Application of lncRNA12385 in inhibiting aquatic invasive growth of alligator weed

Abstract

The application belongs to the technical field of biological prevention and control of invasive plants, and particularly relates to application of lncRNA12385 in inhibiting water-invasive growth of alligator weed. The application screens a key lncRNA for regulating waterlogging adaptability of alligator weed, and establishes a TRV-VIGS system based on the lncRNA to inhibit water reproduction of alligator weed. LncRNA transcriptome sequencing proves that lncRNA12385 is specifically induced and up-regulated under waterlogging stress of alligator weed. A recombinant virus silencing vector pTRV2-lncRNA12385 is constructed, and endogenous lncRNA12385 can be efficiently knocked down by agrobacterium-mediated infection of plants. Waterlogging phenotype test shows that knocking down lncRNA12385 can significantly inhibit elongation of apical stem nodes of alligator weed, and greatly weaken waterlogging tolerance and asexual reproduction invasive potential of alligator weed. The application can be used for basic analysis of waterlogging adaptation regulation mechanism of invasive plants, and can provide a core target for development of low-off-target and environment-friendly RNA biological pesticides.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology for invasive plants, specifically involving the functional identification of plant long non-coding RNAs (lncRNAs) and the application of virus-induced gene silencing (VIGS), and particularly the application of lncRNA12385 in inhibiting the aquatic invasive growth of Alternanthera philoxeroides. Background Technology

[0002] Alternanthera philoxeroides, native to South America, is one of the major invasive alien plants listed in my country. It has widely invaded habitats such as rivers, paddy fields, wetlands, and landscape water bodies, causing serious harm to my country's aquatic ecosystems and agricultural production. The plant's core invasive advantage lies in its unique Low-Oxygen Escape Strategy (LOES): when completely submerged, its stem segments rapidly elongate, allowing the terminal leaves to quickly extend above the water surface for gas exchange, thus tolerating prolonged underwater low-oxygen environments. Simultaneously, it relies on stem segments for highly efficient asexual reproduction, enabling rapid dispersal and population reconstruction within a short period.

[0003] Currently, the main control methods for Alternanthera philoxeroides include physical removal, chemical weeding, biological control, and RNA interference, but all have limitations to varying degrees. Physical removal methods (such as manual harvesting and mechanical cutting) can only remove the above-ground parts; the stem segments remaining in the water or soil are very easy to regrow, resulting in a high recurrence rate and huge costs associated with repeated operations. Regarding chemical control, broad-spectrum herbicides, such as glyphosate, have some effect, but they easily leave residues in water and accumulate in the soil, causing non-target toxicity to native economic plants and aquatic plants such as rice, reeds, and cattails. Long-term application may also induce herbicide resistance in weeds, further exacerbating the difficulty of control. In terms of biological control, the introduced flea beetle (Agasicles hygrophila) is currently a widely used natural enemy insect, but its weed control effect is significantly limited by temperature and geographical conditions. Its activity decreases significantly in low-temperature areas or during seasonal low-temperature periods, making it difficult to achieve long-term, comprehensive control. In recent years, RNA interference control strategies, such as the silencing system targeting the conserved miR162, have shown inhibitory effects under laboratory conditions. However, miR162 is highly conserved in rice, soybeans, and various aquatic plants. When applied in the field, it is prone to off-target effects due to sequence homology, causing unexpected silencing damage to non-target native plants. This poses a significant risk to ecological safety and is not yet ready for practical application.

[0004] Long non-coding RNAs (lncRNAs) are a class of non-coding RNA molecules with transcripts longer than 200 nucleotides and lacking complete protein-coding open reading frames. Recent studies have shown that lncRNAs play crucial roles in plant growth, reproduction, metabolism, and responses to biotic and abiotic stresses. Existing transcriptomic studies have confirmed that a large number of lncRNAs are differentially expressed under waterlogging stress in Alternanthera philoxeroides. However, the specific lncRNAs that directly mediate waterlogging-induced stem elongation and support its aquatic invasion capabilities remain unclear in functional identification reports. Furthermore, gene silencing control systems based on specific lncRNA targets are still lacking in Alternanthera philoxeroides.

[0005] Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) technology has been proven to achieve efficient and stable knockdown of target genes in Alternanthera philoxeroides. It has advantages such as simple operation, short experimental cycle, and no need for a stable transgenic system, and is a mature tool for conducting functional analysis of stress-related genes and verification of control targets in this species. Summary of the Invention

[0006] This invention addresses the shortcomings of existing control technologies for Alternaria alternifolia and the lack of functional targets for lncRNAs, providing the following solutions:

[0007] We have provided a key molecular target, lncRNA12385, that mediates the waterlogging-adaptive growth of Alternanthera philoxeroides. This lncRNA is specifically and significantly upregulated only when Alternanthera philoxeroides is subjected to waterlogging stress, and its function is directly associated with the stem elongation response to hypoxia, filling a research gap in the mechanism of lncRNA regulation of waterlogging tolerance in invasive plants.

[0008] A TRV-VIGS recombinant silencing vector pTRV2-lncRNA12385 targeting lncRNA12385 was constructed, along with a pTRV1 helper vector. Through Agrobacterium-mediated infection, endogenous lncRNA12385 was efficiently knocked down in Alternanthera philoxeroides plants, with a clear and reproducible silencing effect.

[0009] This invention first screened and obtained the key lncRNA that regulates the waterlogging adaptation of Alternanthera philoxeroides, namely lncRNA12385;

[0010] A method based on the TRV-VIGS system to target this lncRNA and inhibit the aquatic reproduction of Alternanthera philoxeroides was established. This invention confirmed through lncRNA transcriptome sequencing that lncRNA12385 is specifically upregulated under waterlogging stress in Alternanthera philoxeroides. Therefore:

[0011] This invention provides the application of lncRNA12385 as a molecular target in the preparation of silencing vectors or nucleic acid control agents for inhibiting the waterlogged adaptive growth of Alternanthera philoxeroides and blocking its aquatic asexual reproduction. The sequence of lncRNA12385 is shown in SEQ.ID.NO1.

[0012] Transcriptome sequencing analysis of this invention confirmed that lncRNA12385 showed a significant upregulation after the young stem tissues of Alternanthera philoxeroides were subjected to complete water submersion stress for 3 hours.

[0013] This invention significantly inhibits waterlogged-induced stem elongation by knocking down endogenous lncRNA12385 expression using the TRV-VIGS system, preventing the plant from escaping the underwater hypoxic environment through elongation growth and thus losing its ability to survive underwater.

[0014] In this invention, lncRNA12385 is a specific long non-coding RNA derived from Alternanthera philoxeroides. Its transcript length is greater than 200 nt, it does not have a complete protein-coding open reading frame, and it is specifically activated and expressed only under waterlogging stress conditions.

[0015] This invention also relates to a TRV-VIGS recombinant silencing vector targeting lncRNA12385, wherein the recombinant silencing vector uses pTRV2 as a backbone vector and inserts a specific targeting fragment of lncRNA12385 therein; the recombinant silencing vector is named pTRV2-lncRNA12385; the auxiliary vector used in conjunction with this vector system is pTRV1.

[0016] The present invention also relates to a method for constructing the recombinant silencing vector, comprising the following steps:

[0017] (1) Using the cDNA of young stems of Alternanthera philoxeroides treated with water as a template, a specific target fragment of lncRNA12385 was obtained by PCR amplification;

[0018] (2) The obtained amplified fragment was ligated with the pTRV2 vector, transformed into competent Escherichia coli cells, and screened to obtain the positive recombinant plasmid pTRV2-lncRNA12385;

[0019] (3) The recombinant plasmid pTRV2-lncRNA12385 and the helper plasmid pTRV1 were transformed into Agrobacterium GV3101 competent cells to obtain strains for infection.

[0020] This invention relates to a VIGS silencing method for inhibiting the growth of Alternanthera philoxeroides under flooded conditions. Specifically, it involves using a mixture of Agrobacterium-mediated recombinant silencing vector pTRV2-lncRNA12385 and helper vector pTRV1 to infect Alternanthera philoxeroides seedlings. The specific operational steps are as follows:

[0021] (1) Agrobacterium carrying pTRV1 and pTRV2-lncRNA12385 were cultured separately, and the bacterial concentration was adjusted to OD. 600 =0.5, and mix them in an equal volume ratio to prepare a mixed infection solution;

[0022] (2) Using a needleless syringe, the mixed infection solution was applied to the lower epidermal tissue of the leaves of Alternanthera philoxeroides seedlings;

[0023] (3) After infection, the plants were placed in a dark and moist environment for 48 hours, and then transferred to a completely submerged environment for 10 days.

[0024] (4) The expression of endogenous lncRNA12385 is silenced through the above-mentioned infection, thereby effectively inhibiting the stem elongation induced by water flooding and blocking the adaptive growth process of Alternanthera philoxeroides in water.

[0025] During the infection process with the mixed infection solution, the lower epidermis of each seedling leaf is soaked with an area of ​​1-2 cm², and the amount of infection solution used is ~1.0 mL / seedling.

[0026] The light-protected and moisture-humidified culture conditions are: temperature 22-25°C, relative humidity 80%-90%, and dark culture; the completely submerged culture conditions are: water temperature 22-26°C, and water depth submerging the top of the plant 3-5 cm.

[0027] In the above method, plants infected with the pTRV2 empty vector were set up as a negative control group to evaluate the specificity and effectiveness of the silencing effect; at the same time, wild-type plants without any infection treatment were set up as a blank control group to assess the basic growth phenotype of plants under flooding conditions.

[0028] The above methods can effectively knock down the expression of endogenous lncRNA12385, significantly inhibit waterlogging-induced stem elongation, and cause the plant to lose its ability to escape and survive underwater.

[0029] In summary, this invention provides a complete operational system for inhibiting the water-flood-adaptive growth of Alternanthera philoxeroides using the aforementioned silencing vector, encompassing engineered bacteria preparation, infection procedures, light-protected and moisture-controlled culture, and evaluation of the complete water-flood phenotype. Experiments have demonstrated that knockdown of lncRNA12385 significantly inhibits water-flood-induced stem elongation, preventing the plant from extending above the water surface to complete gas exchange, thereby effectively blocking its aquatic asexual reproduction and invasive spread capabilities.

[0030] Compared to traditional miRNA-targeting control strategies, lncRNA12385, the subject of this invention, is a specific molecular target of Alternanthera philoxeroides. It has no homologous conserved sequence in major crops and native aquatic plants, which can effectively avoid off-target effects and the risk of damage to non-target species. This lays the core target foundation for the development of RNA nucleic acid biopesticides with low environmental burden and high species selectivity, and has good ecological safety. Attached Figure Description

[0031] Figure 1 Detection of lncRNA12385 expression level in Alternanthera philoxeroides and Alternanthera pilosa under flooded conditions.

[0032] Figure 2 RT-qPCR was used to detect the knockdown effect of lncRNA12385.

[0033] Figure 3 Comparison of plant height after knocking down lncRNA12385. Scale bar is 5 cm.

[0034] Figure 4 Statistical analysis of internode elongation rate after waterlogging treatment. Five plants were analyzed in each group, with each point representing one internode. **** indicates P < 0.0001, *** indicates P < 0.001, ** indicates P < 0.01, * indicates P < 0.05, and ns indicates no significant difference; Student's t-test was used for analysis. Detailed Implementation

[0035] The methods involved in this invention are described in detail below:

[0036] Example 1: RT-qPCR identification of water-flood-specific upregulated lncRNAs

[0037] (1) Experimental materials and treatments: Alternanthera pungens, a closely related species to Alternanthera hygrophila, cannot effectively escape under waterlogging stress through adaptive phenotypes such as stem elongation, and eventually rots and dies underwater. Given that its invasive adaptability is much lower than that of Alternanthera hygrophila, it is often used as a control species. Alternanthera hygrophila and Alternanthera pungens with similar growth were selected for subsequent treatments. The experiment set up a terrestrial control group (normal water supply) and a completely submerged treatment group (the top of the plant was submerged in water). At six time points after the submerged treatment, 0 h (control), 3 h, 24 h, 48 h, 72 h and 96 h, the tender stem tissue of the first and second nodes of the top of the plant was collected as sequencing samples, and three independent biological replicates were set up at each time point.

[0038] (2) Stem tissue samples of Alternanthera philoxeroides and Alternanthera pilosa were collected at each time point and ground into a fine powder in liquid nitrogen. Total RNA was extracted by adding 1 mL of Trizol lysis buffer to every 50–100 mg of tissue. The integrity of the obtained RNA samples was assessed by agarose gel electrophoresis, and the concentration and purity were determined by micro-spectrophotometer. Genomic DNA digestion was then performed.

[0039] (3) RT-qPCR detection of lncRNA expression levels: 1 μg of total RNA after DNase digestion was used as a template for reverse transcription to synthesize cDNA, which was then used as a template for RT-qPCR detection. UBC was used as an internal reference gene, and three technical replicates were set up for each sample. -ΔΔCt The relative expression levels of each lncRNA were calculated. RT-qRCR results showed that, compared with the 0 h control group, lncRNA12385 showed a significantly upregulated expression pattern only in *Alternanthera philoxeroides*, and a rapid response was observed in the early stages of flooding (3 h / 24 h); while in *Alternanthera philoxeroides*, the expression level of lncRNA12385 was not significantly upregulated throughout the flooding treatment, and no obvious stress response characteristics were observed. Figure 1 This specific expression difference further suggests that lncRNA12385 may be a waterlogging response regulator unique to Alternanthera philoxeroides, playing a key regulatory role in mediating waterlogging stress-induced stem elongation.

[0040] Example 2: Construction of pTRV2-lncRNA12385 recombinant vector targeting lncRNA12385

[0041] (1) Template preparation: Take the tender stem tissue of Alternanthera philoxeroides after complete water submersion, extract total RNA, and synthesize first-strand cDNA by reverse transcription, which will serve as the template for subsequent gene cloning.

[0042] (2) Primer design and PCR amplification: Upstream and downstream amplification primers were designed for the specific region of lncRNA12385, with an expected amplified fragment length of 280 bp. PCR amplification was performed using the above cDNA as a template. After separation of the amplified products by agarose gel electrophoresis, the target band was excised and purified by gel extraction.

[0043] (3) Construction of recombinant plasmid: The purified PCR product and the empty pTRV2 (silencing vector) vector were digested with restriction endonucleases. The digested products were purified by gel extraction, mixed at an appropriate molar ratio, and homologous recombinase was added. The mixture was ligated at 50°C for 40 min. The ligation product was transformed into E. coli DH5α competent cells, plated on plates containing Kans antibiotic for antibiotic resistance screening, and positive single clones were selected for sequencing identification. The sequencing results showed that the inserted fragment sequence was completely consistent with the target fragment of lncRNA12385, proving that the recombinant plasmid pTRV2-lncRNA12385 was constructed correctly.

[0044] (4) Preparation of Agrobacterium engineered bacteria: The above recombinant plasmid pTRV2-lncRNA12385 and helper plasmid pTRV1 were transformed into Agrobacterium GV3101 competent cells, respectively. Positive transformants were screened by plating on plates containing the corresponding antibiotics. Single colonies were picked and cultured, and then stored at -80°C with glycerol for later use.

[0045] Example 3: Verification of TRV-VIGS system infection efficiency of Alternanthera philoxeroides and lncRNA12385 knockdown

[0046] (1) Preparation of Agrobacterium infection culture: GV3101 Agrobacterium carrying pTRV1 and pTRV2-lncRNA12385 were inoculated into LB liquid medium containing the corresponding antibiotics for activation culture. The bacterial culture was collected, and the bacterial pellet was collected by centrifugation at 5000 rpm. The supernatant was discarded, and the pellet was resuspended in infection buffer (containing 10 mM MES, 10 mM MgCl2, and 200 μM acetylsylcholine) and adjusted to the final concentration at OD. 600 =0.5. Two bacterial cultures carrying pTRV1 and pTRV2-lncRNA12385 were mixed in a 1:1 volume ratio to obtain the mixed infection solution. Simultaneously, an equal volume mixture of empty pTRV2 vector and pTRV1 was set up as a negative control group.

[0047] (2) Plant infection and culture: Seedlings of Alternanthera philoxeroides with uniform growth and 4-5 pairs of mature leaves were selected as infection materials. The mixed infection solution was drawn up using a needleless syringe and injected from the lower epidermis of the seedling leaves to all the leaves. After infection, the plants were placed in the dark and moist (relative humidity 80%-90%) for 48 hours. Then the infected plants were divided into a terrestrial normal culture group and a completely submerged treatment group, and cultured for another 10 days.

[0048] (3) Knockdown efficiency detection: Young stem tissue from the tips of plants in each treatment group was collected, total RNA was extracted, reverse transcribed, and the relative expression level of lncRNA12385 was detected by real-time quantitative PCR (RT-qPCR). Results showed that the expression level of lncRNA12385 in the pTRV2-lncRNA12385 treatment group was significantly lower than that in the control group, with a knockdown efficiency exceeding 60%. Figure 2 The results showed that the TRV-VIGS silencing vector constructed in this invention can achieve efficient knockdown of endogenous lncRNA12385 in Alternanthera philoxeroides.

[0049] Example 4: The inhibitory effect of knocking down lncRNA12385 on stem elongation growth of Alternanthera philoxeroides under flooded conditions.

[0050] (1) Experimental grouping and phenotypic observation: The infected plants were divided into two groups: an empty vector control group and a pTRV2-lncRNA12385 silencing group. Each group was completely submerged in water. After 10 days of continuous submerged culture, the phenotypic characteristics and growth indicators of the two groups of plants were observed and measured.

[0051] (2) The phenotypic observation results are as follows: Under flooded conditions, the internodes of the control group plants elongated rapidly, the overall plant height increased significantly, the apical internodes were significantly elongated, and the plant apex was able to extend above the water surface to complete gas exchange and maintain normal growth; while the overall plant height of the lncRNA12385 silenced group plants was significantly reduced ( Figure 3 Further analysis of internode elongation revealed that the length of the first and second apical internodes (the main elongation nodes under flooded conditions) was significantly shorter than that of the control group. Most plants were unable to extend their apex above the water surface, indicating severe growth inhibition under prolonged flooding conditions. Figure 4 ).

[0052] (3) Statistical analysis: Five biological replicates were set up in each group, and the length of the first to fourth internodes of the plants was measured. Student's t-test was used to statistically compare the data of the two groups. The results showed that the difference in internode length elongation between the silent group and the control group was statistically significant (P<0.001).

[0053] Based on the above experimental results, lncRNA12385 is a key regulatory factor in Alternanthera philoxeroides' response to flooding stress, driving rapid stem elongation to achieve hypoxia escape. Knocking down this lncRNA using the TRV-VIGS system significantly inhibited flooding-induced stem elongation, causing the plant to lose its ability to escape underwater and thus effectively weakening the aquatic invasion potential of Alternanthera philoxeroides.

Claims

1. Application of lncRNA12385 as a molecular target in the preparation of silencing vectors or nucleic acid control agents that inhibit the waterlogged adaptive growth of Alternanthera philoxeroides and block its aquatic asexual reproduction.

2. The application according to claim 1, characterized in that, Transcriptome sequencing analysis confirmed that lncRNA12385 was specifically and significantly upregulated in the young stem tissues of Alternanthera philoxeroides after 3 hours of complete waterlogging stress.

3. The application according to claim 1, characterized in that, After knocking down the expression of endogenous lncRNA12385 using the TRV-VIGS system, waterlogging-induced stem elongation was significantly inhibited, preventing the plant from escaping the underwater hypoxic environment through elongation growth and thus losing its ability to survive underwater.

4. The application according to claim 1, characterized in that, The lncRNA12385 is a specific long non-coding RNA derived from Alternanthera philoxeroides. Its transcript length is greater than 200 nt, it does not have a complete protein-coding open reading frame, and it is specifically activated and expressed only under waterlogging stress conditions.

5. A TRV-VIGS recombinant silencing vector targeting lncRNA12385, characterized in that, The recombinant silencing vector uses pTRV2 as its backbone vector, into which a specific targeting fragment of lncRNA12385 is inserted; the recombinant silencing vector is named pTRV2-lncRNA12385; the auxiliary vector used in conjunction with this vector system is pTRV1.

6. The method for constructing the recombinant silencing vector according to claim 5, characterized in that, Includes the following steps: (1) Using the cDNA of young stems of Alternanthera philoxeroides treated with water as a template, a specific target fragment of lncRNA12385 was obtained by PCR amplification; (2) The obtained amplified fragment was ligated with the pTRV2 vector, transformed into competent Escherichia coli cells, and screened to obtain the positive recombinant plasmid pTRV2-lncRNA12385; (3) The recombinant plasmid pTRV2-lncRNA12385 and the helper plasmid pTRV1 were transformed into Agrobacterium GV3101 competent cells to obtain strains for infection.

7. A VIGS silencing method for inhibiting the growth of drought-loving Alternanthera philoxeroides under flooded conditions, characterized in that, The Agrobacterium-based mixture of the recombinant silencing vector pTRV2-lncRNA12385 and the helper vector pTRV1 described in claim 5 was used to infect Alternanthera philoxeroides seedlings; the method includes the following steps: (1) Agrobacterium carrying pTRV1 and pTRV2-lncRNA12385 were cultured separately, and the bacterial concentration was adjusted to OD. 600 =0.5, and mix them in an equal volume ratio to prepare a mixed infection solution; (2) Using a needleless syringe, the mixed infection solution was applied to the lower epidermal tissue of the leaves of Alternanthera philoxeroides seedlings; (3) After infection, the plants were placed in a dark and moist environment for 48 hours, and then transferred to a completely submerged environment for 10 days. (4) The expression of endogenous lncRNA12385 was silenced through the above-mentioned infection, which effectively inhibited the stem elongation induced by water flooding and blocked the adaptive growth process of Alternanthera philoxeroides in water.

8. The silencing method according to claim 7, characterized in that: During the infection process, the lower epidermis of the leaves of each seedling was soaked with 1-2 cm², and the amount of infection solution used was ~1.0 mL / seedling.

9. The silencing method according to claim 7, characterized in that: The light-protected and moisture-retaining culture conditions are a temperature of 22–25°C, a relative humidity of 80%–90%, and dark culture; the completely submerged culture conditions are a water temperature of 22–26°C and a water depth that submerges the top of the plant by 3–5 cm.

10. The silencing method according to claim 7, characterized in that: The method also sets up plants infected with the pTRV2 empty vector as a negative control group to evaluate the specificity and effectiveness of the silencing effect; at the same time, it sets up wild-type plants without any infection treatment as a blank control group to assess the basic growth phenotype of plants under flooding conditions.

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