Application of abscisic acid in increasing lettuce latex yield

Abstract

This invention relates to the field of biotechnology, specifically to the application of abscisic acid (ABA) in increasing lettuce latex yield. This invention uses ABA as the active ingredient, acting on lettuce plants to significantly increase the yield of lettuce latex and rubber hydrocarbons, while further increasing or significantly increasing the content of sucrose, acetyl-CoA, isopentenyl pyrophosphate, and rubber hydrocarbons in lettuce. This invention uses ABA to increase the yield of rubber hydrocarbons in dry latex, and also increases the content of rubber hydrocarbons per unit weight of dry latex. This indicates that ABA treatment can produce high-quality latex with superior quality and high yield, possessing higher economic value. The application of ABA is simple and requires only a small amount; a relatively low concentration of ABA can significantly increase the yield of lettuce latex, sucrose, acetyl-CoA, isopentenyl pyrophosphate, and rubber hydrocarbons. It is low-cost, effective, and does not cause new toxic side effects to the environment, making it suitable for widespread application.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of abscisic acid in increasing the yield of lettuce latex. Background Technology

[0002] Lettuce (Lactuca sativa), as a self-pollinating crop, has significant advantages such as a short growth cycle (only 60-90 days from sowing to harvest), a clear genetic background, and mature genetic transformation technology. Lettuce latex is a secondary metabolite rich in natural active ingredients. These active ingredients (such as sesquiterpene lactones and triterpenoids) exhibit anti-inflammatory, analgesic, antibacterial, and antioxidant pharmacological properties, and can be used to prepare drugs for treating inflammation, pain, and infectious diseases, as well as for the development of medical dressings. Certain components in lettuce latex (such as cucurbitacin) have repellent effects on pests and pathogens, and can be used as raw materials for green pesticides. Exogenous application of latex extracts may activate crop defense mechanisms, improving plant disease resistance and drought resistance. Lettuce latex contains rubber precursors such as polyisoprene, which can serve as a supplementary source of natural rubber for the manufacture of elastic materials (such as gloves and tubing). The antioxidant components in latex can extend the shelf life of food or be used as functional ingredients in health foods.

[0003] Despite the broad application prospects of lettuce latex, its natural yield is extremely low, extraction costs are high, and there is a lack of efficient yield-increasing technologies, which limits its commercial application. Therefore, developing new biotechnologies or cultivation methods to increase lettuce latex yield is key to promoting its industrialization. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide the application of abscisic acid in increasing the yield of lettuce latex. The present invention provides a method for increasing the yield of lettuce latex by spraying the leaves and stems of lettuce plants with abscisic acid. The method is simple to operate, low in cost, and suitable for promotion and application to improve economic benefits.

[0005] This invention provides the use of abscisic acid in increasing the latex yield of lettuce and / or in preparing formulations that increase the latex yield of lettuce.

[0006] Compared with other plant growth hormones, the abscisic acid provided by this invention is more suitable for improving the gum production performance of lettuce. The two are highly compatible, and the fresh latex produced has a high content of effective components, thus achieving better technical results.

[0007] In some specific embodiments, increasing the latex yield of lettuce means increasing the latex yield in the lettuce stem.

[0008] This invention provides the application of abscisic acid in increasing latex yield in lettuce stems and in at least one of the following (1) to (4):

[0009] (1) Increase the content and / or yield of rubber hydrocarbons in lettuce stems;

[0010] (2) Increase the sucrose content in the stems and leaves of lettuce;

[0011] (3) Increase the content of acetyl-CoA in lettuce leaves;

[0012] (4) Increase the content of isopentenyl pyrophosphate in lettuce leaves and stems.

[0013] This invention provides the use of abscisic acid in the preparation of formulations that increase latex yield in lettuce stems and at least one of the following (5) to (8):

[0014] (5) Increase the content and / or yield of rubber hydrocarbons in lettuce stems;

[0015] (6) Increase the sucrose content in the stems and leaves of lettuce;

[0016] (7) Increase the content of acetyl-CoA in lettuce leaves;

[0017] (8) Increase the content of isopentenyl pyrophosphate in lettuce leaves and stems.

[0018] This invention provides a method for increasing the yield of lettuce latex, including treating lettuce with abscisic acid.

[0019] This invention provides a method for increasing the yield of lettuce latex while simultaneously improving at least one of the following (9) to (12), characterized in that:

[0020] (9) The content and / or yield of rubber hydrocarbons in the stems of lettuce;

[0021] (10) Sucrose content in the stems and leaves of lettuce;

[0022] (11) The content of acetyl-CoA in lettuce leaves;

[0023] (12) The content of isopentenyl pyrophosphate in lettuce leaves and stems;

[0024] The method includes treating lettuce with abscisic acid.

[0025] In this invention, the content refers to the percentage of a substance in a unit weight of fresh or dry latex, and the yield refers to the weight of the substance.

[0026] In some embodiments, the method involves spraying lettuce leaves and / or stems with a reagent containing abscisic acid.

[0027] In some embodiments, the working concentration of the abscisic acid is 50~150 μM.

[0028] In some specific embodiments, the working concentration of the abscisic acid is 100 μM.

[0029] In some embodiments, the reagent further includes ethanol at a working concentration of 0.01 vol% to 1 vol%.

[0030] In some embodiments, the reagent further includes ethanol at a working concentration of 0.1 vol%.

[0031] In some embodiments, increasing lettuce latex yield means increasing the latex yield in the lettuce stem.

[0032] Compared with the prior art, the beneficial effects of the present invention include:

[0033] This invention uses abscisic acid (ABA) as the active ingredient, which acts on lettuce plants to significantly increase the yield of lettuce latex and rubber hydrocarbons. It also further increases or significantly improves the content of sucrose, acetyl-CoA, isopentenyl pyrophosphate, and rubber hydrocarbons in lettuce. Compared with indoleacetic acid, the ABA provided by this invention is more suitable for improving the latex production performance of lettuce, and the two are highly compatible. Furthermore, compared with the commonly used latex-promoting hormone ethephon, the ABA used in this invention can increase the yield and content of rubber hydrocarbons in dry latex. This indicates that ABA treatment can produce high-quality latex with superior quality and high yield, possessing higher economic value. In application, ABA is simple to use and requires only a small amount. A relatively low concentration of ABA can significantly increase the yield and content of lettuce latex, sucrose, acetyl-CoA, isopentenyl pyrophosphate, and rubber hydrocarbons, resulting in low cost, good effect, and no new toxic side effects on the environment, making it suitable for widespread application. Attached Figure Description

[0034] Figure 1 The results of latex yield testing in lettuce stems are shown.

[0035] Figure 2 The results of the latex content test in lettuce stems are shown.

[0036] Figure 3 This shows the test results of rubber hydrocarbon content in latex from lettuce stems;

[0037] Figure 4 This shows the test results for sucrose content in lettuce leaves;

[0038] Figure 5 This shows the results of the determination of sucrose content in latex in lettuce stems;

[0039] Figure 6 The results of the determination of acetyl-CoA content in lettuce leaves are shown.

[0040] Figure 7 This shows the results of the determination of acetyl-CoA content in latex in lettuce stems;

[0041] Figure 8 The results of IPP content determination in lettuce leaves are shown.

[0042] Figure 9 The results of IPP content determination in lettuce stem latex are shown. Detailed Implementation

[0043] This invention provides the application of abscisic acid in increasing lettuce latex yield. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0044] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0045] Example 1: Effect of abscisic acid on the yield of lettuce latex

[0046] 1. Hormone treatment methods

[0047] 1.1 Preparation and treatment methods of abscisic acid

[0048] A 100 μM / L abscisic acid solution (ABA, purchased from Shanghai Maclean, catalog number 21293-29-8) was prepared. 30 mg / L indoleacetic acid and 180 μM / L ethephon were prepared as controls. 0.1% (v / v) ethanol was used for the solutions and blank control treatment (CK).

[0049] 1.2 Hormone Treatment Methods

[0050] 2. Germinate lettuce seeds (variety pi, from Rr. Dae-Kyun Ro's Lab) in sterilized dishes with moistened absorbent paper at the bottom, keeping the solution moist. After germination, sow the seeds in the original pots pre-filled with sterilized growing medium (peat, vermiculite, and coconut coir), controlling the temperature (26 °C daytime, 22 °C nighttime). Simulate natural light using red, blue, and incandescent light, with a 16-hour light cycle. During the lettuce's growth and development, apply a water-soluble fertilizer (Hua Wu Que, product number: LS-HWQ-9528) every two weeks. When the lettuce reaches bolting stage after approximately two months, spray each plant with a sprayer, applying the hormone solution three times to both the leaves and stems. Simultaneously, control plants are treated with a 0.1% (v / v) ethanol solution using the same method. Ten plants are treated with each hormone, applied every 12 hours, for a total of four treatments and 48 hours. Latex and leaves are collected immediately afterward for data measurement. Latex should be collected from the middle of the stem. For leaves, collect the 6th to 8th leaves from the top.

[0051] 2. Determination Method

[0052] 2.1 Latex Production

[0053] Make a 0.5-1 cm incision at a 45° angle to the horizontal line on the lettuce stem using a blade to facilitate the smooth flow of latex, allowing it to drip into a pre-weighed centrifuge tube (m0). Repeat the incision process multiple times until no more latex flows out of the lettuce stem. Weigh the centrifuge tube containing fresh latex and record the weight as m1. Discount the weight of the empty centrifuge tube; m1 - m0 represents the yield of fresh latex per plant (g). Each treatment is repeated three times. Results are shown below. Figure 1 As shown.

[0054] 2.2 Dry content (total solids content)

[0055] Take the centrifuge tube containing fresh latex that was weighed as described in "2.1", record the weight of the fresh latex in the centrifuge tube as m1-m0, and then place it in an oven at 40℃ for 48 hours to remove moisture until the weight of the dried latex no longer changes. Record the weight as m2. Subtract the weight of the empty centrifuge tube from this weight to obtain the weight of the dried latex as m2-m0. That is, dry treatment, with 6 replicates for each treatment, and the results are as follows: Figure 2 As shown.

[0056] 2.3 Rubber hydrocarbon content and yield

[0057] Weigh the dry gum of each lettuce plant (approximately 15-20 mg) into a pre-weighed glass bottle. Add 5 mL of ddH2O to fully dissolve the water-soluble components in the dry gum. Centrifuge at 5000 rpm for 5 min at room temperature, discard the supernatant, and repeat three times until the water-soluble components are completely clear. Allow the dissolved dry gum to evaporate naturally. Add 5 mL of acetone and dissolve for 1 h. After dissolution, centrifuge at 5000 rpm for 5 min at room temperature, discard the supernatant, and repeat three times. Place the glass bottle containing the acetone-dissolved product in a fume hood to air dry naturally. Add 5 mL of tetrahydrofuran (THF) and dissolve at 70°C for 6 h, following the same procedure as with the acetone. Transfer the supernatant to a pre-weighed clean glass bottle and record the weight m0. Repeat three times. Then weigh the total weight m1 of the glass bottle and the THF-dissolved product. Subtract the weight of the glass bottle and record the weight m1-m0 of the THF-dissolved product, which is the yield of lettuce rubber hydrocarbons. (m1-m0) / m×100% is the hydrocarbon content of the rubber, and the result is as follows: Figure 3 As shown.

[0058] 2.4 Determination of sucrose content

[0059] 2.4.1 Determination of sucrose content in leaves

[0060] Weigh approximately 0.2 g of hormone-treated leaves (removing petioles), grind them in liquid nitrogen, add 1 mL of distilled water and grind again. Transfer the entire crude extract to a new centrifuge tube and centrifuge at 12,000 rpm at room temperature. Collect the supernatant for analysis. Instruments used: Full-wavelength microplate reader (Thermo Fisher Scientific); Reagents used: Sucrose (hexokinase method) assay kit (G0545W, Gree). Sucrose is converted to glucose by a specific enzyme, and then further converted to NADP by a hexokinase complex enzyme. + The sucrose content was calculated by measuring the increase in NADPH at 340 nm after reconstitution to NADPH. One control tube (M, N) and one assay tube (M, N) were set up for each measurement. All measurements were performed in 96-well plates. Assay tube M contained 10 μL of sample, 10 μL of reagent I, 10 μL of reagent II, and 160 μL of reagent III; assay tube N contained 10 μL of sample, 10 μL of reagent II, and 170 μL of reagent III; control tube M contained 10 μL of reagent I, 10 μL of reagent II, and 170 μL of reagent III; and control tube N contained 10 μL of reagent II and 180 μL of reagent III. The reagents in each assay tube were mixed thoroughly, incubated at 30°C for 5 min, and the A1 value was read at 340 nm. After the measurement, 10 μL of reagent IV was added to each of the test tubes M, N, control tube M, and control tube N. The mixture was thoroughly mixed, incubated at 30°C for 30 min, and the A2 value of each tube was read at 340 nm. The calculation formula is as follows:

[0061] ΔA glucose = (A2 - A1) / (test tube N - (A2 - A1) / control tube N (2-1)

[0062] ΔAsucrose = [(A2 - A1)test tube M - (A2 - A1)control tube M] - Δglucose (2-2)

[0063] Sucrose content (mg / g FW) = 2.1733 * ΔA sucrose / W × D (2-3)

[0064] In the formula, W represents the sample mass in g; D represents the dilution factor, with undiluted being 1. The result is as follows: Figure 4 As shown.

[0065] 2.4.2 Determination of latex sucrose content

[0066] The hormone-treated latex was directly dropped into a centrifuge tube containing distilled water and centrifuged at 12,000 rpm at room temperature. The supernatant was collected for analysis. The determination and calculation methods were the same as in 2.4.1, and the results are as follows: Figure 5 As shown.

[0067] 2.5 Determination of Acetyl-CoA Content

[0068] 2.5.1 Leaf Acetyl-CoA

[0069] Weigh approximately 0.2 g of hormone-treated leaves (remove petioles), grind them in liquid nitrogen, add 1 mL of acetyl-CoA extraction buffer, grind again, and transfer the entire crude extract to a new centrifuge tube. Centrifuge at 12,000 rpm at room temperature, and collect the supernatant for testing. Instruments used: Full-wavelength microplate reader (Thermo Fisher Scientific); Reagents used: Acetyl-CoA ELISA kit (Vancovel) F7921-A. Add 10 μL of sample and 40 μL of sample diluent to each well, and incubate at 37 ℃ for 30 min. After incubation, discard the liquid and wash with washing buffer for 30 s, repeating 5 times, and then spin dry. Add 50 μL of acetyl-CoA enzyme-labeled reagent to each well, except for the blank wells. Incubate at 37 ℃ for 30 min, and wash as above. After washing, add 50 μL of chromogenic reagent A and 50 μL of chromogenic reagent B to each well, vortex to mix, and incubate at 37 ℃ in the dark for 10 min. The reaction was then terminated by adding 50 μL of stop solution to each well. The absorbance of each well was measured at 450 nm using a blank well as the zeroing function. The OD value of the sample was substituted into the standard equation to calculate the acetyl-CoA concentration of the sample, as shown in the figure. Figure 6 As shown.

[0070] 2.5.2 Latex Acetyl-CoA

[0071] The hormone-treated latex was directly added dropwise to a centrifuge tube containing the extract, centrifuged at 12,000 rpm at room temperature, and the supernatant was collected for analysis. The determination and calculation methods are the same as in 2.5.1, and the results are as follows. Figure 7 As shown.

[0072] 2.6 IPP content determination

[0073] 2.6.1 Determination of IPP content in leaves

[0074] Weigh approximately 0.2 g of hormone-treated leaves (remove petioles), grind them in liquid nitrogen, add 1 mL of acetyl-CoA extraction buffer and grind again. Transfer the entire crude extract to a new centrifuge tube and centrifuge at 12,000 rpm at room temperature. Collect the supernatant for testing. Instruments used: Full-wavelength microplate reader (Thermo Fisher Scientific); Reagents used: Isoprene pyrophosphate (IPP) ELISA kit 96T (competitive method) (Vancovel) F0082-OA. Add 10 μL of sample, 40 μL of sample diluent, and 50 μL of IPP enzyme-labeled reagent to each sample well, except for the blank wells. Incubate at 37℃ for 1 h. After incubation, discard the liquid and wash with washing buffer for 30 s, repeating 5 times. Shake dry the liquid. After washing, add 50 μL of chromogenic reagent A and 50 μL of chromogenic reagent B to each well, vortex to mix, and incubate at 37℃ in the dark for 10 min. Then add 50 μL of stop solution to each well to terminate the reaction. Zero the sample using a blank well and measure the absorbance at 450 nm. Then, substitute the sample OD value into the standard equation to calculate the sample IPP (isopentene pyrophosphate) concentration. The results are as follows. Figure 8 As shown.

[0075] 2.6.2 Determination of Latex IPP Content

[0076] The hormone-treated latex was directly added dropwise to a centrifuge tube containing the extract, centrifuged at 12,000 rpm at room temperature, and the supernatant was collected for analysis. The determination and calculation methods are the same as in 2.6.1, and the results are as follows. Figure 9 As shown.

[0077] The biosynthesis of lettuce latex follows the following metabolic pathway:

[0078] Sucrose → Acetyl-CoA → IPP (isopentyl pyrophosphate) → Rubber hydrocarbons → Latex dry content → Latex yield. Increased levels of any intermediate product in this pathway can promote the eventual accumulation of latex.

[0079] Combination Figures 1-9 This invention significantly improves the yield and synthesis efficiency of lettuce latex by optimizing plant hormone treatment strategies. Specific conclusions are as follows:

[0080] 1. Abscisic acid treatment significantly improves the quality of fresh latex.

[0081] Compared with the control group and indoleacetic acid treatment, abscisic acid treatment significantly increased the yield of fresh lettuce latex. Figure 1 The total solids content also increased slightly. Figure 2 The content of rubber hydrocarbons in the abscisic acid-treated group was significantly higher than that in the ethephon-treated group, but slightly lower than that in the ethephon-treated group. Further comparison of the rubber hydrocarbon content per unit weight of dry latex revealed that the abscisic acid-treated group had a significantly higher rubber hydrocarbon content than the ethephon-treated group and other groups. Figure 3 Fresh latex contains both water and solids, with the solids including rubber hydrocarbons, proteins, sugars, inorganic salts, and all other non-water components. Figures 1-3 It can be seen that although the yield of fresh latex and the total solids content per unit weight of lettuce treated with abscisic acid were lower than those of the ethephon-treated group, the content of rubber hydrocarbons in the dry latex of lettuce treated with abscisic acid was significantly increased, and the rubber hydrocarbons per unit weight of dry latex were highly enriched. This means that the relative content of water and other non-rubber solids (such as proteins and sugars) in the fresh latex after abscisic acid treatment was lower. The level of rubber hydrocarbon content is a core indicator for measuring the purity and performance of rubber materials, and directly determines the main properties of natural rubber. The significant increase in the rubber hydrocarbon content in the dry latex of lettuce treated with abscisic acid precisely reflects the significant improvement in its latex quality. The total yield of rubber hydrocarbons in the dry latex after treatment with the three hormones is further tested, as shown in Table 1 below. The results also show that the latex produced by lettuce treated with abscisic acid has a higher total rubber hydrocarbon yield. The above data fully demonstrates that lettuce treated with abscisic acid can produce high-quality latex with excellent quality and high yield, and has higher economic value.

[0082] Table 1 Comparison of rubber hydrocarbon yield under different treatments

[0083] Grouping Rubber hydrocarbon production (mg / plant) abscisic acid 2.27 Indoleacetic acid 0.49 Ethephon 0.92

[0084] 2. Abscisic acid treatment promotes the accumulation of key precursors in latex synthesis.

[0085] Abscisic acid treatment significantly enhances the supply of precursor substances by regulating the latex synthesis pathway through multiple targets:

[0086] sucrose( Figure 4 and Figure 5 The increased sucrose content in the stems and leaves provides a sufficient carbon source for the production of acetyl-CoA abscisic acid.

[0087] IPP (isopentene pyrophosphate), Figures 8-9 The IPP content in leaves and stems is significantly increased, directly promoting the extension of the rubber hydrocarbon chain.

[0088] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of abscisic acid in increasing latex yield in lettuce.

2. Application of abscisic acid in the preparation of formulations that increase the latex yield of lettuce.

3. The application according to claim 1 or 2, characterized in that, The improvement of latex yield in lettuce refers to increasing the latex yield in the lettuce stem.

4. Abscisic acid increases latex yield in lettuce stems while simultaneously improving at least one of the following applications (1) to (4): (1) The content and / or yield of rubber hydrocarbons in lettuce stems; (2) Sucrose content in the stems and leaves of lettuce; (3) The content of acetyl-CoA in lettuce leaves; (4) The content of isopentenyl pyrophosphate in the leaves and stems of lettuce.

5. The use of abscisic acid in the preparation of formulations that increase latex yield in lettuce stems while simultaneously improving at least one of the following (5) to (8): (5) The content and / or yield of rubber hydrocarbons in the stems of lettuce; (6) Sucrose content in the stems and leaves of lettuce; (7) The content of acetyl-CoA in lettuce leaves; (8) The content of isopentenyl pyrophosphate in the leaves and stems of lettuce.

6. A method for increasing lettuce latex yield, characterized in that, This includes treating lettuce with abscisic acid.

7. A method for increasing the yield of lettuce latex while simultaneously improving at least one of the following (9) to (12), characterized in that, in: (9) The content and / or yield of rubber hydrocarbons in the stems of lettuce; (10) Sucrose content in the stems and leaves of lettuce; (11) The content of acetyl-CoA in lettuce leaves; (12) The content of isopentenyl pyrophosphate in lettuce leaves and stems; The method includes treating lettuce with abscisic acid.

8. The method according to claim 6 or 7, characterized in that, The method involves spraying lettuce leaves and / or stems with a reagent containing abscisic acid.

9. The method according to claim 8, characterized in that, The working concentration of the abscisic acid is 50~150μM.

10. The method according to claim 8, characterized in that, The reagent also includes ethanol with a working concentration of 0.01 vol% to 1 vol%.

11. The method according to any one of claims 6 or 7, characterized in that, The increase in lettuce latex yield refers to increasing the latex yield in the lettuce stem.

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