CN101643779B - Rice cytochrome P450 gene special primer for assisting in identifying herbicide residue in plant culture matrix - Google Patents
Rice cytochrome P450 gene special primer for assisting in identifying herbicide residue in plant culture matrix Download PDFInfo
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Abstract
The invention discloses a rice cytochrome P450 gene special primer for assisting in identifying herbicide residue in plant culture matrixes. The primer consists of a sequence 1 and a sequence 2 in a sequence table. A method disclosed by the invention comprises: transplanting rice cultured in a plant culture matrix containing no herbicide into a to-be-detected plant culture matrix; taking cDNA obtained through the reverse transcription of total RNA of the rice as a template; using the primer to perform real-time fluorescence quantitative PCR amplification; and calculating the ratio of the initial copy number of specific amplification products in the total RNA of the rice in the to-be-detected plant culture matrix to the initial copy number of the specific amplification products in the total RNA of the rice in the plant culture matrix containing no herbicide through an amplification curve, wherein the ratio is greater than 2.0 +/- 0.05, and the to-be-detected plant culture matrix is a candidate plant culture matrix with herbicide residue. The method has the advantage of ensuring fast easy detection of the herbicide residue in the plant culture matrix.
Description
Technical field
The present invention relates to the residual of weedicide in the auxiliary differential plant culture medium of rice cytochrome P 450 gene special primer.
Background technology
Utilizing the biotech development ecological agricultural chemical is an important directions of modern agriculture research, can alleviate peasant burden, reduces ecological environmental pollution and reduce the human health risk that causes because of a large amount of pesticide residue.Cytochrome P 450 enzymes system is to be combined in the oxydase that contains the mercaptan protoheme on the endoplasmic reticulum.There is very strong species diversity in the Cytochrome P450 supergene family, the vegeto-animal all kinds of vital movements of wide participation,, detoxifcation synthetic such as biomolecules and drug metabolism approach.Utilize functional genomics and information biology means, the function information of the Cytochrome P450 family in the biological metabolism approach is integrated in research, and the paddy rice P450 gene relevant with degrading pesticide residues predicted in research, will help the ecological agricultural chemical of production China autonomy.Is that the agricultural compound design can be carried out in the basis with frizzled receptor with transcribing the pathway component analysis, in conjunction with the Chemoinformatics method, can identify the potential chemical ingredients that is used for sterilant and weedicide.
Summary of the invention
The purpose of this invention is to provide the residual of weedicide in the auxiliary differential plant culture medium of rice cytochrome P 450 gene special primer.
The primer of herbicide residue in the auxiliary differential plant culture medium provided by the present invention, a pair of primer of forming by the nucleotide sequence of the nucleotide sequence of sequence in the sequence table 1 and sequence 2.This primer is to according to the rice cytochrome P 450 gene sequences Design, cytochrome P450 gene called after CYP81A6, and its NCBI location number is NP_001051342, NCBI gene number (GENE ID) is Os03g0760200, DBJ number is AK104825, and TIGR number is LOC_Os03g55240, and full length cDNA sequence length is 2199 Nucleotide, be positioned No. three karyomit(e) from 31,379,697bp to 31,384,722bp, the albumen of this genes encoding has 732 amino acid.The CYP81A6 expression of gene is subjected to inducing of weedicide, and described weedicide is quinclorac, bentazone, metsulfuronmethyl, clomazone or methyl viologen.
Another object of the present invention provides the method for herbicide residue in a kind of auxiliary differential plant culture medium.
The method of herbicide residue in the auxiliary differential plant culture medium provided by the present invention, be that the rice transplanting that will cultivate in the plant culture matrix that does not contain weedicide is in plant culture matrix to be measured, the cDNA that obtains with the total RNA reverse transcription of described paddy rice is a template, classify primer as with the nucleotide sequence of sequence in the sequence table 1 and the nucleotides sequence of sequence 2, carry out the real-time fluorescence quantitative PCR amplification, the ratio of the initial copy number of specific amplification products among the initial copy number that calculates specific amplification products among total RNA of paddy rice of plant culture matrix to be measured by amplification curve and the total RNA of the paddy rice of the plant culture matrix that does not contain weedicide, described ratio is greater than 2.0 ± 0.05, and plant culture matrix to be measured is candidate's plant culture matrix that herbicide residue is arranged.
But described plant culture matrix can be the liquid or solid matrix of any culturing plants, as nutrient solution, soil, vermiculite etc.
When described plant culture matrix to be measured is liquid, can be with the rice transplanting in the plant culture matrix that does not contain weedicide, cultivated in the plant culture matrix to be detected 6 hours-24 hours; When described plant culture matrix to be measured is solid, can collect leach liquor with plant culture matrix to be measured with 5ml/100ml methyl-sulphoxide aqueous solution soaking; The rice transplanting that in the plant culture matrix that does not contain weedicide, cultivate in the plant culture matrix to be measured 6 hours-24 hours.The paddy rice that carries out described transplanting can be in the two leaf stage-four leaf phase.Described weedicide is quinclorac, bentazone, metsulfuronmethyl, clomazone or methyl viologen.
By the method for herbicide residue in the assistant identification plant culture matrix provided by the invention, but whether herbicide residue is arranged in the preliminary evaluation plant culture matrix, can determine in conjunction with existing instrument analytical method whether herbicide residue is arranged in the plant culture matrix again.
Herbicide residue in the quick assistant identification plant culture matrix for convenience,, the present invention also provides a kind of PCR kit for fluorescence quantitative of special use, this PCR kit for fluorescence quantitative comprises a pair of primer, and this primer is to being made up of the nucleotide sequence of sequence in the sequence table 1 and the nucleotide sequence of sequence 2.
The present invention has designed a pair of primer according to a gene order that multiple weedicide is had a rice cytochrome P 450 gene of obviously replying feature, but with this primer to whether herbicide residue is arranged in the auxiliary detection plant culture matrix.Utilize the method for herbicide residue in the assistant identification plant culture matrix of the present invention to carry out preliminary examination to plant culture matrix, the candidate's plant culture matrix that sifts out is determined to have or not herbicide residue with existing instrument analytical method again, the working strength of lowering apparatus analysis makes the detection of herbicide residue in the plant culture matrix fast and convenient greatly.
Description of drawings
Fig. 1 is a rice cytochrome P 450 gene, CYP81A6, position view on the rice genome sequence and gene structure.
Fig. 2 is that the CYP81A6 gene is expressed variation under the treatment condition of quinclorac.
Fig. 3 is that the CYP81A6 gene is expressed variation under the treatment condition of multiple weedicide.
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
Rice varieties among the following embodiment is from national germplasm resource bank.
Analyze by Blast, gene locus in named rice cytochrome P 450 gene and the TIGR paddy rice database is mapped, the corresponding LOC_Os03g55240 of CYP81A6, be positioned at karyomit(e) the 31st, 379 No. three, 697bp to 31,384,722bp has five exons and four introns, and the structure of this gene as shown in Figure 1.According to the mRNA sequence of CYP81A6, use the primer3 instrument, near 3 ' end a pair of special primer ChemR3-1 of design and ChemR3-2, the sequence of primer ChemR3-1 and ChemR3-2 is respectively sequence 1 and the sequence 2 in the sequence table in the mRNA of CYP81A6 sequence.
With the 5ml/100ml methyl-sulphoxide aqueous solution be solvent respectively compound concentration be that 1.65mM dichloroquinoline acid solution, concentration are that 19.98mM bentazone solution, concentration are that 1.31mM metsulfuronmethyl solution, concentration are that 1.75mM clomazone solution and concentration are 10 μ M methyl viologen solution.
(temperature is 28 ℃/25 ℃ under the normal growth condition, relative humidity 83%, 12 hours diurnal cycle) the fine seedling of the paddy rice of growth Japan, in one heart stage usefulness 1.65mM dichloroquinoline acid solution, 19.98mM bentazone solution, 1.31mM metsulfuronmethyl solution, 1.75mM clomazone solution and the processing of 10 μ M methyl viologen solution sprays respectively of two leaves, to handle in contrast with the spraying of the 5ml/100ml methyl-sulphoxide aqueous solution, experiment repeats 3 times, each re-treatment 10-20 strain paddy rice.
Extract 1.65mM quinclorac solution-treated 3 hours, 6 hours and 24 hours and methyl-sulphoxide handled total RNA of the fine seedling of paddy rice Japan of 3 hours, 6 hours and 24 hours, and reverse transcription becomes cDNA; With reverse transcription cDNA is template, and ChemR3-1 and ChemR3-2 are the expression level that primer passes through real-time fluorescence quantitative PCR technology for detection CYP81A6.
Get 6 hours paddy rice of the above-mentioned 5 kinds of herbicide treatment fine seedling of Japan and contrast seedling and extract total RNA respectively, reverse transcription becomes cDNA; With reverse transcription cDNA is template, and ChemR3-1 and ChemR3-2 are the expression level that primer passes through real-time fluorescence quantitative PCR technology for detection CYP81A6.
The quantitative fluorescent PCR reaction conditions is as follows:
As interior mark, carry out real-time fluorescence quantitative PCR reaction, reaction system 10 μ L:10 * PCR Buffer 1 μ L, 25mmol/L Mg with 18S rRNA by following condition
2+1 μ L, 10mmol/L dNTPs 0.5 μ L, each 0.25 μ L of 25 μ mol/L upstream and downstream primers, cDNA template 1 μ L, Taq enzyme (Promega) 0.25 μ L, 20 * SYBR GreenI, 0.5 μ L, ddH2O 6.25 μ L.
Detect the reaction conditions of the expression level real-time fluorescence quantitative PCR of CYP81A6: 95 ℃ of sex change 3min, circulate 40 times: ℃ annealing 25s → 72,95 ℃ of sex change 40s → 61 ℃ extension 1min 20s, melt 95 ℃ of 0s (20 ℃/s) → 71 ℃ 15s (20 ℃/s → 95 ℃ 0s (0.1 ℃/s), cool off 40 ℃ of 30s (20 ℃/s).All quantitative fluorescent PCR reactions are all carried out on LightCycler (Roche).
Real-time fluorescence quantitative PCR is the result show, the expression level of 1.65mM dichloroquinoline acid treatment beginning in 3 hours CYP81A6 just has been higher than contrast, and the copy number ratio of the CYP81A6 of 1.65mM quinclorac treatment group and control group is 8.32 at this moment; 1.65mM dichloroquinoline acid treatment 24 hours, the expression level of CYP81A6 still is higher than contrast, and the copy number ratio of the CYP81A6 of 1.65mM quinclorac treatment group and control group is 29.31 at this moment.(Fig. 2)
" 1 " representative was handled 3 hours with methyl-sulphoxide among Fig. 2, " 2 " represented the dichloroquinoline acid treatment 3 hours, " 3 " representative was handled 6 hours with methyl-sulphoxide, " 4 " represented the dichloroquinoline acid treatment 6 hours, " 5 " representative was handled 24 hours with methyl-sulphoxide, and " 6 " represented the dichloroquinoline acid treatment 24 hours.
Though the processing CYP81A6 of different weedicides replys difference, but after 6 hours quincloracs, bentazone, metsulfuronmethyl, clomazone and methyl viologen are handled, the expression level of CYP81A6 is all apparently higher than control group, at this moment, the copy number ratio of the CYP81A6 of quinclorac treatment group and control group is 8.90; The copy number ratio of the CYP81A6 of bentazone treatment group and control group is 5.23; The copy number ratio of the CYP81A6 of metsulfuronmethyl treatment group and control group is 2.61; The copy number ratio of the CYP81A6 of clomazone treatment group and control group is 12.33; The copy number ratio of the CYP81A6 of methyl viologen treatment group and control group is 12.04.Wherein, the inducing action of clomazone and methyl viologen is the strongest, and quinclorac takes second place, and bentazone and metsulfuronmethyl be weak (Fig. 3).
Sequence table
＜110〉China Agricultural University
＜120〉weedicide residual in the auxiliary differential plant culture medium of rice cytochrome P 450 gene special primer.
<130>CGGNARW81583
<160>2
<210>1
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
gctagcaatc?gctctttgag?a 21
<210>2
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
gaaattgaaa?tacctggcgt?gt 22
Claims (5)
1. the primer of herbicide residue in the auxiliary differential plant culture medium, a pair of primer of forming by the nucleotide sequence of the nucleotide sequence of sequence in the sequence table 1 and sequence 2.
2. primer according to claim 1 is characterized in that: described weedicide is quinclorac, bentazone, metsulfuronmethyl, clomazone or methyl viologen.
3. the PCR kit for fluorescence quantitative of herbicide residue in the auxiliary differential plant culture medium, this test kit comprises a pair of primer, it is characterized in that: described primer is to being made up of the nucleotide sequence of sequence in the sequence table 1 and the nucleotide sequence of sequence 2.
4. test kit according to claim 3 is characterized in that: described weedicide is quinclorac, bentazone, metsulfuronmethyl, clomazone or methyl viologen.
5. the described primer of claim 1 has or not the application in the herbicide spraying in identifying paddy growth.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2008101180218A CN101643779B (en) | 2008-08-06 | 2008-08-06 | Rice cytochrome P450 gene special primer for assisting in identifying herbicide residue in plant culture matrix |
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| CN2008101180218A CN101643779B (en) | 2008-08-06 | 2008-08-06 | Rice cytochrome P450 gene special primer for assisting in identifying herbicide residue in plant culture matrix |
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| CN101643779B true CN101643779B (en) | 2011-10-05 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101063134A (en) * | 2007-04-26 | 2007-10-31 | 浙江大学 | Rice cytochrome P450 gene and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101063134A (en) * | 2007-04-26 | 2007-10-31 | 浙江大学 | Rice cytochrome P450 gene and its application |
Non-Patent Citations (2)
| Title |
|---|
| Gang Pan et al.，.Map-based cloning of novel rice cytochrome P450 gene CYP81A6 that confers resistance to two different classes of herbicides.《Plant Mol Biol》.2006,第61卷933-943. * |
| 朱剑等.细胞色素P450与除草剂代谢.《植物生理学通讯》.2007,第43卷(第1期),9-15. * |
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