CN116735320A - Determination method for activity of rice tillering buds and detection method for active oxygen level in tillering buds - Google Patents
Determination method for activity of rice tillering buds and detection method for active oxygen level in tillering buds Download PDFInfo
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Abstract
The invention discloses a method for measuring activity of rice tillering buds and a method for detecting active oxygen level in the tillering buds. The determination method is used for determining the activity of the rice tillering buds by detecting the Reactive Oxygen Species (ROS) level in the tillering buds of the rice seedlings; the level of Reactive Oxygen Species (ROS) in tillering buds of the rice seedlings is in negative correlation with the activity of the tillering buds, and when the Reactive Oxygen Species (ROS) accumulate more, the activity of the tillering buds of the rice is poor; when Reactive Oxygen Species (ROS) accumulate little, the activity of rice tillering buds is strong. The method for detecting the level of Reactive Oxygen Species (ROS) in the tillering buds comprises the steps of culturing rice seedlings, obtaining tillering bud tissues, performing Reactive Oxygen Species (ROS) staining observation and measuring and analyzing the content of superoxide anions and hydrogen peroxide of Reactive Oxygen Species (ROS) components, and evaluating the level of the Reactive Oxygen Species (ROS) in the tillering buds of the rice in qualitative and quantitative aspects. The invention adds a new method for researching the growth and development (tillering bud vitality) of tillering buds of rice and other crops, and also provides a new thought and insight for explaining the morphological establishment of rice plants.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for measuring activity of rice tillering buds and a method for detecting the level of Reactive Oxygen Species (ROS) in the tillering buds.
Background
Tillering is one of the important agronomic traits of rice, and formation of tillers mainly comprises formation of tillers and elongation of tillers. An axillary bud, i.e., a tillering bud, is usually formed in the axillary region of each leaf of rice. Whether the tillering bud is dormant or active is influenced by a range of environmental factors and hormonal signals.
Reactive Oxygen Species (ROS) are a class of signal molecules in plants, including O 2- 、H 2 O 2 And OH, etc., controls the growth and development process of plants. There are many methods of detecting ROS, including spectrophotometry, i.e., the action based on the reaction between free radicals and redox species, the difference in absorbance between substrate and product at different wavelengths allowing semi-quantification of the free radicals; fluorescence staining, namely utilizing a fluorescent probe to enter cells to generate fluorescent products, and judging information about free radical generation under the stimulation; chromatography, i.e., reaction of OH with a specific reagent to form a stable compound that can be detected by chromatographic analysis; and other detection methods, etc. Dormancy of tillering buds can lead to effective tillering reduction of rice plants, ultimately affecting yield.
At present, whether the accumulation of ROS affects the normal growth of rice tillers and how to detect the ROS content in rice tillers have not been studied.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for measuring the activity of rice tillering buds and a method for detecting the level of active oxygen in the tillering buds, which are used for judging the influence of the level of the active oxygen ROS on the growth of the rice tillering buds.
In order to solve the technical problems, the invention adopts the following technical scheme:
in one aspect, the invention provides a method for determining the activity of rice tillering buds, which comprises the steps of detecting the level of Reactive Oxygen Species (ROS) in the tillering buds of rice seedlings to determine the activity of the rice tillering buds; the level of Reactive Oxygen Species (ROS) in tillering buds of the rice seedlings is in negative correlation with the activity of the tillering buds, and when the Reactive Oxygen Species (ROS) accumulate more, the activity of the tillering buds of the rice is poor; when Reactive Oxygen Species (ROS) accumulate little, the activity of rice tillering buds is strong.
As a further improvement of the invention, the activity of the tillering buds of different rice is compared and measured by detecting and comparing the Reactive Oxygen Species (ROS) level in the tillering buds of different rice seedlings, so as to judge the quantity of the tillers of the rice.
On the other hand, the invention also provides a method for detecting the Reactive Oxygen Species (ROS) level in the tillering buds in the method for measuring the activity of the tillering buds of the rice, which comprises the steps of culturing rice seedlings, obtaining tillering bud tissues, carrying out Reactive Oxygen Species (ROS) staining observation and Reactive Oxygen Species (ROS) component superoxide anion and hydrogen peroxide content measurement and analysis, and evaluating the Reactive Oxygen Species (ROS) level in the tillering buds of the rice from two aspects of qualitative and quantitative aspects.
As a further improvement of the invention, the reactive oxygen species ROS staining observations are performed using NBT staining and/or H2DCFDA fluorescent chromogenic solution.
The Reactive Oxygen Species (ROS) staining observation comprises the following steps:
(1) Seedling growing in field for 21 days and being strong;
(2) The rice seedlings are obtained, and the obtained material part comprises an upper part stem basal tissue and an underground part root cap tissue;
(3) Slightly loosening the leaf sheath at the outermost layer of the basal part by forceps before placing the sampled sample tissue into the staining solution, so that tillering buds wrapped in the leaf sheath can fully contact the staining solution, but the leaf sheath is not damaged;
(4) Putting the sample tissue obtained by taking materials into NBT (nitrile-butadiene-styrene) dyeing or H2DCFDA (direct-to-FDA fluorescent color development) liquid for dyeing treatment;
(5) The results were observed.
In the step (2), the material taking part comprises stem basal tissues of 2 cm of aerial parts and root cap tissues of 1 cm of underground parts;
and/or, in the step (3), in the sampled tissue, a layer of leaf sheath is tightly wrapped, so that the dye liquor cannot contact with tillering buds and cannot be dyed, and the tissue is used as a negative control.
The step (4) is to put the sample tissue obtained by taking materials into NBT staining or H2DCFDA fluorescent color development liquid, and comprises the following steps:
1) Placing the sample tissue obtained by taking materials into NBT staining solution and H2DCFDA fluorescent color development solution respectively, placing into a vacuum pump under a shading condition, vacuumizing for 10 minutes under the pressure condition of 0.08Mpa, uniformly shaking once after one time, ensuring that the sample tissue is fully soaked and the tillering buds and nearby gas components are removed, and then repeatedly vacuumizing for 1 time;
2) Placing the sample tissue subjected to vacuum pumping in a 28 ℃ incubator for dark culture for 6 hours; and (5) performing light-shielding operation in the whole experiment process.
In the step (5), transferring the NBT-dyed tissue into absolute ethyl alcohol with the concentration of 99.5%; washing the H2DCFDA stained tissue with PBS to remove excess H2DCFDA residue;
the result after NBT dyeing is observed in a body type mirror and photographed and recorded;
h2DCFDA stained tissue was observed under a fluorescence microscope at excitation/emission wavelengths of 488nm/545nm, respectively, and recorded by photography.
As a further improvement of the invention, in the determination and analysis of the superoxide anion and hydrogen peroxide content of the reactive oxygen species ROS component, a sample material is prepared according to the following method:
the seedling of 1.5 months is adopted, the first leaf is stripped, the first tillering bud is taken, then the second leaf is stripped, the second tillering bud is taken, the taken tillering buds are mixed together and then divided into two parts on average, and the two parts are respectively used for measuring the content of superoxide anions and hydrogen peroxide.
The superoxide anion and hydrogen peroxide content determination comprises:
carrying out ice bath homogenization by adopting a super-oxygen anion extracting solution PBS (phosphate buffer solution) with the pH of 0.01M and the pH of 7.4, centrifuging for 20 minutes at the temperature of 4 ℃ and extracting supernatant for measuring the super-oxygen anion content at the rotation speed of 10000 g;
the other part adopts hydrogen peroxide extracting solution acetone to carry out ice bath homogenization, and the supernatant is extracted for measuring the hydrogen peroxide content under the condition of the rotating speed of 8000g and the temperature of 4 ℃ for 10 minutes.
The invention not only verifies that the Reactive Oxygen Species (ROS) level can influence the normal growth of rice tillering buds, but also summarizes a method for detecting the Reactive Oxygen Species (ROS) level in the rice tillering buds through multiple test attempts, and adds a new method for research on the growth and development of the rice and other crop tillering buds.
Drawings
The foregoing is merely an overview of the present invention, and the present invention is further described in detail below with reference to the accompanying drawings and detailed description.
FIG. 1 is a graph showing the result of NBT staining of tillering buds of a medium flower 11 plant, wherein tillering buds indicated by gray arrows are immersed in NBT staining solution, and tillering buds indicated by black arrows are wrapped by leaf sheaths and are not immersed in the staining solution, and the scale is 2mm.
FIG. 2 is a graph showing the result of H2DCFDA staining of tillering buds of a medium flower 11 plant, wherein BF is bright field, merged is the result of integration of bright field and light-emitting field, and the scale is 2mm.
FIG. 3 is a graph of NBT staining results of 9311 plants and mutant ngr5, wherein A and B are graphs of NBT staining results of 9311 plants (A) and mutant ngr (B), respectively, with a scale of 2mm, the grey arrow indicates that the tillered shoots are immersed in NBT dye liquor, and the black arrow indicates that the tillered shoots are covered by leaf sheaths and are not immersed in the dye liquor.
FIG. 4 is a graph showing the results of H2DCFDA staining of the 9311 plant and the mutant ngr5 tillering bud, wherein A and B are the results of H2DCFDA staining of the 9311 plant (A) and the mutant ngr (B), respectively, and the scale is 10cm.
FIG. 5 is a graph showing the measurement results of the content of superoxide anions and hydrogen peroxide in 9311 and mutant ngr5 tillered buds, wherein A is a graph showing the measurement results of the content of superoxide anions; b is a graph of the measurement result of the hydrogen peroxide content.
FIG. 6 is a plot of the tillering conditions for 9311 and mutant ngr5, wherein A and B are the tillering phenotypes of 9311 plants (A) and mutant ngr (B), respectively; c is a comparison graph of tiller number of 9311 and mutant ngr; d is a graph of comparison of dormant and elongated shoots of 9311 and tillered shoots of mutant ngr, 2nd-11th represent second to eleventh leaves of rice, respectively (from bottom to top).
FIG. 7 is a graph of ROS-related gene expression profiles of 9311 and mutant ngr5.
Detailed Description
The following examples further illustrate the invention but should not be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. Unless otherwise indicated, all biochemical reagents, carriers, consumables and the like used in the examples were commercially available products.
The invention mainly utilizes a strategy of combining tissue staining and active oxygen ingredient measurement to study the level of Reactive Oxygen Species (ROS) in rice tillering buds and application thereof, and the specific experimental technique comprises the following steps:
1. preparation of rice sample material
1) Selecting rice seedlings with the seedling age of 21 days in a field, pulling out the rice seedlings from the soil, cleaning the roots of the rice seedlings, and removing redundant water by using absorbent paper.
2) The material-taking part is stem basal tissue of rice seedling, which comprises stem basal tissue of 2 cm above ground and root cap tissue of 1 cm below ground.
3) And cutting the material in the target area by a surgical knife for later use.
2. Preparation of Reactive Oxygen Species (ROS) staining solution
1) Preparation of nitro tetrazolium blue chloride (Nitrotetrazolium Blue chloride, NBT for short) staining solution: the NBT staining solution was formulated into 0.05% NBT staining solution with 0.01m PBS with ph=7.6 for subsequent staining experiments.
2) 2, 7-dichloro fluorescein diacetate (2 ',7' -Dichlorodihydro fluorescein diacetate, H2DCFDA for short) solution preparation; 10mM of H2DCFDA mother liquor was prepared with DMSO, and then 10mM of Tris-HCl with pH=7.2 was used to dilute to 10. Mu.M of H2DCFDA solution for the subsequent fluorescence development experiment.
3) The NBT staining solution and the H2DCFDA fluorescent color developing solution are wrapped by tin paper and stored at the temperature of 4 ℃ in a dark place, and the shelf life is three months.
3. Reactive Oxygen Species (ROS) staining observation analysis
1) The prepared sample tissue material is respectively placed into NBT staining solution and H2DCFDA solution, placed into a vacuum pump (VOS-30A, STIK) under the shading condition, vacuumized for 10 minutes under the pressure condition of 0.08Mpa, uniformly shaken once after the completion of the vacuum treatment, ensuring that the tissue material is fully soaked and the tillering buds and nearby gas components are removed, and then vacuumized for 1 time repeatedly.
2) The tissue sample after the evacuation was placed in an incubator at 28℃for dark culture for 6 hours.
3) The NBT stained tissue samples were transferred to 99.5% absolute ethanol and subsequently recorded by photographing under a split mirror (M205 FA, LEICA).
4) Tissue samples stained with H2DCFDA were washed twice with PBS solution, excess H2DCFDA solution was removed, observed under a fluorescence microscope (Zeiss LSM710, carl Zeiss AG) 488/545nm (excitation/emission wavelength) and photographed for recording.
4. Active oxygen ingredient determination
1) Preparing a second sample material, stripping the first leaf, taking the first tillering bud, stripping the second leaf, taking the second tillering bud, mixing the taken tillering buds together, and dividing the mixture into two parts for superoxide anion O 2- And hydrogen peroxide H 2 O 2 And (5) content measurement.
2) Ice bath homogenization was performed in one portion using super-oxyanion extract PBS (0.01 m, ph=7.4), and the supernatant was centrifuged at 10000G at 4 ℃ for 20 minutes, and the supernatant was used for measuring the super-oxyanion content by referring to the super-oxyanion measuring kit instructions (cat#sa-2-G, kokosmo, china).
3) The other part adopts the prepared hydrogen peroxide extracting solution acetone to carry out ice bath homogenization, the mixture is centrifuged for 10 minutes at the temperature of 4 ℃ at the rotating speed of 8000g, the supernatant is extracted for measuring the hydrogen peroxide content, and the measuring method is referred to the specification of a hydrogen peroxide measuring kit (Cat#H2O2-2-Y, kokokumi, china).
The following is a description of specific embodiments:
example 1 Reactive Oxygen Species (ROS) staining of flower 11 tillering buds in japonica rice varieties
1. Rice material culture and dyeing material preparation
Flower 11 in japonica rice variety is selected as a test object, dormancy breaking treatment is carried out on seeds of the flower 11, and the seeds are treated in a baking oven at 45 ℃ for 28 hours for standby. The treated seeds are used for soaking and accelerating germination, the germinated seeds are sown in a seedling bed prepared in advance to obtain seedlings with seedling ages of 18 days, 21 days, 25 days and 30 days, morphological structure and size of tillering buds of a stem base part of the rice are observed through anatomic experiments, the tillering buds with the seedling ages of 21 days are found to be just in a key stage of elongation, the sizes are proper (the tillering buds with the seedling ages of 18 days are smaller and are not convenient for observation and analysis, and the tillering buds with the seedling ages of 25 days and 30 days show elongation characteristics), and the subsequent Reactive Oxygen Species (ROS) dyeing experiments all adopt the seedlings of the rice with the seedling ages of 21 days.
2. Reactive Oxygen Species (ROS) staining observation analysis
1) Influence of the size of the material on the dyeing
In the process of obtaining the rice stem basal tissue, the retaining length of the aerial part of the stem basal is set to be 0.5, 1, 1.5 and 2 cm, the retaining length of the underground part of the stem basal is set to be 0, 0.5 and 1 cm, the obtained plant tissue is respectively soaked in NBT staining solution and H2DCFDA fluorescent solution, the length of the aerial part of the stem basal is found to be most suitable as a result of preliminary experiments, the length of the aerial part of the stem basal is too short, the wound of the plant is too close to tillering buds to influence the staining and observation, the length of the aerial part of the stem basal occupies the volume of the solution, and reagent waste is caused. The underground part of the stem base is most suitable to be preserved to be 1 cm long, and the plant damage is seriously affected by the too short length.
2) Improvement of dyeing effect
Before the sample tissue is put into the staining solution, the outermost leaf sheath of the stem base is gently plucked loose by forceps, so that tillering buds wrapped in the leaf sheath can fully contact the staining solution, but the leaf sheath is not damaged (in the experimental operation process, the integrity of the tillering buds cannot be guaranteed by damaging the leaf sheath, meanwhile, uneven staining can be caused, and photographing is influenced). The leaf sheath is tightly wrapped on the inner layer, so that the dye liquor cannot contact with tillering buds and cannot be dyed, and therefore, the dye liquor can be used as a negative control as a preferable scheme.
The treated stem basal sample tissue is soaked in NBT staining solution and H2DCFDA solution, and the staining time is set to be 2 hours, 4 hours and 6 hours respectively, and as a result, the NBT staining time is found to be 2 hours, the tissue is not stained, when the staining time is 4 hours, the tissue staining is not obvious, and the effect of staining for 6 hours is best. Whereas both 4 hours and 6 hours of H2DCFDA staining treatment showed fluorescent signals, with 6 hours of treatment as the treatment time for the subsequent experiments.
3) Photographing and observing dyeing result
The NBT-stained tissue was transferred to 99.5% absolute ethanol while the H2 DCFDA-stained tissue was washed with PBS to remove excess H2DCFDA residue.
The results after NBT staining were observed under a split microscope and recorded by photographing, while H2DCFDA stained tissues were observed under a fluorescence microscope at excitation/emission wavelengths of 488nm/545nm, respectively.
The result of NBT staining of tillering buds of the medium flower 11 plants is shown in FIG. 1. The result of H2DCFDA staining of tillering buds of the medium flower 11 plants is shown in FIG. 2. As a result, it was found that the NBT staining time was 2 hours, the tissue was not stained, and when the staining time was 4 hours, the tissue staining was not obvious, and the effect was best when the staining time was 6 hours. Whereas both 4 hours and 6 hours of H2DCFDA staining treatment showed fluorescent signals, with 6 hours of treatment as the treatment time for the subsequent experiments.
Example 2 indica 9311 and few tillering mutant ngr5 tillering bud Reactive Oxygen Species (ROS) staining
1. Rice material culture and dyeing material preparation
Wild indica rice variety 9311 and tillering bud development defect mutant ngr5 are selected as test objects, wherein the mutant ngr plant shows a slightly dwarf and the tillering number is obviously reduced compared with that of the wild type 9311, so that the possibility of change of Reactive Oxygen Species (ROS) in tillering buds of the mutant ngr is preliminarily deduced. The rice seedlings with a seedling age of 21 days were cultivated using the seeds of 9311 and the mutant ngr5, and the specific preparation method was the same as the rice material cultivation and dyeing material preparation method of example 1.
2. Reactive Oxygen Species (ROS) staining assay
1) Selecting strong indica rice 9311 and tillering bud development defect mutant ngr seedling with seedling age of 21 days, cleaning the plant, wiping water with absorbent paper, and obtaining materials for standby. The specific experimental method is the same as the optimized method in Reactive Oxygen Species (ROS) staining analysis in example 1, the plant stem base comprises 1 cm tissue material of the aerial stem base and 1 cm root cap of the underground part, and the leaf sheath at the outermost layer of the stem base is gently plucked loose by forceps without damaging the leaf sheath. Immersing the tissue in NBT staining solution and H2DCFDA solution; vacuum was applied twice/10 minutes each, and then the sample tissue was placed in a 28℃incubator for dark culture for 6 hours and used for subsequent observation and analysis.
3. Photographing observation and result analysis of dyeing result
The photographing of the dyeing result is the same as that of the photographing and observing method of the dyeing result in example 1. The NBT-stained tissue was photographed under a split microscope and recorded, and the Reactive Oxygen Species (ROS) staining results in the tillering buds of indica rice 9311 and mutant ngr were compared according to the tillering depths of the tillering buds, and the results are shown in FIGS. 3A and 3B. The results show that NBT staining in the tillering bud development defect mutant ngr5 tillering buds is significantly deeper than that of the wild indica rice variety 9311, and it is speculated that more superoxide anions can be accumulated in the tillering buds of ngr plants. The results of H2DCFDA fluorescent staining are shown in FIG. 4, and the results show that the fluorescence in the tillering buds of the mutant ngr plant (FIG. 4B) is significantly stronger than 9311 (FIG. 4A), and it is presumed that more ROS accumulate in the tillering buds of the ngr plant.
In conclusion, wild type 9311 tiller bud reactive oxygen species ROS do not accumulate obviously, while tiller bud defect mutant ngr accumulates a large amount of reactive oxygen species ROS in tiller buds, which indicates that the development defect of the tiller buds of mutant ngr5 is directly related to the accumulation of reactive oxygen species in tiller buds, and can be used as an important index for detecting the activity of the tiller buds of rice.
Example 3 determination and analysis of superoxide anion and Hydrogen peroxide content in flower 11 tillering buds of japonica variety
1. Rice material culture and dyeing material preparation
And selecting a japonica rice variety ZH11 as a test object. The ZH11 seeds are used for cultivating rice seedlings with seedling age of 1.5 months, and non-elongated tillering buds in plants with 1.5 months are obtained as study objects. The specific preparation method is the same as that of the rice material culture and dyeing material in example 1. Through a large number of practices, the obtained unextended tillering buds are more in 1.5 months, which is beneficial to experiment development.
2. Determination of content of superoxide anion and hydrogen peroxide of active oxygen component in tillering bud
The outermost leaves are stripped off respectively by ZH11 seedlings, the tillering bud tissue is taken off (first leaves are stripped off, first tillering buds are taken off, then second leaves are stripped off, second tillering buds are taken off), and the two tillering buds are mixed and divided into two parts evenly and are respectively used for measuring the content of superoxide anions and hydrogen peroxide. Specific extraction and measurement methods refer to the aforementioned "step 4. 2) and 3) in the measurement of active oxygen ingredient", and three biological replicates are performed.
As shown in Table 1, the three replicates of the superoxide anions in the ZH11 tillers were measured at values of 38.33, 36.62 and 37.19nmol g-1 FW, respectively. As shown in Table 2, the results of three replicates of the hydrogen peroxide content measurements were 0.19,0.22 and 0.22. Mu. Mol g-1 FW, respectively.
In conclusion, the research results of the invention utilize the measurement and analysis of the super oxygen anions and the hydrogen peroxide of the ROS reactive oxygen components to study the level of the reactive oxygen species ROS of the rice tillering buds, and can be used as an important index to judge the development activity of the rice tillering buds on a quantitative level.
TABLE 1 determination of superoxide anion content in tillering buds of flower 11 plants
TABLE 2 determination of the hydrogen peroxide content in tillering buds of medium flower 11 plants
EXAMPLE 4 determination of superoxide anion and Hydrogen peroxide content in the tillering buds of indica type rice 9311 and mutant ngr5
Indica rice 9311 and mutant ngr were analyzed according to the method of the ZH11 tillering sprout superoxide anion and hydrogen peroxide assay analysis in example 3.
1. Rice material culture and dyeing material preparation
The wild indica rice variety 9311 and the mutant ngr with few tillering bud development defects are selected as test objects, and the preliminary conclusion is that the reactive oxygen species ROS in the tillering buds of the mutant ngr comprise superoxide anions and hydrogen peroxide, and the content of the reactive oxygen species ROS can be possibly changed. Seed cultivation using 9311 and mutant ngr5 to give seedling age
1.5 months of rice seedlings, and obtaining unextended tillered buds in 1.5 months of plants as research objects, wherein the specific preparation method is the same as the preparation method of the rice material culture and staining material in the embodiment 1.
2. Determination of content of superoxide anion and hydrogen peroxide of active oxygen component in tillering bud
Preparing second parts of indica rice 9311 and a tillering bud development defect mutant ngr seedling, wherein the seedling age is 1.5 months, respectively stripping the outermost leaves, taking down tillering bud tissues (stripping the first leaves, taking the first tillering buds, then stripping the second leaves, taking the second tillering buds), and dividing the second parts into two parts for measuring the content of superoxide anions and hydrogen peroxide respectively. Specific extraction and measurement methods refer to the aforementioned "step 4. 2) and 3) in the measurement of active oxygen ingredient", and three biological replicates are performed.
The measurement result shows that the content of superoxide anions and the content of hydrogen peroxide in the tillering bud development defect mutant ngr tillering buds are higher than that of the wild type 9311, the result is shown in figure 5, and the result is consistent with the dyeing results of NBT and H2DCFDA, so that the reliability of the ROS dyeing result is further verified. In conclusion, according to the research results, the invention utilizes two experimental techniques and methods of Reactive Oxygen Species (ROS) staining and ROS Reactive Oxygen Species (ROS) superoxide anion and hydrogen peroxide determination and analysis to study the Reactive Oxygen Species (ROS) level of the tillering buds of rice, and the invention discovers that compared with a wild type, the tillering bud development defect mutant accumulates a large amount of Reactive Oxygen Species (ROS) level, and can be used as an important index to judge the development activity of the tillering buds of rice.
EXAMPLE 5 analysis of tiller conditions and ROS-related Gene expression of 9311 and mutant ngr5
As shown in FIG. 6, wherein A and B are tiller phenotype maps of 9311 plant (A) and mutant ngr (B), respectively; c is a comparison graph of tiller number of 9311 and mutant ngr; d is a comparison plot of dormant versus elongated shoots of the tiller shoots of 9311 and mutant ngr. In summary, mutant ngr showed a significant reduction in tiller number compared to wild type 9311, and it was concluded that the reduction in tiller number was due to problems with tiller bud elongation.
As shown in fig. 7, by quantitatively detecting the expression level of the ROS accumulation related genes, a plurality of key genes involved in ROS accumulation, including OsLOL1, osPLS2, osCATA, oscat b, oscac, etc., were found, and compared with the wild type 9311, the expression level of the above genes was significantly increased in mutant ngr5, confirming that ROS accumulation in rice is closely related to the expression level of the ROS accumulation related genes.
In conclusion, the invention utilizes NBT and H2DCFDA staining solution to carry out staining analysis on rice tillering buds, combines content measurement analysis of active oxygen ROS components of the tillering buds such as superoxide anions and hydrogen peroxide, and the like, establishes the connection between the active oxygen ROS level of the rice tillering buds and the activity of the tillering buds, namely that the higher the active oxygen ROS level of the rice tillering buds is, the lower the activity of the tillering buds is, and finally the number of rice tillers is reduced, so the invention provides a novel technology and method for researching the activity of the rice tillering buds, and provides a novel thought and insight for describing the morphological establishment of rice plants.
The above description is only of the preferred embodiments of the present invention, and is not intended to limit the invention in any way, and some simple modifications, equivalent variations or modifications can be made by those skilled in the art using the teachings disclosed herein, which fall within the scope of the present invention.
Claims (10)
1. A method for measuring the activity of rice tillering buds is characterized in that the activity of the rice tillering buds is measured by detecting the level of Reactive Oxygen Species (ROS) in the tillering buds of rice seedlings;
the level of Reactive Oxygen Species (ROS) in tillering buds of the rice seedlings is in negative correlation with the activity of the tillering buds, and when the Reactive Oxygen Species (ROS) accumulate more, the activity of the tillering buds of the rice is poor; when Reactive Oxygen Species (ROS) accumulate little, the activity of rice tillering buds is strong.
2. The method for measuring the activity of rice tillering buds according to claim 1, wherein the activity of different rice tillering buds is measured in a contrasting manner by detecting and contrasting the level of Reactive Oxygen Species (ROS) in the tillering buds of different rice seedlings, and the method is further used for judging the number of rice tillers.
3. A method for detecting the level of Reactive Oxygen Species (ROS) in tillering buds in the method for measuring the activity of tillering buds of rice according to claim 1 or 2, wherein the level of Reactive Oxygen Species (ROS) in the tillering buds of rice is evaluated qualitatively and quantitatively by culturing seedlings of rice, obtaining tillering bud tissues, performing Reactive Oxygen Species (ROS) staining observation and Reactive Oxygen Species (ROS) component superoxide anion and hydrogen peroxide content measurement analysis.
4. The method for detecting Reactive Oxygen Species (ROS) levels in tillering buds according to claim 3, wherein the Reactive Oxygen Species (ROS) staining is performed by NBT staining and/or H2DCFDA fluorescent chromogenic solution.
5. The method for detecting the level of Reactive Oxygen Species (ROS) in tillering buds according to claim 4, wherein the observation of Reactive Oxygen Species (ROS) staining comprises the steps of:
(1) Seedling growing in field for 21 days and being strong;
(2) The rice seedlings are obtained, and the obtained material part comprises an upper part stem basal tissue and an underground part root cap tissue;
(3) Slightly loosening the leaf sheath at the outermost layer of the basal part by forceps before placing the sampled sample tissue into the staining solution, so that tillering buds wrapped in the leaf sheath can fully contact the staining solution, but the leaf sheath is not damaged;
(4) Putting the sample tissue obtained by taking materials into NBT (nitrile-butadiene-styrene) dyeing or H2DCFDA (direct-to-FDA fluorescent color development) liquid for dyeing treatment;
(5) The results were observed.
6. The method according to claim 5, wherein in the step (2), the material-drawing part comprises a stem basal tissue of 2 cm above ground and a root cap tissue of 1 cm below ground;
and/or, in the step (3), in the sampled tissue, a layer of leaf sheath is tightly wrapped, so that the dye liquor cannot contact with tillering buds and cannot be dyed, and the tissue is used as a negative control.
7. The method for detecting the level of Reactive Oxygen Species (ROS) in tillering buds according to claim 5, wherein the step (4) of placing the sampled tissue in NBT staining or H2DCFDA fluorescent developing solution comprises:
1) Placing the sample tissue obtained by taking materials into NBT staining solution and H2DCFDA fluorescent color development solution respectively, placing into a vacuum pump under a shading condition, vacuumizing for 10 minutes under the pressure condition of 0.08Mpa, uniformly shaking once after one time, ensuring that the sample tissue is fully soaked and the tillering buds and nearby gas components are removed, and then repeatedly vacuumizing for 1 time;
2) Placing the sample tissue subjected to vacuum pumping in a 28 ℃ incubator for dark culture for 6 hours; and (5) performing light-shielding operation in the whole experiment process.
8. The method for detecting Reactive Oxygen Species (ROS) levels in tillering buds according to claim 5, wherein in step (5), the NBT stained tissue is transferred to absolute ethanol at 99.5%; washing the H2DCFDA stained tissue with PBS to remove excess H2DCFDA residue;
the result after NBT dyeing is observed in a body type mirror and photographed and recorded;
h2DCFDA stained tissue was observed under a fluorescence microscope at excitation/emission wavelengths of 488nm/545nm, respectively, and recorded by photography.
9. The method for detecting the level of Reactive Oxygen Species (ROS) in tillering buds according to claim 3, wherein in the assay for measuring the content of superoxide anions and hydrogen peroxide in the Reactive Oxygen Species (ROS), a sample material is prepared as follows:
the seedling of 1.5 months is adopted, the first leaf is stripped, the first tillering bud is taken, then the second leaf is stripped, the second tillering bud is taken, the taken tillering buds are mixed together and then divided into two parts on average, and the two parts are respectively used for measuring the content of superoxide anions and hydrogen peroxide.
10. The method for detecting the level of Reactive Oxygen Species (ROS) in tillering buds according to claim 9, wherein the determination of superoxide anion and hydrogen peroxide content comprises:
carrying out ice bath homogenization by adopting a super-oxygen anion extracting solution PBS (phosphate buffer solution) with the pH of 0.01M and the pH of 7.4, centrifuging for 20 minutes at the temperature of 4 ℃ and extracting supernatant for measuring the super-oxygen anion content at the rotation speed of 10000 g;
the other part adopts hydrogen peroxide extracting solution acetone to carry out ice bath homogenization, and the supernatant is extracted for measuring the hydrogen peroxide content under the condition of the rotating speed of 8000g and the temperature of 4 ℃ for 10 minutes.
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