A MuGA20ox1 gene and its application in regulating the efficient propagation of multi-segmented stem segments of *Hydrilla verticillata*.

Abstract

This invention belongs to the field of bio-agricultural technology, specifically relating to a MuGA20ox1 gene and its application in regulating the efficient propagation of multi-segmented stem segments of *Hydrilla verticillata*. The nucleotide sequence of the coding region of the MuGA20ox1 gene provided by this invention is shown in SEQ ID NO:2. It is a double mutant obtained by mutating asparagine at position 299 to aspartic acid and simultaneously mutating phenylalanine at position 302 to alanine, based on the wild-type GA20ox1 gene. This invention transforms *Hydrilla verticillata* with the double mutant gene using Agrobacterium-mediated transformation, resulting in transgenic plants with a significantly increased number of stem segments and a greatly improved budding rate. This effectively solves the problems of low stem segment count and low propagation efficiency in wild-type *Hydrilla verticillata*, and can be widely applied to the efficient propagation of multi-segmented stem segments of *Hydrilla verticillata*, showing promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of bio-agricultural technology, specifically relating to a MuGA20ox1 gene and its application in regulating the efficient propagation of multi-segmented stem segments of *Hydrilla verticillata*. Background Technology

[0002] *Hydrilla verticillata*, belonging to the genus *Hydrilla* in the family Hydrocharitaceae, is a monocotyledonous perennial submerged herbaceous plant with slender, erect stems, also known as "jointed grass" or "king of aquatic plants." It is widely distributed in freshwater areas such as ponds, lakes, ditches, and paddy fields, exhibiting extremely strong adaptability and reproductive capacity. Each stem segment can sprout adventitious roots and new buds, with one segment forming one plant, and broken branches can regenerate. Currently, domestic and international research on *Hydrilla verticillata* mainly focuses on its stress resistance mechanisms, water purification efficiency, and heavy metal accumulation, but research reports on its colonization and propagation capabilities are relatively few.

[0003] Gibberellins (GA) primarily regulate internode cell elongation and intercalary meristem activity in plants, promoting the elongation of existing stem nodes without altering the number of nodes. However, the response of stem nodes to GA exhibits significant species specificity among different plants. The aquatic plant *Hydrilla verticillata* is a multi-segmented tillering, stem segment vegetative propagation type of aquatic plant, whose growth core depends on stem segment differentiation and moderate internode elongation. Endogenous GA homeostasis is the core factor regulating the occurrence and propagation efficiency of its multi-segmented stem segments, and GA20ox1 is a key rate-limiting gene for gibberellin synthesis.

[0004] Site-directed mutagenesis of GA20ox1 can edit key structural domains to avoid excessive GA-induced excessive single-segment elongation and reduced effective stem segment number, resulting in an ideal plant type of *Hydrilla verticillata* with many segments, moderate internode length, robust stems, and strong tillering ability, fundamentally increasing the yield of effective multi-segment stem segments per plant. However, no such research has been reported to date, making the study of GA20ox1 site-directed mutagenesis to improve the stem segment number of *Hydrilla verticillata* of significant research and economic value. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention analyzes the GA20ox1 gene and protein, and screens for site-directed mutagenesis at N299D and F302A sites to reduce steric hindrance of the side chain, stabilize protein folding, and enhance the structural rigidity of the catalytic center, thereby increasing the number of stem nodes and budding rate of *Hydrilla verticillata*.

[0006] On one hand, the present invention provides a MuGA20ox1 gene, characterized in that the coding region sequence of the MuGA20ox1 gene is shown in SEQ ID NO:2.

[0007] Furthermore, the MuGA20ox1 gene is characterized in that it encodes a mutated GA20ox1 protein, the amino acid sequence of which is shown in SEQ ID NO:3.

[0008] Furthermore, the MuGA20ox1 gene is characterized in that it is obtained by site-directed mutagenesis based on the wild-type GA20ox1 gene shown in SEQ ID NO:1, wherein the site-directed mutagenesis involves mutating asparagine at position 299 to aspartic acid and phenylalanine at position 302 to alanine.

[0009] Also provided is a mutant GA20ox1 protein, the amino acid sequence of which is shown in SEQ ID NO:3.

[0010] Also provided is a recombinant expression vector containing the MuGA20ox1 gene described in this invention.

[0011] It also provides a host cell containing the recombinant expression vector described in this invention.

[0012] Also provided is a method for cultivating transgenic *Hydrilla verticillata*, the method comprising transforming *Hydrilla verticillata* using the recombinant expression vector described in this invention to integrate the MuGA20ox1 gene described in this invention into its genome.

[0013] Furthermore, in the method, the transformation is carried out by Agrobacterium-mediated genetic transformation.

[0014] It also provides the application of the MuGA20ox1 gene or the mutated GA20ox1 protein described in this invention in regulating the efficient propagation of multi-segmented stem segments of *Hydrilla verticillata*.

[0015] Furthermore, in the application described, the regulation manifests as increasing the number of stem nodes and / or budding rate of *Hydrilla verticillata*.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The N299D and F302A double mutant GA20ox1 protein provided by this invention significantly increased the number of stem nodes in transgenic plants after transformation into *Hydrilla verticillata*. Cutting experiments showed that the double mutant in Example 1 had a total of 3.25 and 5.75 nodes on the 7th and 14th days after cutting, respectively, while the wild-type control had only 1.45 and 2.75 nodes. The number of stem nodes in the double mutant was more than 109% higher than that in the wild type, effectively solving the problem of insufficient stem nodes in wild-type *Hydrilla verticillata*.

[0018] (2) The double mutant of this invention exhibits excellent germination rate. The germination rate reached 35% on the 5th day after cutting and 75% on the 7th day, while the germination rate of the wild-type control was only 15% and 30% at the same time. The germination rate on the 7th day was 150% higher than that of the wild type, which greatly accelerated the nutrient propagation process of *Hydrilla verticillata* and provided key technical support for large-scale and efficient production.

[0019] (3) This invention demonstrates, through a single mutant comparative experiment (Example 2 uses the N299D single mutant, and Example 3 uses the F302A single mutant), that the N299D and F302A double mutants produce a synergistic effect in promoting the increase of stem node number and budding rate. The synergistic effect of the double mutants on stem node number and budding rate is significantly higher than the sum of the synergistic effects of the two single mutants, which is an unexpected technical effect that cannot be achieved by a single site mutation. Detailed Implementation

[0020] The technical solution of the present invention will be described below with reference to the embodiments. However, the present invention is not limited to the following embodiments.

[0021] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments, but the embodiments are not intended to limit the present invention.

[0022] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0023] Example 1

[0024] The MuGA20ox1 gene undergoes N299D and F302A mutations in the wild-type GA20ox1 gene as shown in SEQ ID NO:1. The mutated amino acid sequence is shown in SEQ ID NO:3. The specific sequence is as follows:

[0025] MLVPHHPSMLIAPQNTSAENHDNMEGQKQLHHFDESLMPNQSNIPSQFIWPDHEKPCLTPPELHIPPIDLKAFLSGDPQAVSAICAEANEACKKHGFFLVVN

[0026] HGVDRKLIAQAHKLIDDFFCMQLSQKQKAQRKIGEHCGYANSFIGRFSSKLPWKETLSFHYSADKSSKSVEDYFLNVMGEDFRKFGSVFQEYCEAMSKLSLGIMELLGMTLGVGRECFRDFFEGNESVMRLNYYPPCQKP ELALGTGPHCDPTSLTILHQDQVEGLQVFVDGRWYSVAPKEDAFVVNIGDTFMALSDGLAKSCMHRAVVNNKIVRKSLAFFLCPNRDKVVTPPKDLISNENPRTYPDFTWPSLLEFTQKHYRSDTETLDAFSRWLLEKNN.

[0027] 1. Vector construction and introduction of mutations

[0028] Overlap Extension PCR

[0029] 1.1 First round of PCR:

[0030] Fragment A: Using a vector carrying the wild-type GA20ox1 gene as shown in SEQ ID NO:1 as a template, fragment A was obtained by PCR amplification using primer pairs GA20ox1-BamHI-F and MuGA20ox1-R.

[0031] Fragment B: Using a vector carrying the wild-type GA20ox1 gene as shown in SEQ ID NO:1 as a template, fragment B was obtained by PCR amplification using primer pairs MuGA20ox1-F and GA20ox1-HindIII-R.

[0032] The sequence information of the above PCR primers is shown in Table 1.

[0033] Table 1 Primer sequence information

[0034]

[0035] The first round of reaction system is shown in Table 2:

[0036] Table 2 First-round PCR reaction system

[0037]

[0038] Perform PCR according to the following procedure:

[0039] Initial denaturation stage: 98℃ pre-denaturation for 2-5 min, 1 cycle.

[0040] Cyclic amplification phase (28 cycles in total):

[0041] Denaturation: 98℃, 15s

[0042] Annealing: 55-61℃, 30s

[0043] Extension: 72℃, 15s

[0044] Final extension stage: 72℃, incubate for 5 min, store the amplification product at 4℃ for later use.

[0045] PCR amplification products were detected by agarose gel electrophoresis to confirm the amplification of single bands of the correct size. Fragment A and fragment B of the PCR amplification products were purified and recovered separately using a gel extraction kit, and the concentrations of the purified fragments A and B were determined.

[0046] 1.2 Overlap Extension PCR Fusion

[0047] The purified fragments A and B were mixed and subjected to 18 cycles of PCR without primers. Annealing and extension of the overlapping regions resulted in the full-length mutant gene. Then, external primers GA20ox1-BamHI-F and GA20ox1-HindIII-R were added for a second round of PCR amplification of the full-length product, yielding the full-length PCR product carrying the mutation site (Mu-GA20ox1).

[0048] 1.3 Enzyme digestion and ligation

[0049] The PCR product and the pCAMBIA1300 empty vector were digested with BamHI and HindIII. The mixture was ligated, transformed into E. coli, and single clones were selected for sequencing verification.

[0050] 2. Validation of mutant plasmids

[0051] Transformation and screening: The ligation product was transformed into DH5α competent Escherichia coli cells, plated on LB agar plates containing kanamycin antibiotic, and positive clones were screened.

[0052] Colony PCR verification: Select a single colony for preliminary PCR identification.

[0053] Sequencing verification: Positive colonies were sent for sequencing. Bidirectional sequencing was performed using universal primers for the vector and internal primers for the gene. The sequencing results of Mu-GA20ox1 are shown in SEQ ID NO:3. The entire coding region of the Mu-GA20ox1 gene is completely correct. In particular, the N299D and F302A sites have been successfully mutated to the target amino acids, and no other unexpected mutations have been introduced.

[0054] Select positive clones and prepare sequencing samples

[0055] Clones: Select at least 5-10 single colonies from the transformed plate and inoculate them into liquid LB medium containing the corresponding antibiotics, and culture them overnight in small batches.

[0056] Plasmid extraction: Colony plasmid DNA was extracted using a commercially available small-batch plasmid extraction kit.

[0057] Sample preparation and sequencing: Dilute the extracted plasmid to 100 ng / μL and send it to a biotechnology company for sequencing.

[0058] The plasmid is considered successfully constructed when the sequencing results meet the following two conditions:

[0059] The expected N299D (AAT->GAT) and F302A (TTC->GCT) mutations were successfully introduced; the rest of the inserted gene was completely identical to the wild-type sequence (SEQ ID NO:1) with no other nucleotide variations.

[0060] 3. Genetic transformation and mutant cultivation of *Hydrilla verticillata* using Agrobacterium-mediated genetic transformation.

[0061] Agrobacterium preparation: The recombinant plasmid that was verified to be correct by sequencing was introduced into EHA105 Agrobacterium tumefaciens competent cells using the freeze-thaw method.

[0062] Explant preparation: Cut a 3-5cm tender stem segment from the top of *Hydrilla verticillata* and make slight scratches.

[0063] Co-culture: Explants of *Hydrilla verticillata* were cultured with *Agrobacterium* strain EHA105 containing the recombinant plasmid pCAMBIA1300-Mu-GA20ox1 (OD). 600 =0.6) were co-inoculated on MS co-medium (pH 5.8) supplemented with 2 mg / L 6-BA, 30 g / L sucrose and 8 g / L agar, and cultured in the dark at 25°C for 3 days, so that Agrobacterium could integrate T-DNA containing the mutant Mu-GA20ox1 gene and selection marker into the genome of Hydrilla verticillata.

[0064] Debacterialization and selection culture: The co-cultured young stem segments of *Hydrilla verticillata* were transferred to a debacterialization medium containing temenine antibiotic to remove *Agrobacterium*, and then transferred to a differentiation medium containing 50 mg / L hygromycin screening agent for the induction and selection of resistant callus.

[0065] Plant regeneration: resistant callus tissue was transferred to budding and rooting media to induce the differentiation of buds and roots, and regenerated into complete resistant seedlings.

[0066] Transplanting and acclimatization: Transplant well-rooted tissue culture seedlings into sterilized substrate and acclimatize them under greenhouse conditions.

[0067] 4. Identification of mutant plants

[0068] RNA was extracted from mutant plants and cDNA was obtained by reverse transcription. Using the cDNA as a template, PCR amplification was performed using primer pairs GA20ox1-JD-F and GA20ox1-JD-R. The PCR amplification products were sent for sequencing. The sequencing results showed that the expected N299D (AAT->GAT) and F302A (TTC->GCT) mutations were successfully introduced. The rest of the inserted gene was completely consistent with the wild-type sequence (SEQ ID NO:1).

[0069] Western Blot analysis: Plant protein samples were prepared, and the expression and size of the Mu-GA20ox1 mutant protein were detected using specific antibodies.

[0070] Example 2

[0071] The N299D gene mutation was performed on the wild-type GA20ox1 gene as shown in SEQ ID NO:1, and the mutant cultivation process is as described in Example 1.

[0072] Example 3

[0073] The F302A gene mutation was performed on the wild-type GA20ox1 gene as shown in SEQ ID NO:1. The mutant cultivation process is as described in Example 1.

[0074] Comparative Example: Wild-type *Hydrilla verticillata* from the same experimental field without mutant cultivation.

[0075] Test case

[0076] This test case uses mutant plants prepared in Examples 1-3 and wild-type *Hydrilla verticillata* from the comparative examples to evaluate the number of stem segments and propagation ability of different types of plants.

[0077] The mutant plants prepared in Examples 1-3 and the wild-type *Hydrilla verticillata* of the comparative examples were selected. The selected plants were uniformly growing, free from pests and diseases, and undamaged. Healthy stem segments from the 2nd-3rd node downwards from the apex were chosen, with a uniform length of 4 cm and one complete stem node retained. Lateral buds, adventitious roots, and residual leaves were removed to ensure no significant difference in the initial phenotypic characteristics of the materials. Each group consisted of 15-20 plants.

[0078] Culture container: a uniformly sized sterile glass tank with a 5cm thick layer of sterile quartz sand as the substrate; the culture water is 1 / 2 Hoagland nutrient solution, with a water depth of 12cm, and cultured in a sterile environment.

[0079] Environmental parameters: temperature 25±1℃, light cycle 16h light / 8h darkness, 1 / 2 volume of sterile nutrient solution replaced every 3 days, maintaining stable dissolved oxygen and pH in the water, and free from pests and diseases throughout the process.

[0080] During the trial, all procedures were handled under routine management, with consistent management across all treatment groups.

[0081] The total number of nodes in each group was observed on the 7th and 14th day after cutting. The number of all complete and visible stem nodes on the main stem was counted, excluding the number of lateral branch nodes. The test results are shown in Table 3.

[0082] Table 3. Statistical results of the number of stem nodes after stem segment propagation of different plants.

[0083]

[0084] Note: ** The differences between the representative and the wild-type plants in the comparative examples were extremely significant.

[0085] The germination rate was calculated on the 5th and 7th day after cutting. Germination rate (%) = number of germinating plants / total number of plants in each group × 100. The test results are shown in Table 4.

[0086] Table 3 shows that Example 1 is a mutant of N299D and F302A sites, Example 2 is an N299D mutant, and Example 3 is an F302A mutant. From the total number of stem nodes on day 7 and day 14 after cutting in Example 3 and the comparative example, it can be seen that the F302A mutation has no significant effect on the increase in the number of stem nodes. Example 2, being an N299D mutant, can increase the number of stem nodes compared to the comparative example, but the increase is small. Example 1, being a mutant of N299D and F302A sites, shows a more than 100% increase in the number of stem nodes compared to the comparative example, suggesting that the N299D and F302A sites synergistically enhance the number of stem nodes.

[0087] Table 4. Statistical results of budding rate after stem cuttings of different plants

[0088]

[0089] Note: * The representative wild-type plants differed significantly from the comparative examples; ** The differences between the representative and the wild-type plants in the comparative examples were extremely significant.

[0090] Table 4 shows that Example 1 is a mutant of N299D and F302A sites, Example 2 is a mutant of N299D, and Example 3 is a mutant of F302A. From the germination rates of Example 3 and the comparative example on the 5th and 7th day after cutting, it can be seen that the F302A mutation significantly improves the germination rate, but its increase is much lower than that of Example 1 with the mutant of N299D and F302A sites. This indicates that the N299D and F302A mutants also have a synergistic effect in promoting germination. This suggests that the synergistic mutation of N299D and F302A optimizes endogenous gibberellin homeostasis and can be applied to the efficient propagation of multi-segmented stem segments of *Hydrilla verticillata*.

[0091] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A MuGA20ox1 gene, characterized in that, The coding region sequence of the MuGA20ox1 gene is shown in SEQ ID NO:

2.

2. The MuGA20ox1 gene according to claim 1, characterized in that, The MuGA20ox1 gene encodes a mutated GA20ox1 protein, the amino acid sequence of which is shown in SEQ ID NO:

3.

3. The MuGA20ox1 gene according to claim 1, characterized in that, The MuGA20ox1 gene was obtained by site-directed mutagenesis based on the wild-type GA20ox1 gene shown in SEQ ID NO:

1. The site-directed mutagenesis involved mutating asparagine at position 299 to aspartic acid and phenylalanine at position 302 to alanine.

4. A mutant GA20ox1 protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

3.

5. A recombinant expression vector comprising the MuGA20ox1 gene as described in claim 1.

6. A host cell comprising the recombinant expression vector of claim 5.

7. A method for cultivating transgenic *Hydrilla verticillata*, characterized in that, This includes transforming *Hydrilla verticillata* using the recombinant expression vector of claim 5 to integrate the MuGA20ox1 gene of claim 1 into its genome.

8. The method according to claim 7, characterized in that, The transformation was carried out using Agrobacterium-mediated genetic transformation.

9. The application of the MuGA20ox1 gene of claim 1 or the mutated GA20ox1 protein of claim 4 in regulating the efficient propagation of multi-segmented stem segments of *Hydrilla verticillata*.

10. The application according to claim 9, characterized in that, The regulation is manifested in increasing the number of stem nodes and / or budding rate of *Hydrilla verticillata*.

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