Application of walnut JrGA20ox gene in improving drought resistance of walnut
By regulating or silencing the expression of the walnut JrGA20ox gene, the problems of long breeding cycles and insufficient drought resistance in walnuts have been solved, suitable walnut germplasm resources have been cultivated, and the drought resistance and industrial efficiency of walnuts under arid conditions have been improved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG FORESTRY UNIVERSITY
- Filing Date
- 2026-04-29
- Publication Date
- 2026-05-29
AI Technical Summary
Walnut breeding has a long cycle and low efficiency. Under drought conditions, walnut yields decrease and quality deteriorates. Existing technologies are insufficient to effectively improve its drought resistance.
By constructing transgenic plants that overexpress or silence the walnut JrGA20ox gene, the gene can be used to regulate the gibberellin synthesis pathway, affecting plant height and drought resistance, thereby cultivating standard or semi-dwarf plants and enhancing or reducing drought resistance.
This has enabled targeted improvement of walnut traits, cultivated walnut germplasm resources with suitable plant type and strong stress resistance, improved the production efficiency and resource utilization efficiency of the walnut industry, and especially enhanced drought resistance under arid conditions.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular biology breeding technology, and particularly relates to walnuts. JrGA20ox Application of genes in improving the drought resistance of walnuts. Background Technology
[0002] Walnut( Juglans regia Walnut (Juglans regia) is a deciduous tree belonging to the genus Juglans of the family Juglandaceae. It is one of the important woody oilseed tree species, possessing high nutritional and economic value, and is known as the "King of Woody Oilseeds." my country is one of the major producers of walnuts, ranking among the world's top in both cultivation area and yield. With the walnut processing industry developing towards refinement, market demand for walnuts and their deep-processed products continues to grow, and its industrial value is constantly increasing.
[0003] However, the development of the walnut industry still faces many constraints. On the one hand, walnut trees are tall and mostly planted in mountainous or hilly areas, making field management and fruit harvesting difficult. On the other hand, walnuts are quite sensitive to environmental conditions, especially under abiotic stresses such as drought, which can easily lead to growth restriction, yield decline, and quality deterioration, thus limiting the further expansion of their suitable growing area and industry scale. In addition, walnut breeding mainly relies on traditional hybridization and selection methods, which suffer from long breeding cycles and low efficiency. Summary of the Invention
[0004] The purpose of this invention is to provide walnuts JrGA20ox The application of genes in improving the drought resistance of walnuts aims to solve the problems of long breeding cycles and low efficiency in the selection and breeding of high-quality walnut germplasm resources in existing technologies, as well as the reduction in walnut yield and quality deterioration under drought conditions.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides walnuts JrGA20ox The application of genes in regulating plant height and / or drought resistance, in the walnut JrGA20ox The nucleotide sequence of the gene is shown in SEQ ID NO.1.
[0006] This invention also provides an amplification of walnuts JrGA20ox The primer pairs for the gene, wherein the nucleotide sequences of the primer pairs are shown in SEQ ID NO.3-4; The walnut JrGA20ox The nucleotide sequence of the gene is shown in SEQ ID NO.1.
[0007] This invention also provides an overexpression of walnut JrGA20ox Methods for constructing transgenic plants from walnut trees. JrGA20oxGenes were ligated into an empty vector to obtain an overexpression recombinant plasmid. The overexpression recombinant plasmid was then transformed into bacteria from the empty vector to obtain overexpression recombinant bacteria. The overexpression recombinant bacteria were then transformed into plant somatic embryos to obtain overexpression walnuts. JrGA20ox Genetically modified plants.
[0008] Preferably, the overexpression recombinant plasmid uses pCAMBIA1300 as the empty vector and walnut as the target vector. JrGA20ox The gene is an inserted gene; The walnut JrGA20ox The nucleotide sequence of the gene is shown in SEQ ID NO.1.
[0009] Preferably, the overexpression recombinant bacteria includes the overexpression recombinant plasmid and the empty vector bacteria; The empty carrier bacteria is Agrobacterium tumefaciens.
[0010] This invention also provides the application of the aforementioned construction method in cultivating standard-sized plants and / or constructing drought-sensitive model plants.
[0011] This invention also provides a silent walnut. JrGA20ox Methods for constructing gene-edited plants targeting walnuts JrGA20ox Injecting antisense oligonucleotide combinations of genes into plant somatic embryos yields silent walnuts. JrGA20ox Gene-edited plants.
[0012] Preferably, the nucleotide sequence of the antisense oligonucleotide combination is shown in SEQ ID NO.5-8.
[0013] Preferably, the concentration of the antisense oligonucleotide combination is 5–15 μmol / L.
[0014] The present invention also provides the application of the described construction method in the cultivation of semi-dwarf plants and / or in improving the drought resistance of plants.
[0015] The present invention has the following technical effects and advantages: This invention clarifies the effects of drought stress treatment and gene expression analysis on walnuts. JrGA20ox Genes are key targets for regulating plant height and drought resistance, influencing these traits by regulating gibberellin synthesis pathways. Negative regulation reduces gibberellin content, leading to dwarfing or enhanced drought resistance; positive regulation increases gibberellin content, which can be used to construct tall plant types or drought-sensitive models. This provides new targets and methods for molecular breeding of walnuts, and is of great significance in achieving targeted improvement of walnut traits, cultivating walnut germplasm resources or new varieties with suitable plant types and strong stress resistance, especially in creating new drought-resistant walnut germplasm and improving the production efficiency and resource utilization efficiency of the walnut industry. Attached Figure Description
[0016] Figure 1 For walnuts JrGA20ox Positive validation results of gene overexpression lines; Figure 2 For walnuts JrGA20ox Positive verification results of gene-edited strains and schematic diagram of target knockout base sites; Figure 3 For walnuts JrGA20ox Results of gene regulation of walnut plant height, where A represents the phenotypic results of each line, B represents the plant height of each line, and C represents the internode length of each line. Figure 4 For walnuts JrGA20ox Phenotypic results of gene regulation of plant drought resistance; Figure 5 The results of index determination for each plant strain are shown below. A represents the POD activity determination result, B represents the SOD activity determination result, C represents the CAT activity determination result, D represents the proline content determination result, E represents the superoxide anion free radical content determination result, F represents the hydrogen peroxide content determination result, and G represents the malondialdehyde content determination result. Detailed Implementation
[0017] This invention provides walnuts JrGA20ox The application of genes in regulating plant height and / or drought resistance, in the walnut JrGA20ox The nucleotide sequence of the gene is shown in SEQ ID NO.1.
[0018] This invention also provides walnuts JrGA20ox The application of genes in regulating gibberellin content in plants, specifically in walnuts. JrGA20ox The nucleotide sequence of the gene is shown in SEQ ID NO.1.
[0019] In this invention, the plant is preferably a walnut.
[0020] This invention also provides an amplification of walnuts JrGA20ox The primer pairs for the gene, wherein the nucleotide sequences of the primer pairs are shown in SEQ ID NO.3-4; The walnut JrGA20ox The nucleotide sequence of the gene is shown in SEQ ID NO.1.
[0021] This invention also provides an overexpression of walnut JrGA20ox Methods for constructing transgenic plants from walnut trees. JrGA20ox Genes were ligated into an empty vector to obtain an overexpression recombinant plasmid. The overexpression recombinant plasmid was then transformed into bacteria from the empty vector to obtain overexpression recombinant bacteria. The overexpression recombinant bacteria were then transformed into plant somatic embryos to obtain overexpression walnuts. JrGA20ox Genetically modified plants.
[0022] In this invention, the overexpression recombinant plasmid uses pCAMBIA1300 as the empty vector and walnut as the target vector. JrGA20ox The gene is an inserted gene; The walnut JrGA20ox The nucleotide sequence of the gene is shown in SEQ ID NO.1.
[0023] In this invention, the overexpression recombinant bacteria include the overexpression recombinant plasmid and the empty vector bacteria; The empty carrier bacteria is Agrobacterium tumefaciens, preferably Agrobacterium GV3101 strain.
[0024] This invention also provides the application of the aforementioned construction method in cultivating standard-sized plants and / or constructing drought-sensitive model plants.
[0025] This invention also provides a silent walnut. JrGA20ox Methods for constructing gene-edited plants targeting walnuts JrGA20ox Injecting antisense oligonucleotide combinations of genes into plant somatic embryos yields silent walnuts. JrGA20ox Gene-edited plants.
[0026] In this invention, the nucleotide sequences of the antisense oligonucleotide combinations are shown in SEQ ID NO.5-8.
[0027] In this invention, the concentration of the antisense oligonucleotide combination is 5–15 μmol / L, preferably 10 μmol / L.
[0028] The present invention also provides the application of the described construction method in the cultivation of semi-dwarf plants and / or in improving the drought resistance of plants.
[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0030] In this invention, the test plants are tissue culture seedlings of the "Kiyo Walnut" variety and walnut somatic embryos from Zhejiang Agriculture and Forestry University. The walnut somatic embryos are differentiated from a complete walnut somatic embryo and cultured at 25±2℃.
[0031] Example 1: Walnut JrGA20ox Construction of gene overexpression vectors RNA was extracted from well-developed walnut somatic embryos and reverse transcribed into walnut cDNA for use in the walnut genome website ( Juglans Walnuts obtained from Genome-Genome Projects JrGA20oxPrimers were designed using the gene CDS sequence as a template (as shown in Table 1). PCR amplification was performed using walnut cDNA as a template. The amplification system consisted of 25 μL of the mixture, including 12.5 μL of 2×PrimeSTAR Max high-fidelity enzyme premix and 1 μL of walnut cDNA. JrGA20ox -F 1μL, JrGA20ox -R 1μL and the remainder water, the amplification program is: 94℃ pre-denaturation for 2 min → (98℃ denaturation for 10 s → 55℃ annealing for 30 s → 72℃ extension for 20 s) × 35 cycles → 72℃ final extension for 5 min, to obtain walnut JrGA20ox Gene; after ligation with the empty vector pCAMBIA1300, transformation into E. coli DH5α competent cells, single-clone colony culture, and sequencing, plasmid was extracted to obtain the overexpression recombinant plasmid pCAMBIA1300- JrGA20ox -GFP.
[0032] Walnut JrGA20ox The nucleotide sequence of the gene is shown in SEQ ID NO.1.
[0033] SEQ ID NO.1: Walnut JrGA20ox The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.12.
[0034] SEQ ID NO.2: MAIDCITSIPSMPAHPPKVDQRTEQKDLVFDASVLRYQPNIPKQFIWPDEEKPNAPELSVPLVDLGGFLSGDPAAAATEASRLVGEACRKHGFFLVVNHGVDDNLIADAHGCMDYFFGLPLSTKQRAQRKIGEHCGYASSFTGRFSSKLPWKETLSFRYSADHTSSHVVREYLCNTMGPDFEQFGRVYQ DYCEAMNTLSLGIMELLGMSLGVNRAHFRGYFEENDSIMRLNYYPPCQKPDLTLGTGPHCDPTSLTILHQDQVGGLQVFVDEWRSIIPNFNAFVVNIGDTFMALSNGRYKSCLHRAVVNSKSPRKSLAFFLCPRNDKVVSPPSELVDSTVCPRIYPDFTWSMLLEFTQKHYRADMNTLEVFSNWLQQKNS Table 1 Expanding walnut JrGA20ox Primer sequences of genes
[0035] Example 2: Walnut JrGA20ox Gene knockout vector construction The walnuts obtained in Example 1 JrGA20ox The gene CDS sequence was submitted to the CRISPR PLANT website (http: / / www.genome.arizona.edu / crispr / CRISPRsearch.html) for target screening, and then off-target effects were evaluated on the Cas-OFFinder website (http: / / www.rgenome.net / cas-offinder / ). The sequence was then sent to Beijing Qingke Biotechnology Co., Ltd. for sequence synthesis, resulting in an antisense oligonucleotide combination (as shown in Table 2). The combination was dissolved and diluted with ultrapure water to 10 μmol / L, which included the first antisense oligonucleotide (as shown in SEQ ID NO. 5-6) and the second antisense oligonucleotide (as shown in SEQ ID NO. 7-8).
[0036] Table 2 Antisense Oligonucleotide Sequences
[0037] Four-primer PCR amplification was performed using pCBC-DT1T2 plasmid diluted 100-fold as a template. The amplification system was 50 μL, comprising 25 μL of 2×Prime STAR Max high-fidelity enzyme premix, 1 μL of pCBC-DT1T2, 10 μL of DT1-BsF, 1 μL of DT1-F0, 1 μL of DT2-R0, 10 μL of DT2-BsR, and the remainder water. The amplification program was: 94℃ pre-denaturation for 2 min → (98℃ denaturation for 10 s → 55℃ annealing for 30 s → 72℃ extension for 20 s) × 35 cycles → 72℃ final extension for 5 min to obtain the amplified product. The amplified product was ligated and transformed with the empty vector pHEC401 to obtain the gene editing vector pHEC401-. JrGA20ox DT-Cas9.
[0038] Example 3: Construction of transgenic lines and gene-edited lines (1) The overexpression recombinant plasmid pCAMBIA1300- obtained in Example 1 was used. JrGA20ox -GFP was transformed into Agrobacterium GV3101 competent cells to obtain overexpressed recombinant bacteria, which were cultured at 28℃ and 200 rpm for 16 h until OD. 600nm =0.6, genetically transformed into walnut somatic embryos, and identified by fluorescence detection and PCR gel electrophoresis (primers used were...). JrGA20ox -F / GFP-R), to obtain overexpressed walnut JrGA20ox Transgenic cotyledonary embryos; (2) The gene editing vector pHEC401- obtained in Example 2 was used. JrGA20ox DT-Cas9 was transformed into Agrobacterium GV3101 competent cells to obtain gene-silenced recombinant bacteria, which were then transformed into walnut somatic embryos according to step (1). The embryos were identified by fluorescence detection and PCR gel electrophoresis (using primers [missing information]). JrGA20ox - Cas9F / R), obtained the Silent Walnut JrGA20ox Gene editing of cotyledon embryos; The primer sequences required for PCR gel electrophoresis identification are shown in Table 3.
[0039] Table 3 Primer sequences required for PCR gel electrophoresis identification
[0040] Experimental Example 1: Phenotypic Analysis of Transgenic and Gene-Edited Lines Successfully identified transgenic cotyledonary embryos and gene-edited cotyledonary embryos were transferred to light culture and germinated into regenerated plants. Phenotypic characteristics, plant height, and internode length were measured in walnut wild-type lines (WT), overexpression lines (OE), and gene-edited lines (M1–M7) with consistent growth. Plants from each line were cut 2cm from the terminal bud downwards (from the terminal bud to the 4th-6th leaf) and placed in DKW solid medium containing 5% PEG-8000 to simulate drought stress for 2 weeks. DKW solid medium without 5% PEG-8000 served as a control. Leaves from each line at 0d, 7d, and 14d under drought stress were collected for qRT-PCR quantitative analysis. The results are as follows: Figures 1-4 As shown.
[0041] The results showed that, compared with WT, the OE lines had an average plant height increase of 46.5% and internode length increase of 74.4%. The plant height of the M1-M7 lines was only 74%, 72%, 74%, 64%, 61%, 73%, and 83% of that of WT, respectively, and the internode length was 67%, 66%, 70%, 52%, 50%, 0.67%, and 73% of that of WT, respectively. This indicates that compared with WT, walnut... JrGA20ox The overexpression of the gene resulted in taller plants with longer internodes, classifying them as standard-sized lines; walnut JrGA20ox Gene-edited lines are shorter and have shorter internodes, making them semi-dwarf lines; Both the WT and OE lines showed a gradual yellowing of leaves in the culture medium with prolonged treatment, and wilting and leaf drop began after 14 days of stress. Compared to the WT line, the OE line exhibited wilting earlier. The gene-edited lines showed good growth, especially the M4 and M5 lines, whose leaves remained green after 14 days of treatment, indicating that the walnut... JrGA20ox The gene-edited lines showed significantly improved drought resistance. Therefore, the WT, OE, M4, and M5 lines were selected for subsequent index determination.
[0042] Experimental Example 2: Determination of Indicators for Transgenic and Gene-Edited Lines Leaves from the WT, OE, M4, and M5 lines after drought stress treatment in Experiment Example 1 were used to determine SOD activity, POD activity, CAT activity, proline content, malondialdehyde (MDA) content, hydrogen peroxide (H2O2) content, and superoxide anion radical (O2) content. - ) content, results as follows Figure 5 As shown.
[0043] The results showed that before drought stress, the activities of SOD, POD, and CAT, as well as the levels of H2O2 and O2, were different among the various lines. - There were no significant differences in the contents of SOD, POD, and proline among the different lines. After drought stress, compared with WT, the activities of SOD, POD, and CAT and the content of proline in the M4 and M5 lines increased significantly, while those in the OE line decreased significantly. Meanwhile, the contents of H2O2 and O2... - The MDA content, on the other hand, shows the opposite trend. This indicates that overexpressed walnuts... JrGA20oxThe gene significantly increases the accumulation of ROS in walnut leaves, thereby reducing the walnut's ability to resist drought, while knocking out walnuts... JrGA20ox Genes can reduce the accumulation of ROS in leaves, thereby improving the walnut's ability to resist drought.
[0044] As can be seen from the above embodiments, the present invention provides walnut JrGA20ox Application of genes in improving drought resistance in walnuts. The walnut of this invention... JrGA20ox Genes are key targets for regulating plant height and drought resistance. They can affect plant height and drought resistance by regulating gibberellin synthesis pathways, thus providing new targets and methods for molecular breeding of walnuts. This is of great significance for achieving targeted improvement of walnut traits, cultivating walnut germplasm resources or new varieties with suitable plant type and strong stress resistance, especially in creating new drought-resistant walnut germplasm and improving the production efficiency and resource utilization efficiency of the walnut industry.
[0045] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. Walnuts JrGA20ox The application of genes in regulating plant height and / or drought resistance is characterized by, The walnut JrGA20ox The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
2. Expand walnut JrGA20ox A primer pair for a gene, characterized in that, The nucleotide sequences of the primer pairs are shown in SEQ ID NO. 3-4; The walnut JrGA20ox The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
3. A method for overexpressing walnut JrGA20ox A method for constructing transgenic plants containing genes, characterized in that, Walnuts JrGA20ox Genes were ligated into an empty vector to obtain an overexpression recombinant plasmid. The overexpression recombinant plasmid was then transformed into bacteria from the empty vector to obtain overexpression recombinant bacteria. The overexpression recombinant bacteria were then transformed into plant somatic embryos to obtain overexpression walnuts. JrGA20ox Genetically modified plants.
4. The construction method according to claim 3, characterized in that, The overexpression recombinant plasmid used pCAMBIA1300 as the empty vector and walnut as the target vector. JrGA20ox The gene is an inserted gene; The walnut JrGA20ox The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
5. The construction method according to claim 4, characterized in that, The overexpression recombinant bacteria include the overexpression recombinant plasmid as described in claim 3 and the empty vector bacteria; The empty carrier bacteria is Agrobacterium tumefaciens.
6. The application of the construction method according to any one of claims 3 to 5 in the cultivation of standard plants and / or the construction of drought-sensitive model plants.
7. A Silent Walnut JrGA20ox A method for constructing gene-edited plants, characterized in that, Target walnuts JrGA20ox Injecting antisense oligonucleotide combinations of genes into plant somatic embryos yields silent walnuts. JrGA20ox Gene-edited plants.
8. The construction method according to claim 7, characterized in that, The nucleotide sequences of the antisense oligonucleotide combinations are shown in SEQ ID NO.5-8.
9. The construction method according to claim 8, characterized in that, The concentration of the antisense oligonucleotide combination is 5–15 μmol / L.
10. The application of the construction method according to any one of claims 7 to 9 in the cultivation of semi-dwarf plants and / or the improvement of plant drought resistance.