CN117402944B - Application of cytochrome P450D 7 in rapid detection of cadmium pollution - Google Patents

Application of cytochrome P450D 7 in rapid detection of cadmium pollution Download PDF

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CN117402944B
CN117402944B CN202311716723.7A CN202311716723A CN117402944B CN 117402944 B CN117402944 B CN 117402944B CN 202311716723 A CN202311716723 A CN 202311716723A CN 117402944 B CN117402944 B CN 117402944B
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cytochrome
gene
tobacco
pollution
heavy metal
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CN117402944A (en
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张海平
周瑾
刘昕雨
张欢
赵晓军
刘海
曾皓
康峥
唐卫迪
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 cytochrome P45071D7 protein in rapid detection of cadmium pollution. The invention utilizes the change of the expression quantity of the tobacco cytochrome P45071D7 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 cytochrome P450D 71 as a biomarker, 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 cytochrome P450D 7 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 cytochrome P45071D7 protein 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+ By inhibiting the enzyme activity and interfering the endogenous hormone balance and the membrane permeability of the plant, the plant can be subjected to toxic action, and the physiological metabolic process of the plant can be destroyed. At present, heavy metal cadmium is listed as a first-class cancerogenic substance, cd, by the International agency for research on cancer (IARC) 2+ After being absorbed by plants, the plant can enter human body for enrichment through food chains, 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, and provides application of cytochrome P450D 7 in rapid detection of cadmium pollution. The cytochrome P450D 7 molecule has high sensitivity and high speed response to heavy metal cadmium pollutants, and can be used as early pollution early warning molecules.
The technical scheme of the invention is as follows:
use of cytochrome P450D 7 in rapid detection of cadmium pollution.
Preferably, the cytochrome P450D 7 comprises a cytochrome P450D 71D7 gene and a cytochrome P450D 7 protein; the amino acid sequence of the cytochrome P45071D7 protein is shown in SEQ ID NO:3 is shown in the figure; the nucleotide sequence of the cytochrome P45071D7 gene is shown in SEQ ID NO: 1.
The invention also provides a method for rapidly detecting the cadmium pollution degree of the plant soil, which adopts a method based on fluorescence quantitative PCR to detect the expression quantity of the cytochrome P450D 7 protein coding gene in the sample to be detected, and if the expression quantity of the cytochrome P450D 7 protein coding gene is up-regulated, the heavy metal pollution of the sample to be detected is prompted.
Preferably, in the fluorescent quantitative PCR method, the forward primer, the reverse primer and the premix of the cytochrome P450D 7 protein coding gene are adopted to perform fluorescent quantitative PCR experiments, and an internal reference gene 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 TBGreen Premix Ex Taq II (2X) 5 μl; primer F (10. Mu.M) 0.4. Mu.l; primerR (10. Mu.M) 0.4. Mu.l; ROX Reference Dye II (50×) 0.2 μl;1 μl of cDNA template; sterilized water 3. Mu.l;
and/or the amplification procedure of fluorescent quantitative PCR is: 94 ℃ for 30s;94 ℃ for 5s;59.3℃for 34s;40 cycles.
The invention also provides a kit for detecting the expression quantity of the cytochrome P450D 7 protein coding gene, which comprises a primer for quantitatively detecting the cytochrome P45071D7 protein coding gene and 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 provides application of the reagent for inhibiting the expression of cytochrome P450D 7 protein coding genes in construction of heavy metal cadmium pollution-sensitive plants.
Preferably, the method of use is virus-induced gene silencing.
The invention has the beneficial effects that:
the invention provides application of cytochrome P450D 7 in rapid detection of heavy metal cadmium pollution. Experiments show that the cytochrome P45071D7 gene can generate a forward response when being stressed by cadmium ions by taking tobacco as a plant representative, 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 cytochrome P45071D7 gene in the tobacco is knocked out, and the result shows that the tobacco with the gene knocked out shows intolerance to cadmium ion stress. As can be seen, cytochrome P450D 7 responds to heavy metal cadmium stress, and appears to up-regulate expression when heavy metal cadmium is stressed, while plants from which the gene is knocked out are intolerant to heavy metal cadmium. Therefore, the cytochrome P45071D7 can be used as a biomarker for rapidly detecting the pollution of heavy metal cadmium, and the problem that the current method for detecting the heavy metal cadmium in plant soil is long in period is solved; experimental results show that the detection of the cytochrome P45071D7 gene is utilized to detect heavy metal cadmium in plants, so that the repeatability is good, the sensitivity is high, and the problem that the sensitivity of the existing plant soil heavy metal cadmium detection method is low is solved.
The invention utilizes the detection of cytochrome P45071D7 gene, 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 role in improving the current detection technology of heavy metal cadmium pollution of 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 5 week old wild type K326 tobacco at 50mM Cd 2+ Plant height phenotype comparison after 20 days of treatment, and B is tobacco plant height change before and after cadmium stress treatment.
FIG. 2 shows that 5 week old wild type K326 tobacco was at 50mM Cd 2+ Phenotype observation of tobacco root system after stress treatment for two months。
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 cytochrome P45071D7 gene expression in tobacco.
FIG. 5 shows the detection of gene silencing efficiency of cytochrome P450D 7 in gene silencing plants.
FIG. 6 is a phenotypic observation after cadmium stress of gene-silenced plants.
Detailed Description
The invention provides an application of cytochrome P450D 7 in rapid detection of cadmium pollution; the cytochrome P450D 7 comprises a cytochrome P450D 71D7 gene and a cytochrome P45071D7 protein. The gene locus number of the coding gene of the cytochrome P45071D7 is LOC107788281, the length of mRNA is 1694bp, and the nucleotide sequence of the mRNA of the cytochrome P450D 7 gene is preferably shown in SEQ ID NO: 1.
The nucleotide sequence of the CDS sequence of the cytochrome P450D 7 protein coding gene is shown in SEQ ID NO: 2.
The amino acid sequence of the cytochrome P45071D7 protein is shown in 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
Use of cytochrome P450D 7 in rapid detection of cadmium pollution.
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), illuminated for 16h, watered once for 3 days, and fertilized once for 14 days (for phenotypic observation of tobacco after 20 days of cadmium stress treatment).
2. Sample preparation
By means of 1mol/LCdCl 2 The solution was configured as 50mmol/L CdCl 2 And (3) treating liquid.
3. Pollution treatment
By means of CdCl of 50mmol/L 2 The solution was treated every three days, 20ml each time.
4. Phenotype observation of tobacco after 20 days of cadmium treatment
It was observed that the plant height of K326 tobacco (shown in FIG. 1B) was measured after 20 days of continuous treatment with cadmium ions. The root systems are damaged (as shown in fig. 1 and A and fig. 2).
5. Physiological index detection after cadmium stress
(1) Hydrogen peroxide (H) 2 O 2 ) Detection of
By means of Solarbio hydrogen peroxide (H) 2 O 2 ) Content detection kit BC3595 for measuring H 2 O 2 The content, the operation steps are as follows:
(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.
Reagents two, three and four (reagents were all from Solarbio hydrogen peroxide (H) 2 O 2 ) The components in the content detection kit BC 3595) are placed in a water bath at 25 ℃ for more than 10min.
1mmol/mL of acetone standard solution was diluted to 2. Mu. Mol/mL of standard solution with a 96-well plate, and 1 mmol/acetone standard solution was diluted to 1. Mu. Mol/mL of standard solution with a microglass cuvette. Reagents shown in Table 1 were added sequentially to the EP tube.
Note that: reagents one, two, three and four were all from Solarbio hydrogen peroxide (H 2 O 2 ) The components in the content detection kit BC 3595.
Reagent four (from Solarbio hydrogen peroxide (H) 2 O 2 ) Content detection kitThe components in BC 3595) were dissolved and precipitated, and then allowed to stand at room temperature for 5min, 200. Mu.L of the precipitate was transferred to a micro-glass cuvette or 96-well plate to measure the absorbance at 415nm (1 in a blank tube, 3 in an experimental group). Calculated according to the following formulaAnd->。
③H 2 O 2 And (3) content calculation: the specific calculation formula is as follows according to the tissue quality calculation:
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; and (5) centrifuging at 4 ℃ for 10min at 8000g, 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, and ΔA (A1-A2) was calculated.
(3) CAT activity calculation: calculated as sample mass, the following is specific:
definition of units: every gram of tissue catalyzes 1 mu mol of H per minute in a reaction system 2 O 2 Degradation is defined as one unit of enzyme activity.
CAT(U/g) = 764.5 × ΔA ÷ W
Wherein W represents the sample mass in g.
(3) Malondialdehyde (MDA) detection
The MDA content is determined by using a 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 the temperature of 8000g for 10min at 4 ℃, taking supernatant, and placing on ice for testing.
(2) Measurement procedure
The visible spectrophotometer/enzyme-labeled instrument 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 from the component of 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. Respectively calculating delta A, delta A 532 ，ΔA 600 The calculation formula is as follows:
ΔA 532 = A 532 determination of-A 532 Blank space
ΔA 600 = A 600 Determination of-A 600 Blank space
ΔA = ΔA 532 - ΔA 600
Wherein the blank tube was measured 1 time and the experimental group was 3 times.
(3) MDA content calculation: MDA content is calculated according to the sample mass, and the calculation formula is as follows:
MDA content (nmol/g) = 32.258 ×ΔA ≡W×F
Wherein: w represents the sample mass in g; f represents the dilution factor by which the hyperlipidemia or lipid sample is multiplied.
(4) Reduced Glutathione (GSH) detection
The content of superoxide anions is determined by using a 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, and adding 1mL of extracting solution for ice bath homogenization; 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 and third reagents are from the components in the Solarbio reduced Glutathione (GSH) content detection kit BC 1290.
(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 the standard tube. From the standard curve, ΔA (y, ΔA) was taken into the following formula to calculate the sample concentration (x, μg/mL).
The formula is as follows, 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.
(5) And (3) result judgment:
reactive Oxygen Species (ROS) broadly refer to oxygen-derived free and non-free radicals, including superoxide anions (O2) - ) Hydrogen peroxide (H) 2 O 2 ) Hydroxyl radical (OH) - ) Ozone (O) 3 ) And singlet oxygen (1O) 2 ). After the plants are stressed by heavy metal cadmium ions, oxidation stress can be rapidly caused on the plants due to the large accumulation of intracellular Reactive Oxygen Species (ROS), so that the oxidation damage of 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 (figure 3), and hydrogen peroxide (H) in tobacco treated by cadmium stress 2 O 2 ) 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 cytochrome P45071D7 genes under cadmium stress at different treatment times.
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 day, 20 days, 40 days, 60 days, 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 with enzyme-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 lysis buffer solution RL in advance, adding 500 mu l of the lysis buffer solution RL into the ground powdery sample, and repeatedly blowing with a pipette until no obvious precipitate exists in the lysis buffer solution RL;
(3) Centrifuging the lysis buffer RL obtained in the step (2) at 12,000rpm and 4 ℃ for 5min;
(4) Carefully aspirate the supernatant into a fresh 1.5ml rnase free centrifuge 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 mixture to an RNA purification column (containing a 2ml liquid collection tube);
(7) Centrifuging at 12,000rpm for 1min, and discarding the filtrate; the RNA purification column was placed back into a 2ml liquid collection tube;
(8) 500 μl of buffer RWA was added to the RNA purification column, centrifuged at 12,000rpm for 30s, and the filtrate was discarded;
(9) 600 μl of buffer RWB was added to the RNA purification column, centrifuged at 12,000rpm for 30s, and the filtrate was discarded; 70ml of absolute ethyl alcohol needs to be added into the buffer solution RWB in advance;
(10) Repeating step (9);
(11) The RNA purification column was re-placed on a 2ml liquid collection tube and centrifuged at 12,000rpm for 2min;
(12) Placing the RNA purification column on a 1.5ml centrifuge tube without RNase, adding 50 μl of enzyme-free water at the center of the RNA purification 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:
(1) 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 according to Table 6, and the mixture was prepared in an amount of +2 for the reaction to ensure 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
To be used forNtUbcAs an internal reference gene, the first strand cDNA is used as a template, and the SYBR fluorescent quantitative PCR method is used for detecting the expression level of the cytochrome P450D 7 gene (the cDNA sequence of the cytochrome P45071D7 gene is shown as SEQ ID NO: 2) in a control group and an experimental group.
Wherein, the reference geneNtUbcThe cytochrome P45071D7 gene specific primers are:
cytochrome P45071D7 upstream primer:
5’-TTGGAAACAGGTTCTGCAGTG-3’(SEQ ID NO:4)；
cytochrome P45071D7 downstream primer:
5’-CCTGCTGCATGATTCTCTTGG-3’(SEQ ID NO:5)；
reference geneNtUbcAn upstream primer:
5’-CTGGACAGCAGACTGACATC-3’(SEQ ID NO:6)；
reference geneNtUbcA downstream primer:
5’-CAGGATAATTTGCTGTAACAGATTA-3’(SEQ ID NO:7)。
and (3) PCR amplification: 5 μl of SYBR Green dye reagent was used, the reaction system was TB Green Premix Ex Taq II (2×); primer F (10. Mu.M) 0.4. Mu.l; primer R (10. Mu.M) 0.4. Mu.l; ROX Reference Dye II (50×) 0.2 μl;1 μl of cDNA template; sterilized water 3. Mu.l; a total of 10 μl. 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;59.3℃for 34s;40 cycles.
7. Calculation of relative expression level
By usingMethod for calculating cytochrome P45071D7 gene and internal referenceNtUbcThe relative expression amount of the gene in each concentration group is shown in Table 7, and the histogram of the expression amount is shown in FIG. 4.
8. Result determination
As shown in Table 7, cytochrome P45071D7 gene was up-regulated on different days of cadmium exposure, indicating that it was Cd-exposed 2+ The contaminated soil can promote the up-regulation of the expression of the gene of the cytochrome P450D 7 of the tobacco, which proves that the expression condition of the gene of the cytochrome P45071D7 of the tobacco can be used for monitoring the heavy metal Cd in the soil 2+ Pollution level.
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 cytochrome P45071D7 protein genes in a sample to be detected, and if the expression quantity of the cytochrome P45071D7 protein genes is up-regulated, the sample to be detected is prompted to be polluted by heavy metal cadmium.
The experiment of fluorescent quantitative PCR was performed using the forward and reverse primers and premix of the cytochrome P45071D7 protein gene, using the internal reference gene as an internal standard.
Preparing a fluorescent quantitative PCR reaction system by adopting SYBR Green dye reagent, wherein the reaction system comprises TB Green Premix Ex Taq II (2X) 5 μl; primer F (10. Mu.M) 0.4. Mu.l; primerR (10. Mu.M) 0.4. Mu.l; ROX Reference Dye II (50×) 0.2 μl;1 μl of cDNA template; sterilized water 3. Mu.l; the amplification procedure of the fluorescent quantitative PCR is as follows: 94 ℃ for 30s;94 ℃ for 5s;59.3℃for 34s;40 cycles.
Example 3
A kit for detecting the expression quantity of a cytochrome P45071D7 protein coding gene comprises a primer for quantitatively detecting the cytochrome P45071D7 protein coding gene and a fluorescent quantitative PCR detection premix; 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 nucleotide sequences of the forward primer and the reverse primer for quantitatively detecting the cytochrome P450D 7 coding gene in the kit are preferably shown as SEQ ID NO:4 (5'-TTGGAAACAGGTTCTGCAGTG-3') and seq id NO:5 (5'-CCTGCTGCATGATTCTCTTGG-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 cytochrome P45071D7 protein coding gene expression 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.
The cytochrome P45071D7 gene is silenced by using a virus-induced gene silencing technology, and cadmium stress treatment is carried out on the silenced 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 alpha competent cells and Agrobacterium tumefaciensAgrobacterium 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 volume 25ml; weigh 2.54125 g MgCl 2 ·6H 2 O, using distilled water to fix the volume to 25ml; 0.1965g of acetosyringone is weighed and dissolved in DMSO of 1ml to prepare mother liquor, and the mother liquor is stored for standby in a dark place. When in use, 1ml MES and 1ml MgCl are taken 2 ·6H 2 O, 10. Mu.l acetosyringone, was used as a working fluid by adjusting pH to 5.5 with distilled water to a volume of 50 ml.
(2) Other related reagents: rifampicin (10 mg/ml); kanamycin (50 mg/ml), LB liquid medium: weighing 10g of tryptone, 5g of yeast powder and 5g of NaCl, fixing the volume to 1L by using distilled water, sterilizing by high-pressure steam, slightly cooling after the temperature is reached, and packaging into flat plates;
MS solid medium (1L): 12 g of agar, 4.43 and g of MS basal medium and 30g of sucrose, using distilled water to fix the volume to 1L, adjusting the pH to 5.8-5.9, sterilizing by high-pressure steam, cooling slightly after the temperature is reached, 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. Cytochrome P450D 71D7 specific segment search and primer design
According to the CDS sequence (SEQ ID NO: 2) of the cytochrome P45071D7 gene, the specific segment sequence of this gene is shown in 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 cytochrome P45071D7 gene specific segment primers are as follows:
cytochrome P45071D7 gene specific segment upstream primer:
5’-agaaggcctccatggggatccATGGAGCTTCAATCTTCTCCTTTC-3’（SEQ ID NO：9）；
cytochrome P45071D7 gene specific segment downstream primer:
5’-cgtgagctcggtaccggatccTGCAAAAGCGAGATCATGAGTT-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. at the temperature of 2min; 94. 30℃, s; 60. 30℃, s; 72. 30S at the temperature; 72. at the temperature of 5min; 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 instructions of the Omega BIO-TEK Gel Extraction Kit kit:
(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 solution into the gel 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 at 12000 rpm for 60s, pouring out waste liquid in a collecting pipe, and placing the adsorption column into the collecting pipe;
(4) Adding 300 μl of binding buffer into the adsorption column, centrifuging at 12000 rpm for 60s, pouring out the waste liquid in the collection tube, and placing the adsorption column into the collection tube;
(5) Adding 600 μl of a rinse solution SPW elution buffer into the adsorption column, centrifuging at 12000 rpm for 60s, pouring out the waste liquid in the collection tube, and placing the adsorption column into the collection tube;
(6) Repeating the operation step (5);
(7) Placing the adsorption column back into a collecting pipe, centrifuging at 12000 rpm for 2min, and removing the rinse liquid as much as possible; placing the adsorption column at room temperature for several minutes, and thoroughly airing;
(8) Placing the adsorption column into a clean centrifuge tube, suspending and dripping 30 μl of elution buffer into the middle of the adsorption membrane, standing at room temperature for 2min, centrifuging at 12000 rpm for 2min, and collecting DNA solution.
8. Linearization carrier
First, pTRV2 vector was digested. Restriction enzymes QuickCut BamHI restriction enzymes were purchased from Takara and were formulated according to 20. Mu.L of the reaction system according to the instructions of use (see Table 9). The temperature is kept at 37 ℃ for 30min. The digested product was subjected to agarose gel electrophoresis.
9. Ligation of the Gene of interest to the vector
The target gene and the digestion vector were ligated using Vazyme ClonExpress. II One Step Cloning Kit (C112). The reaction solutions were prepared according to the reaction systems shown in Table 10. The mixture is kept at 37 ℃ for 30min and then is placed at 4 ℃ or immediately placed on ice for cooling for standby.
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 60s in 42 deg.c water bath;
(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. at the temperature of 2min; 94. 30℃, s; 60. 30℃, s; 72. 30S at the temperature; 72. at the temperature of 5min; 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 using software such as Snapgene and the like.
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 the solution I is used, adding RNase A (all RNase A provided in the kit is added), mixing, and storing at 2-8deg.C;
(2) Before first use, absolute ethyl alcohol is added into the HBC buffer solution according to the specification of a reagent bottle label;
(3) Taking 1-5 ml overnight cultured bacterial liquid, adding the bacterial liquid into a 1.5ml centrifuge tube, centrifuging at 12,000rpm for 1 minute, absorbing supernatant as much as possible, and preheating an elution buffer in a water bath at 65 ℃;
(4) Adding 250 μl of solution I into the centrifuge tube with the bacterial pellet left, and thoroughly suspending the bacterial pellet using a pipette or vortex shaker;
(5) Adding 250 μl of solution II into the centrifuge tube, mixing, and allowing the bacterial cells to be fully cracked, wherein the bacterial liquid becomes clear and viscous;
(6) Adding 350 μl of solution III into the centrifuge tube, mixing, 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 of 3M NaOH,12,000 rpm into an adsorption column loaded into a collecting pipe, centrifuging for 1min, pouring out waste liquid in the collecting pipe, and putting the adsorption column back into the collecting pipe;
(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 12,000rpm 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 12,000rpm 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 12,000rpm 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;
placing the adsorption column in a new centrifuge tube, suspending and dripping 30 μl of eluent into the middle part of the adsorption membrane, standing at room temperature for 1min, centrifuging at 12,000rpm for 1min, collecting plasmid solution into the centrifuge tube, and preserving plasmid 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 ℃: pTRV1, pTRV2: LOC107788281 were inoculated respectively into 2ml of LB liquid medium containing rifampicin (10. Mu.g/ml) and kanamycin (50. Mu.g/ml), cultured at 220rpm,28℃with shaking for 12 hours;
(2) Inoculating the primary bacteria shaken in the step (1) into a 30ml LB liquid culture medium containing rifampicin (10 mu g/ml) and kanamycin (50 mu g/ml) according to the ratio of 1:20, and culturing at 220rpm 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 5ml 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 at room temperature in a shaker (50 rpm.) protected from light for 3h;
(7) pTRV1 was mixed with equal volumes of pTRV2, LOC107788281, pTRV2.
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 lower 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 LOC107788281, pTRV1+pTRV2. One week later, the injection silencing was boosted.
16. Real-time fluorescent quantitative PCR (polymerase chain reaction) verification of cytochrome P45071D7 gene silencing efficiency
mRNA from cytochrome P45071D7 gene-silenced plants was extracted and reverse transcribed (total RNA extraction and reverse transcription steps are shown in Experimental example 2, steps 4 and 5, in example 1).
To be used forNtUbcAs an internal reference gene, the first strand cDNA is used as a template, and the SYBR fluorescent quantitative PCR method is used for detecting the expression level of the cytochrome P450D 7 gene (the cDNA sequence of the cytochrome P45071D7 gene is shown as SEQ ID NO: 2) in the gene silencing plant.
Wherein, the reference geneNtUbcThe cytochrome P45071D7 gene specific primers are:
cytochrome P45071D7 upstream primer:
5’-TTGGAAACAGGTTCTGCAGTG-3’(SEQ ID NO:4)；
cytochrome P45071D7 downstream primer:
5’-CCTGCTGCATGATTCTCTTGG-3’(SEQ ID NO:5)；
reference geneNtUbcAn upstream primer:
5’-CTGGACAGCAGACTGACATC-3’(SEQ ID NO:6)；
reference geneNtUbcA downstream primer:
5’-CAGGATAATTTGCTGTAACAGATTA-3’(SEQ ID NO:7)。
(1) PCR amplification
5 μl of SYBR Green dye reagent was used, the reaction system was TB Green Premix Ex Taq II (2×); primer F (10. Mu.M) 0.4. Mu.l; primer R (10. Mu.M) 0.4. Mu.l; ROX Reference Dye II (50×) 0.2 μl;1 μl of cDNA template; 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;59.3℃for 34s;40 cycles.
(2) Calculation of relative expression level
By usingMethod for calculating cytochrome P45071D7 gene and internal referenceNtUbcThe histogram of the relative expression of the genes in infecting empty plants and infecting target gene plants is shown in figure 5.
(3) Result determination
Compared with the infected empty pTRV2 control plant, the expression level of the cytochrome P45071D7 gene in the silencing plant is obviously reduced, namely the cytochrome P45071D7 gene is 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, cytochrome P45071D7 gene silencing plants show obvious wilting and obvious leaf yellowing, which indicates sensitivity 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 cytochrome P450D 71 gene responds to cadmium ions, and the cytochrome P450D 71D7 gene silences the cadmium ions, so that the gene is more sensitive to stress of the cadmium ions, 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 (7)

1. The application of the tobacco cytochrome P450D 7 gene in the rapid detection of the cadmium pollution of tobacco soil is characterized in that the nucleotide sequence of the cytochrome P45071D7 gene is shown as SEQ ID NO: 1.
2. A method for rapidly detecting cadmium pollution of tobacco soil is characterized in that a fluorescence quantitative PCR-based method is adopted to detect the expression quantity of cytochrome P45071D7 genes in a sample to be detected, if the expression quantity of the cytochrome P45071D7 genes is up-regulated, the existence of heavy metal cadmium pollution is indicated, and the nucleotide sequence of the cytochrome P45071D7 genes is shown as SEQ ID NO:1, wherein the sample to be tested is tobacco.
3. The method for rapidly detecting cadmium pollution in tobacco soil according to claim 2, wherein the experiment of fluorescent quantitative PCR is performed by using a forward primer, a reverse primer and a premix of cytochrome P450D 7 gene, and an internal reference gene is used as an internal standard.
4. The method for rapidly detecting cadmium pollution in tobacco soil according to claim 2, wherein the reaction system of fluorescent quantitative PCR is prepared by SYBR Green dye reagent and comprises 2x TB Green Premix Ex Taq II 5 μl; 10. mu.M Primer F0.4. Mu.l; 10. mu.M Primer R0.4. Mu.l; 50x ROX Reference Dye II 0.2 μl;1 μl of cDNA template; sterilized water 3. Mu.l.
5. The method for rapidly detecting cadmium pollution in tobacco soil according to claim 2, wherein the amplification procedure of the fluorescent quantitative PCR is: 94 ℃ for 30s;94 ℃ for 5s;59.3℃for 34s;40 cycles.
6. Application of a reagent for inhibiting cytochrome P45071D7 gene expression in construction of tobacco sensitive to heavy metal cadmium pollution, wherein the nucleotide sequence of the cytochrome P45071D7 gene is shown in SEQ ID NO: 1.
7. The use of an agent for inhibiting cytochrome P450D 7 gene expression according to claim 6 in the construction of tobacco susceptible to heavy metal cadmium pollution, wherein the method of use is virus-induced gene silencing.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022144095A1 (en) * 2020-12-30 2022-07-07 Fundacion Azti/Azti Fundazioa Biosystems to detect chemical contaminants

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022144095A1 (en) * 2020-12-30 2022-07-07 Fundacion Azti/Azti Fundazioa Biosystems to detect chemical contaminants

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
Cadmium stimulates transcription of the cytochrome p450 side chain cleavage gene in genetically modified stable porcine granulosa cells;Andrea D Smida等;Biol Reprod;20040131;第70卷(第1期);25-31 *
Functional characterization of CYP71D443, a cytochrome P450 catalyzing C-22 hydroxylation in the 20-hydroxyecdysone biosynthesis of Ajuga hairy roots;Yuki Tsukagoshi等;Phytochemistry;20160731;第127卷;23-28 *
RNA-Seq of Agropyron mongolicum Keng in response to drought stress;Yanhong Ma等;Grassland Science;20171119;第64卷(第1期);第11页左栏第3段、第12页左栏第1段 *
栉孔扇贝在镉污染胁迫下消化盲囊组织的转录组分析;张辉等;中国水产科学;20170731;第24卷(第4期);802-810 *
紫苏叶片响应镉胁迫的蛋白质差异表达分析;谢惠玲等;中国生态农业学报;20141031;第22卷(第10期);1207-1213 *
细胞色素P450CYP3A65斑马鱼模型建立及对环境污染物的生物响应;李春杰等;中国药理学与毒理学杂志;20141231;第28卷(第6期);870-877 *
细胞色素P450在茄科作物中的研究进展;张昊等;作物研究;20070615(第02期);132-136 *
重金属Cd胁迫下三七主要药效成分与其合成关键酶DS和P450基因表达相关性研究;朱美霖等;中草药;20161231;第47卷(第24期);4428-4432 *
镉胁迫对盐芥根木栓质代谢的影响;陈宁美等;河南农业科学;20181015;第47卷(第10期);105-110 *

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