CN114303836B - Determination method of optimization strategy for stress resistance regulation in rice - Google Patents

Abstract

The invention discloses a method for determining a rice stress-resistance regulation and control optimization strategy, which applies different nitrogen source application schemes to rice seedlings; constructing a matrix I for cultivating rice stress resistance regulation and control by different nitrogen sources in rice tissues, and processing the matrix I to obtain a matrix II-a matrix V; respectively comparing the values in the matrix I to obtain the contribution of the inorganic nitrogen and the organic nitrogen which are applied in combination to the free Pro; comparing the values in the matrix II, the matrix III and the matrix V to obtain the contribution of the inorganic nitrogen or the organic nitrogen which is independently applied to the free Pro; according to the method, a strategy model for regulating and controlling chromium stress of rice is obtained by screening different nitrogen sources by taking free Pro as a biomarker, and stress resistance regulation and control optimization strategies of the rice at different growth stages are determined. The method for determining the rice stress-resistance regulation and control optimal strategy simplifies the various experimental processes of botany by constructing the strategy model, and provides a scientific and effective strategy determination method for the field of rice stress-resistance regulation and control.

Description

Determination method of optimization strategy for stress resistance regulation in rice

technical field

The invention belongs to the technical field of botany and relates to a method for determining an optimal strategy for stress resistance regulation of rice.

Background technique

As an important industrial raw material, chromium (Cr) is widely used in petroleum refining, leather tanning, textile manufacturing, electroplating, dye processing and other industries. According to statistics, the current average annual global Cr consumption is about 12.5 million tons, while the accumulated Cr in the previous period is about 1,105.4 billion tons, leading to increasingly serious Cr pollution in the environment. According to the "National Soil Pollutant Survey Bulletin" issued by the Ministry of Environmental Protection on April 17, 2014, the total over-standard rate of soil pollution in the country was 16.1%, of which the point over-standard rate of Cr was 1.1%. Plants are the producers in the food chain and the most important part of the ecosystem. The use of exogenous green regulators to regulate plant growth and development under pollution stress is a hot issue that everyone pays attention to.

Nitrogen is an essential constant element for plant growth, among which nitrate nitrogen (NO ₃ ^- ) and ammonium nitrogen (NH ₄ ⁺ ) are the most easily absorbed inorganic nitrogen by plants. Proline (Pro) is an amino acid with multiple functions in plants. It can not only alleviate biotic and abiotic stress, but also play an important role in maintaining amino acid balance in plants. Therefore, Pro is also often used as a biomarker to respond to plant stress intensity. Glutamic acid (Glu) and arginine (Arg) are precursors in the two synthetic Pro pathways in plants, and the assimilation of NO ₃ ^- and NH ₄ ⁺ in plants is also closely related to the inventory of Glu and Arg.

The present invention uses free Pro as a biomarker to screen different nitrogen sources under chromium stress (inorganic nitrogen sources: NO ₃ ^- and NH ₄ ⁺ ; organic nitrogen sources: Glu and Arg) to cultivate optimal strategies for stress-resistance regulation of rice, for rice to adapt to chromium Pollution provides selectivity.

Contents of the invention

In order to achieve the above object, the present invention provides a method for determining the optimal strategy for rice stress resistance regulation, using free proline as a biomarker to screen the optimal strategy for cultivating rice stress resistance regulation with different nitrogen source combinations under chromium stress, so as to alleviate the impact of pollutants on plants. Toxicity solves the problems existing in the prior art.

The technical scheme adopted in the present invention is a method for determining the optimal strategy for stress resistance regulation of rice, comprising the following steps:

Step 1: Under the stress of various concentrations of Cr(III), implement different nitrogen source application schemes in rice seedlings; the nitrogen source application schemes include: no organic nitrogen treatment, arginine treatment, glutamic acid treatment, no inorganic nitrogen Nitrogen treatment, nitrate nitrogen treatment, ammonium nitrogen treatment, any one or a combination of any two; measure the free Pro content in rice seedling tissue in each nitrogen source application scheme;

Step 2: Combining any two combinations of organic nitrogen-free treatment, arginine treatment, glutamic acid treatment, inorganic nitrogen-free treatment, nitrate nitrogen treatment, and ammonium nitrogen treatment to construct rice tissues with different nitrogen sources to cultivate rice resistance The matrix I of inverse regulation is shown in the following matrix:

The rows and columns of the matrix are denoted by r _i and c _j respectively, i, j = 1, 2, 3; the specific elements of the matrix are denoted by a _ij , i, j = 1, 2, 3; Combined application; in matrix I, a ₁₁ represents the free Pro content without organic nitrogen treatment and without inorganic nitrogen treatment; a ₁₂ represents the free Pro content without arginine pretreatment and inorganic nitrogen treatment; a ₁₃ represents glutamic acid pretreatment and free Pro content without inorganic nitrogen treatment; a ₂₁ means free Pro content without organic nitrogen treatment and nitrate nitrogen treatment; a ₂₂ means free Pro content when arginine pretreatment and nitrate nitrogen treatment are added; a ₂₃ means The content of free Pro in glutamic acid pretreatment and nitrate nitrogen treatment; a ₃₁ indicates the free Pro content in the absence of organic nitrogen treatment and ammonium nitrogen treatment; a ₃₂ indicates the free Pro content in arginine pretreatment and ammonium nitrogen treatment Pro content; a ₃₃ represents the free Pro content when glutamic acid pretreatment and ammonium nitrogen were added;

Step 3: Perform r ₂ -r ₁ , r ₃ -r ₁ , r ₃ -r ₂ three elementary row transformations on matrix I to obtain matrix II; then perform c ₂ -c ₁ , c ₃ -c ₁ on matrix I, c ₃ -c ₂ three elementary column transformations to obtain matrix III; subtract matrix II from matrix III to obtain matrix IV; matrix V is composed of a ₁₁ , a ₁₂ , a ₂₁ , and a ₂₂ in matrix IV;

Step 4: Compare the values of a ₂₂ , a ₂₃ , a ₃₂ , and a ₃₃ in matrix I respectively to obtain the contribution of combined application of inorganic nitrogen and organic nitrogen to free Pro;

Comparing the values of element a _3j in the third row of matrix II, element a _i3 in the third column of matrix III, and a ₁₁ , a ₁₂ , a ₂₁ , and a ₂₂ in matrix V respectively, it is obtained to compare the effect of applying inorganic nitrogen or organic nitrogen alone on free Pro the size of the contribution;

Step 5: According to the method of step 4, the contribution of nitrogen source to free Pro in rice seedling tissue under different stress concentrations is obtained; According to the size of the contribution, the strategy model of using free Pro as a biomarker to screen different nitrogen sources to cultivate rice to regulate chromium stress was obtained. According to the contribution model, the optimal strategy for stress resistance regulation in different growth stages of rice was determined.

Further, in step 3, matrix II is specifically as follows:

In matrix II, r ₁ represents the contribution of nitrate nitrogen treatment to free Pro; r ₂ represents the contribution of ammonium nitrogen treatment to free Pro; r ₃ represents the contribution of the difference of free Pro between ammonium nitrogen treatment and nitrate nitrogen treatment.

Further, in step 3, matrix III is as follows:

In matrix III, c ₁ represents the contribution of arginine treatment to free Pro; c ₂ represents the contribution of glutamate treatment to free Pro; c ₃ represents the contribution of the difference of free Pro between glutamate treatment and arginine treatment .

Further, in step 3, the matrix IV is as follows:

In matrix IV, a ₁₁ represents the contribution of arginine treatment and nitrate nitrogen treatment to the difference of free Pro; a ₁₂ represents the contribution of glutamic acid treatment and nitrate nitrogen treatment to the difference of free Pro; a ₁₃ represents glutamic acid The contribution of treatment, arginine treatment and nitrate nitrogen treatment to the difference of free Pro; a ₂₁ indicates the contribution of arginine treatment and ammonium nitrogen treatment to the difference of free Pro; a ₂₂ indicates the contribution of glutamic acid treatment and ammonium nitrogen treatment The contribution of treatment to the difference of free Pro; a ₂₃ represents the contribution of glutamic acid treatment and arginine treatment, ammonium nitrogen treatment to the difference of free Pro; a ₃₁ represents the contribution of arginine treatment and nitrate nitrogen treatment, ammonium nitrogen treatment The contribution of treatment to the difference of free Pro; a ₃₂ represents the contribution of glutamic acid treatment and nitrate nitrogen treatment, ammonium nitrogen treatment to the difference of free Pro; a ₃₃ represents the contribution of glutamic acid treatment and arginine treatment, nitrate nitrogen treatment The contribution of treatment and ammonium nitrogen treatment to the difference of free Pro.

Further, in step 3, the matrix V is as follows:

Further, in step 4, the numerical values of the element a _3j in the third row of matrix II, the element a _i3 in the third column of matrix III and a ₁₁ , a ₁₂ , a ₂₁ , and a ₂₂ in matrix V are respectively compared to obtain a comparison of the inorganic The contribution of nitrogen or organic nitrogen to free Pro, specifically:

According to the positive and negative values of the elements in the third row of matrix II, compare the contribution of ammonium nitrogen and nitrate nitrogen alone to free Pro; according to the positive and negative values of the elements in the third column of matrix III, compare the application of glutamic acid alone The contribution of acid and arginine alone to free Pro; according to the positive or negative value of a ₁₁ of matrix V, compare the contribution of arginine alone and nitrate nitrogen alone to free Pro; according to the value of a ₁₂ of matrix V According to the positive and negative values of the value, compare the contribution of glutamic acid alone and nitrate nitrogen alone to free Pro; according to the positive or negative of the a ₂₁ value of matrix V, compare the contribution of arginine alone and ammonium nitrogen alone to free Pro According to the positive or negative value of a ₂₂ of matrix V, compare the contribution of glutamic acid alone and ammonium nitrogen alone to free Pro;

According to the contribution of ammonium nitrogen and nitrate nitrogen alone to free Pro, the contribution of glutamic acid and arginine alone to free Pro, the contribution of arginine and nitrate nitrogen alone to free Pro The contribution of Pro, the contribution of glutamate alone and nitrate nitrogen alone to free Pro, the contribution of arginine alone and ammonium nitrogen alone to free Pro, the contribution of glutamate alone and ammonium alone The contribution of state nitrogen to free Pro can be obtained from the contribution of inorganic nitrogen or organic nitrogen to free Pro.

The beneficial effects of the present invention are: in the embodiment of the present invention, free proline is used as a biomarker, and single or combined regulators are simply and efficiently selected to greatly alleviate the toxicity of pollutants to plants. Only one test of nine treatments is enough. Construct the matrix I of stress-resistance regulation of rice cultivated by different nitrogen sources in rice tissue, and perform matrix transformation and processing such as elementary row transformation, elementary column transformation, and matrix subtraction on matrix I, and then according to the size of the values in the transformed matrix, The positive and negative analysis of the contribution of each nitrogen source to the regulation of stress resistance of rice in each nitrogen source application scheme was carried out, and then a strategy model was obtained to screen different nitrogen sources to regulate chromium stress by using free Pro as a biomarker. Stress-resistance regulation optimal strategy in growth stage. The method for determining the optimal strategy for rice stress resistance regulation in the embodiment of the present invention simplifies the various test procedures in botany through the construction of a strategy model, effectively improves the efficiency of rice stress resistance regulation, and provides a scientific and effective strategy determination method for the field of rice stress resistance regulation.

detailed description

The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

1. Experimental protocol

1.1 Preparation of rice seedlings

The rice seeds are soaked in water for 12 hours, and then put into a disposable cup for sand cultivation. The planting density is about 30-40 seeds/cup. The cultivation was carried out in an artificial climate box with a temperature of 25° C., a humidity of 65%, and a light intensity of 20,000 lux. Water the modified ISO8692 nutrient solution (Table 1). After the paddy rice grows for 16 days, the sandy soil at its root is cleaned, and the paddy seedlings with the same individual size are selected for subsequent use.

Table 1 Modified ISO8692 Nutrient Solution Formula

serial number Reagent concentration serial number Reagent concentration 1 KNO₃ 2823.9μmol/L 8 H₃BO₃ 2992.1nmol/L 2 MgCl₂ 6H₂O 59.0μmol/L 9 MnCl₂ 4H₂O 2097.0nmol/L 3 CaCl₂ 2H₂O 122.4μmol/L 10 Na₂MoO₄ 2H₂O 28.9nmol/L 4 MgSO₄ 7H₂O 60.9μmol/L 11 CuSO₄ 2H₂O 0.1nmol/L 5 KH₂PO₄ 246.0μmol/L 12 ZnSO₄ 22.0nmol/L 6 NaHCO₃ 1785.5μmol/L 13 CoCl₂ 6H₂O 6.3nmol/L 7 Fe-EDTA 10.0μmol/L

1.2 Experimental design

_In this experiment, CrCl3 was selected as the main pollutant, and the screened rice seedlings were cultured in the following treatment solutions:

(1) Cr(III)-N treatment [(-N _o )&(-N _I )]: rice seedlings were placed in 50mL nitrogen-free nutrient solution (nitrogen starvation, nitrogen-free nutrient solution refers to removal of KNO ₃ The concentration of Cr(III) in nitrogen-free nutrient solution: 0, 12.0, 24.0 and 40.0 mg Cr/L in the Erlenmeyer flask of the modified ISO8692 nutrient solution).

(2) Cr(III)-N+Arg treatment [(+N _Arg )&(-N _I )]: rice seedlings were placed in 3mM Arg solution for 12h, and then placed in 50mL nitrogen-free nutrient solution In the Erlenmeyer flask, the concentration of Cr(III) in the nitrogen-free nutrient solution: 0, 12.0, 24.0 and 40.0 mg Cr/L.

(3) Cr(III)-N+Glu treatment [(+N _Glu )&(-N _I )]: rice seedlings were placed in 10mM Glu solution for 12h, and then placed in 50mL nitrogen-free nutrient solution In the Erlenmeyer flask, the concentration of Cr(III) in the nitrogen-free nutrient solution: 0, 12.0, 24.0 and 40.0 mg Cr/L.

将水稻幼苗放置于50mL含KNO₃ (39.5mgN/L)营养液的锥形瓶中，含KNO₃营养液中Cr(III)的浓度：0、12.0、 24.0和40.0mg Cr/L。(4) Cr(III)+NO ₃ ^-treatment

The rice seedlings were placed in 50 mL Erlenmeyer flasks containing KNO ₃ (39.5 mgN/L) nutrient solution, and the concentrations of Cr(III) in the KNO ₃ nutrient solution were: 0, 12.0, 24.0 and 40.0 mg Cr/L.

将水稻幼苗放置于3mM 的Arg溶液中培育12h，然后再放置于50mL含KNO₃(39.5mg N/L)营养液的锥形瓶中，含KNO₃营养液中Cr(III)的浓度：0、12.0、24.0和40.0mg Cr/L。(5) Cr(III)+NO ₃ ^- +Arg treatment

Rice seedlings were placed in 3mM Arg solution and incubated for 12h, and then placed in a 50mL Erlenmeyer flask containing KNO ₃ (39.5mg N/L) nutrient solution, the concentration of Cr(III) in the KNO ₃ nutrient solution: 0 , 12.0, 24.0 and 40.0mg Cr/L.

将水稻幼苗放置于10mM 的Glu溶液中培育12h，然后将再放置于50mL含KNO₃(39.5mg N/L)营养液的锥形瓶中，含KNO₃营养液中Cr(III)的浓度：0、12.0、24.0和40.0mg Cr/L。(6) Cr(III)+NO ₃ ^- +Glu treatment

The rice seedlings were placed in 10mM Glu solution and cultivated for 12h, and then placed in a 50mL Erlenmeyer flask containing KNO ₃ (39.5mg N/L) nutrient solution. The concentration of Cr(III) in the KNO ₃ nutrient solution was: 0, 12.0, 24.0 and 40.0 mg Cr/L.

将水稻幼苗放置于50mL含 NH₄Cl(39.5mg N/L)营养液的锥形瓶中，含NH₄Cl营养液中Cr(III)的浓度： 0、12.0、24.0和40.0mg Cr/L。(7) Cr(III)+NH ₄ ⁺ treatment

Place the rice seedlings in a 50mL Erlenmeyer flask containing NH ₄ Cl (39.5mg N/L) nutrient solution, the concentration of Cr(III) in the NH ₄ Cl nutrient solution: 0, 12.0, 24.0 and 40.0mg Cr/L .

将水稻幼苗放置于3mM 的Arg溶液中培育12h，然后再放置于50mL含NH₄Cl(39.5mg N/L)营养液的锥形瓶中，含NH₄Cl营养液中Cr(III)的浓度：0、12.0、24.0和40.0mg Cr/L。(8) Cr(III)+NH ₄ ⁺ +Arg treatment

Rice seedlings were placed in 3mM Arg solution and incubated for 12h, and then placed in a 50mL Erlenmeyer flask containing NH ₄ Cl (39.5mg N/L) nutrient solution, the concentration of Cr(III) in the NH ₄ Cl nutrient solution : 0, 12.0, 24.0 and 40.0 mg Cr/L.

将水稻幼苗放置于10mM 的Glu溶液中培育12h，然后再放置于50mL含NH₄Cl(39.5mg N/L)营养液的锥形瓶中，含NH₄Cl营养液中Cr(III)的浓度：0、12.0、24.0和40.0mg Cr/L。锥形瓶都用锡箔纸包裹做避光处理，最大限度地减少水分流失并抑制藻类生长。每个处理有4个生物学重复，暴露时间为3天。(9) Cr(III)+NH ₄ ⁺ +Glu treatment

The rice seedlings were placed in 10mM Glu solution and cultivated for 12h, and then placed in a 50mL Erlenmeyer flask containing NH ₄ Cl (39.5mg N/L) nutrient solution, the concentration of Cr(III) in the NH ₄ Cl nutrient solution : 0, 12.0, 24.0 and 40.0 mg Cr/L. Erlenmeyer flasks are wrapped in foil to protect from light to minimize water loss and inhibit algae growth. Each treatment had 4 biological replicates with 3 days of exposure.

1.3 Content of free proline in different tissues of rice

After each treatment group was stressed for 3 days, a certain amount of plant tissue samples were accurately weighed (the weighing weight of root tissue (fresh weight) was 0.2 grams, and the weighing fresh weight of leaf tissue (fresh weight) was 0.2 grams), placed in In the pre-cooled mortar, first add 2.5mL sulfosalicylic acid aqueous solution (the mass percentage of sulfosalicylic acid is 3%), after grinding and homogenizing, move into a 10mL centrifuge tube, and use 2.5mL sulfosalicylic acid aqueous solution ( The mass percentage of sulfosalicylic acid is 3%) to clean the mortar, pour the cleaning solution into a centrifuge tube, and centrifuge (11000rpm) at low temperature (4°C) for 15min; take 2mL of the supernatant in a 10mL glass tube, and add 2mL of ice Acetic acid and 2mL acidic ninhydrin solution (1.25g ninhydrin dissolved in 30mL glacial acetic acid and 20mL phosphoric acid, heated to dissolve at 70°C and stored in the refrigerator for later use), in boiling water bath for 1h, then in ice bath for 5min, add 4mL of toluene, shake well After standing still for 30min, get the supernatant at 520nm for colorimetry, toluene is a blank control, calculate and obtain the free proline content of plant samples, the numerical unit of free proline content is (μg/g FW, FW refers to fresh Heavy.

2. Experimental results

Table 2 shows the changes of free Pro content in rice cultivated with different nitrogen sources under Cr(III) stress.

Cr(III)-N treatment: the content of free Pro in roots and leaves increased;

Cr(III)-N+Arg treatment: the content of free Pro in roots and leaves increased;

Cr(III)-N+Glu treatment: the content of free Pro in roots and leaves increased;

Cr(III)+NO ₃ ^-treatment : the content of free Pro in leaves increased, but the content of free Pro in roots did not change significantly;

Cr(III)+NO ₃ ^- +Arg treatment: the content of free Pro in roots and leaves decreased;

Cr(III)+NO ₃ ^- +Glu treatment: the content of free Pro in leaves decreased, and the content of free Pro in roots increased;

Cr(III)+NH ₄ ⁺ treatment: the content of free Pro in roots and leaves increased;

Cr(III)+NH ₄ ⁺ +Arg treatment: the content of free Pro in the leaves decreased, and the changes in the roots were not obvious;

Cr(III)+NH ₄ ⁺ +Glu treatment: the content of free Pro in the leaves decreased, but the changes in the roots were not obvious.

From the above experimental results, it is not possible to quickly determine which nitrogen source (individual nitrogen source: NO ₃ ^- , NH ₄ ⁺ , Arg, Glu; or combined nitrogen source: NO ₃ ^- +NH ₄ ⁺ , NO ₃ ^- +Arg, NO ₃ ^- +Glu, NH ₄ ⁺ +Arg, NH ₄ ⁺ +Glu, Arg+Glu are the optimal nitrogen sources for regulating the content of free Pro in rice seedlings under Cr(III) stress. Therefore, construct a screening system for Cr(III) stress It is very necessary to optimize the strategy model for stress-resistance regulation of rice under different nitrogen sources.

Table 2 Changes of free Pro content in rice cultivated with different nitrogen sources under Cr(III) stress

3. Using free proline as a biomarker to screen the optimal strategy model for cultivating rice with different nitrogen sources to regulate chromium stress

3.1 Modeling process of optimization strategies for rice stress resistance regulation under different nitrogen sources under chromium stress

The content of free Pro in plants is closely related to the type of exogenous nitrogen supply. For example, the content of free Pro in rice seedlings cultivated in NO ₃ ^- or NH ₄ ⁺ will be significantly different, and the supply of exogenous Glu and Arg also has a significant impact on the content of free Pro in rice seedlings. The establishment of this model is based on the effect of different nitrogen sources alone or in combination on the change of free Pro content in rice seedlings, simulating the changes of different nitrogen sources alone or in combination on the change of free Pro content in rice seedlings under different Cr(III) stress concentrations, To explore the optimal strategy of using free Pro as a biomarker to screen different nitrogen sources (inorganic nitrogen sources: NO ₃ ^- and NH ₄ ⁺ ; organic nitrogen sources: Glu and Arg) to regulate Cr(III) stress by cultivating rice alone or in combination.

铵态氮处理

则得到矩阵①：Under different concentrations of Cr(III) stress, different nitrogen sources were applied alone or in combination in rice seedlings: no organic nitrogen treatment (-NO ), arginine treatment ( ₊ N _Arg ), glutamic acid treatment (+N _Glu ), non-inorganic nitrogen treatment (-N _I ), nitrate nitrogen treatment

Ammonium nitrogen treatment

Then get the matrix ①:

The rows and columns of the matrix are denoted by r _i and c _j (i,j=1,2,3) respectively, the specific elements of the matrix are denoted by a _ij (i,j=1,2,3) respectively, in the matrix “&"Indicates the combined application of two nitrogen sources, then in the matrix at this time:

a ₁₁ represents free Pro content when there is no organic nitrogen treatment and no inorganic nitrogen treatment;

a ₁₂ represents free Pro content when arginine is pretreated and treated without inorganic nitrogen;

a ₁₃ represents the free Pro content during glutamic acid pretreatment and no inorganic nitrogen treatment;

a ₂₁ represents the free Pro content when there is no organic nitrogen treatment and nitrate nitrogen is added;

a ₂₂ represents the free Pro content when arginine is pretreated and nitrate nitrogen is added;

a ₂₃ represents the free Pro content when glutamic acid is pretreated and nitrate nitrogen is added;

a ₃₁ represents the free Pro content when there is no organic nitrogen treatment and ammonium nitrogen is added;

a ₃₂ represents the free Pro content when arginine is pretreated and ammonium nitrogen is added;

a ₃₃ represents the free Pro content when glutamic acid was pretreated and ammonium nitrogen was added.

For comparison, three elementary row transformations are performed on matrix ① respectively: r ₂ -r ₁ , r ₃ -r ₁ , r ₃ -r ₂ , and three matrices of matrix ②, matrix ③, and matrix ④ are obtained:

Combine r ₁ in matrix ②, r ₁ in matrix ③, and r ₂ in matrix ④ to get matrix ⑤:

At this time in the matrix ⑤

r ₁ represents the contribution of nitrate nitrogen treatment to free Pro;

r ₂ represents the contribution of ammonium nitrogen treatment to free Pro;

r ₃ represents the contribution of ammonium nitrogen treatment and nitrate nitrogen treatment to the difference of free Pro.

Then perform three elementary column transformations on matrix ① respectively: c ₂ -c ₁ , c ₃ -c ₁ , c ₃ -c ₂ , to obtain three matrices, and combine them in the same way to obtain matrix ⑥:

At this time in the matrix ⑥

c ₁ represents the contribution of arginine treatment to free Pro;

c2 represents the contribution of glutamate treatment to free Pro _;

c ₃ represents the contribution of glutamate treatment and arginine treatment to the difference of free Pro.

Subtract matrix ⑤ from matrix ⑥ to get matrix ⑦:

Among them, -N _o and -N _I are set to 0.

Extract the parts that need to be analyzed: a ₁₁ , a ₁₂ , a ₂₁ , a ₂₂ form a new matrix ⑧:

At this time in matrix ⑧

a ₁₁ represents the contribution of arginine treatment and nitrate nitrogen treatment to the difference of free Pro;

a ₁₂ represents the contribution of glutamic acid treatment and nitrate nitrogen treatment to the difference of free Pro;

a ₂₁ represents the contribution of arginine treatment and ammonium nitrogen treatment to the difference of free Pro;

a ₂₂ represents the contribution of glutamic acid treatment and ammonium nitrogen treatment to the difference of free Pro.

If you want to compare the contribution of inorganic nitrogen or organic nitrogen to free Pro, you only need to compare the element a _3j in the third row of matrix ⑤, the element a _i3 in the third column of matrix ⑥ with a ₁₁ , a ₁₂ , a ₂₁ , a The numerical value of ₂₂ .

3.2 Modeling results

The results of free Pro content determination in rice seedling leaves and roots are shown in Table 3 and Table 4 respectively:

Table 3 Free Pro content in leaves of rice seedlings under different concentrations of Cr(Ⅲ) stress (μg/g FW)

Table 4 Content of free Pro in roots of rice seedlings under different concentrations of Cr(Ⅲ) stress (μg/g FW)

Take the data of the three control groups (Control) in Table 3 as an example to establish a matrix and compare it. The matrix ①' is as follows:

From the matrix ①', it can be seen that when there is no Cr(III) stress, the contribution of combined application of inorganic nitrogen and organic nitrogen to free Pro is as follows:

Perform three elementary row transformations on matrix ①': r ₂ -r ₁ , r ₃ -r ₁ , r ₃ -r ₂ , combine r 1 in matrix ②', r ₁ in matrix ③', and r ₁ in matrix ④' _r2 to get the matrix ⑤':

对矩阵①’分别进行三次初等列变换：c₂-c₁，c₃-c₁， c₃-c₂，得到三个矩阵，同理组合得到矩阵⑥’：From the third row of matrix ⑤', it can be seen that the contribution of inorganic nitrogen to free Pro in the absence of Cr(III) stress:

Perform three elementary column transformations on matrix ①': c ₂ -c ₁ , c ₃ -c ₁ , c ₃ -c ₂ to obtain three matrices, and combine them in the same way to obtain matrix ⑥':

From the third column of matrix ⑥', it can be seen that the contribution of organic nitrogen to free Pro when no Cr(III) stress is added: N _Arg >N _Glu ; matrix ⑥' is subtracted from matrix ⑤' to obtain matrix ⑦', from The parts we need are proposed in the matrix ⑦': a ₁₁ , a ₁₂ , a ₂₁ , a ₂₂ , forming a new matrix ⑧':

From the matrix ⑧', we can see that the contribution of inorganic nitrogen or organic nitrogen to free Pro in the absence of Cr(III) stress:

按照这个方法，得到其它胁迫浓度下根和叶中组合或单独施用无机氮和有机氮对游离Pro的贡献如下表5。In summary, the contribution of inorganic nitrogen or organic nitrogen alone to free Pro in leaves when no stress was added was:

According to this method, the contribution of inorganic nitrogen and organic nitrogen to free Pro in roots and leaves under other stress concentrations or alone is obtained in Table 5.

Table 5 Contribution of combined or single application of inorganic nitrogen and organic nitrogen to rice seedling Pro under different concentrations of Cr(Ⅲ) stress

According to the contribution of nitrogen sources alone and in combination to free Pro in rice seedling tissues under various stress concentrations, a strategy model for screening different nitrogen sources to regulate chromium stress in rice seedlings with free Pro as a biomarker was obtained, and the different growth rates of rice were determined according to the contribution model. Stress-resistance regulation optimization strategy for stage.

+N_Arg这个N源组合对游离Pro的贡献均最大，可作为未来实验及实际应用中优选组合N源；而单独施用氮源试验组，

对Cr(Ⅲ)胁迫水稻幼苗叶中游离Pro的贡献最大，N_Arg对Cr(Ⅲ)胁迫水稻幼苗根中游离Pro的贡献最大，因此可在水稻不同生长阶段采用不同的施加氮源策略，以解决其应对 Cr(Ⅲ)胁迫的需求。It can be seen from Table 5 that no matter in rice roots or leaves, or under different Cr(Ⅲ) stress concentrations,

The N source combination +N _Arg has the largest contribution to free Pro, which can be used as the preferred combination of N sources in future experiments and practical applications; while the nitrogen source test group alone,

The contribution of free Pro in the leaves of rice seedlings under Cr(Ⅲ) stress is the largest, and N _Arg is the largest contribution to the free Pro in the roots of rice seedlings under Cr(Ⅲ) stress. Therefore, different nitrogen source strategies can be adopted at different growth stages of rice to To solve its needs to cope with Cr(Ⅲ) stress.

Each embodiment in this specification is described in a related manner, the same and similar parts of each embodiment can be referred to each other, and each embodiment focuses on the differences from other embodiments.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present invention are included in the protection scope of the present invention.

Claims (2)

1. The method for determining the rice stress-resistance regulation and control optimization strategy is characterized by comprising the following steps of:

step 1: applying different nitrogen source application schemes to the rice seedlings under the stress of Cr (III) with various concentrations; the nitrogen source administration regimen comprises: determining the content of free Pro in the rice seedling tissue in each nitrogen source application scheme by combining any two of organic nitrogen-free treatment, arginine treatment, glutamic acid treatment, inorganic nitrogen-free treatment, nitrate nitrogen treatment and ammonium nitrogen treatment;

and 2, step: constructing a matrix I for cultivating rice stress resistance regulation and control by using any two combinations of organic nitrogen-free treatment, arginine treatment, glutamic acid treatment, inorganic nitrogen-free treatment, nitrate nitrogen treatment and ammonium nitrogen treatment, wherein the matrix I comprises the following components:

；

in matrix I, the rows and columns of the matrix are each denoted by r _i And c _j It is shown that,i, j =1,2,3; a specific element of the matrix is represented by _ij It is shown that the process of the present invention,i, j = 1, 2, 3；&represents the combined administration of two nitrogen sources; in matrix I, a ₁₁ Indicates the free Pro content in the absence of organic nitrogen treatment and in the absence of inorganic nitrogen treatment; a is a ₁₂ Represents the free Pro content in arginine pretreatment and no inorganic nitrogen treatment; a is a ₁₃ Represents the free Pro content in glutamic acid pretreatment and no inorganic nitrogen treatment; a is a ₂₁ Indicates the free Pro content in the case of treatment without organic nitrogen and treatment with nitrate nitrogen; a is a ₂₂ Represents the free Pro content when arginine pretreatment and nitrate nitrogen addition treatment are carried out; a is ₂₃ Represents the free Pro content when the glutamic acid is pretreated and the nitrate nitrogen is added; a is ₃₁ Indicates the free Pro content in the case of treatment without organic nitrogen and treatment with ammonium nitrogen; a is ₃₂ Represents the free Pro content in arginine pretreatment and ammonium nitrogen addition treatment; a is ₃₃ Represents the free Pro content when glutamic acid is pretreated and ammonium nitrogen is added;

and step 3: r is performed on the matrix I ₂ -r ₁ ，r ₃ -r ₁ ，r ₃ -r ₂ Performing three times of primary row transformation to obtain a matrix II; c is performed on the matrix I ₂ -c ₁ ，c ₃ -c ₁ ，c ₃ -c ₂ Performing three times of primary column transformation to obtain a matrix III; subtracting the matrix II from the matrix III to obtain a matrix IV; from a in the matrix IV ₁₁ 、a ₁₂ 、a ₂₁ 、a ₂₂ Forming a matrix V;

and 4, step 4: comparing matrix I with a respectively ₂₂ 、a ₂₃ 、a ₃₂ 、a ₃₃ The magnitude of the numerical values, resulting in the magnitude of the contribution of the combined administration of inorganic nitrogen and organic nitrogen to free Pro;

comparing respectively the third row elements a of the matrix II _3j Matrix III third column element a _i3 And a in matrix V ₁₁ 、a ₁₂ 、a ₂₁ 、a ₂₂ The amount of free Pro compared to the amount of contribution of inorganic nitrogen or organic nitrogen alone;

and 5: according to the method of the step 4, obtaining the contribution size of the nitrogen source to the free Pro by the single and combined application in the rice seedling tissues under different stress concentrations; according to the contribution of the nitrogen sources to free Pro by independent and combined application in rice seedling tissues under various stress concentrations, a strategy model for screening different nitrogen sources to culture rice to regulate chromium stress by taking the free Pro as a biomarker is obtained, and stress resistance regulation and optimization strategies of the rice at different growth stages are determined according to the contribution model;

in step 3, the matrix ii is specifically as follows:

；

in matrix II, r ₁ Represents the contribution of nitrate nitrogen treatment to free Pro; r is ₂ Represents the contribution of ammonium nitrogen treatment to free Pro; r is ₃ Represents the contribution of the difference in free Pro from the ammonium nitrogen treatment and the nitrate nitrogen treatment;

in step 3, the matrix iii is as follows:

；

in matrix III, c ₁ Indicates the contribution of arginine treatment to free Pro; c. C ₂ Represents the contribution of glutamate treatment to free Pro; c. C ₃ Represents the contribution representing the difference in free Pro from glutamate treatment and arginine treatment;

in step 3, the matrix iv is as follows:

；

wherein-N _o and-N _I Set to 0;

in the matrix IV, a ₁₁ Represents the contribution of arginine treatment to the difference in free Pro from nitrate nitrogen treatment; a is a ₁₂ Represents the contribution of glutamic acid treatment and nitrate nitrogen treatment to the difference of free Pro; a is ₁₃ Represents the contribution of glutamic acid treatment to the difference of free Pro from arginine treatment and nitrate nitrogen treatment; a is a ₂₁ Represents the contribution of arginine treatment to the difference in free Pro from ammonium nitrogen treatment; a is ₂₂ Represents the contribution of glutamate treatment and ammonium nitrogen treatment to the difference in free Pro; a is a ₂₃ Represents the contribution of the difference of free Pro by glutamic acid treatment, arginine treatment and ammonium nitrogen treatment; a is ₃₁ Represents the contribution of arginine treatment, nitrate nitrogen treatment and ammonium nitrogen treatment to the difference of free Pro; a is ₃₂ Represents the contribution of glutamic acid treatment, nitrate nitrogen treatment and ammonium nitrogen treatment to the difference of free Pro; a is a ₃₃ Represents the contribution of glutamic acid treatment to the difference of free Pro from arginine treatment, nitrate nitrogen treatment and ammonium nitrogen treatment;

in step 3, the matrix v is as follows:

。

2. the method for determining the rice stress-resistance control optimization strategy as claimed in claim 1, wherein in step 4, the third row element a of the respective comparison matrix IIis _3j Matrix III third column element a _i3 And a in matrix V ₁₁ 、a ₁₂ 、a ₂₁ 、a ₂₂ The amount of contribution of inorganic nitrogen or organic nitrogen to free Pro compared to the amount of contribution of inorganic nitrogen or organic nitrogen administered alone is obtained by:

comparing the contribution of the ammonium nitrogen alone and the nitrate nitrogen alone to the free Pro according to the positive and negative of the numerical values of the elements in the third row of the matrix II; comparing the contribution of glutamic acid alone to arginine alone to free Pro according to the positive and negative values of the elements in the third column of matrix III; according to a of matrix V ₁₁ Positive and negative values, comparing the contribution of arginine alone to free Pro versus nitrate nitrogen alone; according to a of matrix V ₁₂ The magnitude of the contribution to free Pro of glutamic acid alone versus nitrate nitrogen alone; according to a of matrix V ₂₁ Positive and negative values, comparing the amount of contribution of arginine alone to free Pro versus ammonium nitrogen alone; according to a of matrix V ₂₂ The magnitude of the contribution to free Pro compared to glutamic acid alone versus ammonium nitrogen alone;

the magnitude of the contribution of inorganic or organic nitrogen alone to free Pro is obtained from the magnitude of the contribution of ammonium nitrogen alone and nitrate nitrogen alone to free Pro, the magnitude of the contribution of glutamic acid alone and arginine alone to free Pro, the magnitude of the contribution of arginine alone and nitrate nitrogen alone to free Pro, the magnitude of the contribution of glutamic acid alone and nitrate nitrogen alone to free Pro, the magnitude of the contribution of arginine alone and ammonium nitrogen alone to free Pro, and the magnitude of the contribution of glutamic acid alone and ammonium nitrogen alone to free Pro.