A method for increasing lignin content in rice
Patent Information
- Application Number
- CN202510557044.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-29
- Publication Date
- 2026-10-30
AI Technical Summary
[0036]本发明研究发现，将水稻根系浸泡于卵磷脂溶液，能够显著提高木质素合成通路关键基因的表达水平，水稻内的木质素含量显著增加。说明卵磷脂可以显著促进水稻木质素的合成，从而提高水稻的木质素含量。其中，卵磷脂对OsBet v1过表达水稻的促进木质素的合成效果更佳。另外，随着卵磷脂处理时间的延长，水稻的木质素含量呈现出增加趋势。
Smart Images
Figure CN122996986A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology. More specifically, it relates to a method for increasing the lignin content of rice. Background Technology
[0002] Lignin is a complex phenolic polymer formed from three alcohol monomers (p-coumarol, coniferyl alcohol, and sinigrin), and is one of the core components of the secondary cell wall of vascular plants. As an important component of the cell wall, lignin maintains the rigidity and mechanical strength of the cell wall, providing the basis for structural stability and physical resistance in plants.
[0003] During plant root development, lignin deposition directly affects the mechanical properties and physiological functions of root tissues. Studies have shown that increased lignin content can strengthen cell wall rigidity, providing greater mechanical strength to the roots, making the plant more stable, and reducing lodging caused by external factors. Furthermore, lignin plays multiple regulatory roles in root stress response: on the one hand, highly lignified roots can maintain the structural integrity of water transport channels, reducing non-stomatal water loss under drought stress, and simultaneously inhibiting excessive salt ion accumulation by regulating transmembrane ion transport, thus synergistically enhancing the plant's drought resistance and salt tolerance; on the other hand, as the first line of defense against biological stress, lignin's physical barrier effect can effectively inhibit pathogen infection and pest feeding behavior, thereby enhancing the plant's systemic resistance to diseases and pests.
[0004] Studies have shown that the lignin content of rice is related to its growth performance, as well as its resistance to pests and diseases, drought, and salinity. Therefore, developing a technology that can promote lignin synthesis and increase lignin content in rice can help improve its growth performance and stress resistance, which is of great practical significance for sustainable agricultural development and food security. Summary of the Invention
[0005] The present invention aims to provide a method for increasing the lignin content of rice by using lecithin to promote the synthesis of lignin in rice, thereby improving the rice's resistance to lodging, drought, and pests and diseases.
[0006] The purpose of this invention is to provide the application of lecithin in promoting lignin synthesis in rice.
[0007] The purpose of this invention is to provide a method for increasing the lignin content of rice.
[0008] The above-mentioned objective of this invention is achieved through the following technical solution:
[0009] This invention has found that soaking rice roots in a lecithin solution significantly increases the expression levels of key genes in the lignin synthesis pathway, resulting in a significant increase in lignin content within the rice. This indicates that lecithin can significantly promote lignin synthesis in rice, thereby increasing its lignin content. Therefore, the following method is claimed for protection:
[0010] This invention provides the application of lecithin in promoting lignin synthesis in rice.
[0011] This invention provides the application of lecithin in the preparation of products that promote the synthesis of lignin in rice.
[0012] This invention provides the application of lecithin in promoting the expression of genes related to lignin biosynthesis in rice.
[0013] This invention provides the application of lecithin in the preparation of products that promote the expression of genes related to the biosynthesis of lignin in rice.
[0014] This invention provides the application of lecithin in increasing the lignin content of rice.
[0015] This invention provides the application of lecithin in the preparation of products that increase the lignin content of rice.
[0016] Preferably, the rice is rice with OsBet v1 gene overexpression.
[0017] As an alternative implementation, in the above application, the lecithin is used in the form of a lecithin solution with a concentration of 2-6 mM (preferably 4 mM).
[0018] The present invention also provides a method for increasing the lignin content of rice by treating rice seedlings with a lecithin solution.
[0019] As an alternative implementation, the concentration of the lecithin solution is 2-6 mM.
[0020] As an alternative implementation, the concentration of the lecithin solution is 4 mM.
[0021] As an alternative implementation, the treatment involves soaking rice roots in a lecithin solution.
[0022] As an alternative implementation, the soaking time is 24-144 hours.
[0023] Preferably, the soaking time is 48-144 hours.
[0024] More preferably, the soaking time is 72-144 hours.
[0025] As an alternative implementation, the lecithin solution described above comprises lecithin, water, a cosolvent, a thickener, and a surfactant.
[0026] As an alternative implementation, the cosolvent is dimethyl sulfoxide (DMSO), the thickener is glycerol, and the surfactant is polysorbate-20.
[0027] As an alternative implementation, the lecithin solution is prepared by dissolving lecithin in water to form a solution (the final concentration of lecithin in the solution is 4mM), adding 1% by total volume of dimethyl sulfoxide (DMSO), 0.5% by total volume of glycerol, and 0.1% by total volume of polysorbate-20 (Tween20) to the solution, and mixing thoroughly.
[0028] As an alternative implementation, during the soaking process, the rice is cultivated under light for 12-16 hours at a temperature of 24-28℃, a humidity of 30-50%, and a light intensity of 12,000-16,000 lumens; or under dark conditions for 8-12 hours at a temperature of 22-26℃ and a humidity of 30-50%.
[0029] This invention also found that, compared to wild-type rice, lecithin treatment has a better effect on promoting lignin production in rice overexpressing the OsBet v1 gene. Therefore, preferably, in the above-described method for promoting lignin synthesis in rice, the rice is rice overexpressing the OsBet v1 gene.
[0030] As an alternative implementation, the method for increasing the lignin content of rice specifically involves immersing sterile rice seedlings in a lecithin solution.
[0031] As an alternative implementation, the method for preparing sterile rice seedlings involves disinfecting rice seeds, placing the disinfected rice seeds in a culture medium, and culturing for 7-10 days to obtain sterile rice seedlings.
[0032] As an alternative implementation, the disinfection method is as follows: rice seeds are disinfected using 70-80% alcohol and 1-2% NaClO solution for 40-50 minutes.
[0033] As an alternative implementation, in the above-mentioned method for preparing sterile rice seedlings, the culture medium is B5 medium.
[0034] As an alternative implementation method, the above-mentioned method for preparing sterile rice seedlings involves hardening off the sterile rice seedlings with clean water for 2-3 days.
[0035] The present invention has the following beneficial effects:
[0036] This invention found that soaking rice roots in a lecithin solution significantly increased the expression levels of key genes in the lignin synthesis pathway, resulting in a significant increase in lignin content within the rice. This indicates that lecithin can significantly promote lignin synthesis in rice, thereby increasing its lignin content. Specifically, lecithin showed a more pronounced effect on promoting lignin synthesis in rice overexpressing OsBet v1. Furthermore, the lignin content of the rice showed an increasing trend with prolonged lecithin treatment time.
[0037] The lecithin used in this invention is a naturally occurring compound that is environmentally friendly and harmless. The method provided by this invention simply involves soaking rice roots in a lecithin solution to significantly increase lignin content. It is simple to operate and easy to promote and apply. This method can be widely used for seedling treatment to enhance the lignin content of rice in advance, thereby improving the rice's resistance to lodging, drought, and pests and diseases, demonstrating excellent application prospects and value. Attached Figure Description
[0038] Figure 1 Figure 1 shows the results of lignin content determination in the roots of wild-type rice, OsBet v1 overexpressing rice, and OsBet v1 mutant rice.
[0039] Figure 2 The results show the expression levels of key genes for lignin synthesis in the roots of wild-type rice, OsBet v1-overexpressing rice, and OsBet v1 mutant rice after 24 hours of lecithin treatment.
[0040] Figure 3 Results of lignin content determination in roots of wild-type rice, OsBet v1 overexpressing rice, and OsBet v1 mutant rice after 24 h of lecithin treatment.
[0041] Figure 4 The results show the determination of lignin content in rice treated with lecithin at different times. Detailed Implementation
[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way.
[0043] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.
[0044] In the following examples, the tested rice varieties were wild-type japonica rice “Nipponbare” (WT) and Nipponbare OsBet v1 mutant (KO) and Nipponbare OsBet v1 overexpression strain (OE) based on Nipponbare.
[0045] The instruments and materials used in the following examples are: LED artificial climate incubator, NanoDrop2000 micro spectrophotometer, and disposable plastic cups (size: 2.5cm×2.5cm×8cm).
[0046] Lecithin is soybean lecithin, CAS number: 8002-43-5, structural formula:
[0047]
[0048] The Lignin Content Determination Kit (Acetylation Method) is from Shanghai Yuanye Biotechnology Co., Ltd., and its catalog number is R32846-48T.
[0049] Preparation method of B5 culture medium: Weigh 3.21g of B5 powder, 28g of glucose and 4g of plant gel, mix them evenly in a magnetic stirrer, and sterilize at 115℃ for 25min. The B5 powder was purchased from Haibo Biotechnology Co., Ltd., product number 20230729.
[0050] Example 1: Method for treating rice seedlings with lecithin
[0051] In this embodiment, wild-type rice, OsBet v1 mutant rice, and OsBet v1 overexpressing rice were used as the tested rice varieties (3 lines).
[0052] I. Preparation of Lecithin Solution
[0053] Preparation of lecithin (100mM) solution: Add 1.5g of lecithin powder to 20mL of sterile water, then add 1% DMSO, 0.5% glycerol, and 0.1% Tween 20 by total volume. Finally, sonicate in an ultrasonic instrument for 20min and store at -20℃ protected from light. When using, dilute with sterile water to a 4mM lecithin solution according to the ratio.
[0054] II. Lecithin Treatment of Rice Seedlings
[0055] (1) Take rice seeds, peel off the rice husks, disinfect with 75% alcohol for 1 min, wash with ddH2O for 30 s, disinfect with 1-2% NaClO solution for 40-50 min, and wash with sterile ddH2O 5-6 times in a clean bench.
[0056] (2) Place the sterilized seeds in B5 culture medium and culture them in an artificial climate incubator for 7-10 days to obtain rice seedlings.
[0057] (3) Wash the rice seedlings off the B5 culture medium and harden them with clean water for 2-3 days.
[0058] (4) Add 30 mL of 4 mM lecithin solution to a plastic cup (2.5 cm × 2.5 cm × 8 cm). Transfer wild-type rice, OsBet v1 mutant rice and overexpression rice that have undergone hardening to the 4 mM lecithin solution (lecithin treatment). The control group is soaked in water (water treatment). Incubate in an artificial climate incubator for 24 h, 48 h, 72 h and 144 h. Set up 3 replicates for each treatment.
[0059] Example 2: Lignin content in rice roots of different strains
[0060] I. Experimental Methods
[0061] The lignin content of wild-type japonica rice Nipponbare and OsBet v1 mutant and overexpressing rice roots was determined by taking rice roots that had just been washed from the culture medium in step (3) of Example 1, setting up 3 replicates for each line, and using a lignin content assay kit (acetylation method) to determine the lignin content.
[0062] II. Experimental Results
[0063] The lignin content of the roots of different strains was determined, and the results are as follows: Figure 1 As shown, the results indicated that the average lignin content in wild-type rice roots was 318.96 mg / g, the average lignin content in OsBet v1 overexpressing rice roots was 415.66 mg / g, and the lignin content in OsBet v1 mutant rice roots was 267.70 mg / g. This suggests that OsBet v1 can increase the lignin content in rice roots.
[0064] Example 3: Changes in the expression levels of key genes for lignin synthesis in rice roots after lecithin treatment.
[0065] I. Experimental Methods
[0066] Rice roots treated with 4mM lecithin and water for 24 hours in Example 1 were collected. Three replicates were set up for each line and treatment. RNA was extracted using an RNA extraction kit and then reverse transcribed using a reverse transcription kit. The obtained cDNA was subjected to real-time quantitative PCR to detect changes in key genes (PAL gene, C4H gene, CAD gene, and POD gene) in the rice lignin synthesis pathway. The primers used for real-time quantitative PCR are shown in Table 1.
[0067] Table 1 Primer list for quantitative real-time PCR
[0068]
[0069] II. Experimental Results
[0070] The expression levels of lignin synthesis-related genes after lecithin treatment were determined using real-time quantitative PCR. Figure 2 The results showed that after 24 hours of lecithin treatment, compared with H2O treatment, the expression levels of the PAL gene in OsBet v1 overexpressing rice, OsBet v1 mutant rice, and wild-type rice increased by 8.3, 2.1, and 4.8 times, respectively; the expression levels of the C4H gene increased by 2.3, 1.3, and 1.4 times, respectively; the expression levels of the CAD gene increased by 12.1, 2.2, and 3.8 times, respectively; and the expression levels of the POD gene increased by 7.2, 3.1, and 4.9 times, respectively. This indicates that lecithin can promote the lignin synthesis pathway in rice and increase lignin content.
[0071] Example 4: Effect of lecithin treatment on lignin content in rice roots
[0072] I. Experimental Methods
[0073] Rice roots treated with 4 mM lecithin and water for 24 hours in Example 1 were collected. Three replicates were set up for each line and treatment. The lignin content was determined using a lignin content assay kit (acetylation method).
[0074] II. Experimental Results
[0075] The results of root lignin content determination after 24 hours of lecithin treatment are as follows: Figure 3 As shown, the results indicated that after 24 h of lecithin treatment, the lignin contents of OsBet v1-overexpressing rice, OsBet v1 mutant rice, and wild-type rice were 481.6 mg / g, 207.2 mg / g, and 332.3 mg / g, respectively; after 24 h of H2O treatment, the lignin contents of OsBet v1-overexpressing rice, OsBet v1 mutant rice, and wild-type rice were 381.2 mg / g, 187.2 mg / g, and 280.3 mg / g, respectively. Lecithin treatment significantly increased the lignin content in OsBet v1-overexpressing rice and wild-type rice, indicating that OsBet v1 can positively regulate rice lignin synthesis by degrading lecithin.
[0076] Example 5: Effect of lecithin treatment time on lignin content in rice
[0077] I. Experimental Methods
[0078] Wild-type rice roots treated with 4mM lecithin and water at different time points in Example 1 were used to determine the lignin content using a lignin content assay kit (acetylation method).
[0079] II. Experimental Results
[0080] The lignin content in rice roots at different time points after lecithin treatment was determined, and the results are as follows: Figure 4 As shown, the results indicated that after lecithin treatment for 24 h, 48 h, 72 h, and 144 h, the lignin content of rice was 332.5 mg / g, 344.6 mg / g, 369.8 mg / g, and 409.3 mg / g, respectively. After H2O treatment for 24 h, 48 h, 72 h, and 144 h, the lignin content of rice showed no significant difference, ranging from 278.1 mg / g to 288.0 mg / g. These results demonstrate that extending the lecithin treatment time can increase the accumulation of lignin in rice.
[0081] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. Application of lecithin in promoting lignin synthesis in rice.
2. Application of lecithin in the preparation of products that promote lignin synthesis in rice.
3. Application of lecithin in promoting the expression of genes related to lignin biosynthesis in rice.
4. Application of lecithin in the preparation of products that promote the expression of genes related to the biosynthesis of lignin in rice.
5. Application of lecithin in increasing the lignin content of rice.
6. Application of lecithin in the preparation of products with increased lignin content in rice.
7. A method for increasing the lignin content of rice, characterized in that, Rice seedlings were treated with lecithin solution.
8. The method according to claim 7, characterized in that, The concentration of the lecithin solution is 2-6 mM.
9. The method according to claim 7 or 8, characterized in that, The treatment involves soaking rice roots in a lecithin solution.
10. The method according to claim 9, characterized in that, The soaking time is 24-144 hours.