Nls-megrxc4 gene for promoting cassava stem adventitious root formation and application thereof
Patent Information
- Application Number
- CN202611144967.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-30
- Publication Date
- 2026-10-27
- Estimated Expiration
- 2046-07-30
AI Technical Summary
[0017]本发明将核定位信号（NLS）融合至MeGRXC4蛋白后，通过调控木薯MeGRXC4蛋白亚细胞定位以促进植物不定根发生，可显著促进茎段产生根原基，能够在木薯茎的节间和节上形成不定根，增加生根数量，提高生根效率，在木薯育苗及栽培上具有如下的应用价值：
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant molecular biology and crop genetic improvement, specifically involving an NLS-MeGRXC4 gene that promotes the formation of adventitious roots in cassava stems and its application. Background Technology
[0002] Propagation by cuttings utilizes the ability of plant vegetative organs to produce adventitious buds and roots. Part or all of the roots, stems, leaves, or buds are cut off and inserted into a substrate, where, under suitable environmental conditions, they root and sprout, thus becoming a complete and independent new plant. In crops like cassava that rely on cuttings, tissue culture, and regeneration systems for propagation, rooting ability directly affects the survival rate. Therefore, improving the rooting ability of cassava during cutting and tissue culture propagation is crucial for the planting industry. Summary of the Invention
[0003] This invention provides an NLS-MeGRXC4 gene that promotes the formation of adventitious roots in cassava stems and its application. Overexpression of the NLS-MeGRXC4 gene in cassava can promote the occurrence of adventitious roots, increase the rooting speed and rooting rate of cassava, and improve the efficiency of cassava seedling cultivation.
[0004] The technical solution of this invention is implemented as follows:
[0005] An NLS-MeGRXC4 gene that promotes the formation of adventitious roots in cassava stems, the nucleotide sequence of which is shown in SEQ ID NO: 1. The NLS-MeGRXC4 gene is obtained by fusing a nuclear localization signal (NLS) at the N-terminus of the MeGRXC4 protein, the nucleotide sequence of which is shown in SEQ ID NO: 2.
[0006] A recombinant vector containing the NLS-MeGRXC4 gene.
[0007] Furthermore, the original vector of the recombinant vector is the pCAMBIA1300-35S vector.
[0008] Host bacteria or expression cassettes containing the coding region of the NLS-MeGRXC4 gene.
[0009] The application of the NLS-MeGRXC4 gene, the recombinant vector, the host bacteria, or the expression cassette in promoting the formation of adventitious roots in cassava.
[0010] Furthermore, overexpression of the NLS-MeGRXC4 gene in cassava plants promotes the formation of adventitious roots in cassava.
[0011] Furthermore, adventitious roots form in the internodes and / or nodes of the cassava stem.
[0012] Furthermore, when the NLS-MeGRXC4 gene is located in the cell nucleus, adventitious roots are formed in both the internodes and nodes of the cassava stem.
[0013] Furthermore, when the NLS-MeGRXC4 gene is located in the cytoplasm and nucleus, adventitious roots form at the nodes of the cassava stem.
[0014] The NLS-MeGRXC4 gene, or the recombinant vector, or the host bacteria or expression cassette, are used to promote the production of root primordia in cassava stem segments, increase the number of adventitious roots on cassava stems, promote rooting of cassava stem cuttings, promote rooting of cassava tissue culture seedlings, promote cassava root development, improve cassava drought resistance, improve cassava resistance to poor soil, and / or shorten the cassava seedling cultivation cycle.
[0015] A transgenic cassava obtained by overexpressing the NLS-MeGRXC4 gene in cassava.
[0016] Beneficial effects:
[0017] This invention fuses the nuclear localization signal (NLS) into the MeGRXC4 protein, thereby regulating the subcellular localization of the cassava MeGRXC4 protein to promote adventitious root formation in plants. This significantly promotes the production of root primordia in stem segments, enabling the formation of adventitious roots at the internodes and nodes of cassava stems, increasing the number of roots and improving rooting efficiency. It has the following application value in cassava seedling cultivation and planting:
[0018] 1. Improve asexual reproduction efficiency
[0019] In crops such as cassava that rely on cutting propagation, rooting ability directly affects the survival rate. Unlike conventional mature cassava which only produces adventitious roots at the base, this experiment allows adventitious roots to grow at the stem segments and internodes, significantly increasing the rooting rate and improving the efficiency of asexual reproduction in plants.
[0020] 2. Shorten the seedling cycle
[0021] By forming root primordia at stem segments and internodes, seedlings produce a large number of adventitious roots, promoting seedling growth and development, reducing seedling time, and making it suitable for large-scale seedling production systems.
[0022] 3. Improve stress resistance and survival rate
[0023] The formation of adventitious roots helps expand the plant's root system and enhances root development. Increased root development increases the absorption area, improves the plant's ability to absorb water and nutrients, helps the plant adapt to environments such as drought and poor soil, and increases the survival rate after transplanting.
[0024] 4. Expand the utilization of materials that are difficult to root.
[0025] This method provides a novel molecular regulation approach to promote rooting in varieties or materials that are difficult to root, and has significant application potential.
[0026] 5. Applications in tissue culture and regeneration systems
[0027] It can be used to improve the efficiency of callus differentiation into roots, optimize the regeneration system, and reduce the cost of hormone use. Attached Figure Description
[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0029] Figure 1 This is a PCR electrophoresis image of the NLS-MeGRXC4 gene. Note: M is a 2000bp DNA marker.
[0030] Figure 2 This is the Southern Blot result for the NLS-MeGRXC4 gene. Note: M stands for 2000bp DNA Marker.
[0031] Figure 3 The results of RT-qPCR for the NLS-MeGRXC4 gene are shown.
[0032] Figure 4 The results are from a Western blot analysis of the NLS-MeGRXC4 gene.
[0033] Figure 5 Subcellular localization of the NLS-MeGRXC4 gene.
[0034] Figure 6 Phenotype of tissue culture seedling stem segments.
[0035] Figure 7 The phenotype of root primordia growth in potted seedlings.
[0036] Figure 8 This describes the adventitious root growth phenotype of potted seedlings. Detailed Implementation
[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments to better understand the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.
[0038] Example 1
[0039] Molecular identification of cassava genetically modified materials
[0040] The NLS-MeGRXC4 gene, with its nucleotide sequence shown in SEQ NO: 1, was obtained by fusing a nuclear localization signal (NLS) to the N-terminus of the MeGRXC4 protein. The nucleotide sequence encoding the MeGRXC4 protein is shown in SEQ NO: 2 (with a SalI restriction enzyme cleavage site at the 5' end). GTCGAC- The 3' end contains a BamHI restriction enzyme cleavage site. GGATTC- 3'). Using the pCAMBIA1300-35S vector as the backbone, the vector was constructed using homologous recombination technology.
[0041] High-fidelity DNA polymerase was used to amplify the MeGRXC4 and NLS-MeGRXC4 target fragments, as well as the linearized pCAMBIA1300-35S vector backbone. The PCR products were purified by agarose gel electrophoresis and then homologous recombination was performed using the ClonExpressII One Step Cloning Kit according to the manufacturer's instructions. The MeGRXC4 or NLS-MeGRXC4 fragment was seamlessly inserted upstream of the GFP coding sequence, maintaining the same open reading frame as GFP, thus obtaining the MeGRXC4-GFP and NLS-MeGRXC4-GFP fusion expression vectors.
[0042] The recombinant product was transformed into DH5α competent cells, and positive clones were obtained after antibiotic screening. Colony PCR and Sanger sequencing were used to identify the recombinant vector, confirming that the target fragment was correctly inserted and that there were no base mutations. The correctly sequenced recombinant plasmids were named pG1300-35S:MeGRXC4 and pG1300-35S:NLS-MeGRXC4, respectively. Subsequently, the recombinant vector was introduced into Agrobacterium LBA4404 competent cells, and after PCR verification of positive strains, it was used for cassava genetic transformation and subcellular localization analysis.
[0043] Using Agrobacterium-mediated transformation, pG1300-35S:NLS-MeGRXC4:GFP and pG1300-35S:MeGRXC4:GFP overexpression vectors were genetically transformed into cassava cv.60444 embryogenic callus. After resistance induction (CAM+10 mg / L Hyg+500 mg / L Carb), MeGRXC4 transgenic cassava resistant plants were obtained.
[0044] (1) PCR identification of transgenic cassava
[0045] A suitable amount of transgenic resistant cassava leaves were taken, and genomic DNA was extracted from the transgenic cassava using a kit (Magen Biosciences, D3018-02). After PCR amplification and electrophoresis, the products with the correct band size were sent for sequencing analysis. The results showed that the band of the MeGRXC4-OE line was approximately 700 bp, and that of the NLS-MeGRXC4-OE line was approximately 720 bp, which was consistent with the target band size. Figure 1 ).
[0046] PCR identification primers (SEQ NO: 3 and SEQ NO: 4):
[0047] primer F: 5'-GCAAGTGGATTGATGTGAT-3'
[0048] Primer R: 5'-CTTGCCGGTGGTGCAGATGAACTT-3'.
[0049] PCR reaction system:
[0050]
[0051] PCR reaction procedure:
[0052]
[0053] (2) Molecular detection of transgenic cassava using Southern Blot
[0054] After PCR amplification, the target fragment was recovered and sequenced, and the probe was labeled with DIG (Roche, 11745832910). Genomic DNA was extracted in large quantities from transgenic cassava, digested with enzymes, and then subjected to electrophoresis (40V, 12h) and membrane transfer (siphon method), and incubated overnight. The copy number of the target gene in the genome was detected using a Southern blot colorimetric kit (Roche, 11585762001).
[0055] Southern blot analysis results ( Figure 2 The results showed that specific hybridization signals were detected in all different transgenic materials. Each transgenic line exhibited a distinct band around 10 kb. Specifically, the MeGRXC4-OE line showed a clear single or near-single-copy band; while the NLS-MeGRXC4-OE-2, NLS-MeGRXC4-OE-3, and NLS-MeGRXC4-OE-5 lines showed a main band at approximately 10 kb. The differences in the number and location of bands among the different lines indicate slight differences in the integration mechanism of the exogenous gene into the genome, but all were successfully integrated into the host genome.
[0056] (3) RT-qPCR molecular detection of transgenic cassava
[0057] Based on the nucleotide sequence characteristics of the target gene and the endogenous control gene, bioinformatics design of sequence-specific amplification primers was performed using the Primer 6.0 primer design platform. RT-qPCR detection primers were designed using the Primer-BLAST tool in the NCBI database (https: / / www.ncbi.nlm.nih.gov).
[0058] Primer sequences (SEQ NO: 5 and SEQ NO: 6):
[0059] MeGRXC4-qPCR-F: 5'-ATGGATGCGGTGAACGAGTTG-3'
[0060] MeGRXC4-qPCR-R: 5'-GGCTCCAAATCCACATATGAGTGA-3'.
[0061] Reaction system: SYBR ® The Premix Ex Taq™ II Kit (Takara) was used for quantitative qPCR, and the detection instrument was the StepOne™ Real-Time PCR System (Applied Biosystems, USA). MeActin was used as the internal control gene for qPCR. Specific sample loading instructions are shown in the table below.
[0062] Real-time quantitative PCR reaction system
[0063]
[0064] RT-qPCR reaction procedure:
[0065]
[0066] The results are as follows Figure 3 As shown, compared with the wild-type line WT, the expression level of the target gene was significantly increased in the overexpression lines MeGRXC4-OE, NLS-MeGRXC4-OE-2, and NLS-MeGRXC4-OE-5. The expression levels among different lines were significantly different, indicating that different transformation events or insertion sites have an impact on gene expression. However, the expression level of NLS-MeGRXC4-OE-3 was almost zero.
[0067] (4) Western Blot molecular detection of genetically modified cassava
[0068] Fresh cassava leaves were rapidly frozen and ground in liquid nitrogen. An appropriate amount (approximately 100 mg) of powder was weighed and lysed in a pre-cooled centrifuge tube for extraction. The protein concentration of the sample was determined using the BCA method. After SDS-PAGE electrophoresis, membrane transfer, blocking, incubation with antibody (GFP), washing, and development (ECL chemiluminescence), the signal was collected.
[0069] Western blot results ( Figure 4 The results showed that specific expression signals of the target protein were detected in all transgenic lines. Clear bands were visible at approximately 39–40 kDa, consistent with the theoretical molecular weight range of the target protein. Specifically, the MeGRXC4-OE line showed a strong signal at 39 kDa, indicating successful overexpression of MeGRXC4-GFP protein; the NLS-MeGRXC4-OE-2 and NLS-MeGRXC4-OE-5 lines showed corresponding bands at 40 kDa, while the NLS-MeGRXC4-OE-3 did not show the corresponding target band, consistent with the transcriptional trend. The Ponceau S staining results below showed that the total protein loading in each lane was basically consistent, indicating good comparability of the Western blot results.
[0070] (5) Fluorescence observation
[0071] Young stem segments of MeGRXC4 overexpression transgenic seedlings with consistent growth were carefully peeled off using tweezers and placed in the center of a clean glass slide. A suitable amount of sterile distilled water was then added to keep the material moist, and a coverslip was slowly placed over it, avoiding air bubbles and mechanical damage. Immediately after preparation, fluorescence signals were observed using a Nikon laser scanning confocal microscope. The corresponding channels were set according to the excitation and emission wavelength parameters of the fluorescent protein; the excitation wavelength for GFP fluorescence was set to 488 nm, and the emission wavelength range was 500-550 nm. The fluorescence distribution of the fusion protein in the cells was detected and imaged. Bright-field and fluorescence images were simultaneously acquired during observation and overlaid for analysis to determine the localization of the target protein in the cell nucleus and cytoplasm.
[0072] Results of observation by laser confocal microscopy ( Figure 5The results showed that the MeGRXC4 protein exhibited significantly different subcellular localization patterns with and without nuclear localization signal (NLS) addition. In the MeGRXC4-OE line, the MeGRXC4 protein was mainly localized in the cytoplasm and nucleus, while in the NLS-MeGRXC4-OE-2 and NLS-MeGRXC4-OE-5 lines, the fluorescence signal was localized only in the nucleus. Furthermore, no fluorescence was detected in the NLS-MeGRXC4-OE-3 line, consistent with Western blotting results. Although NLS-MeGRXC4-OE-3 carried the same transgenic vector, its phenotype reverted to wild-type levels due to exogenous gene silencing. Therefore, it can eliminate interference from genetic transformation and tissue culture while maintaining the same transformation background. This approach is more beneficial for elucidating the effects of NLS-MeGRXC4 expression itself than directly using the wild-type as a control, and can be used as a transgenic control in subsequent phenotypic analysis experiments.
[0073] Example 2
[0074] Phenotypic analysis of adventitious root formation in tissue culture seedlings
[0075] Observation of tissue culture seedlings cultured in MS medium for 4 months revealed that NLS-MeGRXC4-OE-2 and NLS-MeGRXC4-OE-5 plants formed numerous adventitious roots at nodes and internodes, while MeGRXC4-OE and NLS-MeGRXC4-OE-3 (NC) exhibited the same phenotype as the wild type, with adventitious roots only appearing at the nodes and none at the internodes. Figure 6 Therefore, we believe that strictly confining MeGRXC4 within the nucleus leads to the growth of adventitious roots in the internodes.
[0076] Example 3
[0077] Rooting phenotype of potted seedling stem segments
[0078] MeGRXC4 transgenic seedlings grown on MS medium for 2 months were hardened off and then transplanted into pots for 3 months. It was observed that the NLS-MeGRXC4-OE-3 and MeGRXC4-OE lines did not form root primordia at the stem position, while the NLS-MeGRXC4-OE-2 and NLS-MeGRXC4-OE-5 lines showed root primordia at both nodes and internodes. Figure 7 The results were consistent with the phenotype of tissue-cultured seedlings. Adventitious roots developed approximately two weeks after stem segments with long root primordia were placed close to the soil surface. Figure 8 ).
[0079] The above results demonstrate that, in this invention, regulating the subcellular localization of the cassava MeGRXC4 protein can promote adventitious root formation in plants. Incorporating nuclear localization signals (NLS) into the MeGRXC4 protein significantly promotes the production of root primordia at both internodes and nodes, increasing the number of roots and improving rooting efficiency. This technology has production value in the following areas:
[0080] 1. Improve asexual reproduction efficiency
[0081] In crops such as cassava that rely on cutting propagation, rooting ability directly affects the survival rate. Unlike conventional mature cassava which only produces adventitious roots at the base, this experiment allows adventitious roots to grow in the internodes of stem segments, significantly increasing the rooting rate and improving the efficiency of asexual reproduction in plants.
[0082] 2. Shorten the seedling cycle
[0083] By forming root primordia at stem segments and internodes, seedlings produce a large number of adventitious roots, promoting seedling growth and development, reducing seedling time, and making it suitable for large-scale seedling production systems.
[0084] 3. Improve stress resistance and survival rate
[0085] The formation of adventitious roots helps expand the plant's root system and enhances root development. Increased root development increases the absorption area, improves the plant's ability to absorb water and nutrients, helps the plant adapt to environments such as drought and poor soil, and increases the survival rate after transplanting.
[0086] 4. Expand the utilization of materials that are difficult to root.
[0087] This method provides a novel molecular regulation approach to promote rooting in varieties or materials that are difficult to root, and has significant application potential.
[0088] 5. Applications in tissue culture and regeneration systems
[0089] It can be used to improve the efficiency of callus differentiation into roots, optimize the regeneration system, and reduce the cost of hormone use.
[0090] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.
Claims
1. The application of the NLS-MeGRXC4 gene, or a recombinant vector containing the NLS-MeGRXC4 gene, a host bacterium, or an expression cassette in promoting the formation of adventitious roots in cassava, characterized in that, Overexpression of the NLS-MeGRXC4 gene in cassava plants promotes the formation of adventitious roots in cassava. The nucleotide sequence of the NLS-MeGRXC4 gene is shown in SEQ ID NO:
1.
2. The application as described in claim 1, characterized in that, The original vector for the recombinant vector was the pCAMBIA1300-35S vector.
3. The application as described in claim 1, characterized in that, When the NLS-MeGRXC4 gene is located in the cell nucleus, adventitious roots are formed in both the internodes and nodes of the cassava stem.
4. The application as described in claim 1, characterized in that, When the NLS-MeGRXC4 gene is located in the cytoplasm and nucleus, adventitious roots form at the nodes of the cassava stem.
5. The application of the NLS-MeGRXC4 gene, or a recombinant vector, host bacterium, or expression cassette containing the NLS-MeGRXC4 gene, in promoting the production of root primordia in cassava stem segments, increasing the number of adventitious roots in cassava stems, promoting rooting of cassava stem cuttings, and / or promoting rooting of cassava tissue culture seedlings by overexpressing the NLS-MeGRXC4 gene, wherein the nucleotide sequence of the NLS-MeGRXC4 gene is shown in SEQ ID NO:
1.
6. The application as described in claim 5, characterized in that, The original vector for the recombinant vector was the pCAMBIA1300-35S vector.