Method for improving plant resistance by exogenously applying MEcPP

Abstract

The invention belongs to the field of molecular biology and plant stress resistance, and particularly relates to a method for improving plant resistance by exogenously applying 2-C-methyl-D-erythritol 2, 4-cyclic diphosphate (MEcPP). The method for improving the plant resistance by exogenously applying the MEcPP comprises the step of spraying an MEcPP solution in a plant seedling stage to improve the plant resistance. The invention provides a method for improving plant resistance, which is simple, easy to operate, time-saving and wide in application range, and provides a strategy and a method for improving the tolerance of plants to stress.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and plant stress resistance, and particularly relates to a method for improving plant resistance by exogenously applying MEcPP. Background Art

[0002] In recent years, bryophytes have attracted widespread attention due to their advantages such as easy cultivation, short life cycle, and use in landscape construction and environmental monitoring. Studies have shown that bryophytes differentiated about 470 million years ago and have evolved a certain degree of tolerance to adversity stress in the long-term process of adapting to environmental changes, making them a high-quality material for stress resistance research. The genome of Physcomitrium patens has been sequenced, with a clear genetic background, and many of its genes are involved in plant stress responses.

[0003] 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate (Methylerythritol cyclodiphosphate, MEcPP) is an intermediate metabolite of the methylerythritol phosphate (MEP) pathway. In the prior art, the endogenous MEcPP content of plants is usually increased by means of stress and genetic improvement, but the effects of the above methods are not ideal. For example, stress can affect the growth and development of plants and the yield and quality of crops; the genetic improvement cycle is long and some plants do not have a suitable genetic transformation system.

[0004] Therefore, how to provide a method for improving plant resistance that is widely applicable, convenient and quick has become an urgent problem to be solved in this field. Summary of the invention

[0005] In order to solve the problems of long improvement cycle and lack of suitable genetic transformation system in the process of improving plant stress resistance, the present invention provides a method of exogenous application of MEcPP with short genetic improvement cycle, applicable to most plants and capable of improving plant resistance. The present invention provides the following technical solutions:

[0006] The invention provides application of 2-C-methyl-D-erythritol 2,4-cyclic diphosphate in improving plant resistance. The 2-C-methyl-D-erythritol 2,4-cyclic diphosphate is a 2-C-methyl-D-erythritol 2,4-cyclic diphosphate solution. The concentration of the 2-C-methyl-D-erythritol 2,4-cyclic diphosphate solution is 10 μM to 50 μM.

[0007] The present invention also provides a method for improving plant resistance by exogenously applying 2-C-methyl-D-erythritol 2,4-cyclodiphosphate, comprising the following steps:

[0008] The 2-C-methyl-D-erythritol 2,4-cyclodiphosphate solution was sprayed on the leaves of the seedling plants.

[0009] Preferably, the 2-C-methyl-D-erythritol 2,4-cyclodiphosphate solution is sprayed until the 2-C-methyl-D-erythritol 2,4-cyclodiphosphate solution drips on the leaf surface.

[0010] Preferably, the improving plant resistance is improving the plant's high temperature resistance.

[0011] Preferably, the plant is Physcomitrella patens.

[0012] The present invention also provides a method for cultivating a high temperature resistant plant, comprising the following steps:

[0013] After treating the plant leaves for 1 hour according to the method described above, the plants are subjected to high temperature resistance culture, and then the plants are subjected to recovery culture to obtain high temperature resistant plants.

[0014] Preferably, the plant is a seedling plant.

[0015] Preferably, the operation of the high temperature resistant cultivation is: culturing the plants in an environment of 40-45°C for 4-5 hours.

[0016] Preferably, the operation of recovery cultivation is: culturing the plants in an environment of 25°C.

[0017] Preferably, the plant is Physcomitrella patens.

[0018] The beneficial effects of the present invention are:

[0019] The present invention provides a method for improving plant resistance by exogenously applying MEcPP, which is achieved by spraying a MEcPP solution on leaves of seedling plants. The method of the present invention for improving plant resistance by exogenously applying MEcPP is simple and easy to operate, saves time, has a wide range of applications, and is economical. After exogenously applying MEcPP, the resistance of the plant is significantly improved, and has an ideal application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0021] Figure 1 This is a flow chart of the present invention for cultivating high temperature resistant plants by exogenously applying MEcPP;

[0022] Figure 2 This is a schematic diagram of the effects of different groups in Example 2 on improving the tolerance of Physcomitrella patens gametophyte to high temperature;

[0023] Figure 3It is a schematic diagram of the effect of different groups in Example 2 on improving the survival rate of Physcomitrella patens gametophyte during high temperature recovery;

[0024] Note: Figure 3 The survival rate is Figure 2 The ratio of the pixel area of ​​the green area in the gametophyte cluster of Physcomitrella patens to the total pixel area of ​​the gametophyte cluster of Physcomitrella patens;

[0025] Figure 4 The RNA-seq test results in Example 3;

[0026] Figure 5 The RNA-seq test results in Example 3;

[0027] Figure 6 This is the RT-qPCR detection result in Example 3. DETAILED DESCRIPTION

[0028] The invention provides an application of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcPP) in improving plant resistance. The MEcPP is a MEcPP solution, and the concentration of the MEcPP solution is 10 μM to 50 μM.

[0029] As an embodiment, the concentration of the MEcPP solution can be any one of 10 μM, 20 μM, 30 μM, 40 μM and 50 μM; in a specific embodiment, the concentration of the MEcPP solution is 10 μM.

[0030] The present invention also provides a method for improving plant resistance by exogenously applying MEcPP, comprising the following steps:

[0031] The MEcPP solution was sprayed on the leaves of seedling plants.

[0032] As an embodiment, when the MEcPP solution is sprayed on the leaves of the seedling plant, the concentration of the MEcPP solution is 10 μM to 50 μM. As an embodiment, the spraying amount of the MEcPP solution is: spray the MEcPP solution until the MEcPP solution drips on the leaf surface. As an embodiment, the concentration of the MEcPP solution can be any one of 10 μM, 20 μM, 30 μM, 40 μM and 50 μM; as an embodiment, the plant includes Physcomitrella patens. As an embodiment, when the plant is Physcomitrella patens, the amount of MEcPP solution used is 100 μL to 200 μL. As an embodiment, when the plant is Physcomitrella patens, the application amount of the MEcPP can be any one of 100 μL, 110 μL, 120 μL, 130 μL, 140 μL, 150 μL, 160 μL, 170 μL, 180 μL, 190 μL and 200 μL. In a specific embodiment, the plant is Physcomitrella patens, the concentration of MEcPP is 10 μM, the application amount is 100 μL, and the application method is to spray the MEcPP solution on the leaves of the seedling plant.

[0033] As an embodiment, the plant resistance is the plant's high temperature resistance, and the plant is a Physcomitrella patens gametophyte cultivated for 3 weeks.

[0034] The present invention also provides a method for cultivating a high temperature resistant plant, comprising the following steps:

[0035] After treating the plant leaves for 1 hour according to the method described above, the plants are subjected to high temperature resistance culture, and then the plants are subjected to recovery culture to obtain high temperature resistant plants.

[0036] The present invention treats plant leaves for 1 hour according to the method as described above to obtain treated plant materials. As one embodiment, the plant is a seedling plant; as another embodiment, the plant is a Physcomitrella patens gametophyte cultivated for 3 weeks. As one embodiment, treating the plant leaves for 1 hour according to the method as described above is to place the plant at a temperature of 25°C, a humidity of 40%, a photoperiod of 16h / 8h, and a light intensity of 60-80μmol photons m -2 s -1 Culture in a light incubator for 1 h.

[0037] After obtaining the treated plant material, the present invention performs high temperature culture on the treated plant material to obtain the plant material after high temperature culture. As an embodiment, the operation of the high temperature resistant culture is: culturing the plant in a dark environment of 40 to 45°C for 4 to 5 hours. As an embodiment, the temperature of the high temperature resistant culture can be any one of 40°C, 41°C, 42°C, 43°C, 44°C and 45°C; as an embodiment, the time of the high temperature resistant culture can be any one of 4h, 4.5h and 5h.

[0038] After obtaining the plant material after high temperature culture, the present invention performs recovery culture on the plant material after high temperature culture to obtain a high temperature resistant plant. As an embodiment, the operation of the recovery culture is: the temperature is 25°C, the humidity is 40%, the photoperiod is 16h / 8h, and the light intensity is 60-80μmolphotons m -2 s -1 cultured in an incubator.

[0039] In a specific embodiment, the present invention places the Physcomitrella patens gametophyte at a temperature of 25°C, a humidity of 40%, a photoperiod of 16h / 8h, and a light intensity of 70 μmolphotons m -2 s -1 After culturing in a light incubator for 1 hour, the treated Physcomitrella patens gametophyte material was obtained, and then the treated Physcomitrella patens gametophyte material was placed in a dark incubator at 45°C for high temperature incubation for 4 hours to obtain the Physcomitrella patens gametophyte material after high temperature incubation. Finally, the Physcomitrella patens gametophyte material after high temperature incubation was placed in a temperature of 25°C, a humidity of 40%, a photoperiod of 16h / 8h, and a light intensity of 70μmolphotons m -2 s -1 The culture was restored in an incubator to obtain high-temperature resistant Physcomitrella patens gametophyte plants.

[0040] In order to further illustrate the present invention, a method for improving plant resistance by exogenously applying MEcPP provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0041] In the present invention, the MEcPP is purchased from Echelon and is numbered I-M054. The plastic centrifuge tubes, gun tips, etc. used in the examples are sterilized at 121°C and dried for later use. The reagents and consumables used for RNA extraction and reverse transcription are sterile and enzyme-free test consumables; the mortar and metal spoon are wiped and disinfected with alcohol cotton.

[0042] Example 1

[0043] Method for obtaining high temperature resistant Physcomitrella patens gametophyte by exogenous application of MEcPP

[0044] The gametophyte shoot tips of Physcomitrella patens with uniform growth were selected and aseptically cultured in BCD medium for 3 weeks to obtain the gametophyte clusters of Physcomitrella patens cultured for 3 weeks as plant materials. The culture conditions were as follows: temperature 25°C, humidity 40%, photoperiod 16h / 8h, light intensity 70μmolphotons m -2 s -1 In a light incubator, divide the Physcomitrella patens gametophyte clusters into 24 equal parts, with 8 parts forming one group, for a total of 3 groups, for later use.

[0045] MEcPP was diluted to 50 μM and 10 μM respectively using sterile double distilled water, and sterilized by filtration using a sterile 0.22 μm filter membrane to ensure sterility before use.

[0046] In the clean bench, 100 μL of sterile double distilled water (hereinafter referred to as Mock), 100 μL of 10 μM MEcPP (hereinafter referred to as lc_MEcPP) and 100 μL of 50 μM MEcPP (hereinafter referred to as hc_MEcPP) were carefully and evenly added to a single Physcomitrella patens gametophyte cluster that had been cultured for 3 weeks using a pipette tip until MEcPP solution or sterile double distilled water dripped on the leaf surface. The Physcomitrella patens gametophyte cluster was transferred to a light incubator and cultured for 1 hour. Culture conditions: temperature 25 °C, humidity 40%, photoperiod 16h / 8h, light intensity 60-80 μmolphotons m -2 s -1 .

[0047] After culturing MEcPP for 1 hour, a portion of each Physcomitrella patens gametophyte cluster was quickly frozen in liquid nitrogen and stored at -80°C. The remaining Physcomitrella patens gametophyte clusters were transferred to a dark 45°C incubator and incubated at high temperature for 4 hours. During the high temperature incubation process, the plant material was rotated 90° or moved in the incubator every 30 minutes to ensure uniform heating of the plant material.

[0048] After high temperature incubation, the Physcomitrella patens gametophyte clusters were transferred to a temperature of 25°C, a humidity of 40%, a photoperiod of 16 h / 8 h, and a light intensity of 70 μmolphotons m -2 s -1 The recovery culture was carried out in a light incubator for 7 days.

[0049] Example 2

[0050] The phenotypes of Physcomitrella patens gametophyte clusters in the Mock group, lc_MEcPP group, and hc_MEcPP group in Example 1 that were not cultured at high temperature and recovered after culture at high temperature for 7 days were observed and photographed using a stereo microscope. One sample was randomly displayed for each group. The results are shown in Figure 1. Figure 2 shown. Figure 2 The first row is a schematic diagram of the growth status of the Physcomitrella gametophyte in different groups of Physcomitrella gametophyte clusters that have not been cultured at high temperature, and the second row is a schematic diagram of the growth status of the Physcomitrella gametophyte in different groups of Physcomitrella gametophyte clusters after recovery from high temperature culture.

[0051] Depend on Figure 2 It can be seen that the degree of pseudo-leaf whitening of the Physcomitrella patens gametophyte clusters with exogenous application of lc_MEcPP is much lower than that of the control group (Mock and hc_MEcPP), which can significantly improve the survival rate of Physcomitrella patens gametophytes and improve their tolerance to high temperature. This shows that this method is simple, easy to operate and has significant effects.

[0052] ImageJ software was used to count the eight Physcomitrella patens gametophyte clusters in the Mock group, lc_MEcPP group, and hc_MEcPP group in Example 1, and the high-temperature survival rate of Physcomitrella patens gametophyte in the Physcomitrella patens gametophyte clusters in the recovery period was counted. The results are shown in Table 1. Figure 2 The pictures of Physcomitrella patens gametophyte clusters in the Mock group, lc_MEcPP group, and hc_MEcPP group were shown in the figure. The survival rate was calculated according to the calculation method of survival rate = pixel area of ​​the green area in the Physcomitrella patens gametophyte cluster / total pixel area of ​​the Physcomitrella patens gametophyte cluster. The results are shown in Figure 2. Figure 3 As shown in Table 1.

[0053] Table 1 Exogenous application of lc_MEcPP improves the survival rate of Physcomitrella patens during high temperature recovery

[0054]

[0055] Depend on Figure 3 As shown in Table 1, the group with lc_MEcPP addition can significantly improve the survival rate of Physcomitrella patens during high temperature recovery, and the survival rate is much higher than that of the Mock group and the hc_MEcPP group.

[0056] Example 3

[0057] Taking the Physcomitrella patens gametophyte clusters stored at -80°C in Example 1 as an example, the above-mentioned Physcomitrella patens gametophyte clusters were taken out respectively, ground into powder in liquid nitrogen, and about 0.2 g of the powder was weighed and placed in a 1.5 mL sterile enzyme-free centrifuge tube containing 1 mL Trizol for RNA extraction. A portion was sent to BGI for transcriptome sequencing (RNA-seq), and RNA from the Physcomitrella patens gametophyte materials of the Mock group and the lc_MEcPP group after 1 hour of treatment was extracted respectively. The extracted RNA was subjected to transcriptome sequencing, and the FPKM of the gene was obtained after analysis. Data was extracted from the FPKM, and the results are as follows: Figures 4-5 The remainder was reverse transcribed using a cDNA synthesis kit as indicated.

[0058] The reverse transcription operation is as follows: After the reverse transcribed cDNA is diluted 20 times, the reaction is carried out using fluorescent quantitative PCR enzyme (2×qPCRmix) according to the RT-qPCR reaction system in Table 2:

[0059] Table 2 RT-qPCR reaction system

[0060] Reactants Volume (μL) cDNA 2 Primer F 0.4 Primer R 0.4 2×qPCRmix 5 <![CDATA[DEPC-ddH 2 The]]> 2.5

[0061] The "three-step method" was used for RT-qPCR. The reaction procedure of the "three-step method" is shown in Table 3:

[0062] Table 3 PCR reaction program

[0063]

[0064] Adoption 2 -ΔΔT The RT-qPCR data were analyzed by the method of ACT7 gene in Table 4 as the internal reference gene. The ACT7 gene and other genes and primer sequences used in RT-qPCR are shown in Table 4. The expression calculation results of the quantitative internal reference gene and the gene to be tested are shown in Figure 6 As shown:

[0065] Table 4 Gene and primer sequences in RT-qPCR

[0066]

[0067]

[0068] Note: The gene numbers mentioned in Table 4 are all gene accession numbers in the Phytozome 13 database.

[0069] according to Figures 4-5 It can be seen that the gene expression of heat shock transcription factor HSF and heat shock protein HSPs in Physcomitrella patens with exogenous lc_MEcPP was significantly increased. Figure 6 It can be seen that the gene expression of the heat shock transcription factor HSF of the Physcomitrella patens with exogenous application of lc_MEcPP was significantly increased. Exogenous application of lc_MEcPP can increase the survival rate of Physcomitrella patens in high temperature and thus improve its tolerance to high temperature. This may be because plants will adopt certain adaptive strategies to cope with high temperature stress during the long-term evolution process, which mainly involve the activity of the heat shock transcription factor HSFA1 and the accumulation of heat shock proteins HSPs. In this example, it is related to the activation of the gene expression of HSF and HSPs in the Physcomitrella patens.

[0070] In summary, the method according to the present invention can improve the tolerance of plants to high temperatures, avoid the serious impact of increasing the endogenous MEcPP content of plants through adverse stress on plant growth and development and crop yield quality, avoid the shortcomings of long genetic improvement cycle and lack of suitable genetic transformation system for some plants, etc., and has the advantages of simplicity, easy operation, time saving and wide application range, etc., provides an effective strategy and method for plants to adapt to severe external environment, and has certain innovation and great application value.

[0071] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments like this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of 2-C-methyl-D-erythritol 2,4-cyclic diphosphate in improving plant resistance, characterized in that: The 2-C-methyl-D-erythritol 2,4-cyclic diphosphate is a 2-C-methyl-D-erythritol 2,4-cyclic diphosphate solution, and the concentration of the 2-C-methyl-D-erythritol 2,4-cyclic diphosphate solution is 10 μM to 50 μM.

2. A method for improving plant resistance by exogenously applying 2-C-methyl-D-erythritol 2,4-cyclodiphosphate, characterized in that: The steps include: The 2-C-methyl-D-erythritol 2,4-cyclodiphosphate solution was sprayed on the leaves of the seedling plants.

3. The method according to claim 2, characterized in that The 2-C-methyl-D-erythritol 2,4-cyclodiphosphate solution is sprayed until the 2-C-methyl-D-erythritol 2,4-cyclodiphosphate solution drips on the leaf surface.

4. The method according to claim 2, characterized in that The improving of plant resistance is to improve the high temperature resistance of plants.

5. The method according to claim 2, characterized in that The plant is Physcomitrella patens.

6. A method for cultivating high temperature resistant plants, characterized in that: The steps include: After treating the plant leaves for 1 hour according to the method as described in any one of claims 2 to 5, the plant is subjected to high temperature resistant cultivation, and then the plant is subjected to recovery cultivation to obtain a high temperature resistant plant.

7. The cultivation method according to claim 6, characterized in that The plants are seedling plants.

8. The cultivation method according to claim 6, characterized in that The operation of the high temperature resistant cultivation is: culturing the plants in an environment of 40-45° C. for 4-5 hours.

9. The cultivation method according to claim 6, characterized in that: The operation of the recovery culture is: culturing the plants in an environment of 25°C.

10. The cultivation method according to claim 6, characterized in that: The plant is Physcomitrella patens.

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