Method for increasing pectin content of plant by regulating DELLA gene
By regulating the DELLA gene family of Arabidopsis, a multi-gene mutant was constructed, which significantly increased the pectin content, solved the problem of high production costs of traditional pectin, and achieved low-cost and environmentally friendly pectin production.
Patent Information
- Application Number
- CN202510469645.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-15
- Publication Date
- 2025-07-04
AI Technical Summary
The traditional pectin industry relies on raw materials such as citrus peel or apple pomace, resulting in high production costs. There is a lack of a solution to systematically optimize pectin synthesis from the genetic regulation level. The direct regulatory effect of DELLA protein on pectin synthesis has not been reported.
By regulating the RGA, GAI, RGL1, RGL2, and RGL3 genes in the Arabidopsis DELLA gene family, multigene mutants, including double mutant dellaD, tetra mutant dellaQ and five mutant dellaP, significantly increasing the pectin content.
It significantly increased the pectin content of Arabidopsis seedlings and seeds, reduced production costs, opened up efficient and low-cost pectin production channels, and promoted the sustainable development of the pectin industry.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of plant genetic engineering, and specifically relates to a method for constructing multi-gene mutant plants by directionally knocking out DELLA gene family members, significantly increasing the pectin content in plants, and its applications in agriculture and industry. Background Art
[0002] As a key structural component of the plant cell wall, pectin not only endows the plant cell wall with its unique physical and chemical properties, but also plays an important role in cell recognition, signal transduction, and plant growth and development. In the industrial field, pectin is widely used in food thickeners, pharmaceutical excipients, industrial gelling agents, etc. However, traditional industrial production of pectin mainly relies on extraction from plant raw materials such as citrus peels or apple pomace. This method has obvious raw material dependence, resulting in high production costs and limiting the large-scale application and further development of pectin.
[0003] In plants, the synthesis of pectin is finely regulated by multiple factors, among which gene regulation plays a crucial role. In-depth understanding of the molecular mechanism of pectin synthesis is of great significance for optimizing pectin production by genetic engineering means. At present, although there have been some research reports on genes related to pectin synthesis, there is still a lack of a systematic optimization plan for pectin synthesis at the gene regulation level.
[0004] DELLA proteins, as negative regulators of the gibberellin (GA) signaling pathway, play a key role in plant growth and development. Research has shown that DELLA proteins regulate plant development by interacting with multiple transcription factors or affecting the light signaling pathway. Functional redundant genes refer to two or more genes in an organism that can perform the same or similar functions. These redundant genes can complement each other to ensure that when one gene loses its function, another gene can compensate for its function, thus maintaining the normal physiological functions of the organism. There are a total of five DELLA genes in the Arabidopsis thaliana genome, namely RGA , GAI , RGL1 , RGL2 and RGL3 . Existing research has pointed out that DELLA the co-knockout of multiple genes can significantly change the metabolic pathway of the plant cell wall, thereby affecting plant growth and development. However, there is currently no report on the direct regulatory effect of DELLA proteins on pectin synthesis. This provides a new research direction and potential breakthrough for optimizing pectin synthesis by gene regulation means. Summary of the Invention
[0005] Object of the Invention: The present invention aims to propose an innovative strategy to significantly increase the pectin content in plants by precisely targeting and knocking out multiple DELLA genes. Without negatively affecting plant growth, it opens up a new, efficient, and low-cost resource route for industrial pectin production. This method will provide more competitive plant raw materials for large-scale pectin production and promote the sustainable development of the pectin industry.
[0006] Technical Solution: To achieve the above object of the invention, the technical solution adopted by the present invention is as follows: A method for increasing the pectin content in plants by regulating DELLA genes, wherein the DELLA genes are 5 functionally redundant genes in the DELLA family of Arabidopsis thaliana: RGA , GAI , RGL1 , RGL2 , RGL3 , and the gene information is as follows: RGA(AT2G01570), GAI (AT1G14920), RGL1(AT1G66350), RGL2(AT3G03450), RGL3(AT5G17490) . The method for increasing the pectin content in plants is to simultaneously silence two or more DELLA genes as target genes.
[0007] Preferably, the 5 functionally redundant genes in the DELLA family of Arabidopsis thaliana: RGA , GAI , RGL1 , RGL2 , RGL3 are simultaneously knocked out to further increase the pectin content in plants.
[0008] The present invention also provides a method for simultaneously silencing target genes, that is, using mutants to knock out DELLA family genes, and the method includes the following steps: Constructing an RGA gene mutant by EMS chemical mutagenesis rga-24 , and respectively constructing single-gene mutants of Arabidopsis thaliana RGA , GAI , RGL1 and RGL2 genes by Ds transposon insertion technology, that is, rga-t2 , gai-t6 , rgl1-1 and rgl2-1 , and constructing a mutant of RGL3 gene by T-DNA insertion technology rgl3-1 .
[0009] Then, through genetic hybridization, double mutants dellaD ( rga-24 x gai-t6 ), quadruple mutants dellaQ ( rga-t2 xgai-t6 x rgl1-1 x rgl2-1 ) and the penta-mutant dellaP ( dellaQ x rgl3-1 ).
[0010] The present invention also provides a method for determining the pectin content of Arabidopsis thaliana, and the method includes determining the pectin content of seedlings and seeds, and includes the following steps: 1. Pectin extraction (1) Pectin extraction from seedlings: After grinding Arabidopsis thaliana seedlings, sequentially extract with solvents such as ethanol, chloroform-methanol, and acetone to remove impurities, and finally extract pectin with ammonium oxalate in a water bath to obtain a pectin extract.
[0011] (2) Pectin extraction from seeds: Divide Arabidopsis thaliana seeds into outer mucilage and inner mucilage for separate extraction, and after combining the extracts, perform dialysis, drying, and weighing.
[0012] 2. Standard curve preparation Operate on standard GalA solutions with different concentrations according to the sample treatment and determination steps for subsequent quantitative analysis of pectin content.
[0013] 3. Pectin content determination Treat the sample with sodium tetraborate / sulfuric acid solution and m-hydroxybiphenyl solution, measure the absorbance value at 520 nm and compare it with the standard curve to calculate the pectin content.
[0014] The pectin content of wild-type (WT) seedlings is 89.7 ± 2.2 μg / mg. dellaP The pectin content of seedlings is 95.6 ± 2.2 μg / mg, which is about 6.5% higher than that of WT. It can be seen that dellaP the mutant has increased the pectin content of seedlings.
[0015] The pectin content of wild-type (WT) seeds is about 98 μg / mg. The pectin content of dellaD seeds is about 114 μg / mg. The pectin content of dellaQ seeds is about 125 μg / mg. The pectin content of dellaP seeds is about 127 μg / mg, which is 29.7% higher than that of WT. It can be seen that della the pectin content of mutant seeds is significantly higher than that of WT, especially dellaP the mutant, whose pectin content is 29.7% higher than that of WT.
[0016] Meanwhile, the present invention also provides a method for measuring the pectin thickness of Arabidopsis thaliana. The method is to stain Arabidopsis thaliana seeds with ruthenium red staining solution, remove the outer mucilage, observe the thickness of the inner mucilage under a microscope, and measure and calculate the mucilage thickness using ImageJ software. The results show that the pectin layer thickness of the wild-type (WT) seed coat is 0.08 ± 0.006 mm; dellaD The pectin layer thickness of the mutant seed coat is 0.10 ± 0.007 mm, which is about 28% higher than that of the WT; dellaQ The pectin layer thickness of the mutant seed coat is 0.12 ± 0.006 mm, which is about 56% higher than that of the WT; dellaP The pectin layer thickness of the mutant seed coat is 0.12 ± 0.006 mm, which is about 52% higher than that of the WT. It can be seen that della the pectin layer thickness of the mutant seeds is significantly increased, especially dellaQ and dellaP mutants, which are about 56% and 52% higher than the WT respectively.
[0017] Beneficial effects: By precisely regulating the DELLA gene family, the present invention has achieved a significant increase in the pectin content of plants, providing high-value-added raw materials for agricultural production and industrial applications. This method breaks through the dependence on single raw materials such as citrus and apples in traditional pectin production, opens up an efficient utilization path for herbaceous plants and seed resources, and has wide universality. It can be extended to important economic crops such as tomatoes and soybeans, providing new ideas and methods for the improvement of various crops. By reducing the dependence on chemical extraction raw materials, this technology reduces production costs, promotes a green production mode, reduces environmental impacts, and contributes to sustainable development. In addition, the present invention also reveals the molecular mechanism of DELLA genes in regulating plant pectin synthesis, providing original theoretical support for the fields of plant developmental biology and cell wall engineering, and opening up new research directions and application prospects for the pectin industry and agricultural improvement. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 Pentamutant dellaP Comparison chart of pectin content between pentamutant and wild-type (WT) seedlings; Figure 2 Comparison of pectin content in the mucilage of mutant seeds Figure 3 Phenotypic comparison of pectin layer thickness in the mucilage of wild-type and mutant Arabidopsis thaliana plants Figure 4 Statistical chart of pectin layer thickness of the mucilage released from mutant seeds DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention will be further described below in conjunction with specific embodiments. For the molecular biology experimental methods not specifically described in the following embodiments, reference can be made to the methods listed in "Molecular Cloning: A Laboratory Manual" (Third Edition) by J. Sambrook or the conventional methods in the art, or carried out according to the kits and product specifications.
[0020] Example 1: DELLA Construction of gene mutants 1. Selection of target genes Five genes with redundant functions in the Arabidopsis DELLA family ( RGA, GAI, RGL1, RGL2, RGL3 ) were selected as target genes for knockout. RGA(AT2G01570), GAI(AT1G14920), RGL1(AT1G66350), RGL2(AT3G03450), RGL3(AT5G17490) , and the gene-related information can be obtained from the TAIR website.
[0021] 2. Construction of mutants Arabidopsis mutant materials are key tools for studying plant gene functions. Currently, methods for artificially obtaining plant mutants through genetic manipulation include Agrobacterium transfer DNA (T-DNA) insertion technology, Ds transposon insertion technology, EMS chemical mutagenesis technology, and CRISPR / Cas9 gene editing technology, etc.
[0022] (1) Obtaining single-gene mutants: Using wild-type Arabidopsis as the background, mutants of Arabidopsis DELLA genes were constructed respectively, among which RGA gene mutant rga-24 was obtained through EMS chemical mutagenesis technology. Through Ds transposon insertion technology, single-gene mutants of Arabidopsis RGA, GAI, RGL1 and RGL2 genes were constructed respectively, namely rga-t2 , gai-t6 , rgl1-1 and rgl2-1 . In addition, a mutant of RGL3 gene rgl3-1 was constructed using T-DNA insertion technology. For details of the method, refer to the third edition of Molecular Cloning.
[0023] (2) Obtaining multi-gene mutants: Through genetic mating, different single mutants were genetically crossed, and the offspring of multi-gene mutants were screened to obtain homozygotes. Double mutants dellaD (rga-24 × gai-t6) , quadruple mutants dellaQ (rga-t2 x gai-t6 x rgl1-1 x rgl2-1) and quintuple mutants dellaP ( rga-t2 × gai-t6 × rgl1-1 × rgl2- 1 × rgl3-1 ) were gradually constructed through hybridization; among them dellaP ( CS16298) It can be purchased from the ABRC mutant library. Information about the mutants can be found on the ABRC website.
[0024] Example 2: Determination of pectin content in seedlings 1. Pectin extraction (1) Disinfect the WT and della mutant seeds and sow them on 1 / 2 MS medium. Culture for 10 days at 22 °C under a 16-hour light / 8-hour dark condition, and collect the seedlings.
[0025] (2) Put the Arabidopsis seedlings into a mortar and grind them using liquid nitrogen.
[0026] (3) Extract with 70% ethanol for 2 hours. After extraction, centrifuge at 5,000 g for 15 minutes, discard the supernatant, and repeat 3 times.
[0027] (4) Add chloroform:methanol (1:1, v / v) and extract for 2 hours. After extraction, centrifuge at 5,000 g for 15 minutes, discard the supernatant, and repeat 2 times.
[0028] (5) Add 100% acetone and extract for 2 hours. After extraction, centrifuge at 10,000 g for 15 minutes, discard the supernatant, and repeat 2 times.
[0029] (6) Finally, suspend the precipitate with 100% acetone and dry it at 55 °C. Collect the dried solid powder and treat it with α-amylase to remove starch to obtain the cell wall powder.
[0030] (8) Weigh 2 mg of the cell wall powder and add 1 mL of 0.5% ammonium oxalate. Heat in a water bath at 100 °C for 1 hour, shake intermittently to mix evenly, centrifuge, transfer the supernatant to a new tube. Add 1 mL of 0.5% ammonium oxalate to the precipitate again, heat in a water bath at 100 °C for 30 minutes, shake intermittently to mix evenly, centrifuge, and combine the two supernatants to obtain the pectin extract (a total of 2 mL).
[0031] (9) Weigh 2 mg of the cell wall powder and add 1 mL of 0.5% ammonium oxalate. Heat in a water bath at 100 °C for 1 hour and shake intermittently to mix evenly.
[0032] (10) Centrifuge the mixture at 10,000 g for 5 minutes and then transfer the supernatant to a new tube.
[0033] (11) Add 1 mL of 0.5% ammonium oxalate to the precipitate again, heat in a water bath at 100 °C for 30 minutes, and shake intermittently to mix evenly.
[0034] (12) Centrifuge the mixture at 10,000 g for 5 minutes and transfer the supernatant to a new tube. Combine the supernatants obtained from the two centrifugations to obtain the pectin extract (a total of 2 mL).
[0035] 2. Standard curve preparation (1) Dilute the 10 mg / mL GalA standard solution to the following concentrations: 0, 0.01, 0.02, 0.03, 0.04, 0.05 μg / μL.
[0036] (2) Take 100 μL of each of the above-diluted standard solutions and place them in 2 mL centrifuge tubes.
[0037] (3) Operate according to the sample treatment and determination steps.
[0038] 3. Determination of pectin content (1) Take an appropriate amount of pectin extract and place it in a 2 mL centrifuge tube.
[0039] (2) Dilute with water to 100 μL (to make its concentration approximately 0.2 - 10 μg / 100 μL; for primary cell wall starch-free AIR (Alcohol Insoluble Residue), after extraction according to the above steps, dilute 5 - 10 times).
[0040] (3) Add 0.6 mL of 0.0125 M sodium tetraborate / sulfuric acid solution (operate on ice) to the above standard solution and sample tubes respectively, and mix well.
[0041] (4) Place the tubes in a boiling water bath and heat for 5 minutes, then cool in an ice bath.
[0042] (5) Add 10 μL of 1.5 mg / ml m-hydroxybiphenyl solution (prepared with 5 mg / ml NaOH solution), mix well and shake for 5 minutes (showing a pink color), and let stand.
[0043] (6) Pipette 200 μL of the reaction solution into an ELISA plate and measure the absorbance at 520 nm.
[0044] (7) Compare the measured values with the standard curve and calculate the pectin content of the sample.
[0045] The results show dellaP The pectin content of the seedlings is 95.6 ± 2.2 μg / mg, which is about 6.5% higher than that of WT (89.7 ± 2.2 μg / mg) (attached Figure 1 )
[0046] Example 3: Determination of seed pectin content Seed pectin is divided into inner pectin mucilage and outer pectin mucilage. The determination of seed pectin content is divided into the following steps: 1. Obtaining of Arabidopsis thaliana seeds: The wild type (WT) and dellaThe mutant seeds were sown on 1 / 2 MS medium after disinfection and cultured in an artificial climate chamber at 22 °C (photoperiod set to 16 h light / 8 h dark). After maturation, the seeds were harvested and dried for use.
[0047] Accurately weigh 100 mg of mature and dry Arabidopsis seeds and put them into a 15 mL centrifuge tube.
[0048] 2. Extraction of outer mucilage: (1) Add 5 mL of distilled water.
[0049] (2) Oscillate at 220 rpm and 16 °C for 1 hour.
[0050] (3) After centrifugation, transfer the supernatant to a 50 mL centrifuge tube.
[0051] (4) Wash the precipitate once with 2 mL of distilled water and combine the supernatants to obtain the outer mucilage extract.
[0052] 3. Extraction of inner mucilage: (1) Resuspend the precipitate with 5 mL of distilled water.
[0053] (2) Perform ultrasonic treatment (200 W, about 2 minutes).
[0054] (3) After centrifugation, transfer the supernatant to the same 50 mL centrifuge tube.
[0055] (4) Wash the precipitate once with 2 mL of distilled water and combine the supernatants to obtain the inner mucilage extract.
[0056] 4. Dialysis: Combine the inner and outer layer extracts, transfer them to a dialysis bag (molecular weight cut-off 1000 Da), and dialyze against running water for 72 hours.
[0057] 5. Drying and weighing: Transfer the dialyzed extract to a new 50 mL centrifuge tube, freeze-dry it, and weigh it.
[0058] It was determined that the pectin content in wild-type (WT) seeds was approximately 98 μg / mg, dellaD the pectin content in the seeds was approximately 114 μg / mg, dellaQ the pectin content in the seeds was approximately 125 μg / mg, dellaP the pectin content in the seeds was approximately 127 μg / mg. dellaP The pectin yield was 127 ± 4.4 mg / g of seeds, a 29.7% increase compared to WT (98 ± 4 mg / g) (Appendix Figure 2 ).
[0059] Example 4: Observation and measurement of the thickness of the seed coat pectin layer 1. Sow the wild type (WT) and della mutant seeds after disinfection on 1 / 2 MS medium, and place them in an artificial climate chamber at 22 °C (photoperiod: 16 h light / 8 h dark) for cultivation. After maturity, harvest the seeds and dry them for later use.
[0060] 2. Put the dry and mature Arabidopsis seeds into a centrifuge tube, add 0.01% ruthenium red staining solution and stain for 1 hour, while shaking at 200 rpm at room temperature to remove the outer mucilage, and observe the thickness of the inner mucilage under a microscope. It was observed under the microscope that the thickness of the mucilage pectin layer released by the mutant seeds was significantly higher than that of the WT (attached Figure 3 ).
[0061] 3. Measurement and statistics.
[0062] Measure the mucilage thickness with Image J software, and the results show that dellaD the thickness of the pectin layer of the mutant seed coat is 0.10 ± 0.007 mm, which is about 28% higher than that of the WT (0.08 ± 0.006 mm). dellaQ the thickness of the pectin layer of the mutant seed coat is 0.12 ± 0.005 mm, which is about 56% higher than that of the WT. dellaP the thickness of the pectin layer of the mutant seed coat is 0.12 ± 0.006 mm, which is about 52% higher than that of the WT (attached Figure 4 ); statistical analysis shows that dellaD, dellaQ, dellaP the differences between the mutants and the WT all reached a significant level, proving that the loss of function of the della gene can significantly promote the biosynthesis of the seed coat pectin layer.
[0063] In summary, the examples of the present invention show that by simultaneously silencing multiple members of the DELLA gene family, the pectin content of Arabidopsis can be significantly increased, especially the pectin content in seeds. In particular, knocking out five genes of the Arabidopsis DELLA family simultaneously (RGA, GAI, RGL1, RGL2, RGL3) achieved the maximum increase in pectin content. This indicates that by regulating multiple members of the DELLA gene family, the pectin content of Arabidopsis can be effectively regulated.
[0064] The regulatory technology of DELLA polygenes shows great application potential in the agricultural and industrial fields. By knocking out DELLA polygenes, not only can the pectin yield be significantly increased, but also higher-quality raw materials can be provided for agricultural production. In addition, this technology also shows good universality in other plants. By silencing homologous genes of della genes, the effect of increasing pectin content can also be achieved. The increase in pectin content can not only enhance the stress resistance of plants, but also improve the overall agricultural efficiency and food security. Through this method, new raw material sources for low-cost and environmentally friendly pectin production are opened up, reducing the dependence on traditional production methods and promoting the development of green industries such as biofuels at the same time.
[0065] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts among the various embodiments, reference can be made to each other.
Claims
1. A method for increasing the pectin content of plants by regulating DELLA genes, characterized in that Enhancing pectin content by silencing more than two members of a plant DELLA gene family.
2. The method for increasing the pectin content of plants by regulating the DELLA gene according to claim 1, wherein The plant is Arabidopsis thaliana.
3. The silence according to claim 1, characterized in that Genes with redundant functions in the DELLA family are used as target genes for knockout.
4. According to claim 1, DELLA a gene family member, characterized in that The gene described is RGA (AT2G01570), GAI(AT1G14920), RGL1(AT1G66350), RGL2(AT3G03450), RGL3(AT5G17490) .
5. The method according to claim 1, wherein: The members of the DELLA gene family knocked out are RGA and GAI.
6. The method according to claim 1, wherein: The members of the DELLA gene family knocked out are RGA, GAI, RGL1 and RGL2.
7. The method according to claim 1, wherein: The members of the DELLA gene family knocked out are RGA, GAI, RGL1, RGL2 and RGL3.
8. The method according to claim 3, wherein: The Arabidopsis mutant plants are obtained by knocking out the DELLA gene, which is achieved by the following methods: Use EMS mutagenesis to construct the RGA gene mutant rga-24; Use Ds transposon insertion technology to construct mutants of RGA (rga-t2), GAI (gai-t6), RGL1 (rgl1-1), RGL2 (rgl2-1); Use T-DNA insertion technology to construct the RGL3 gene mutant rgl3-1; Obtain multi-gene mutant plants through genetic hybridization.
9. The Arabidopsis mutant plant according to claim 8, wherein: The plant is a double mutant dellaD (rga-24 x gai-t6) , quadruple mutant dellaQ (rga-t2 x gai-t6 x rgl1-1 x rgl2-1) or quintuple mutant dellaP (rga-t2 x gai-t6 x rgl1-1 x rgl2-1 x rgl3-1) .
10. Use of the Arabidopsis mutant plant according to claim 9 in pectin extraction, characterized in that: The pectin content of the seeds of the plant is increased by 29.7% compared with the wild type, and the pectin layer thickness is increased by more than 50%.