CN117604088B - Application of abscisic acid and environmental stress induced protein TAS14 in rapid detection of cadmium pollution - Google Patents

Application of abscisic acid and environmental stress induced protein TAS14 in rapid detection of cadmium pollution Download PDF

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CN117604088B
CN117604088B CN202410094621.4A CN202410094621A CN117604088B CN 117604088 B CN117604088 B CN 117604088B CN 202410094621 A CN202410094621 A CN 202410094621A CN 117604088 B CN117604088 B CN 117604088B
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tas14
abscisic acid
gene
environmental stress
tobacco
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CN117604088A (en
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陈雅琼
周瑾
刘昕雨
张欢
罗程
李明波
李卓衡
郑吉云
田华
代瑾然
陈穗云
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Yunnan University YNU
Kunming Company of Yunnan Tobacco Co
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Kunming Company of Yunnan Tobacco Co
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Abstract

The invention relates to the technical field of molecular biology detection of environmental pollution, in particular to application of abscisic acid and an environmental stress induced protein TAS14 in rapid detection of cadmium pollution. The invention utilizes the change of the expression quantity of tobacco abscisic acid and the environmental stress induced protein TAS14 gene to rapidly detect the pollution degree of heavy metal cadmium in plant soil, and monitors the pollution of plants in short term and long term. The detection method of the invention uses abscisic acid and environmental stress induced protein TAS14 as biomarkers, and can sensitively monitor the pollution degree and the hazard degree of heavy metal cadmium pollution. The method disclosed by the invention uses tobacco as an experimental object, and a novel biomarker is screened out for rapidly detecting heavy metal cadmium, so that a great improvement effect on the detection technology of the heavy metal cadmium pollution of the current soil is achieved, and the method has a good popularization and application prospect.

Description

Application of abscisic acid and environmental stress induced protein TAS14 in rapid detection of cadmium pollution
Technical Field
The invention relates to the technical field of molecular biology detection of environmental pollution, in particular to application of abscisic acid and an environmental stress induced protein TAS14 in rapid detection of cadmium pollution.
Background
With the development of economy and the improvement of productivity, heavy industries such as chemical industry, mining, metallurgy, steel and the like rapidly develop, and the environment is invaded by 'three wastes' which are unreasonably discharged in order to pursue economic benefits, a large amount of toxic heavy metals are accumulated in nature due to large-scale use of pesticides and random discarding and stacking of a large amount of garbage and wastes, and heavy metal pollution in soil is serious.
Heavy metal cadmium (Cd) is a hazardous substance widely existing in the environment, which poses serious threat to plant and human health. Cadmium accumulation can cause growth limitation of plant root systems, dysplasia of stems and leaves, and morphological abnormality such as leaf atrophy, leaf edge scorch and the like. In plants, cd 2+ acts as a poison to plants by inhibiting enzymatic activity, interfering with the endogenous hormone balance and membrane permeability of the plant, disrupting the physiological metabolic processes of the plant. At present, heavy metal cadmium is listed as a first-level cancerogenic substance by the international cancer research Institution (IARC), cd 2+ enters human body for enrichment through a food chain after being absorbed by plants, and various diseases can be caused to a certain extent, for example, long-term exposure to cadmium can increase the risk of various cancers such as colon, prostate, lung and the like. In addition, cadmium accumulated in human body can interfere balance of minerals such as calcium, phosphorus and the like, so that bone diseases such as osteoporosis, fracture and the like are caused, health of human body is seriously endangered, and the health-care food has the characteristics of long-term property, concealment, irreversibility and the like.
In order to prevent the pollution from being aggravated, the pollution needs to be detected and pre-warned in the early stage of the pollution, and corresponding effective measures are adopted to reduce the pollution and prevent the pollution from being aggravated. The existing traditional plant soil heavy metal detection method has the defects of long period and low sensitivity, pollution cannot be timely early-warned, corresponding adjustment measures can not be made, and the crop yield is negatively influenced.
Disclosure of Invention
The invention solves the problems of long period and low sensitivity of the existing plant soil heavy metal cadmium detection method, provides application of abscisic acid and environment stress induced protein TAS14 in rapid detection of cadmium pollution, has high sensitivity and high-speed reaction of molecules of the abscisic acid and the environment stress induced protein TAS14 on heavy metal cadmium pollutants, and can be used as early pollution early warning molecules.
The technical scheme of the invention is as follows:
Application of abscisic acid and environmental stress induced protein TAS14 in rapid detection of cadmium pollution is provided.
Preferably, the amino acid sequences of the abscisic acid and the environmental stress induction protein TAS14 are shown in SEQ ID NO:3 is shown in the figure; the nucleotide sequences of the abscisic acid and the environmental stress induction protein TAS14 gene are shown in SEQ ID NO: 1.
The invention also provides a method for rapidly detecting the cadmium pollution degree of plant soil, which adopts a fluorescence quantitative PCR-based method to detect the expression quantity of abscisic acid and an environmental stress induction protein TAS14 gene in a sample to be detected, and if the expression quantity of the abscisic acid and the environmental stress induction protein TAS14 gene is up-regulated, the cadmium pollution of the sample to be detected is prompted.
Preferably, in the fluorescent quantitative PCR method, a forward primer and a reverse primer of an abscisic acid and an environmental stress induction protein TAS14 encoding gene and a premix are adopted to carry out fluorescent quantitative PCR experiment, and an internal reference gene NtUbc is used as an internal standard.
Preferably, the following reaction system and procedure are used in the fluorescent quantitative PCR method:
And/or preparing a fluorescent quantitative PCR reaction system by adopting SYBR Green dye reagent, wherein the fluorescent quantitative PCR reaction system comprises TB Green Premix Ex Taq II（2×）5 μl;Primer F (10 μM) 0.4 μl;Primer R (10 μM)0.4 μl;ROX Reference Dye II（50×） 0.2 μl;cDNA template 1 μl; sterilized water 3. Mu.l;
And/or the amplification procedure of fluorescent quantitative PCR is: 94 ℃ for 30s;94 ℃ for 5s;57.8 ℃,34s;40 cycles.
The invention also discloses a kit for detecting the expression quantity of the abscisic acid and the environmental stress induced protein TAS14 coding gene, which comprises a primer for quantitatively detecting the abscisic acid and the environmental stress induced protein TAS14 coding gene and a fluorescent quantitative PCR detection premix.
Preferably, the nucleotide sequence of the primer in the kit is shown as SEQ ID NO:4 and SEQ ID NO: shown at 5.
The invention also discloses application of the reagent for inhibiting the expression of the abscisic acid and the environmental stress induced protein TAS14 coding gene in constructing heavy metal cadmium pollution sensitive plants.
Preferably, the method of use of the above application is virus-induced gene silencing.
The beneficial effects are that:
The invention provides an application of abscisic acid and an environment stress induced protein TAS14 as biomarkers in rapid detection of cadmium pollution. Experiments show that when tobacco is used as a plant representative, the abscisic acid and the environmental stress induction protein TAS14 gene generate forward response when being stressed by cadmium ions, and the expression level of the gene is obviously up-regulated, so that the heavy metal cadmium pollution condition of the plant is reflected. Meanwhile, the result of knocking out abscisic acid and an environmental stress induction protein TAS14 gene in tobacco shows that the tobacco after gene knocking out shows intolerance to cadmium ion stress. Therefore, abscisic acid and the environmental stress induction protein TAS14 respond to heavy metal cadmium stress, and are expressed as up-regulated expression when the heavy metal cadmium stress occurs, and the plant with the gene knocked out is intolerant to the heavy metal cadmium. Therefore, the abscisic acid and the environmental stress induction protein TAS14 can be used as biomarkers for rapidly detecting the pollution of heavy metal cadmium, and the problem of long period of the existing plant soil heavy metal cadmium detection method is solved; experimental results show that the detection of abscisic acid and the environmental stress induced protein TAS14 gene is used for detecting heavy metal cadmium in plants, so that the repeatability and the sensitivity are good, and the problem that the sensitivity of the existing plant soil heavy metal cadmium detection method is low is solved.
The method utilizes the detection of the abscisic acid and the environmental stress induction protein TAS14 gene to detect the heavy metal cadmium in the plants, can shorten the detection period, improve the detection sensitivity, has the advantages of high reliability, good repeatability, high sensitivity, lower cost and the like, plays a great promotion role in the current detection technology of the heavy metal cadmium pollution of the plants, and has better popularization and application prospects.
Drawings
FIG. 1 is a plant height phenotype observation of tobacco after cadmium stress treatment, wherein A is plant height phenotype comparison of 5-week-old wild-type K326 tobacco after 20 days of treatment with 50mM Cd 2+, and B is plant height change of tobacco before and after cadmium stress treatment.
FIG. 2 is a tobacco root phenotype observation after 5 week old wild type K326 tobacco is treated for two months with 50mM Cd 2+ stress.
FIG. 3 shows the change of various physiological indexes before and after the cadmium stress treatment of wild K326 tobacco.
FIG. 4 shows the effect of cadmium stress on the expression level of abscisic acid and the environmental stress-inducing protein TAS14 gene in tobacco.
FIG. 5 shows the detection of gene silencing efficiency of gene silencing plant abscisic acid and the environmental stress inducible protein TAS 14.
FIG. 6 is a phenotypic observation after cadmium stress of gene-silenced plants.
Detailed Description
The invention provides an application of abscisic acid and an environmental stress induced protein TAS14 in rapid detection of cadmium pollution; the coding locus number of the abscisic acid and the environmental stress induction protein TAS14 is LOC107819915, the length of mRNA is 881bp, and in the specific embodiment of the invention, the nucleotide sequence of the abscisic acid and the environmental stress induction protein TAS14 coding gene is shown as SEQ ID NO: 1.
The nucleotide sequence of the CDS sequence of the abscisic acid and the environmental stress induction protein TAS14 coding gene is shown in SEQ ID NO: 2.
The amino acid sequences of abscisic acid and an environmental stress induction protein TAS14 are shown as SEQ ID NO: 3.
The present invention will be described in detail with reference to examples, but they should not be construed as limiting the scope of the invention.
Example 1
Application of abscisic acid and environmental stress induced protein TAS14 in rapid detection of cadmium pollution is provided.
Experimental example 1
1. Experimental organisms
Wild type K326 tobacco of 5 weeks of age was used as the experimental organism. Experimental tobacco was grown in an experimental greenhouse at 28/21 ℃ (day/night) with an illumination time of 16 h. Watering once in 3 days and fertilizing once in 14 days (for phenotypic observation of tobacco after 20 days of cadmium stress treatment).
2. Sample preparation
A CdCl 2 treatment solution of 50mmol/L was prepared using a 1mol/LCdCl 2 solution.
3. Pollution treatment
Treatment was performed once every three days with 50mmol/L CdCl 2 solution, 20ml each time.
4. Phenotype observations after 20 days cadmium treatment
It was observed that after 20 days of continuous treatment with cadmium ions, the plant height of K326 tobacco (shown in FIG. 1B) and the root system were damaged (shown in FIG. 1A and FIG. 2).
5. Physiological index detection after cadmium stress
(1) Hydrogen peroxide (H 2O2) detection
The H 2O2 content is determined by utilizing Solarbio hydrogen peroxide (H 2O2) content detection kit BC3595, and the following operation steps are adopted:
① Sample processing
About 0.1g of tobacco leaf tissue subjected to cadmium stress treatment is weighed, and 1mL of extracting solution is added for ice bath homogenization. 8000g, centrifuging at 4 ℃ for 10min, taking supernatant, and placing on ice for testing.
② Measurement procedure
Preheating a spectrophotometer or an enzyme-labeled instrument for more than 30min, adjusting the wavelength to 415nm, and zeroing distilled water.
The second, third and fourth reagents (all from Solarbio hydrogen peroxide (H 2O2) content detection kit BC 3595) were placed in a 25 ℃ water bath for more than 10 min.
1Mmol/mL of acetone standard solution was diluted to 2. Mu. Mol/mL of standard solution with a 96-well plate, and 1mmol/mL of acetone standard solution was diluted to 1. Mu. Mol/mL of standard solution with a microglass cuvette. The reagents of Table 1 were added sequentially to the EP tube.
Note that: reagents one, two, three and four were all from Solarbio hydrogen peroxide (H 2O2) content detection kit BC 3595.
After adding reagent IV (component from Solarbio hydrogen peroxide (H 2O2) content detection kit BC 3595) to dissolve and precipitate, standing at room temperature for 5min, transferring 200 μl into a micro-glass cuvette or 96-well plate, and measuring absorbance at 415nm (blank tube measurement 1 time, experimental group setting 3 replicates). Calculated according to the following formulaAnd/>：
③H2O2 And (3) content calculation: the method is calculated according to the tissue quality, and the method comprises the following specific formula:
wherein W represents tissue mass in g.
(2) Catalase (CAT) assay
CAT activity was determined using Solarbio Catalase (CAT) activity assay kit BC0205, comprising the following steps:
① Sample processing:
Weighing about 0.1g of tobacco leaf tissue subjected to cadmium stress treatment, and adding 1mL of extracting solution for ice bath homogenization; 8000g, centrifuging at 4 ℃ for 10min, taking supernatant, and placing on ice for testing.
② Measurement procedure
Preheating a spectrophotometer or an enzyme-labeled instrument for more than 30min, adjusting the wavelength to 240nm, and then zeroing by distilled water.
And (3) placing CAT detection working solution in a water bath at 25 ℃ for more than 10min before the detection.
10. Mu.L of the sample and 190. Mu.L of the working solution were added to a 96-well plate, immediately mixed and timed, and the initial absorbance at 240nm A1 and absorbance A2 after 1min were recorded. ΔA (A1-A2) was calculated.
③ CAT activity calculation: calculated as sample mass, the following is specific:
Definition of units: the degradation of 1 mu mol H 2O2 per gram of tissue in the reaction system is defined as an enzyme activity unit.
CAT(U/g) = 764.5×ΔA÷W
Wherein: w represents the sample mass in g.
(3) Malondialdehyde (MDA) detection
MDA content is determined by using Solarbio Malondialdehyde (MDA) content detection kit BC0025, and the operation steps are as follows:
① Sample processing
Weighing about 0.1g of tobacco leaf tissue subjected to cadmium stress treatment, and adding 1mL of extracting solution for ice bath homogenization; and (5) centrifuging at 4 ℃ for 10min at 8000g, taking supernatant, and placing on ice for testing.
② Measurement procedure
The visible spectrophotometer/microplate reader was preheated for more than 30min and then zeroed with distilled water. The components shown in Table 2 were added in order.
Note that: reagent III was derived from components in Solarbio Malondialdehyde (MDA) content detection kit BC 0025.
The mixed solution is kept in a water bath at 100 ℃ for 60min, then is placed in an ice bath for cooling to 10000g, and is centrifuged for 10min at normal temperature. 200. Mu.L of the supernatant was pipetted into a glass microtitre or 96-well plate and the absorbance of each sample was measured at 532nm and 600 nm. Δa 532,ΔA600 is calculated separately, specifically by the following formula:
ΔA532 = A532 measurement - A532 Blank space
ΔA600 = A600 measurement - A600 Blank space
ΔA = ΔA532 - ΔA600
the blank tube is only needed to be made 1-2 times, and the experimental group is made 3 times.
③ MDA content calculation: the MDA content is calculated according to the sample mass, and is specifically calculated as follows:
MDA content (nmol/g) = 32.258 XΔA ≡W × F
Wherein: w: sample mass, g; f: dilution factors the dilution factors are multiplied by the samples of hyperlipidemia or greases.
(4) Reduced Glutathione (GSH) detection
The content of superoxide anions is determined by utilizing Solarbio reduced Glutathione (GSH) content detection kit BC1290, and the operation steps are as follows:
① Sample processing:
Weighing about 0.1g of tobacco leaf tissue subjected to cadmium stress treatment, adding 1mL of extracting solution for ice bath, and homogenizing; and (5) centrifuging at 4 ℃ for 10min at 8000g, and taking supernatant and placing the supernatant at 4 ℃ for detection.
② Measurement procedure
Preheating a spectrophotometer or an enzyme-labeled instrument for more than 30min, adjusting the wavelength to 412nm, and zeroing by using distilled water.
Preparation of a standard: the standard solution (10 mg/mL) was aspirated, and diluted with distilled water to 300. Mu.g/mL, 200. Mu.g/mL, 100. Mu.g/mL, 50. Mu.g/mL, 25. Mu.g/mL.
The standard dilution table is shown in table 3:
The reagents shown in Table 4 were added to each of the 96-well plates.
Note that: the second reagent and the third reagent come from Solarbio reduced Glutathione (GSH) content detection kit BC1290.
③ GSH content calculation:
Drawing a standard curve: a standard curve y=kx+b was established based on the concentration (x, μg/mL) and absorbance Δa standard (y, Δa Standard of ) of the standard tube. From the standard curve, ΔA (y, ΔA) was taken into the following formula to calculate the sample concentration (x, μg/mL).
Calculated according to the sample mass: GSH content (μg/g) =x/W
Wherein: w represents the sample mass in g; x represents the sample concentration in μg/mL.
Reactive Oxygen Species (ROS) broadly refer to oxygen-derived radicals and non-radicals, including superoxide anions (O 2 -), hydrogen peroxide (H 2O2), hydroxyl radicals (OH-), ozone (O 3), and singlet oxygen (1O 2). After being stressed by heavy metal ions, plants can rapidly cause oxidative stress on plant bodies due to the large accumulation of intracellular Reactive Oxygen Species (ROS), so that oxidative damage at the cellular level is caused. Catalase (CAT) in plant cells belongs to antioxidant enzymes, and is involved in the regulation of intracellular Reactive Oxygen Species (ROS) for reducing excessive Reactive Oxygen Species (ROS), and reduced Glutathione (GSH) is also involved in the process of reducing Reactive Oxygen Species (ROS) in plant cells. The physiological index detection result shows (shown in figure 3) that the content of hydrogen peroxide (H 2O2) in the tobacco subjected to cadmium stress treatment is obviously increased, and meanwhile, the content of Malondialdehyde (MDA) is increased, and the Malondialdehyde (MDA) can cause peroxidation damage to membrane lipid and damage to cell membrane structure; ascorbate Peroxidase (APX), catalase (CAT) and reduced glutathione activities and levels of cellular Reactive Oxygen Species (ROS) are significantly reduced. In conclusion, the tobacco subjected to cadmium stress treatment reduces the function of an antioxidant system, and active oxygen accumulation in cells is increased, so that the damage to plant cells is caused.
Experimental example 2
And detecting the expression quantity of abscisic acid and an environmental stress induction protein TAS14 gene at different treatment times under cadmium stress.
1. Experimental organisms
4 Week old K326 tobacco was used as the experimental organism. Experimental tobacco was grown in an experimental greenhouse at a room temperature of 28/21 ℃ (day/night) with 16 h light.
2. Sample preparation
The experimental samples were 0,20, 40, 60 days of cadmium-treated tobacco, respectively, and the control group was 0 day of treatment with K326 tobacco, each treatment being repeated 3 times.
3. Pollution treatment
The cadmium ion treatment concentration was 50mM, 30ml was added each time, the treatment was performed every 3 days, and samples were taken at 20, 40 and 60 days for the treatment time, respectively, and other culture conditions were the same.
4. Total RNA extraction
The experimental tobacco did not die during the trial. The experimental tobacco was sampled at 0, 20, 40, 60 days of treatment, and 3 tobacco leaves in each parallel group were mixed and sampled for a total of 12 samples. Total RNA was extracted and measured for purity and concentration by conventional methods, and sample RNA concentrations were adjusted to be consistent using RNA FREE WATER. The RNA extraction experiment steps are as follows:
(1) Rapidly transferring a fresh tobacco leaf sample into a liquid nitrogen tank (the mass of the sample is 50 mg-100 mg), adding 3 grinding beads into a grinding tube, and placing the sample into a high-speed low-temperature tissue grinding instrument to be ground into powder;
(2) Adding 50 XDTT Solution into the cracking Buffer RL in advance, adding 500 mu l of Buffer RL into the ground powdery sample, and repeatedly blowing with a pipetting gun until no obvious precipitate exists in the cracking Solution;
(3) Centrifuging the lysate at 12,000rpm and 4deg.C for 5min;
(4) Carefully aspirate the supernatant into a new 1.5ml RNase Free Tube;
(5) Adding the absolute ethyl alcohol with the volume of 1/2 of that of the previous step, and uniformly mixing the solution by using a pipette;
(6) Immediately transferring the whole mixed solution into RNA Spin Column (containing 2ml Collection Tube);
(7) Centrifuging at 12,000rpm for 1min, and discarding the filtrate; placing the RNA Spin Column back into 2ml Collection Tube;
(8) 500 μl Buffer RWA was added to RNA Spin Column, centrifuged at 12,000rpm for 30 s, and the filtrate was discarded;
(9) 600 μl of Buffer RWB was added to RNA Spin Column, centrifuged at 12,000rpm for 30 s, and the filtrate was discarded; 70ml of absolute ethyl alcohol needs to be added into Buffer RWB in advance;
(10) Repeating step (9);
(11) The RNA Spin Column was re-placed on 2ml Collection Tube and centrifuged at 12,000rpm for 2min;
(12) Placing RNA Spin Column on 1.5ml RNASE FREE Collection Tube, adding 50 μl RNASE FREE DH 2 O at the center of RNA Spin Column membrane, and standing at room temperature for 5min;
(13) Centrifuging at 12,000rpm for 2min to elute RNA, and repeating the process again;
(14) And (3) RNA quality detection: RNA integrity was checked by 1.5% agarose gel electrophoresis, and after detection, stored at-80℃for further use.
5. Reverse transcription
The 12 samples of total RNA were each reverse transcribed using a reverse transcription reagent. The reverse transcription procedure is as follows:
Preparing a reaction solution for removing genome DNA: the reaction mixture was prepared on ice according to the ingredients of Table 5, and the genomic DNA reaction was removed, and the RNA content was not more than 1. Mu.g.
(2) After the sample is added, the mixture is placed for reaction in a metal bath at 42 ℃ for 2min, and after the reaction is finished, the mixture is placed for preservation at 4 ℃.
(3) The reverse transcription reaction solution was prepared on ice as shown in Table 6, mixed in an amount of +2 for the purpose of ensuring accuracy, and then 10. Mu.l was dispensed into each reaction tube.
The reaction procedure for reverse transcription is: 15min at 37 ℃; 5sec at 85 ℃.
6. Real-time fluorescent quantitative PCR
And detecting the expression quantity of the abscisic acid and the environmental stress induction protein TAS14 gene (the cDNA sequences of the abscisic acid and the environmental stress induction protein TAS14 gene are shown as SEQ ID NO: 2) in a control group and an experimental group by using NtUbc as an internal reference gene and a first-chain cDNA as a template and adopting a SYBR fluorescent quantitative PCR method.
Wherein, the specific primers of the reference gene NtUbc, abscisic acid and the environmental stress induction protein TAS14 gene are as follows:
abscisic acid and environmental stress-inducible protein TAS14 upstream primer:
5’-AACTGGAGGCGCTACTGATG-3’(SEQ ID NO:4)；
Abscisic acid and environmental stress-inducible protein TAS14 downstream primer:
5’-CGAACACTCACACTCAACGC-3’(SEQ ID NO:5)；
Upstream primer of internal reference gene NtUbc:
5’-CTGGACAGCAGACTGACATC-3’(SEQ ID NO:6)；
Primer downstream of reference gene NtUbc:
5’-CAGGATAATTTGCTGTAACAGATTA-3’(SEQ ID NO:7)。
(1) PCR amplification
Adopting SYBR Green dye reagent, wherein the reaction system is TB Green Premix Ex Taq II（2×）5 μl;Primer F (10 μM) 0.4 μl;Primer R (10 μM) 0.4 μl;ROX Reference Dye II（50×）0.2 μl;cDNA template 1 μl; sterilized water 3. Mu.l; total 10. Mu.l. PCR reactions were performed on an ABI Prism 7500 fluorescent quantifier with the following PCR amplification procedures: pre-denaturation at 94 ℃ for 30s;94 ℃ for 5s;57.8 ℃,34s;40 cycles.
7. Calculation of relative expression level
By usingThe relative expression amounts of abscisic acid, the environmental stress induced protein TAS14 gene and the internal reference NtUbc gene in each concentration group are calculated by the method, the data are shown in table 7, and the histogram of the expression amounts is shown in figure 4.
8. Result determination
As shown in FIG. 4, the relative expression levels of abscisic acid and the environmental stress inducible protein TAS14 gene were up-regulated after cadmium treatment for 20 days, 40 days and 60 days compared with the control group for 0 day.
As shown in Table 7, the relative expression levels of abscisic acid and the environmental stress-induced protein TAS14 gene are up-regulated in the days of different cadmium exposure, which indicates that the soil polluted by Cd 2+ can promote the up-regulated expression of the tobacco abscisic acid and the environmental stress-induced protein TAS14 gene, and the expression conditions of the tobacco abscisic acid and the environmental stress-induced protein TAS14 gene can be used for monitoring the pollution degree of the soil heavy metal Cd 2+.
Example 2
A method for rapidly detecting the cadmium pollution degree of plant soil adopts a fluorescence quantitative PCR-based method to detect the expression quantity of abscisic acid and an environmental stress induction protein TAS14 gene in a sample to be detected, and if the expression quantity of the abscisic acid and the environmental stress induction protein TAS14 gene is up-regulated, the sample to be detected is prompted to have cadmium pollution.
In the fluorescent quantitative PCR method, a forward primer, a reverse primer and a premix of abscisic acid and an environmental stress induced protein TAS14 coding gene are adopted to carry out fluorescent quantitative PCR experiments, and an internal reference gene NtUbc is used as an internal standard.
The fluorescent quantitative PCR method adopts the following reaction system and procedure:
And/or preparing a fluorescent quantitative PCR reaction system by adopting SYBR Green dye reagent, wherein the fluorescent quantitative PCR reaction system comprises TB Green Premix Ex Taq II（2×）5 μl;Primer F (10 μM) 0.4 μl;Primer R (10 μM)0.4 μl;ROX Reference Dye II（50×） 0.2 μl;cDNA template 1 μl; sterilized water 3. Mu.l;
And/or the amplification procedure of fluorescent quantitative PCR is: 94 ℃ for 30s;94 ℃ for 5s;57.8 ℃,34s;40 cycles.
Example 3
A kit for quantitatively detecting the expression quantity of an abscisic acid and an environmental stress induction protein TAS14 coding gene comprises a primer for quantitatively detecting the abscisic acid and the environmental stress induction protein TAS14 coding gene and a fluorescent quantitative PCR detection premix.
The nucleotide sequences of the forward primer and the reverse primer of the coding genes of the abscisic acid and the environmental stress induced protein TAS14 are shown as SEQ ID NO:4 (5'-AACTGGAGGCGCTACTGATG-3') and SEQ ID NO:5 (5'-CGAACACTCACACTCAACGC-3'). The kind of the fluorescent quantitative PCR detection premix is not particularly limited, and a premix method known in the art may be used.
The kit also comprises an internal reference gene primer. The kind of the internal reference gene primer is not particularly limited in the present invention, and internal reference gene primers well known in the art may be used. The nucleotide sequences of the forward primer and the reverse primer of the reference gene are as follows:
SEQ ID NO. 6 (5'-CTGGACAGCAGACTGACATC-3') and
SEQ ID NO. 7 (5'-CAGGATAATTTGCTGTAACAGATTA-3').
Example 4
Application of reagent for inhibiting abscisic acid and expression of environmental stress induced protein TAS14 coding gene in construction of heavy metal cadmium pollution sensitive plants; the application method is virus-induced gene silencing, and the constructed heavy metal cadmium pollution-sensitive plants are used for short-term and long-term pollution monitoring.
Silencing abscisic acid and environmental stress induced protein TAS14 gene by using virus-induced gene silencing technology and cadmium stress treatment of silencing plant
1. Experimental organisms
4 Week old K326 tobacco was used as the experimental organism. Experimental tobacco was grown in an experimental greenhouse at a room temperature of 28/21 ℃ (day/night) with 16 h light.
Vectors and strains
Coli (ESCHERICHIA COLI) DH5 a competent cells and Agrobacterium tumefaciens (Agrobacterium tumefaciens) GV3101 competent cells, and TRV-VIGS vector.
3. Related reagent and culture medium
(1) Agrobacterium induction liquid: 2.665 g MES (morpholinoethanesulfonic acid) was weighed and distilled water was used to determine the volume to 25ml; weighing 2.54125 g MgCl 2·6H2 O, and using distilled water to fix the volume to 25ml; 0.1965g of acetosyringone is weighed and dissolved in DMSO of 1ml to prepare mother solution, and the mother solution is stored for standby in a dark place. When in use, 1ml of MES,1ml of MgCl 2·6H2 O and 10. Mu.l of acetosyringone are taken, the volume is fixed to 50ml by distilled water, and the pH is adjusted to 5.5, and the mixture is used as working solution.
(2) Other related reagents: rifampicin (10 mg/ml); kanamycin (50 mg/ml), LB liquid medium: weighing Tryptone 10 g,Yeast Extract 5g,NaCl 5g, constant volume to 1L with distilled water, sterilizing with high pressure steam, cooling slightly, and packaging into flat plates;
MS solid medium (1L): agar 12 g,M&S Basal Medium with Vitamins 4.43g, sucrose 30g, constant volume to 1L with distilled water, adjusting pH to 5.8-5.9, sterilizing with high pressure steam, cooling slightly, and packaging into flat plates.
4. Total RNA extraction and reverse transcription
The procedure is shown in steps 4 and 5 of Experimental example 2 in example 1.
5. Abscisic acid and environment stress induced protein TAS14 specific segment search and primer design
According to the CDS sequence (SEQ ID NO: 2) of the abscisic acid and environment stress induced protein TAS14 gene, the specific segment sequence of the gene is shown as SEQ ID NO:8, bamHI is selected as a cleavage site on the pTRV2 plasmid, and a specific segment homologous recombination primer with the cleavage site of BamHI is designed.
Wherein, the specific segment primers of the abscisic acid and the environmental stress induction protein TAS14 gene are as follows:
abscisic acid and an environmental stress induction protein TAS14 gene specific segment upstream primer:
5’-agaaggcctccatggggatccCAAGCCAAAACCAAATGCAGC-3’（SEQ ID NO：9）；
Abscisic acid and primers downstream of the specific segment of the environmental stress inducible protein TAS14 gene:
5’-cgtgagctcggtaccggatccTCTCCTTCTTATCATCAGTAGCGC-3’（SEQ ID NO：10）。
6. amplifying specific sequence fragments of a gene of interest
The cDNA obtained by reverse transcription of the total RNA of tobacco K326 is used as a template, and a specific segment of the target fragment to be detected is amplified according to the use instruction of Trans 2X EASY TAP PCR SuperMix enzyme. The amplification system is shown in Table 8.
The PCR amplification procedure was: 94. 2 min deg.c; 94. 30 ℃ s; 60. 30 ℃ s; 72. temperature is 30s; 72. 5min DEG C; 35 cycles. After PCR amplification, 1% agarose gel electrophoresis was performed.
7. Cut gel recovery of fragments of interest
The procedure for recovery of agarose gel was as follows according to the Gel Extraction Kit kit instructions of Omega BIO-TEK:
(1) Cutting off the required DNA fragments after electrophoresis, putting the cut DNA fragments into a clean centrifuge tube, and weighing;
(2) Adding an equal volume of solution Binding buffer into the glue block, placing in a water bath at 60 ℃, and uniformly mixing every 2-3 min;
(3) Adding the mixed solution obtained in the last step into an adsorption column, centrifuging 12000rpm for 60s, pouring out waste liquid in a collecting pipe, and placing the adsorption column into the collecting pipe;
(4) Adding 300 μl Binding buffer into the adsorption column, centrifuging 12000 rpm for 60s, pouring out the waste liquid in the collecting pipe, and placing the adsorption column into the collecting pipe;
(5) Adding 600 μl of a rinsing liquid SPW wash buffer into the adsorption column, centrifuging for 60s at 12000 rpm, pouring out the waste liquid in the collecting pipe, and placing the adsorption column into the collecting pipe;
(6) Repeating the operation step (5);
(7) Placing the adsorption column back into a collecting pipe, centrifuging 12000rpm for 2min, and removing the rinsing liquid as much as possible; placing the adsorption column at room temperature for several minutes, and thoroughly airing;
(8) The adsorption column is placed in a clean centrifuge tube, 30 μl of elution buffer is suspended and dripped into the middle position of the adsorption film, the solution is placed at room temperature for 2 min, and the DNA solution is collected by centrifugation at 12000 rpm for 2 min.
8. Linearization carrier
First, pTRV2 vector was digested. Restriction enzymes QuickCut BamH I were purchased from Takara, formulated according to the instructions of use according to 20. Mu.L reaction system (see Table 9), incubated at 37℃for 30min. The digested product was subjected to agarose gel electrophoresis.
9. Ligation of the Gene of interest to the vector
And carrying out connection on the target gene and the digestion vector by using Vazyme ClonExpress cube II One Step Cloning Kit (C112). The reaction solution was prepared according to the reaction system shown in Table 10, and after the temperature was maintained at 37℃and 30 min, it was left at 4℃or immediately cooled on ice for use.
10. Conversion by heat shock
(1) Thawing DH5 alpha competent cells on ice;
(2) Adding 5 μl of recombinant product into 50 μl of competent cells, mixing with light elastic tube wall, and standing on ice for 30min;
(3) Heat shock in a water bath at 42 ℃ for 60s;
(4) 450 μl of LB medium (without antibiotics) was added, and the mixture was shaken at 37℃for 1h;
(5) And (3) sucking 50 mu l of thalli, adding the thalli onto a plate with response resistance, uniformly coating the plate by using a sterile coating rod, and culturing the plate at 37 ℃ overnight for 12-16 hours.
11. Colony PCR verification of target genes
(1) The recombinant product identification system is shown in Table 11.
The recombinant product identification reaction procedure was: 94. 2 min deg.c; 94. 30 ℃ s; 60. 30 ℃ s; 72. temperature is 30s; 72. DEG C5 min;35 cycles.
(2) Taking 1-3 colonies corresponding to positive strips, inoculating the colonies into LB liquid medium (containing 50 mug/ml kanamycin) for propagation culture, selecting 2-3 positive clones for sequencing according to the identification result of the recombinant product, and analyzing the sequencing result by utilizing Snapgene and other software.
12. Plasmid extraction
Recombinant bacteria with correct sequencing were selected and plasmids were extracted according to the instructions of the plasmid extraction kit (omega, D6943) as follows:
(1) Before use, adding RNase A (all RNase A provided in the kit) into Solution I, and storing at 2-8deg.C;
(2) Before first use, absolute ethyl alcohol is added into the HBC Buffer according to the specification of a reagent bottle label;
(3) Taking 1-5 ml overnight cultured bacterial liquid, adding into a 1.5 ml centrifuge tube, centrifuging at 12,000 rpm for 1min, absorbing supernatant as much as possible, and preheating the eluting buffer in a water bath at 65 ℃;
(4) 250 μl of Solution I is added into the centrifuge tube with the bacterial pellet left, and the bacterial pellet is thoroughly suspended using a pipette or vortex shaker;
(5) Adding 250 mul of Solution II into the centrifuge tube, and uniformly mixing the Solution II upside down to fully crack the thalli, wherein the bacterial liquid becomes clear and sticky at the moment;
(6) Adding 350 μl of Solution III into the centrifuge tube, mixing up and down, and storing at-30deg.C for 5min;
(7) Centrifuging the mixed solution at room temperature at maximum rotation speed for 10min, wherein white flocculent precipitate appears;
(8) Column balancing, namely adding 100 μl 3M NaOH,12,000 rpm into an adsorption Column (Spin Column CM) loaded into a Collection Tube (Collection Tube), centrifuging for 1min, pouring out waste liquid in the Collection Tube, and putting the adsorption Column back into the Collection Tube;
(9) Adding the supernatant obtained in the step 7 into an adsorption column filled into a collecting pipe, taking care not to suck out sediment, centrifuging at 12,000 rpm for 1min, pouring out waste liquid in the collecting pipe, and placing the adsorption column into the collecting pipe;
(10) Adding 500 μl HBC Buffer into the collection tube, centrifuging at 12,000 rpm for 1min, and pouring out the waste liquid in the collection tube;
(11) Repeating step (10);
(12) The adsorption column was replaced in the collection tube, centrifuged at 12,000 rpm for 1min, and the waste liquid in the collection tube was discarded. Placing the adsorption column at room temperature for several minutes to thoroughly dry;
(13) Placing the adsorption column in a new centrifuge tube, suspending and dripping 30 μl of the absorption Buffer into the middle part of the adsorption membrane, standing at room temperature for 1min, centrifuging at 12,000 rpm for 1min, collecting plasmid solution into the centrifuge tube, and preserving plasmids at-20deg.C.
13. Competent transformation of Agrobacterium
(1) Thawing competent cells GV3101 on ice;
(2) 1. Mu.g of plasmid was added to competent cells, mixed and placed on ice for 5min;
(3) Freezing the mixture in liquid nitrogen for 5min;
(4) The mixture was left at 37℃for 5min;
(6) 500 μl LB medium was added to the mixture, and the mixture was shaken at 28℃and 220rpm for 3 hours;
(7) Centrifuging to collect thallus at 3000rpm for 2-5min;
(8) Coating the collected thalli on an LB plate containing corresponding antibiotics, and placing the plate upside down at 28 ℃ for incubation for 2-3 days;
(9) And (3) detection: single colony is picked, amplified and cultured, plasmid is extracted, PCR detection is carried out, and positive bacterial liquid is stored at-80 ℃ with glycerol.
14. Preparation and induction of aggressive dye solutions
(1) Resuscitating and culturing glycerol bacteria stored at-80 ℃: respectively inoculating pTRV1, pTRV2 and pTRV2 LOC107819915 into 2ml LB liquid medium containing rifampicin (10 mug/ml) and kanamycin (50 mug/ml), culturing at 220 rpm and 28 ℃ under shaking for 12h;
(2) Inoculating the primary bacteria shaken in the step (1) into 30 ml LB liquid culture medium containing rifampicin (10 mu g/ml) and kanamycin (50 mu g/ml) according to the proportion of 1:20, and culturing at 220 rpm and 28 ℃ for 5-6h in an oscillating way;
(3) Spectrophotometry (OD 600) measures absorbance of each culture, OD600 should be between 0.5-0.6;
(4) After reaching an OD600 between 0.5 and 0.6, 4000g, centrifuged at room temperature for 5min, the supernatant discarded and the Agrobacterium cells in each culture in the pellet collected;
(5) Resuspending the cells in 5 ml agrobacterium induction buffer;
(6) Absorbance was measured with a spectrophotometer (OD 600), and the absorbance of each culture was adjusted to od600=0.2 to 0.6, and should not exceed 0.7. Then incubated in a shaker (50 rpm) at room temperature for 3h in the absence of light;
(7) pTRV1 was mixed with equal volumes of pTRV2:LOC107819915, pTRV 2.
15. Plant infection injection
Selecting 4-week-old tobacco K326, sucking the mixed bacterial liquid by using a 1ml needleless injector, inoculating to the back of the leaf, injecting 0.5 ml mixed bacterial liquid into each leaf, and inoculating three to four leaves into each plant. Altogether 2 groups were set: pTRV1+pTRV2 LOC107819915, pTRV1+pTRV2. One week later, the injection silencing was boosted.
16. Real-time fluorescent quantitative PCR (polymerase chain reaction) verification of abscisic acid and environmental stress induced protein TAS14 gene silencing efficiency
MRNA of plants genetically silenced with abscisic acid and the environmental stress-inducing protein TAS14 was extracted and subjected to reverse transcription (the total RNA extraction and reverse transcription steps are shown in Experimental example 2, steps 4 and 5, in example 1).
NtUbc is used as an internal reference gene, a first strand cDNA is used as a template, and SYBR fluorescent quantitative PCR method is used for detecting the expression quantity of abscisic acid and an environment stress induced protein TAS14 gene (the cDNA sequences of the abscisic acid and the environment stress induced protein TAS14 gene are shown as SEQ ID NO: 2) in a gene silencing plant.
Wherein, the specific primers of the reference gene NtUbc, abscisic acid and the environmental stress induction protein TAS14 gene are as follows:
abscisic acid and environmental stress-inducible protein TAS14 upstream primer:
5’-AACTGGAGGCGCTACTGATG-3’(SEQ ID NO:4)；
Abscisic acid and environmental stress-inducible protein TAS14 downstream primer:
5’-CGAACACTCACACTCAACGC-3’(SEQ ID NO:5)；
Upstream primer of internal reference gene NtUbc:
5’-CTGGACAGCAGACTGACATC-3’(SEQ ID NO:6)；
Primer downstream of reference gene NtUbc:
5’-CAGGATAATTTGCTGTAACAGATTA-3’(SEQ ID NO:7)。
(1) PCR amplification
Adopting SYBR Green dye reagent, wherein the reaction system is TB Green Premix Ex Taq II（2×）5 μl;Primer F (10 μM) 0.4 μl;Primer R (10 μM) 0.4 μl;ROX Reference Dye II（50×）0.2 μl;cDNA template 1 μl; sterilized water 3. Mu.l; total 10. Mu.l. PCR reactions were performed on an ABI Prism 7500 fluorescent quantifier with the following PCR amplification procedures: 94 ℃ for 30s;94 ℃ for 5s;57.8 ℃,34s;40 cycles.
(2) Calculation of relative expression level
By usingThe relative expression amounts of abscisic acid and the environmental stress induced protein TAS14 gene and internal reference NtUbc gene in the infection of empty plants and the infection of target gene plants are calculated by the method.
(3) Result determination
As shown in figure 5, compared with the infected no-load pTRV2 control plant, the expression level of the abscisic acid and the environmental stress induction protein TAS14 gene in the silent plant is obviously reduced, namely, the abscisic acid and the environmental stress induction protein TAS14 gene are successfully silenced, and the subsequent stress treatment can be carried out.
17. Cadmium ion stress treatment of silenced plants
The cadmium ion treatment concentration is 50mM, 20ml each time, and the phenotype observation after the silence plant cadmium stress is continuously treated for 3 days is shown in figure 6.
After cadmium stress treatment, abscisic acid and an environmental stress induced protein TAS14 gene silencing plant show obvious wilting and leaf yellowing, which shows that the plant is sensitive to cadmium treatment, and pTRV1 is injected: only a small part of leaves of the pTRV2 empty-load plant show wilting and yellowing phenomena, which indicates that the abscisic acid and the environmental stress induced protein TAS14 gene respond to cadmium ions, and the abscisic acid and the environmental stress induced protein TAS14 gene silence the cadmium ion stress of the plant more sensitively, and further indicates that the gene can be used as a biomarker for sensitively monitoring the pollution degree and the hazard degree of heavy metal cadmium pollution.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (8)

1. Application of tobacco abscisic acid and an environmental stress induction protein TAS14 gene in rapid detection of cadmium pollution of tobacco soil, wherein the nucleotide sequences of the abscisic acid and the environmental stress induction protein TAS14 gene are shown as SEQ ID NO: 1.
2. The use according to claim 1, wherein the abscisic acid has the amino acid sequence as set forth in SEQ ID NO: 3.
3. A method for rapidly detecting cadmium pollution of tobacco soil is characterized in that a fluorescent quantitative PCR-based method is adopted to specifically detect the expression level of abscisic acid and an environmental stress induction protein TAS14 gene in a sample to be detected, and if the expression level of the abscisic acid and the environmental stress induction protein TAS14 gene is up-regulated, the presence of cadmium pollution is indicated, and the nucleotide sequences of the abscisic acid and the environmental stress induction protein TAS14 gene are shown as SEQ ID NO:1, wherein the sample to be tested is tobacco.
4. The method for rapidly detecting cadmium pollution in tobacco soil according to claim 3, wherein the experiment of fluorescence quantitative PCR is performed by using the forward primer, the reverse primer and the premix of abscisic acid and the gene encoding the environmental stress induction protein TAS14, and the reference gene NtUbc is used as an internal standard.
5. A method for rapid detection of cadmium pollution in tobacco soil according to claim 3, wherein the reaction system of fluorescent quantitative PCR is formulated with SYBR Green dye reagent, comprising 2 x TB Green Premix Ex Taq II μl; 10. mu.M Primer F0.4. Mu.l; 10. mu.M Primer R0.4. Mu.l; 50 XROX REFERENCE DYE II 0.2.2. Mu.l; 1 μl of cDNA template; sterilized water 3. Mu.l.
6. The method for rapidly detecting cadmium pollution in tobacco soil according to claim 3, wherein the amplification procedure of the fluorescent quantitative PCR is as follows: 94 ℃ for 30s;94 ℃ for 5s;57.8 ℃,34s;40 cycles.
7. Application of a reagent for specifically inhibiting expression of abscisic acid and an environment stress induced protein TAS14 gene in construction of tobacco sensitive to heavy metal cadmium pollution, wherein the nucleotide sequences of the abscisic acid and the environment stress induced protein TAS14 gene are shown as SEQ ID NO: 1.
8. The use of an agent for specifically inhibiting abscisic acid and the expression of the environmental stress-induced protein TAS14 gene in the construction of tobacco susceptible to heavy metal cadmium pollution according to claim 7, characterized in that the method of use is virus-induced gene silencing.
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