene into plants; performing a gain-of-function mutation on RanGAP; promoting RanGAP expression by expressing an enhancing promoter or a tissue-specific promoter; or promoting RanGAP expression by an enhancer.
6. The application as described in any one of claims 1-2 or the method as described in any one of claims 3-5, characterized in that, The legumes mentioned include: birdsfoot root, alfalfa, soybean, pea, peanut, kidney bean, mung bean, red bean, broad bean, cowpea, or milkvetch.
7. The application as described in any one of claims 1-2 or the method as described in any one of claims 3-5, characterized in that, The amino acid sequence of the RanGAP1 protein is selected from the following group: (i) a protein with the amino acid sequence shown in SEQ ID NO:1; (ii) a protein derived from (i) having the regulatory function of the amino acid sequence shown in SEQ ID NO:1, formed by substitution, deletion, or addition of one or more amino acid residues; (iii) a protein having the regulatory function of the amino acid sequence with ≥80% homology to the amino acid sequence shown in SEQ ID NO:1; or (iv) a protein formed by adding a tag sequence or restriction enzyme site sequence to the N or C terminus of the amino acid sequence shown in SEQ ID NO:1, or by adding a signal peptide sequence to its N terminus; or The amino acid sequence of the RanGAP2 protein is selected from the following group: (i') a protein with the amino acid sequence shown in SEQ ID NO:3; (ii') a protein derived from (i') having the regulatory function formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:3; (iii') a protein having the regulatory function with an amino acid sequence that is ≥80% homologous to the amino acid sequence shown in SEQ ID NO:3; or, (iv') a protein formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the protein with the amino acid sequence shown in SEQ ID NO:3, or by adding a signal peptide sequence to its N end.
8. An isolated polynucleotide, having the nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:4, having the function of driving gene-specific expression; preferably, the driving gene is specifically expressed in the root vascular bundle, lateral root / nodule primordium.
9. The application of the isolated polynucleotide of claim 8 for driving gene-specific expression; preferably, the driving gene is specifically expressed in the root vascular bundles and lateral root / nodule primordia; more preferably, the driving gene is specifically expressed in the root vascular bundles and lateral root primordia before infection with rhizobia; and the driving gene is specifically expressed in the root vascular bundles and nodule primordia after infection with rhizobia.
10. An expression construct for specifically expressing a target gene in root vascular bundles, lateral roots / nodule primordia, comprising the isolated polynucleotide of claim 8 as an element driving gene expression; preferably, the expression construct further contains a target gene operatively linked to the isolated polynucleotide.
11. Uses of RanGAP in legumes: as molecular markers for identifying traits in legumes, or as molecular markers for targeted screening of legumes; among which, The RanGAP includes RanGAP1 or RanGAP2; the characteristics include: rhizobium infection and nodulation phenotype.
12. A method for selecting or identifying leguminous plants, comprising: To identify the expression or sequence characteristics of RanGAP protein or its gene in test plants; If the RanGAP protein or its gene is expressed or has low activity in the test plant, it is a plant with inhibited rhizobium infection and a small number of nodules; if the RanGAP protein or its gene is expressed or has high activity in the test plant, it is a plant with high rhizobium infection and a large number of nodules. RanGAP includes RanGAP1 or RanGAP2.
13. A method for screening regulatory reagents that regulate rhizobium infection and nodulation phenotype in leguminous plants, comprising: (1) Add the candidate material to the system containing RanGAP; (2) Detect the system and observe the expression or activity of RanGAP therein. If the expression or activity of RanGAP is increased, it indicates that the candidate substance can be used to promote rhizobium infection and increase the number of nodules. If the expression or activity of RanGAP is decreased, it indicates that the candidate substance can be used to inhibit rhizobium infection and reduce the number of nodules. The RanGAP includes RanGAP1 or RanGAP2.