CN114574616A - Kit and method for detecting cadmium ions in rice plants - Google Patents

Abstract

The invention discloses a kit and a method for detecting cadmium ions in rice plants. The detection method comprises the following steps: carrying out prehybridization on Enzyme strand and Substrate strand to obtain prehybridization solution; mixing the rice tissue to be detected with the pre-hybridization solution, and detecting the content of cadmium by detecting the intensity of a fluorescent signal. The invention takes cadmium ions as molecular biomarkers and is based on Cd²⁺Establishment of Cd by dependence on DNAzyme enzymatic reaction and Fluorescence Resonance Energy Transfer (FRET) mechanism²⁺Detect biosensor, realize Cd in rice plant²⁺The rapid detection of (2). The method has the unique advantages of high sensitivity, good stability, low cost and the like, and has important significance for quickly identifying and breeding the rice variety with low cadmium accumulation.

Description

Kit and method for detecting cadmium ions in rice plants

Technical Field

The invention relates to the technical field of plant protection, in particular to a kit and a method for detecting cadmium ions in rice plants.

Background

Cadmium is widely applied to the industrial fields of electroplating, mining, smelting, batteries and the like, so that farmlands in China are widely polluted by cadmium. Cadmium can enter human body through food chain, and has biological half-life period of 10-30 years in human body. Excessive cadmium accumulation can lead to the development of diseases including kidney damage, osteoporosis and even cancer. Therefore, cadmium is classified as the first cancer by the international agency for research on cancer, and several mandatory standards are established in each country for limiting the cadmium content in food.

The rice is a crop which is easy to enrich cadmium. In recent years, the cadmium rice event in Hunan has attracted much attention. At present, emergency varieties with low cadmium accumulation are mainly screened from the existing rice varieties in production, but the problem of cadmium pollution of rice can be only partially solved, and the rice varieties with low cadmium accumulation which are suitable for being planted in areas with severe cadmium pollution are urgently needed to be produced. Therefore, the screening and the cultivation of the rice variety with low cadmium accumulation which can be planted in the areas with different cadmium pollution degrees become urgent.

According to the regulation of GB/T5009.15, the existing cadmium content detection method mainly comprises the following steps: atomic absorption spectrometry, atomic fluorescence, colorimetry, high performance liquid chromatography, and the like. However, the methods have the disadvantages of long experimental period, high cost, complicated detection steps and the like to different degrees, and are difficult to popularize and use on a large scale. There is a need to find a molecular biomarker based on cadmium ionCd²⁺Establishment of Cd by dependence on DNAzyme enzymatic reaction and Fluorescence Resonance Energy Transfer (FRET) mechanism²⁺Detect biosensor, realize Cd in rice plant²⁺The method of (1) for rapid detection.

Disclosure of Invention

Aiming at the technical problems to be solved, the invention provides a kit for detecting cadmium ions in rice plants and a detection method thereof²⁺Establishment of Cd by dependence on DNAzyme enzymatic reaction and Fluorescence Resonance Energy Transfer (FRET) mechanism²⁺Detect biosensor, realize Cd in rice plant²⁺The rapid detection of (2). The method has the unique advantages of high sensitivity, good stability, low cost and the like, the period of the whole detection process is short, the reaction is rapid, the operation process can be mastered without professional training, the method is convenient to rapidly popularize and use, and the method has important significance for rapidly identifying and breeding the rice varieties with low cadmium accumulation.

In order to achieve the above object, the present invention provides a kit for detecting cadmium ions in rice plants, comprising: enzyme strand and Substrate strand;

the DNA sequence of the Enzyme strand is shown as SEQ ID NO. 1;

the DNA sequence of the Substrate strand is shown as SEQ ID NO. 2.

In the kit, the 3' end of the Enzyme strand is modified with a fluorescence quenching group; the 5' end of the Substrate strand is modified with a fluorescent group.

Further, in the kit, the fluorescence quenching group is BHQ2, and the fluorescent group is ROX.

The kit further comprises a MES buffer. Further, the MES buffer solution comprises 20 to 150mM MES and 10 to 100mM NaCl, and has a pH of 4.5 to 6.5. Further, the MES buffer includes 50mM MES, 25mM NaCl, pH 6.0.

Based on a general technical concept, the invention also provides a detection method of cadmium ions in rice plants, which comprises the following steps:

s1, carrying out prehybridization on Enzyme strand and Substrate strand to obtain a prehybridization solution;

s2, mixing the rice tissue to be detected with the pre-hybridization solution, and detecting the cadmium content by detecting the intensity of the fluorescence signal.

In the detection method, further, the step S1 is specifically:

s1-1, diluting Enzyme strand and Substrate strand dry powder to 10-500 nM by MES buffer solution respectively, and fully mixing according to the volume ratio of 1: 1-10: 1 to obtain a mixed solution;

and S1-2, preserving the temperature of the mixed solution in a water bath kettle at the temperature of 90-97 ℃ for 5-15 min, and naturally cooling overnight to form a complementary double-stranded structure.

In the detection method, further, the step S2 is specifically:

S2-A1, immersing the first leaf of the fully-unfolded leaf of the rice seedling into a pre-hybridization solution;

S2-A2, carrying out vacuum pumping treatment, starting timing at-50 kPa to 100kPa, maintaining for 5 to 30min, and standing overnight;

S2-A3, placing the leaf in a plant living body molecular imaging system to observe the fluorescence spectrum of the leaf.

In the detection method, further, the step S2 is specifically:

S2-B1, cutting the upper part of the roots of the rice seedlings, and collecting bleeding sap flowing out of the cut;

S2-B2, mixing the rice bleeding sap and the pre-hybridization solution to obtain a mixed solution;

S2-B3, placing the mixed solution in a plant living body molecular imaging system to observe a fluorescence spectrum.

In the detection method, further, the step S2 is specifically:

S2-C1, soaking the rice seeds in the prehybridization solution, and standing overnight in a dark environment;

S2-C2, placing the seeds in a plant living body molecular imaging system to observe a fluorescence spectrum.

Compared with the prior art, the invention has the advantages that:

1. the invention provides a testThe kit for detecting cadmium ions in rice plants takes the cadmium ions as molecular biomarkers and is based on Cd²⁺Establishment of Cd by dependence on DNAzyme enzymatic reaction and Fluorescence Resonance Energy Transfer (FRET) mechanism²⁺Detect the biosensor to achieve the aim of detecting Cd in rice leaves²⁺The rapid detection is carried out, thereby realizing the rapid identification and breeding of the rice variety with low cadmium accumulation.

2. The invention provides the application of the kit for detecting the cadmium ions in the rice plants in the detection of the cadmium ions in the rice plants, and the method has the unique advantages of high sensitivity, good stability, low cost and the like, has short period of the whole detection process, is quick in reaction, can master the operation flow without professional training, and is convenient for quick popularization and use. For Cd in rice plants²⁺The rapid detection and the rapid identification and breeding of the rice variety with low cadmium accumulation are of great significance.

Drawings

In order to make the objects, 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 with reference to the drawings in the embodiments of the present invention.

FIG. 1 is a schematic diagram of the kit for detecting cadmium ions according to example 1 of the present invention;

FIG. 2 is a graph showing the result of identifying an actual sample of a rice leaf detected in example 2 of the present invention;

FIG. 3 is a graph showing the result of identifying an actual sample for detecting a bleeding sap of rice in example 3 of the present invention;

FIG. 4 is a graph showing the results of identification of actual samples for detecting rice seeds in example 4 of the present invention.

FIG. 5 is a graph showing the results of sensitivity detection in example 5 of the present invention.

Detailed Description

The invention is further described below with reference to specific preferred embodiments, without thereby limiting the scope of protection of the invention.

Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods. The methods in the following examples are conventional in the art unless otherwise specified.

Examples

The materials and equipment used in the following examples are commercially available.

The first embodiment is as follows:

a kit for detecting cadmium ions in rice plants comprises Enzyme strand chains, Substrate strand chains and MES buffer solution.

Wherein the Enzyme strand and Substrate strand chains are synthesized by Biotechnology engineering (Shanghai) Inc.

The DNA sequence of Enzyme strand is shown as SEQ ID NO.1, and specifically comprises:

5'-gttcgccatcttccttcgatagttaaaatagtgact-BHQ2-3'

the DNA sequence of the Substrate strand is shown as SEQ ID NO.2, and specifically comprises:

5'-ROX-AGTCACTATrA*GGAAGATGGCGAAC-3'。

wherein, T-BHQ2 represents a modified quenching group BHQ2 on the T base at the 3' terminal; ROX-A represents a modified fluorophore at the A base at the 5' end; rA represents phosphorothioated modified Cd²⁺A DNAzyme-dependent cleavage site.

The MES buffer solution is: 100mL MES (2- (N-morpholine) ethanesulfonic acid) solution with the concentration of 50mM is prepared by the following method: 0.1461g of NaCl was weighed into a beaker, and a small amount of 50mM MES solution was added thereto, and after sufficient dissolution, the volume of the solution was adjusted to 100mL with 50mM MES solution, and the pH was adjusted to 6.0 with NaOH solution to obtain MES buffer.

The detection principle of the detection kit of the embodiment is shown in figure 1, and specifically comprises the following steps:

substrate strand and Enzyme strand nucleotide sequences are characterized by complementarity at both ends, and as temperature is lowered, Enzyme strand and Substrate strand anneal sufficiently to form complementary double-stranded structures. The double-stranded structure causes the quenching group (BHQ2) on the Enzyme strand and the fluorescent group (ROX) on the Substrate strand to be close to each other, and the pre-hybridized mixed solution shows no fluorescent signal or only a weak background fluorescent signal under the action of Fluorescence Resonance Energy Transfer (FRET).

If Cd is present in the reaction system²⁺Free Cd²⁺Cd in Enzyme strand²⁺The enzyme chain with catalytic activity is formed by the combination of the dependent DNAzyme enzyme chain. The activated Enzyme chain catalyzes cleavage of the Substrate strand from rA, Enzyme strand/Substrate strand/Cd with loss of intact structure²⁺The complex finally disintegrates to release Cd²⁺Enzyme strand and cleaved Substrate strand. Released Cd²⁺The Enzyme strand which further participates in the next cycle of enzymatic reaction and is changed into a free state again forms a complementary double-stranded structure with a small amount of free Substrate strand in the mixed solution, and the background signal is further reduced under the action of Fluorescence Resonance Energy Transfer (FRET). The cleaved Substrate strand free fragment generates a strong fluorescent signal due to the separation of the fluorophore (ROX) and quencher (BHQ 2).

If Cd is absent in the reaction system²⁺In the process, Enzyme strand and Substrate strand form a stable complementary double-stranded structure, and the mixed solution after prehybridization does not display a fluorescence signal or only has a weak background fluorescence signal under the action of Fluorescence Resonance Energy Transfer (FRET), so that Cd is realized²⁺And (4) identifying.

Cd in the environment²⁺The higher the content of (A), the stronger the fluorescence signal. By comparing the intensity of the fluorescence signal, the rapid identification and breeding of the low cadmium accumulation rice variety are realized.

Example 2:

a method for rapidly detecting cadmium ions in rice plants comprises the following steps of detecting objects of a low cadmium accumulation rice variety, namely Lopa nationality red 3B and a high cadmium accumulation rice variety Huazhan leaf, and specifically detecting the cadmium ions in the rice plants by the method:

(1) enzyme strand and Substrate strand prehybridization: enzyme strand and Substrate strand dry powders in the detection kit of example 1 were diluted to 100nM with MES buffer, respectively, and the diluted Enzyme strand and Substrate strand were mixed well at a volume ratio of 4:1, the mixture was incubated in a 95 ℃ water bath for 7min, and the power was turned off, and allowed to cool naturally overnight to form a complementary double-stranded structure, to obtain a prehybridization solution. The whole process is carried out under the condition of keeping out of the sun.

(2) Culturing rice seedlings:

2.1, 50 full seeds of each of the rice variety with low cadmium accumulation (Lopa L3B) and the rice variety with high cadmium accumulation (Huazhan) are taken, the seeds are soaked in clear water for 48 hours, and the clear water is replaced every 12 hours in the period. Taking out the seeds, wrapping the seeds with a wet towel, putting the wrapped seeds in a 37 ℃ thermostat for germination acceleration, sowing the seeds in a 96-hole plate with an opening at the bottom after the seeds are exposed to white, putting the 96-hole plate in a water culture box, adding clear water until the water surface just submerges the seeds, and putting the seeds in a 37 ℃ light culture chamber for continuous growth.

2.2 after two days of hydroponic growth, the clear water was replaced with a rice hydroponic nutrient solution (using Yoshida rice nutrient solution (dry powder) from Coolaber, Inc., having a stock number of NSP1040-1 kg.) to continue the growth, during which the nutrient solution was replaced every 3 days. After 2 weeks, CdCl was added to the nutrient solution₂The final concentration was adjusted to 4mg/L, and the culture was continued for 1 week.

(3) Rice leaf infection:

3.1, adding 1.5mL of sterile water into a 2mL centrifuge tube, and simultaneously adding a small amount of surfactant (Triton X-100) to fully mix the mixture.

3.2, cutting a long leaf section of 1.5cm from the middle part of the first fully-unfolded leaf of the rice seedling by using a pair of scissors, placing the long leaf section in a centrifuge tube, turning upside down, uniformly mixing for 8min, and transferring the rice leaf to sterile water for 3 times clearly. Taking out the rice leaves, placing the rice leaves on absorbent paper, and placing the rice leaves in a new 2mL centrifuge tube after fully removing surface residual water.

And 3.3, respectively adding the prehybridization liquid obtained in the step 1 into the centrifuge tube to enable the liquid level to be submerged over the top end of the blade.

And 3.4, opening the centrifugal tube cover, placing the centrifugal tube cover in a vacuumizing device for vacuumizing, and starting timing and maintaining for 10min when the pressure reaches-70 kPa. The tube was removed, capped, and placed in the dark overnight.

(4) Rice leaf Cd²⁺And (3) detection: carefully taking out rice leaf with tweezers for next day, placing the leaf on absorbent paper, carefully cleaning surface pre-hybridization solution, observing fluorescence spectrum of the leaf in living plant molecular imaging system, setting excitation spectrum at 520nm, and collecting Cd in the rice leaf²⁺The higher the content, the stronger the fluorescence signal. The rapid identification and breeding of the rice variety with low cadmium accumulation is realized through the strength of the leaf fluorescence signal.

The detection results are shown in fig. 2: the redder the color (shown as darker in the figure) represents Cd²⁺The higher the content, the lighter the color indicates Cd²⁺The lower the content. Wherein CK is not subjected to Cd²⁺Processing, so there is almost no red in the middle of the leaf (the darker part is shown as red in the figure, the small amount in the middle of the leaf and the red at the fracture should be noise); 3B and Huazhan all underwent Cd²⁺Treatment (Cd)²⁺Treatment concentration and time are the same), since 3B is known Cd²⁺Low accumulation of Cd in all leaves of rice²⁺Low content (light color), Huazhan is conventional material (normal absorption Cd)²⁺) And so the color is dark.

Different experimental conditions have certain influence on experimental results. The optimal condition for each reaction is that the corresponding signal-to-noise ratio reaches a maximum. This example is carried out on the leaf under optimum conditions by Cd²⁺The content detection result is as follows: 3B is 0.029 mg/kg; huazhan is 0.331mg/kg, and the experimental effect reaches the best.

Example 3:

a method for rapidly detecting cadmium ions in rice plants comprises the following steps:

(1) enzyme strand and Substrate strand prehybridization:

the Enzyme strand and Substrate strand dry powders in the detection kit of example 1 are respectively diluted to 100nM by MES buffer solution, the diluted Enzyme strand and Substrate strand are fully mixed according to the volume ratio of 4:1, the mixed solution is kept warm for 7min in a water bath kettle at 95 ℃, the power supply is turned off, the mixed solution is naturally cooled overnight, a complementary double-stranded structure is formed, and the whole process is carried out under the condition of keeping out of the sun.

(2) Obtaining a bleeding sap of a rice plant:

2.1, 50 full seeds of each of the rice variety with low cadmium accumulation (Lopa L3B) and the rice variety with high cadmium accumulation (Huazhan) are taken, the seeds are soaked in clear water for 48 hours, and the clear water is replaced every 12 hours in the period. Taking out the seeds, wrapping the seeds with a wet towel, putting the wrapped seeds in a 37 ℃ thermostat for germination acceleration, sowing the seeds in a 96-hole plate with an opening at the bottom after the seeds are exposed to white, putting the 96-hole plate in a water culture box, adding clear water until the water surface just submerges the seeds, and putting the seeds in a 37 ℃ light culture chamber for continuous growth.

2.2, after the water culture growth for two days, replacing the clear water with the water culture nutrient solution of the rice to enable the rice to continue to grow, and replacing the nutrient solution every 3 days in the period. After 2 weeks, CdCl was added to the nutrient solution₂The final concentration was adjusted to 4mg/L, and the culture was continued for 1 week.

2.3, after the culture is finished, cutting off the rice seedlings regularly at a position 1cm away from the roots by using scissors and continuously placing the rice seedlings in a light culture chamber for growing. After half an hour, bleeding-out of bleeding-out liquid at the fracture of the rice seedling can be seen, and the Lopa red 3B and Huazhan bleeding-out liquid are respectively collected by a liquid transfer gun.

(3) Bleeding sap Cd for rice²⁺And (3) detection:

3.1, sucking 50 mul of each of Lonicera japonica Red 3B and Huazhan bleeding sap by a pipette, respectively placing the 50 mul of Lonicera japonica Red 3B and Huazhan bleeding sap into a clean PCR tubule, respectively adding 5 mul of the prehybridization fluid obtained in the step 1.3 into the two tubules, respectively mixing the two tubules uniformly, placing the PCR tubule into a plant living body molecular image system to observe a fluorescence spectrum, setting an excitation spectrum to be 520nm, and placing Cd in the bleeding sap²⁺The higher the content, the stronger the fluorescence signal. The rapid identification and breeding of the rice variety with low cadmium accumulation is realized through the strength of the bleeding liquid fluorescence signal.

The results of the test are shown in FIG. 3. FIG. 3 is a view similar to FIG. 2 showing different tests performed on the same batch of material. Cd [ Cd ]²⁺Cd entering cells from rice root system, transferring from root cells to xylem, transferring from xylem to phloem between rice basal segments, and entering phloem²⁺As the moisture is transported towards the blades. Thus, Cd in bleeding fluids²⁺Content (wt.)Cd capable of reflecting rice root system absorption²⁺And (4) content. FIG. 3 shows the deepest color of Chinese Huazhan, which indicates the Cd absorbed by the root system of Huazhan²⁺The content is the most (the upper half part of the Huazhan bleeding sap tube is red in color, and the lower half part of the Huazhan bleeding sap tube is yellow because the lower end of the PCR tubule is conical and cannot be kept on the same horizontal plane with the upper end, and a camera cannot focus when a picture is taken).

Different experimental conditions have certain influence on experimental results. The optimal condition for each reaction is that the corresponding signal-to-noise ratio reaches a maximum. In the best condition, Cd is carried out on the bleeding fluid²⁺The content detection result is as follows: 3B is 0.017 mg/kg; the Huazhan is 0.0441mg/kg, and the experimental effect is optimal.

Example 4:

a method for rapidly detecting cadmium ions in rice plants is applied to rice seeds obtained by respectively planting a high-cadmium-accumulation rice variety Huazhan in a conventional field and a cadmium ion polluted field, and specifically comprises the following steps:

(1) enzyme strand and Substrate strand prehybridization: enzyme strand and Substrate strand dry powders in the detection kit of example 1 were diluted to 100nM with MES buffer, respectively, and the diluted Enzyme strand and Substrate strand were mixed well at a volume ratio of 4:1, the mixture was incubated in a 95 ℃ water bath for 7min, and the power was turned off, and allowed to cool naturally overnight to form a complementary double-stranded structure, to obtain a prehybridization solution. The whole process is carried out under the condition of keeping out of the sun.

(2) Obtaining rice seeds:

2.1, taking the Huazhan rice seeds to normally accelerate germination and sowing the Huazhan rice seeds in a nursery, taking 25-day-old plants to transplant into pots (the inner diameter of the pot is 266mm, the height of the pot is 190mm), and transplanting ten pots in total for 3 plants in each pot. Wherein five pots are made of conventional soil, and corresponding CdCl is added into the five pots according to the weight of the added soil₂The concentration of cadmium ions in the solution is 1.0 mg/Kg.

And 2.2, after the transplanting is finished, moving the pot into a glass greenhouse to avoid the influence of rainwater on the concentration of cadmium ions. During the period, normal water and fertilizer management is carried out until the rice seeds are ripe and received.

(3) RiceCd²⁺And (3) detection:

3.1, respectively taking the Huazhan rice seeds harvested from the soil with different cadmium ion concentrations, carefully removing the shells by using tweezers, then putting the grains after the shells are removed into 1.5mL centrifuge tubes, then respectively adding the prehybridization liquid obtained in the step 1.3 into the two centrifuge tubes to ensure that the liquid level of the prehybridization liquid is over the seeds, wrapping the mixture by using tin foil paper, and then placing the mixture in a dark environment for standing for 24 hours.

3.2, taking out the seeds, fully absorbing residual moisture on the surfaces of the seeds by using absorbent paper, placing the seeds in a plant living body molecular imaging system to observe a fluorescence spectrum, setting an excitation spectrum to be 520nm, and adding Cd into the seeds²⁺The higher the content, the stronger the fluorescence signal. The detection of the content of cadmium ions in the rice is realized through the strength of the seed fluorescent signal.

The detection results are shown in FIG. 4: cd in Huazhan seed²⁺High content, so the color is red (the upper half of the seed is yellow and the same principle is shown in figure 3), the whole figure 4 is divided into a bright field part and a dark field part, wherein the bright field part is used for taking a picture under a normal light source, and the dark field part is used for taking a picture under an excitation wave of 520 nm.

Different experimental conditions have certain influence on experimental results. The optimal condition for each reaction is that the corresponding signal-to-noise ratio reaches a maximum. This example shows that under the optimum conditions, we carry out Cd on rice²⁺The content detection result is as follows: 3B is 0.029 mg/kg; huazhan is 0.331mg/kg, and the experimental effect reaches the best.

Examine the sensitivity of the detection method of example 5:

using the method of example 5, for different Cd²⁺The concentration (0-450 nM) of the standard was measured and the results are shown in FIG. 5.

As can be seen from the figure, when Cd²⁺Fluorescence (detected) was observed at concentrations as low as 50nM (about 5.62X 10-6g/kg), as specified by the national Standard for Cd in rice²⁺The concentration is not more than 2 x 10^-4g/kg, the lower limit of detection is two orders of magnitude lower than GB or even lower, and the detection method is proved to be high in sensitivity.

The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make many variations and modifications to the disclosed embodiments, or equivalent variations, without departing from the spirit and scope of the invention, using the methods and techniques disclosed above. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention.

Sequence listing

<110> research center for hybrid rice in Hunan province

Hunan Agricultural University

<120> reagent kit for detecting cadmium ions in rice plants and detection method

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gttcgccatc ttccttcgat agttaaaata gtgact 36

<210> 2

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

agtcactatg gaagatggcg aac 23

Claims (10)

1. A kit for detecting cadmium ions in rice plants is characterized by comprising: enzyme strand and Substrate strand;

the DNA sequence of the Enzyme strand is shown as SEQ ID NO. 1;

the DNA sequence of the Substrate strand is shown as SEQ ID NO. 2.

2. The kit according to claim 1, wherein the 3' end of the Enzyme strand is modified with a fluorescence quenching group; the 5' end of the Substrate strand is modified with a fluorescent group.

3. The kit of claim 2, wherein the fluorescence quencher is BHQ2 and the fluorophore is ROX.

4. The kit of claim 1, further comprising a MES buffer.

5. The kit according to claim 4, wherein the MES buffer comprises 20 to 150mM MES and 10 to 100mM NaCl, and has a pH of 4.5 to 6.5.

6. A detection method of cadmium ions in rice plants is characterized by comprising the following steps:

s1, prehybridization of Enzyme strand and Substrate strand in the kit according to any one of claims 1 to 5 to obtain a prehybridization solution;

s2, mixing the rice tissue to be detected with the pre-hybridization solution, and detecting the cadmium content by detecting the intensity of the fluorescence signal.

7. The detection method according to claim 6, wherein the S1 is specifically:

s1-1, diluting Enzyme strand and Substrate strand dry powder to 10-500 nM with MES buffer solution respectively, and fully mixing according to the volume ratio of 1: 1-10: 1 to obtain mixed solution;

and S1-2, preserving the temperature of the mixed solution in a water bath kettle at the temperature of 90-97 ℃ for 5-15 min, and naturally cooling overnight to form a complementary double-stranded structure.

8. The detection method according to claim 6 or 7, wherein the S2 is specifically:

S2-A1, immersing the first leaf of the fully-unfolded leaf of the rice seedling into a pre-hybridization solution;

S2-A2, carrying out vacuum pumping treatment, starting timing at-50 kPa to 100kPa, maintaining for 5 to 30min, and standing overnight;

S2-A3, placing the leaf in a plant living body molecular imaging system to observe the fluorescence spectrum of the leaf.

9. The detection method according to claim 6 or 7, wherein the S2 is specifically:

S2-B1, cutting the upper part of the roots of the rice seedlings, and collecting bleeding sap flowing out of the cut;

S2-B2, mixing the rice bleeding sap and the pre-hybridization solution to obtain a mixed solution;

S2-B3, placing the mixed solution in a plant living body molecular imaging system to observe a fluorescence spectrum.

10. The detection method according to claim 6 or 7, wherein the S2 is specifically:

S2-C1, soaking the rice seeds in the prehybridization solution, and standing overnight in a dark environment;

S2-C2, placing the seeds in a plant living body molecular imaging system to observe a fluorescence spectrum.

Images