Method for detecting high temperature tolerance of rice and detection kit

Abstract

The present invention discloses a method and a detection kit for detecting the high-temperature tolerance of rice. By detecting the dynamic curve of the ROS content in the excised leaf discs of the flag leaves of rice during the heading and flowering stage, it is determined whether the rice plants during the heading and flowering stage are heat-resistant. The detection kit includes L-012, horseradish peroxidase, and Tris-HCl buffer solution. Based on the ROS in the excised leaf discs of the flag leaves of rice during the heading and flowering stage at an environmental temperature of 40°C as a biomarker, the present invention constructs an identification system for the high-temperature tolerance of rice during the heading and flowering stage through the dynamic curve of the ROS content, thereby realizing the rapid identification and breeding of new heat-resistant rice varieties; this method has unique advantages such as a short detection period, reliable results, and low cost. The detection kit has high sensitivity and rapid reaction, and the operation process can be mastered without professional training, which is convenient for rapid popularization and use, and is of great significance for the rapid identification and breeding of heat-resistant rice varieties.

Description

Technical Field

[0001] The invention relates to the technical field of plant breeding, and in particular to a method for detecting high temperature tolerance of rice and a detection kit. Background Art

[0002] Rice growth is affected by climatic factors, and light, heat, and water are the three major climatic factors for rice growth. Among them, high temperature is an important limiting factor for rice growth. Studies have shown that developing anthers (pollen) are most sensitive to high temperature stress. When the daily average temperature is higher than 32°C and the daily maximum temperature is higher than 35°C, the number and activity of rice pollen will decrease, anther dehiscence will be hindered, and even male sterility will occur, ultimately leading to a decrease in rice yield and quality.

[0003] In recent years, with the progress of global industrialization and the explosive growth of population, the global temperature has been rising year by year, and extreme high temperature weather in summer has occurred frequently, which seriously threatens the planting and production of rice in my country. Accelerating the breeding of excellent varieties with strong heat resistance is an effective way to solve this problem. In the breeding of traditional high-temperature resistant rice varieties, greenhouses are often used to artificially heat the rice in the heading and flowering stage. When the rice matures, the fruit setting rate is investigated, and the fruit setting rate is used as the evaluation standard for the high temperature tolerance of rice varieties. The traditional high-temperature resistance identification method requires a large amount of capital investment in the early stage to build greenhouses, and there are certain requirements for the site. In addition, the growth period of different rice varieties is inconsistent, and even the same variety has a time span of about ten days from the initial heading stage to the full heading stage, which makes it difficult for the traditional greenhouse identification method to ensure the high temperature treatment time of different rice ears, resulting in poor repeatability and low accuracy of the identification results. Summary of the invention

[0004] In view of the technical problems to be solved, the present invention provides a method and a detection kit for detecting high temperature tolerance of rice. Based on the ROS in the detached leaf disc of the flag leaf of rice at the heading and flowering stage at an ambient temperature of 40°C as a biomarker, the present invention constructs a high temperature tolerance identification system for rice at the heading and flowering stage through the dynamic curve of ROS content, thereby realizing the rapid identification and breeding of new varieties of high temperature resistant rice. This method abandons the traditional method of identification using greenhouses, and therefore has the unique advantages of short detection cycle, reliable results and low cost. The entire detection process has high sensitivity and rapid response, and the operation process can be mastered without professional training, which is convenient for rapid promotion and use.

[0005] In order to achieve the above object, the present invention provides a method for detecting high temperature tolerance of rice, by detecting the dynamic curve of ROS content in the detached leaf disk of the rice flag leaf at the heading and flowering stage, to judge whether the rice plant is resistant to high temperature at the heading and flowering stage.

[0006] In the above method, further, the criterion for determining whether the rice plants at the heading and flowering stage are heat-tolerant is as follows: when the dynamic curve of the ROS content fluctuates at a relatively low level all the time, it is determined as a heat-tolerant plant at the heading and flowering stage;

[0007] When the dynamic curve of the ROS content first rises rapidly, and then turns to decline rapidly after reaching the peak, showing a parabolic curve, it is determined as a moderately heat-sensitive plant at the heading and flowering stage;

[0008] When the dynamic curve of the ROS content rises rapidly, and after reaching the peak, the ROS content tends to balance and remains at the peak level, it is determined as a heat-sensitive plant at the heading and flowering stage.

[0009] In the above method, further, the method for detecting the dynamic curve of the ROS content in the excised leaf discs of the flag leaves of rice at the heading and flowering stage includes the following steps:

[0010] (1) Mix Tris-HCl buffer, L-012 and horseradish peroxidase to obtain a reaction working solution;

[0011] (2) Treat the excised leaf discs of the flag leaves of rice at the heading and flowering stage with the reaction working solution and detect with an enzyme-labeled instrument;

[0012] (3) Draw the dynamic curve of the ROS content according to the monitored values.

[0013] Based on a general technical concept, the present invention also provides a detection kit for the above method, and the detection kit includes L-012, horseradish peroxidase, and Tris-HCl buffer.

[0014] In the above detection kit, further, the concentration of the L-012 is 20 mM.

[0015] In the above detection kit, further, the concentration of the horseradish peroxidase is 10 mg / mL.

[0016] In the above detection kit, further, the concentration of the Tris-HCl buffer is 50 mM.

[0017] In the above detection kit, further, the pH of the Tris-HCl buffer is 7.5.

[0018] Compared with the prior art, the advantages of the present invention are as follows:

[0019] (1) The present invention provides a method for detecting the heat tolerance of rice. Based on the ROS in the excised leaf discs of the flag leaves of rice at the heading and flowering stage at an environmental temperature of 40°C as a biomarker, a heat tolerance identification system for rice at the heading and flowering stage is constructed through the dynamic curve of the ROS content, so as to realize the rapid identification and breeding of new heat-tolerant rice varieties.

[0020] High temperature is a kind of adversity for plants. Since plants cannot move, they cannot avoid harm and seek benefits. When high temperature comes, plants can only adapt to or resist high temperature through their own metabolic regulation, and this metabolism includes the metabolic reaction of ROS. Under high temperature stress, high temperature-sensitive rice will produce a large amount of ROS in organelles such as chloroplasts, mitochondria, peroxisomes, and endoplasmic reticulum due to high temperature. Excessive ROS will further oxidize cell membranes, lipids, etc., damage cell functions and further produce more ROS, which leads to the dynamic curve of ROS content showing a rapid increase in the early stage. When it reaches a threshold, it tends to be stable and always maintains a relatively high level of ROS content.

[0021] For high-temperature resistant materials, a large amount of ROS will also be generated under high temperature stress. However, due to the strong ROS scavenging ability of high-temperature resistant materials, the ROS content in cells is always maintained at a relatively low level, and there is no obvious upward trend in the dynamic curve of ROS content, always tending to be stable (there is also a possibility here that high-temperature resistant materials may be insensitive to high temperature, plants do not feel high temperature, so they will not generate a large amount of ROS, and thus will not cause damage to cells or tissues).

[0022] For moderately high temperature-sensitive materials, a large amount of ROS will also be generated under high temperature. The dynamic curve of ROS content first rises rapidly, then drops rapidly after reaching the peak, and then tends to be stable and maintains at a relatively low level. The appearance of this situation is presumably that a large amount of ROS is generated in the early stage. When it reaches a certain content, the cell activates the ROS scavenging mechanism, causing the ROS content to drop rapidly. This kind of plant shows moderate high temperature sensitivity.

[0023] (2) The present invention provides a detection kit for the high temperature tolerance of rice, which can quickly and accurately identify the high temperature tolerance of rice in a short period. This method abandons the traditional method of identification using greenhouse greenhouses, thus avoiding expensive equipment investment and site requirements. In addition, this method also has unique advantages such as short detection period, reliable results, and low cost. It has high detection sensitivity, rapid reaction, and the operation process can be mastered without professional training, which is convenient for rapid popularization and use, and is of great significance for the rapid identification and breeding of high temperature resistant rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention.

[0025] Figure 1 It is a statistical chart of the identification results of the actual samples of the excised leaf discs of the flag leaves of rice, mature panicles, and seed setting rates in Example 2 of the present invention.

[0026] Figure 2 This is the statistical chart of the identification results of the actual rice anther samples, mature panicles, and seed setting rates in Example 3 of the present invention. Detailed implementation manners

[0027] The present invention will be further described below in conjunction with specific and preferred embodiments, but the protection scope of the present invention is not limited thereby.

[0028] Unless otherwise defined, all professional terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.

[0029] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or can be prepared by existing methods. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0030] Embodiment

[0031] The instruments and reagents used in the following embodiments are all commercially available.

[0032] Embodiment 1:

[0033] A detection kit for the high-temperature tolerance of rice of the present invention includes 20 mM L-012 (reactive oxygen species probe), 10 mg / mL horseradish peroxidase, and 50 mM Tris-HCl buffer solution with pH = 7.5.

[0034] Preparation of L-012 mother liquor. Weigh 6.23 mg of L-012 powder and dissolve it in sterile water to obtain 20 mM L-012 mother liquor, and store it in a -20°C refrigerator in the dark.

[0035] Preparation of horseradish peroxidase mother liquor. Weigh 10 mg of horseradish peroxidase powder and dissolve it in 1 mL of sterile water to obtain 10 mg / mL horseradish peroxidase mother liquor, and store it in a -20°C refrigerator in the dark.

[0036] Preparation of Tris-HCl buffer solution. Weigh 6.055 g of Tris and place it in a 1 L beaker, add about 800 ml of deionized water and stir to dissolve. After complete dissolution, add concentrated HCl to adjust the pH to 7.5, and finally make up the volume to 1 L with deionized water to obtain 50 mM Tris-HCl buffer solution with pH = 7.5.

[0037] The reagents in the detection kit are mixed before use.

[0038] Embodiment 2:

[0039] Application of a detection kit of Example 1 in detecting the high-temperature tolerance of rice, and the specific object of the application is the excised leaf discs of the flag leaves of rice at the heading and flowering stage, which specifically includes the following steps:

[0040] (1) Cultivation of rice plants:

[0041] 1.1 Select 50 seeds of the rice variety to be detected, soak them in clean water for 48 hours, and change the clean water once during this period. After soaking, drain the excess water, place the seeds in an incubator at 37°C for germination, and directly sow the germinated seeds on the field seedbed for seedling raising.

[0042] 1.2 When the seedling age reaches 25 days, pull out the seedlings for field transplantation, and then carry out normal water and fertilizer management until the rice grows to the heading and flowering stage.

[0043] (2) Pretreatment of rice leaves:

[0044] 2.1 Collect leaf discs on the flag leaves of rice using a punch with a diameter of 5 mm, place the collected leaf discs in a 1.5 mL centrifuge tube, and pre-add 1 mL of sterile water to the centrifuge tube.

[0045] 2.2 After the leaf discs are collected, place the centrifuge tube in the dark at room temperature and incubate overnight without shaking the centrifuge tube.

[0046] (3) Treatment of the detection kit:

[0047] 3.1 Take the detection kit of Example 1, and use a pipette to respectively take 1940 µL of 50 mM Tris-HCl (pH = 7.5) buffer, 40 µL of L-012 mother liquor, and 20 µL of horseradish peroxidase mother liquor, and mix well to obtain the reaction working solution.

[0048] 3.2 Aliquot the prepared reaction working solution into a white 96-well microplate, and add 100 µL of the reaction working solution to each well.

[0049] (4) Detection of the ROS content in rice leaves:

[0050] 4.1 Gently take out the incubated leaf discs with forceps, place them on absorbent paper to remove the residual sterile water on the surface, and then transfer them to the microplate. Place one leaf disc in each detection well, set three replicates for each material, and at the same time set control wells with only the reaction working solution (without leaf discs) and the working solution replaced with sterile water (adding sterile water and leaf discs).

[0051] 4.2. Place the enzyme-linked immunosorbent assay (ELISA) plate in an ELISA reader. Set the detection chamber temperature to 40 °C, the detection method to Luminescence, the detection frequency to once every 30 seconds, and the detection duration to 30 minutes. Draw a dynamic curve of the ROS content based on the monitored values, and use the dynamic curve graph of the ROS content to determine whether the rice plants at the heading and flowering stage are heat-tolerant. If the dynamic curve of the ROS content always fluctuates at a relatively low level, it is determined to be a heat-tolerant plant at the heading and flowering stage; if the dynamic curve of the ROS content first rises rapidly, then turns to decline rapidly after reaching the peak, showing a parabolic curve, it is determined to be a moderately heat-sensitive material at the heading and flowering stage; if the dynamic curve of the ROS content always shows a rapid rise, and after reaching the peak, the ROS content tends to balance and maintain at the peak level, it is determined to be a heat-sensitive material at the heading and flowering stage, thus realizing the rapid identification and breeding of new heat-tolerant rice varieties.

[0052] 4.3. Detect the high-temperature seed setting rate, specifically as follows:

[0053] 4.3.1. Select 50 seeds of each rice variety to be screened, soak them in clean water for 48 hours, and change the clean water once during this period. After soaking, drain the excess water, and place the seeds in an incubator at 37 °C for germination.

[0054] 4.3.2. Sow the seeds that have just shown white tips directly on the field seedbed for seedling raising;

[0055] 4.3.3. When the seedling age reaches 25 days, transplant them to an artificial intelligence greenhouse. Plant three rows for each variety to be screened, with eight plants in each row, for a total of 24 plants.

[0056] 4.3.4. Open the ventilation skylight of the greenhouse and conduct normal water and fertilizer management until the rice grows to the heading and flowering stage;

[0057] 4.3.5. When the rice grows to the heading and flowering stage, use artificial intervention to conduct high-temperature stress treatment on the rice. The temperature control conditions are set as follows: from 0:00 to 6:30, 28 °C; from 6:30 to 8:30, 30 °C; from 8:30 to 10:30, 35 °C; from 10:30 to 14:30, 38 °C; from 14:30 to 16:30, 35 °C; from 16:30 to 18:30, 30 °C; from 18:30 to 24:00, 28 °C, and the humidity is set to 75%. Conduct spike marking on the day of high-temperature stress treatment, that is, select five newly emerged flower spikes for each variety for marking. The high-temperature stress treatment ends until the marked spikes are completely emerged and flowering is completed. Take the average value of five biological replicates for each variety. If the seed setting rate is higher than 80%, it is considered a heat-tolerant rice variety at the heading and flowering stage; if the seed setting rate is lower than 40%, it is a heat-sensitive variety; the rest are moderately sensitive varieties.

[0058] Figure 1The ROS content dynamic curves and the fruit setting rate statistical results of different rice materials drawn according to the monitoring values are shown in the figure. It can be seen from the figure that the ROS content dynamic curve is consistent with the test results of the fruit setting rate, proving that the method of the present invention is highly accurate in detecting the high temperature tolerance of rice.

[0059] Embodiment 3:

[0060] An application of the detection kit of Example 1 in detecting high temperature tolerance of rice, wherein the specific object of application is rice anther tissue, specifically comprises the following steps:

[0061] (1) Rice plant cultivation:

[0062] 1.1. Select 50 seeds of the rice variety to be tested and soak them in clean water for 48 hours, changing the clean water once. After soaking, drain the excess water and place the seeds in a 37℃ constant temperature box for germination. The germinated seeds are directly sown on the field seedling bed for seedling cultivation.

[0063] 1.2. When the seedlings are 25 days old, pull out the seedlings for field transplanting, and then carry out normal water and fertilizer management until the rice grows to the heading and flowering stage.

[0064] (2) Rice anther pretreatment:

[0065] 2.1. Collect rice panicles to be tested from the field on a clear afternoon. Pull out the intact panicles from the rice tillers and quickly insert the lower end of the panicles into a plastic bottle with water.

[0066] 2.2. Return to the laboratory and select the spikelets that are about to open the next day. Use pointed tweezers to gently open the lemma. Use tweezers to grasp the anther silk and take out the complete anther tissue. Place it in a 1.5mL centrifuge tube. Add 1mL of sterile water to the centrifuge tube in advance. Take only one anther from each spikelet and three anther tissues for each material to be tested.

[0067] 2.3. After the anthers are collected, place the centrifuge tube incubated in the dark at room temperature for half an hour. Do not shake the centrifuge tube during this period.

[0068] (3) Handling of test kits:

[0069] 3.1. Take the detection kit of Example 1, and use a pipette to take 1940 µL of 50 mM Tris-HCl (pH = 7.5) buffer, 40 µL of L-012 stock solution, and 20 µL of horseradish peroxidase stock solution, and mix them thoroughly to obtain a reaction working solution.

[0070] 3.2. Dispense the prepared reaction working solution into a white 96-well ELISA plate and add 100uL of reaction working solution to each well.

[0071] (4)Detection of ROS content in rice anthers:

[0072] 4.1 Gently take out the incubated anthers with forceps, place them on absorbent paper to remove the residual sterile water on the surface, and then transfer them to an enzyme-linked immunosorbent assay (ELISA) plate. Place one anther in each detection well, set three replicates for each material, and simultaneously set control wells with only the reaction working solution (without anthers) and wells with the working solution replaced by sterile water (adding sterile water and anthers).

[0073] 4.2 Place the ELISA plate in an enzyme-labeling instrument, set the detection chamber temperature to 40 °C, the detection mode to Luminescence, the detection frequency to 30 seconds per time, and the detection duration to 30 minutes. Draw a dynamic curve of ROS content based on the monitored values, and determine whether the rice plants at the heading and flowering stage are heat-tolerant according to the dynamic curve of ROS content. If the dynamic curve of ROS content always fluctuates at a low level, it is determined as a heat-tolerant plant at the heading and flowering stage; if the dynamic curve of ROS content first rises rapidly, then turns to decline rapidly after reaching the peak, showing a parabolic curve, it is determined as a moderately heat-sensitive material at the heading and flowering stage; if the dynamic curve of ROS content always shows a rapid rise, and after reaching the peak, the ROS content tends to balance and maintain at the peak level, it is determined as a heat-sensitive material at the heading and flowering stage, thus realizing the rapid identification and breeding of new heat-tolerant rice varieties.

[0074] 4.3 Detect the high-temperature seed-setting rate, and the detection method is the same as that in Example 2.

[0075] Figure 2 Dynamic curves of ROS content are drawn for different rice materials according to the monitored values, as well as the detection results of seed-setting rate. It can be seen from the figure that the dynamic curve of ROS content is consistent with the statistical results of seed-setting rate, proving that the method for detecting rice heat tolerance according to the present invention has high accuracy.

[0076] Different experimental conditions have a certain impact on the experimental results. Under the optimal conditions, the experimental effect reaches the best.

[0077] As mentioned above, it is only a preferred embodiment of the present invention, and there is no any formal limitation to the present invention. Although the present invention is disclosed by the preferred embodiment as above, it is not intended to limit the present invention. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes, without departing from the spirit and technical solution of the present invention. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the technical solution of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for detecting the high-temperature tolerance of rice, characterized in that, By detecting the dynamic curve of the ROS content in the excised leaf discs or excised anther tissues of the flag leaves of rice during the heading and flowering stage, it is determined whether the rice plants during the heading and flowering stage are heat-tolerant; When the dynamic curve of the ROS content fluctuates at a relatively low level all the time, it is determined as a heat-tolerant plant during the heading and flowering stage; When the dynamic curve of the ROS content first rises rapidly, and then turns to decline rapidly after reaching the peak, showing a parabolic curve, it is determined as a moderately heat-sensitive plant during the heading and flowering stage; When the dynamic curve of the ROS content rises rapidly, and after reaching the peak, the ROS content tends to balance and maintain at the peak level, it is determined as a heat-sensitive plant during the heading and flowering stage; The method for detecting the dynamic curve of the ROS content includes the following steps: (1) Mix Tris-HCl buffer, L-012 and horseradish peroxidase to obtain a reaction working solution; (2) Treat the excised leaf discs or excised anther tissues of the flag leaves of rice during the heading and flowering stage with the reaction working solution, and detect with an enzyme-labeled instrument. The temperature of the detection chamber is set at 40 °C, the detection frequency is set at 30 seconds / time, and the detection duration is set at 30 minutes; (3) Draw a dynamic curve of the ROS content according to the monitored values.

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