Method for increasing oil accumulation in plants

Abstract

The present application discloses a method for increasing the oil accumulation in plants, aiming to solve the problem that there is a lack of new effective technical approaches for improving oil accumulation in current plants. The present application has found through research that endoplasmic reticulum stress induced by regulating chemical substances (tunicamycin, dithiothreitol, thapsigargin) or gene ( lew1 gene or its homologous gene) mutation can increase the oil content in plant tissues and seeds; increasing the oil accumulation in plants can effectively increase the oil content of oil crops or other plants, effectively increase the oil production of plants, and is of great significance to the production of vegetable oil.

Description

Technical Field

[0001] This application relates to the technical field of plant oil biosynthesis, and specifically relates to a method for increasing the oil accumulation in plants. Background Art

[0002] Oil is a lipid formed by fatty acids and glycerol, namely triacylglycerol. Vegetable oil is extracted from plant tissues (roots, stems, leaves or seeds, usually seeds). Increasing the oil content in plants can effectively improve the yield of vegetable oil, which is of great significance for vegetable oil production.

[0003] The synthesis of plant oil starts from chloroplasts; fatty acids are de novo synthesized in chloroplasts and then transported to the endoplasmic reticulum in the form of acyl-CoA. In the endoplasmic reticulum, fatty acids and glycerol condense to form triacylglycerol (TAG). Previous studies on oil synthesis mainly focused on the biosynthesis of fatty acids, the transport of fatty acids and the biosynthesis process of TAG. Increasing the accumulation of plant oil also mostly focused on the above-mentioned links.

[0004] Tunicamycin (TM) is a chemical substance that interferes with protein glycosylation and induces endoplasmic reticulum stress by interfering with protein glycosylation. In addition, LEW1 encodes a cis-isoprenyltransferase that participates in the synthesis of dolichol, a sugar transporter carrier involved in protein glycosylation. 1ew1 The mutation leads to impaired synthesis of dolichol, a decrease in protein glycosylation level, and thus induces endoplasmic reticulum stress. The synthesis of TAG occurs in the endoplasmic reticulum, and the effect of endoplasmic reticulum stress on TAG synthesis has not been reported.

[0005] The information disclosed in this background art section is only used to deepen the understanding of the background art of the present disclosure, and should not be regarded as an admission or any form of implication that this information constitutes the prior art known to those skilled in the art. Summary of the Invention

[0006] The inventors found through research that endoplasmic reticulum stress induces an increase in the TAG content in plant plants and seeds; and the methods for inducing endoplasmic reticulum stress in plants include: ① through chemical regulation, such as TM, dithiothreitol (DTT) or thapsigargin (TG), to disrupt the protein glycosylation level or the redox level of cells and trigger endoplasmic reticulum stress; ② through gene editing or other technologies to mutate a certain gene (such as LEW1 or its homologous gene) to disrupt the ecological balance of the endoplasmic reticulum and induce endoplasmic reticulum stress. Based on the above methods to induce endoplasmic reticulum stress, the TAG content in plants can be increased.

[0007] According to one aspect of the present disclosure, the present application provides a method for increasing the oil accumulation in plants by inducing endoplasmic reticulum stress in plants.

[0008] In some embodiments of the present disclosure, the method for inducing endoplasmic reticulum stress in plants is at least one of chemical substance regulation, lew1 gene or homologous gene mutation.

[0009] In some embodiments of the present disclosure, the method of chemical substance regulation is: using a chemical substance to disrupt the protein glycosylation level or the redox level in cells to trigger endoplasmic reticulum stress.

[0010] In some embodiments of the present disclosure, the chemical substance is at least one of tunicamycin, dithiothreitol, and thapsigargin.

[0011] In some embodiments of the present disclosure, the lew1 gene or homologous gene mutation causes impaired synthesis of dolichol, a decrease in protein glycosylation level, and induces endoplasmic reticulum stress.

[0012] In some embodiments of the present disclosure, the plant is any one of Arabidopsis thaliana, wheat, corn, rice, peanut, soybean, rapeseed, sesame, cotton, tobacco, and algae.

[0013] According to another aspect of the present disclosure, there is provided a method for cultivating high-oil plant varieties, inducing lew1 gene or homologous gene mutation.

[0014] One or more technical solutions provided in the embodiments of the present application have at least any one of the following technical effects or advantages:

[0015] Based on the endoplasmic reticulum stress induced by chemical substance regulation or / and gene mutation, the oil content in plant tissues and seeds can be increased; increasing the oil accumulation in plants can effectively increase the oil content of oil crops or other plants, and thus effectively increase the oil production of plants, which is of great significance for vegetable oil production. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a comparative graph of lipid liquid chromatography-mass spectrometry analysis of the whole Arabidopsis thaliana plants after TM treatment and lew1 mutation in one embodiment of the present application; CK is the untreated control, and TM is the TM treatment; "*" indicates that the t-test analysis of the two groups of data shows significant differences, p and the p value is less than 0.05.

[0017] Figure 2 This is for one embodiment of the present application after TM treatment or lew1Fluorescent quantitative analysis of the expression of genes induced by mutations in the endoplasmic reticulum stress response; "*" indicates that the t-test analysis of the two groups of data shows significant differences, p and the value is less than 0.05.

[0018] Figure 3 This is a comparative TLC analysis diagram of TAG in Arabidopsis seeds after TM treatment in an embodiment of the present application; among them, CK is the untreated control, and TM is the TM treatment; the displayed bands are the results of the TAG thin-layer chromatography; the numbers marked below the figure are the quantitative results of the gray-scale analysis of the thin-layer chromatography bands using ImageJ software.

[0019] Figure 4 In an embodiment of the present application lew1 This is a TLC analysis map of TAG in mutant seeds; 60 seeds are added with 300 μL of lipid extraction solution; an equal volume of lipid extraction solution is loaded onto a silica gel plate for TLC analysis; the displayed bands are the results of the TAG thin-layer chromatography; the numbers marked below the figure are the quantitative results of the gray-scale analysis of the thin-layer chromatography bands using ImageJ software.

[0020] Figure 5 In an embodiment of the present application, adding FFA helps lew1 to analyze the phenotype of adapting to endoplasmic reticulum stress. Specific embodiments

[0021] Except for the lipid liquid chromatography-mass spectrometry analysis which has only one biological replicate, other experiments have at least three biological replicates. The main experimental methods, reagents, and equipment involved in the following embodiments are described as follows.

[0022] 1. Seed germination treatment and tunicamycin stress treatment

[0023] Take the seeds into a 1.5 mL EP tube, add 1 mL of the prepared seed disinfectant solution (0.5% NaClO and 0.01% Triton), mix well and let stand for 15 min, shaking constantly during this period. Wash with sterilized ddH2O at least 5 times, and then sow them on 1 / 2MS and the medium containing TM or FFA. After vernalization at 4°C for three days, place them in a light incubator to grow, and the photoperiod cycle is 16 h of light and 8 h of darkness.

[0024] 2. RNA extraction and fluorescent quantitative PCR method

[0025] Total RNA of whole seedlings was extracted using the TRIzol method (Life, Invitrogen). 2 μg of RNA was reverse transcribed into cDNA (Hifair® Ⅱ 1st Strand cDNA Synthesis SuperMix for qPCR, gDNA digester plus, 11123ES60). The qPCR procedure was performed using a Thermo Scientific StepOnePlus real-time PCR detection system. The qPCR parameters were as follows: 5 min at 95 °C, 40 cycles of 10 s at 95 °C and 30 s at 60 °C, followed by melting curve analysis. Actin was used as a control.

[0026] 3. Lipid extraction and thin-layer chromatography analysis

[0027] (1)Approximately 60 mg of Arabidopsis thaliana plant leaves at 10 days old were placed in a 1.5 mL EP tube and quickly stored in liquid nitrogen.

[0028] (2)300 μL of lipid extraction buffer (methanol: chloroform: formic acid = 2:1:0.1, V / V / V) was added to each sample and vigorously shaken until the leaves became completely white and transparent.

[0029] (3)After adding 150 μL of 0.2 M H3PO4 - 1 M KCl solution, it was quickly mixed until the liquid turned milky white.

[0030] (4)Centrifuge at 12,000 rpm for 1 min at room temperature, and the lower layer liquid is the extracted lipids.

[0031] (5)Prepare the developing solution. For separating neutral lipids, it is hexane: ether: acetic acid (70:30:1, V / V / V).

[0032] (6)Draw a line with a pencil 1 cm from the bottom of the TLC plate (S1250). Spot the samples every 1 cm, air dry, and place it in a closed chromatography tank containing the developing solution. Pre-saturate for more than half an hour, taking care to avoid the TLC plate contacting the developing solution.

[0033] (7)Put the TLC plate into the developing solution. Take out the TLC plate when the developing solution moves to the topmost position, and place the TLC plate in a fume hood until it is completely dry.

[0034] (8)Carbohydrate treatment: Evenly spray the TLC plate with 5% sulfuric acid ethanol solution, place it in a fume hood to dry, then develop in an oven (105 °C) and take pictures.

[0035] 4. Method of lipid extraction and liquid chromatography - mass spectrometry (LCMS)

[0036] Lipid extraction was performed according to the aforementioned protocol with slight modifications. Ten-day-old seedlings were weighed and inactivated with hot isopropanol. After inactivation, the samples were extracted with a solvent containing chloroform:methanol:300 mM ammonium acetate (30:41.5:3.5) (v / v / v), and incubated with shaking at 150 YPM at room temperature for 24 h. Then the samples were centrifuged to obtain the supernatant. The inactivation and extraction steps were repeated once. The lipid supernatants were pooled and dried in a rotary vacuum dryer. The lipid extracts were stored at -80 °C for use.

[0037] All lipidomic analyses were performed at Lipid All Technologies as described previously. For the normal-phase analysis of polar lipids, a Phenomenex Luna 3μm silica column (inner diameter 150×2.0 mm) was used to separate individual species under the following conditions: mobile phase A (chloroform:methanol:ammonium hydroxide, 89.5:10:0.5) and mobile phase B (chloroform:methanol:ammonium hydroxide:water, 55:39:0.50:5.5). Four replicates were taken for all materials used in lipid liquid chromatography-mass spectrometry.

[0038] To better understand the technical solution of the present application, the above technical solution will be described in detail below in conjunction with the accompanying drawings of the specification and specific embodiments.

[0039] Example 1: Verification test for the increase in TAG content in Arabidopsis thaliana induced by tunicamycin

[0040] Arabidopsis thaliana seedlings grown for 10 days were sprayed with 5 μg / mL tunicamycin (TM), and after 8 h of treatment, lipids were extracted from the whole plants for liquid chromatography-mass spectrometry analysis. The results showed that compared with the condition without TM treatment, the content of TAG increased by 51-94% after TM treatment ( Figure 1 A). After 5 h of TM treatment, fluorescence quantitative PCR showed that the expression levels of the endoplasmic reticulum stress response genes Bip2, bZip28, and bZip60 were greatly induced ( Figure 2 ), indicating that TM treatment caused the plants to be under endoplasmic reticulum stress conditions, and the endoplasmic reticulum stress response genes in the cells were induced to express. The results showed that by spraying the plant with the endoplasmic reticulum stress inducer TM, endoplasmic reticulum stress could be induced, and the content of TAG in the plant could be increased.

[0041] Meanwhile, wild-type Arabidopsis thaliana plants at the full flowering stage were sprayed with 5 μg / mL TM twice (with a one-day interval), and the harvested seeds were used to extract lipids, and thin-layer chromatography analysis was performed on the lipids. The results showed that after TM treatment, the TAG content in the seeds was about 23% higher than that in the untreated seeds ( Figure 3). Considering the increase in TAG content in the whole plant, it was found that spraying TM not only increased the TAG content in the plant but also effectively increased the TAG content in the seeds.

[0042] Example 2: lew1 Verification test on the increase of TAG content induced by mutation in Arabidopsis

[0043] In addition to inducing endoplasmic reticulum stress by spraying chemicals on plants, mutating the dolichol synthase LEW1, a sugar transporter for protein glycosylation, can also induce endoplasmic reticulum stress. In a previous study by the applicant, a lew1 mutant was obtained through EMS mutagenesis screening. Due to impaired protein glycosylation lew1 the plants were constantly under endoplasmic reticulum stress, and the expression levels of endoplasmic reticulum stress response genes Bip2 , bZip28 and bZip60 were also higher in the absence of TM treatment than in the wild type ( Figure 2 ). Lipids from the whole plant of the 1ew1 mutant were extracted for liquid chromatography-mass spectrometry analysis. The results showed that 1ew1 the TAG content in the mutant was 1.845 times that of the wild type, with an average increase in TAG content of 84.5% ( Figure 1 A). At the same time, lipids from the seeds of the lew1 mutant were also extracted and analyzed by thin-layer chromatography. The thin-layer chromatography analysis of lipids showed that lew1 the TAG content in the mutant seeds was also significantly higher than that in the wild type seeds ( Figure 4 ), indicating that endoplasmic reticulum stress induced by gene mutation can also effectively increase the TAG content in plant seeds.

[0044] Similarly, plant lipids were extracted for liquid chromatography-mass spectrometry analysis. The results showed that in addition to the increase in TAG content after TM treatment, the contents of diacylglycerol (DAG), free fatty acids (FFA), and membrane lipids (phosphatidylcholine, PC; phosphatidylethanolamine, PE; phosphatidylserine, PS) also increased ( Figure 1 B, 1C, 1D, 1E, and 1F). Adding FFA to the Arabidopsis culture medium was used to simulate the effect of increased TAG on plant growth. lew1 Due to impaired protein glycosylation resulting in sensitivity to TM, the phenotype of sensitivity to TM was partially restored in the culture medium supplemented with FFA ( lew1 ). Figure 5). Exogenous addition of FFA to simulate the increase in TAG can help plants adapt to ER stress and the increase in the content of membrane lipid components under ER stress, indicating that the accumulation of TAG under ER stress prepares for the increased membrane lipid demand under ER stress and is a response mechanism for plants to adapt to ER stress.

[0045] Meanwhile, it was also found in the study that lew1 The mutant plants were short, the siliques were small, and the number of grains per plant was small; therefore, knocking out LEW1 or its homologous genes by gene editing or other knockout methods to increase the oil content of plants may affect the yield in actual production. However, regulating the oil content in plant tissues or seeds by chemicals such as tunicamycin has no effect on yield production.

[0046] The above examples provide two methods (chemical regulation and lew1 gene mutation) to induce ER stress in plants, both of which can effectively increase the content of TAG in plant tissues and seeds. It should be particularly noted that all methods of chemical regulation or gene editing mutation that disrupt the ER ecological balance can increase the content of plant TAG by inducing ER stress and fall within the scope of the inventive concept of the present invention; in addition, the above examples were based on Arabidopsis thaliana as a model plant. Obviously, in other plants including wheat, corn, rice, peanut, soybean, rapeseed, sesame, cotton, tobacco, algae, etc., the method of inducing ER stress by chemical regulation or gene editing mutation to increase the content of TAG in plant tissues or seeds also belongs to the content of this application.

[0047] Obviously, those skilled in the art can make various changes and modifications to this application without departing from the spirit and scope of the invention of this application. Thus, if these modifications and variations of this application fall within the scope of the claims of this application and their equivalent technologies, this application also intends to include these changes and modifications.

Claims

1. A method for increasing the oil accumulation in plants, characterized in that, By knockout lew1 The synthesis of dolichol is impaired and the level of protein glycosylation is decreased, leading to endoplasmic reticulum stress. The plant is Arabidopsis thaliana and the oil is triacylglycerol.

2. A method for cultivating high-oil plant varieties, characterized in that, Knockout lew1 the gene to impair the synthesis of dolichol and decrease the level of protein glycosylation, wherein the plant is Arabidopsis thaliana and the oil is triacylglycerol.

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