CN102796821A - Method of detecting relative level of tobacco nicotine conversion - Google Patents
Method of detecting relative level of tobacco nicotine conversion Download PDFInfo
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- CN102796821A CN102796821A CN2012103150533A CN201210315053A CN102796821A CN 102796821 A CN102796821 A CN 102796821A CN 2012103150533 A CN2012103150533 A CN 2012103150533A CN 201210315053 A CN201210315053 A CN 201210315053A CN 102796821 A CN102796821 A CN 102796821A
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Abstract
The invention discloses a method of detecting relative level of tobacco nicotine conversion. The method comprises the following steps of: (1) collecting leaves at different stages of same type of tobacco, leaves of different parts or leaves of different types of tobacco; (2) extracting total RNA (Ribonucleic Acid) of the leaves respectively and synthesizing cDNA (complementary Deoxyribonucleic Acid) through reverse transcription; (3) designing a specific primer aiming at a nicotine conversion related gene and performing real-time fluorescent quantitative PCR (Polymerase Chain Reaction) by using the specific primer by taking the cDNA as a template to obtain respective Ct value; and (4) calculating by using a standard curve according to the Ct value to obtain template quantity related to the nicotine conversion in the respective cDNA and judging the relative level of the nicotine conversion between the leaves at different stages of same type of tobacco, leaves of different parts or leaves of different types of tobacco. According to the method, real-time monitoring can be performed on the relative level of the tobacco nicotine conversion, the accuracy is high, the flexibility is high, and bad tobacco conversion strains can be timely eliminated.
Description
Technical field
The invention belongs to technical field of molecular biology, relate in particular to a kind of tobacco smoke alkaloid that detects and transform the method for level relatively.
Background technology
Vegeto-alkali is extensively to be present in the intravital one type of secondary metabolite be made up of the small molecules nitrogenous compound of plant, the existing in the world at present separated evaluation of alkaloids substance that surpasses 12000 kinds.Vegeto-alkali has special status in tobacco and goods thereof, it is not only the important quality key element of tobacco, and has stipulated the speciality of tobacco as a kind of commodity.Nicotine, nornicotine, neonicotine and anatabine are main alkaloid in 4 in the tobacco.It mainly is the nicotine of sucking wherein (nicotine) that people suck tobacco, has another name called Nicotine.Common tobacco belongs to the nicotine accumulation type, and nicotine content accounts for 90～95% of total alkaloid content, and other 3 kinds only account for 5%～10% of vegeto-alkali total content, and nornicotine content generally is no more than 3.5% of total alkaloid content.
Common tobacco is the double recessive genotype (ctctcscs) of isozygotying, and does not have nicotine demethyl ability.But in the cigarette group of hill body of Cultivar, some plant can have nicotine demethyl ability because of transgenation, cause nicotine content significantly to reduce, the then corresponding increase of nornicotine content, even can reach more than 95%.The proterties that this nicotine transforms to nornicotine is called nicotine and transforms, and the cigarette strain with nicotine conversion capability is called transformant (converter).
The rising meeting of nornicotine content directly influences the fragrant jealous quality of tobacco leaf, causes flue-cured tobacco modulation back appearance " cherry is red " phenomenon and fragrance not good.Simultaneously owing to nornicotine is easy in the ageing process after tobacco leaf modulation and modulation biochemical reactions such as oxidation, nitrosification and acidylate take place; Form many objectionable constituent; Like myosmine (myosmine), acidylate nornicotine (acylnornicotines) and nitrosonornicotine (NNN) etc., so also there is disadvantageous effect in the rising of nornicotine content to HUMAN HEALTH.
The nornicotine of control lower level is not only because it has the potential carinogenicity as the precursor substance of many objectionable constituent to human body, also because nornicotine itself also has negative impact to the health of human body.Nornicotine can cause proteinic unusual glycosylation, but also can covalent reaction take place with many steroid medicines commonly used (like retrocortine), thereby changes the drug effect and the toxicity of this type medicine.
Therefore, thus the nornicotine content of in time rejecting the transformant control lower level in the tobacco colony becomes one of hot issue that international tobacco industry circle and academia pay close attention to.
The method that detects the tobacco smoke alkaloid level of conversion at present mainly is to detect its vegeto-alkali component and content through vapor-phase chromatography (GC) and gas chromatography mass spectrometry method (GC-MS), and then calculates the nicotine level of conversion.Though this method is stable, operability is all relatively good; But this method only can analyze usually after gathering ripe tobacco leaf or the modulation after tobacco leaf; And need certain processing after gathering; Comparatively consuming time, can't be in the intravital nicotine level of conversion of the distinct group of whole growing dynamic knowledge different varieties tobacco leaf and same kind, and then can't in time bad transformant be eliminated.
Summary of the invention
The invention provides a kind of tobacco smoke alkaloid that detects and transform the method for level relatively, nicotine transforms level relatively between the blade of different times or different sites or different varieties tobacco to utilize this method can the same article of easy rapid detection to grow tobacco.
A kind of tobacco smoke alkaloid that detects transforms the method for level relatively, comprises the steps:
(1) collects the grow tobacco blade of different times or different sites or different varieties tobacco of same article;
(2) extract total RNA of said blade respectively, rt synthesizes cDNA;
(3) transforming the genes involved design specific primers to nicotine, is template with said cDNA, utilizes said Auele Specific Primer to carry out real-time fluorescence quantitative PCR, obtains Ct value separately;
(4) according to said Ct value, utilize typical curve to calculate the template amount that nicotine among each cDNA transforms genes involved, judge that the nicotine that same article grow tobacco between the blade of different times or different sites or different varieties tobacco transforms level relatively;
Said Auele Specific Primer is:
Upstream primer: 5 '-ACGTGATCCTAAACTCTGGTCTG-3 ';
Downstream primer: 5 '-GCCTGCACCTTCCTTCATG-3 ';
It is nicotine demethylase gene C YP82E4 that said nicotine transforms genes involved, and its full-length cDNA nucleotide sequence is shown in SEQ ID NO.1.
When the blade nicotine that detects same tobacco bred different sites or different tobacco breds transforms relatively level, generally select the ripening stage blade for use.
Nicotine demethylase gene C YP82E4 is the member of CYP82E2 gene family, and mediation nicotine is to the conversion of nornicotine.This gene family especially transcriptional level of nicotine demethylase gene C YP82E4 in the tobacco transformant generally is higher than non-transformant; Use the RNAi perturbation technique to suppress this expression of gene, can effectively reduce the ratio of high nornicotine content tobacco at the tobacco transformant.Therefore, to this gene design method, can accurately detect tobacco smoke alkaloid delicately and transform level relatively.
What real-time fluorescence quantitative PCR of the present invention increased is not the complete sequence that nicotine transforms genes involved, and only is a certain fragment wherein, and its base sequence is shown in SEQ ID No.4.In the rt process, volume and the concentration of each group RNA that gets are identical, through calculating the template amount (concentration) that nicotine transforms genes involved, just can obtain this expression of gene level.
Said real-time fluorescence quantitative PCR adopts the chimeric method of SYBR Green I fluorescence.
The amplification system of said real-time fluorescence quantitative PCR is 20 μ L, wherein: Takara 2 * SYBR Premix EX Taq 10 μ L, each 0.5 μ L of the forward and reverse primer of 10 μ M, cDNA template 2 μ L, sterile purified water 7 μ L.
The response procedures of said real-time fluorescence quantitative PCR is: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 50s, 35 circulations; 72 ℃ are extended 5min.
The preparation method of said typical curve is:
(1) standard substance of one group of concentration in gradient distribution of preparation;
(2) be template with said standard substance, utilize said primer, carry out real-time fluorescence quantitative PCR, record Ct value;
(3) drafting obtains typical curve.
Said standard substance are the recombinant vectors suspension that contains nicotine demethylase gene C YP82E4 full-length cDNA nucleotide sequence, or contain the genetically modified host cell suspension of said recombinant vectors.
The preparation method of said standard substance is:
(1) extracting total RNA of tobacco leaf and reverse transcription is cDNA;
(2) be template with said cDNA, carry out pcr amplification and obtain the purpose fragment;
(3) said purpose fragment is connected into the T carrier, obtains recombinant vectors.
The employed primer of said pcr amplification is:
Upstream primer is: 5 '-ATGCTTTCTCCCATAGAAGCC-3 ';
Downstream primer is: 5 '-TTAATAAAGCTCAGGTGCCAG-3 '.
The quality of said total RNA is most important, should adopt high-quality test kit or reagent to extract, like Qiagen RNeasy Plant Mini kit test kit.
Because RNA is prone to degraded and impurity is difficult to remove, therefore to measure the purity of the RNA that extracts.The OD of the pure article of RNA
260/ OD
280Ratio is 2.0, if this ratio is lower, has explained that albumen or phenol pollute; If this ratio is too high, explain that RNA has degraded in various degree.General OD
260/ OD
280Ratio is that the RNA of 1.8-2.0 can use.
Pcr amplification nicotine demethylase gene C YP82E4 full-length cDNA is to be used to make up recombinant vectors; And real-time fluorescence quantitative PCR is in order to detect this expression of gene amount, therefore when amplification, does not need the complete sequence amplification, but selects for use primer should have better specificity.
The present invention adopts the chimeric method of SYBR Green I fluorescence to carry out real-time fluorescence quantitative PCR, is based on this method and has the following advantages: to the dna profiling non-selectivity, be applicable to any DNA; Needn't the complex design probe, easy to use; Very sensitive; Cheap.
But, make it to be prone to combine to the dna profiling non-selectivity just because of SYBR Green I, produce false positive with non-specific double-stranded DNA.Therefore, after the real-time fluorescence quantitative PCR reaction finishes, to do the melting curve analysis, judge whether amplified production is target fragment.If it is unimodal that curve has only, and to go out the peak position be annealing temperature, then there is not non-specific fluorescence, quantitatively accurately; If assorted peak occurring or going out the peak position is not annealing temperature, then there are other products non-specific fluorescence to occur, quantitatively inaccurate.
In the real-time fluorescence quantitative PCR, log x
0(template amount) and Ct value are linear, can make typical curve through the standard substance of known template amount, according to the Ct value of each sample, just can calculate pairing template amount, and then the nicotine of analyzing each sample transforms level relatively.
Compared with prior art, beneficial effect of the present invention is:
(1) detection tobacco smoke alkaloid according to the invention transforms that the chimeric method tolerance range of level adopted relatively SYBR Green I fluorescence is high, susceptibility is strong; And than the relative quantification method, the present invention adopts standard substance to carry out absolute quantitation, transforms and has bigger advantage aspect horizontal relatively analyzing the grow tobacco nicotine of blade of different times or different sites or different varieties tobacco of same article.
(2) method sampling amount of the present invention is few, compares with conventional detection (GC method and GC-MS method), can not cause obvious injury to sampling cigarette strain, and easy and simple to handle, result accurately, good reproducibility.
(3) existing detection method can only could be analyzed its nicotine level of conversion behind tobacco leaf picking; And can transforming relatively level to the nicotine of different tobacco samples in whole growing, the present invention monitors in real time; Avoided the time limitation of existing detection method, opened up a new effective way for the researchist in time rejects bad transformant with the plantation family.
Description of drawings
Fig. 1 is the detected through gel electrophoresis result of the conventional PCR of nicotine demethylase gene C YP82E4cDNA total length.Wherein, M is Takara DL2000DNAMarker, and 1 is nicotine demethylase gene C YP82E4cDNA total length amplified production.
Fig. 2 makes up the detected through gel electrophoresis result of conventional PCR checking for the reorganization plasmid standard.Wherein, M is Takara DL2000DNA Marker, and 1-6 is the purpose fragment in the different positive colonies.
Fig. 3 is the amplification curve of the recombinant plasmid standard substance real-time fluorescence quantitative PCR of different dilution gradients.
Fig. 4 is the typical curve of the recombinant plasmid standard substance real-time fluorescence quantitative PCR of different dilution gradients.Wherein, X-coordinate is represented the logarithmic value of template number, and ordinate zou is represented the Ct value.
Fig. 5 A is the melting curve of the recombinant plasmid standard substance real-time fluorescence quantitative PCR of different dilution gradients.
Fig. 5 B is the melting peak of the recombinant plasmid standard substance real-time fluorescence quantitative PCR of different dilution gradients.
Fig. 6 is the detected through gel electrophoresis result of the conventional PCR of recombinant plasmid standard substance of different dilution gradients.Wherein, M is Takara 100bp DNA Marker, and 1-7 is followed successively by 1 * 10
5, 1 * 10
6, 1 * 10
7, 1 * 10
8, 1 * 10
9, 1 * 10
10, 1 * 10
11The recombinant plasmid standard substance of copy/mL.
Embodiment
Be example only below with the recombinant plasmid that contains nicotine demethylase gene C YP82E4 full-length cDNA; Illustrate it and detect tobacco smoke alkaloid and transform the effect of level relatively, other recombinant vectors or genetically modified host cell of containing this recombinant vectors that contains all or part of nucleotide sequence of nicotine demethylase gene C YP82E4 full-length cDNA detects tobacco smoke alkaloid, and to transform the effect of level relatively all identical with these standard substance.
(1) tobacco leaf sampling
With burley tobaccos B37 is material, chooses its ripening stage middle part blade 2g, and the bright leaf sample of getting is wrapped and mark with masking foil, puts into the ice chest that liquid nitrogen is housed rapidly and preserves, and is sent to the laboratory fast and carries out further work.
(2) total RNA extraction, assay and reverse transcription reaction
Total RNA extracts: get the tobacco sample 200mg in the above-mentioned masking foil, adopt the RNeasy Plant Mini kit of Qiagen company to extract its total RNA, concrete steps are following:
It is levigate that tobacco leaf is added liquid nitrogen, changes the Eppendorf pipe before the liquid nitrogen volatilization over to and add 450 μ L RLT to extract damping fluid, and the back 56 ℃ of temperature of vibrating are bathed 3min; Change extracting solution over to the purple post, the centrifugal 2min of 10000g, filtrating changes new Eppendorf pipe over to, adds 225 μ L absolute ethyl alcohol mixings; Filtrating changes pink post again over to, and the centrifugal 15s of 8000g abandons filtrating; Add 700 μ L RW1 in pink post, the centrifugal 15s of 8000g abandons filtrating; Add 500 μ L RPE in pink post, the centrifugal 15s of 8000g abandons filtrating, repeats once the centrifugal 2min of blank pipe; Pink post is inserted on the new Eppendorf pipe, and adding 30 μ L does not have RNase water, the centrifugal 1min of 10000g; Extract good RNA sample and put-80 ℃ of preservations.
Total rna content is measured: using the built-in RNA mensuration program of nucleic acid-protein determinator to read rna content is 345.8 μ g/mL, OD
260/ OD
280Value is 1.933, shows that total RNA purity is better, subsequent use.
Reverse transcription reaction: adopting the AMV ThermoScript II of Takara company, is cDNA with the mRNA reverse transcription among total RNA, subsequent use after diluting 10 times.Concrete steps are following:
Reaction system is 20 μ L, wherein: total RNA 2 μ L, 50pmol/ μ L Oligo (dT) 18primer1 μ L, 10mM dNTP Mixture 2.5 μ L, RNase Inhibitor 20U, AMV ThermoScript II 10U, 5 * AMV Buffer, 4 μ L, DEPC H
2O is settled to 20 μ L.Room temperature held 10min moves in 42 ℃ of thermostatic baths and is incubated 1.5h, and cooling 2min gets final product in frozen water.With subsequent use after 10 times of the cDNA that the obtains dilutions.
(3) preparation of standard substance
With above-mentioned cDNA is template, with following primer nicotine demethylase gene C YP82E4 full length cDNA sequence is carried out conventional pcr amplification, and said primer is:
Forward primer sequence: 5 '-ATGCTTTCTCCCATAGAAGCC-3 ';
Reverse primer sequence: 5 '-TTAATAAAGCTCAGGTGCCAG-3 '.
The concrete steps of said conventional pcr amplification are:
1) clone of goal gene and purifying
The PCR reaction system is 50 μ L, wherein: 5U/ μ L Takara LATaq 0.5 μ L, 2.5mMdNTP 8 μ L, LA Taq Buffer (Mg
2+Free) 5 μ L, 25mM MgCl
25 μ L, each 1 μ L of the forward and reverse primer of 20 μ M, cDNA template 1 μ L, sterile purified water 28.5 μ L.
The PCR response procedures is: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 90s, 30 circulations; 72 ℃ are extended 7min.
Get 5 μ L PCR products and carry out 1% agarose gel electrophoresis, electrophoresis result is as shown in Figure 1.The result shows that this amplified production is the purpose fragment about showing that this amplified production size is for 1600bp.Get remaining 45 μ L PCR products and carry out the recovery of 1% agarose gel electrophoresis, utilize Promega glue to reclaim test kit (numbering A9281) and carry out the segmental recovery purifying of purpose.Get 5 μ L behind the recovery purifying and carry out 1% agarose gel electrophoresis, the result shows that the effect that reclaims purifying is better, can be used for T-A or other double digestions clone.
2) T-A clone technology
A, reaction: the purpose fragment that will reclaim purifying is connected with Takara pMDl8-T simple Vector carrier; Linked system is 10 μ L, wherein: purpose fragment 2 μ L, pMD18-T simple Vector carrier 1 μ L, efficiently connect liquid Solution I 5 μ L, dH
2O 2 μ L; 4 ℃ of connections of spending the night.
B, competent cell transform: 10 μ L are connected product be added in the 100 μ L DH5 α competent cells, place 30min on ice; 42 ℃ of heating 45s place 1min again in ice; Add 890 μ L LB substratum, 37 ℃ of shaking culture 60min; Containing incubated overnight on the LB substratum of X-Gal, IPTG and Amp, form single bacterium colony.
3) evaluation of positive colony
A, positive colony checking: 16 single bacterium colonies of white of picking are to the test tube that contains 5mL LB (Amp+) liquid nutrient medium respectively on the substratum of coated plate after conversion, and 37 ℃ of shaken overnight are cultivated.Getting 1 μ L bacterium liquid is template, adopts PCR system and program in the step 1) to carry out pcr amplification, whether has changed the purpose band about 1600bp in the checking plasmid.The result is as shown in Figure 2 in checking.To send order-checking through the bacterium liquid that PCR proves positive colony; Sequencing result is 1554bp; Its positive recombinant plasmid of blast comparison result proof, and nicotine demethylase gene C YP82E4mRNA sequence (GenBank accession number: DQ205656.1 among this recombinant plasmid and the GenBank; DQ131885.1; DQ131886.1) have homology more than 99%, show that plasmid standard makes up successfully.The nucleotide sequence that is carried in this recombinant plasmid is shown in SEQ ID NO.1.
B, extraction plasmid: select to contain the segmental bacterium liquid of purpose, adopt the improved SDS alkaline lysis AxyPrep of Axygen company DNA test kit to extract DNA.Detailed process is following:
Get 3mL bacterium liquid, the centrifugal 1min of 12000g abandons supernatant to the greatest extent; Add 250 μ L S1 damping fluid resuspension bacterial precipitations; Add 250 μ L S2 damping fluids, gentle and fully mixing make the abundant cracking of thalline; Add 350 μ L S3 damping fluids, gentle mixing, the centrifugal 10min of 12000g; Draw supernatant and transfer in the preparation pipe, the centrifugal 1min of 12000g abandons filtrating; Add 500 μ L W1 damping fluids, the centrifugal 1min of 12000g abandons filtrating; Add 700 μ L W2 damping fluids, the centrifugal 1min of 12000g abandons filtrating; In the preparation pipe, add 80 μ L deionized waters, the centrifugal 1min of 12000g;-20 ℃ of preservations are subsequent use.
(4) the recombinant plasmid standard substance detect
1) concentration determination of recombinant plasmid and copy number calculate
Get the plasmid standard 2 μ L of purifying, utilizing the nucleic acid-protein determinator to record its concentration is 94.5 μ g/mL, and OD
260/ OD
280Ratio is 1.888, shows that plasmid standard purity is better.Can calculate its copy number according to plasmid standard concentration, calculation formula is following:
The copy number of DNA=(molar mass of quality/DNA of DNA) * 6.02 * 10
23
1bp=660Da in the double-stranded DNA, the then bp of molar mass=660Da of DNA * (2692+1554).Bring above-mentioned formula into, the copy number that draws plasmid standard is 2.030 * 10
13Copy/mL.
2) real-time fluorescence quantitative PCR detects the scope of recombinant plasmid standard substance
The recombinant plasmid standard substance are carried out 10 times doubling dilution, and each dilutes gradient is 1 * 10
11, 1 * 10
10... 1 * 10
1Copy/mL.Utilize following Auele Specific Primer to carry out real-time fluorescence quantitative PCR and detect, the nucleotides sequence of this primer is classified as:
The forward primer sequence is: 5 '-ACGTGATCCTAAACTCTGGTCTG-3 ';
The reverse primer sequence is: 5 '-GCCTGCACCTTCCTTCATG-3 '.
Adopting the chimeric method of SYBR Green I fluorescence to carry out real-time fluorescence quantitative PCR detects; Reaction system is 20 μ L, wherein: Takara 2 * SYBR Premix EX Taq 10 μ L, each 0.5 μ L of the forward and reverse primer of 10 μ M, plasmid standard 2 μ L, sterile purified water 7 μ L.
The real-time fluorescence quantitative PCR response procedures is: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 50s, 35 circulations; 72 ℃ are extended 5min; 55-95 ℃ slowly heats up, produces melting curve.
Amplification curve is as shown in Figure 3, can be known that by figure the scope of real-time fluorescence quantitative PCR detection recombinant plasmid standard substance is 1 * 10
5-1 * 10
11Copy/mL.
3) drafting of real-time fluorescence quantitative PCR examination criteria curve
Get the recombinant plasmid standard substance (1 * 10 of known 7 doubling dilutions
11, 1 * 10
10, 1 * 10
9, 1 * 10
8, 1 * 10
7, 1 * 10
6, 1 * 10
5Copy/mL), carry out the drafting of real-time fluorescence quantitative PCR examination criteria curve.Each dilution gradient is done 3 repetitions, carries out the real-time fluorescence quantitative PCR reaction, and reaction system and program are with embodiment 1 step (4)-2).Typical curve is as shown in Figure 4, and plasmid standard is 1 * 10
5-1 * 10
10R in the scope of copy/mL
2=0.9937, explain that this system is better linear; Slope is-4.4208, explains that this system amplification efficiency is higher.The result is the average result of 3 revision tests among the figure.
4) melting curve of real-time fluorescence quantitative PCR examination criteria curve
The same step of the measuring method of the melting curve of recombinant plasmid standard substance (4)-2).Measure the result shown in Fig. 5 A and 5B, the Tm value of specific reaction is 81.5 ± 0.5 ℃, and curve is unimodal, explains non-specific fluorescence not occur, and is quantitative accurate.
5) conventional PCR detects the scope of recombinant plasmid standard substance
To dilute gradient is 1 * 10
5-1 * 10
11The recombinant plasmid standard substance of copy/mL carry out conventional PCR and detect.
Conventional PCR reaction system is 50 μ L, wherein, and 5U/ μ L Takara LA Taq 0.5 μ L, 2.5mM dNTP 8 μ L, LA Taq Buffer (Mg
2+Free) 5 μ L, 25mM MgCl
25 μ L, each 1 μ L of the forward and reverse primer of 20 μ M, recombinant plasmid standard substance 1 μ L, sterile purified water 28.5 μ L.
Conventional PCR response procedures is: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 90s, 30 circulations; 72 ℃ are extended 7min.
Get 5 μ L PCR products and carry out 1% agarose gel electrophoresis; Electrophoresis result is as shown in Figure 6; The amplified production size is between 200-300bp, and sequencing result is 238bp, after the blast comparison; Prove that it is the part nucleotide fragments in the reorganization plasmid standard purpose fragment, the sequence of this nucleotide fragments is shown in SEQ ID NO.4.
Transforming two mutants HNC-1 with burley tobaccos B37 and high-nicotine is material, utilizes said primer to detect its nicotine and transforms level relatively, and concrete steps are following:
(1) tobacco leaf sampling
Transforming two mutants HNC-1 with burley tobaccos B37 and high-nicotine is material; Choose each 2g of middle part blade in its group's phase, prosperous long-term and ripening stage respectively; The bright leaf sample of getting is wrapped and mark with masking foil; Put into the ice chest that liquid nitrogen is housed rapidly and preserve, be sent to the laboratory fast and carry out further work.
(2) total RNA extraction, assay and reverse transcription reaction
Total RNA extracts: get the tobacco sample 200mg in the above-mentioned masking foil, adopt the RNeasy Plant Mini kit of Qiagen company to extract its total RNA, concrete steps are following:
It is levigate that tobacco leaf is added liquid nitrogen, changes the Eppendorf pipe before the liquid nitrogen volatilization over to and add 450 μ L RLT to extract damping fluid, and the back 56 ℃ of temperature of vibrating are bathed 3min; Change extracting solution over to the purple post, the centrifugal 2min of 10000g, filtrating changes new Eppendorf pipe over to, adds 225 μ L absolute ethyl alcohol mixings; Filtrating changes pink post again over to, and the centrifugal 15s of 8000g abandons filtrating; Add 700 μ L RW1 in pink post, the centrifugal 15s of 8000g abandons filtrating; Add 500 μ L RPE in pink post, the centrifugal 15s of 8000g abandons filtrating, repeats once the centrifugal 2min of blank pipe; Pink post is inserted on the new Eppendorf pipe, and adding 30 μ L does not have RNase water, the centrifugal 1min of 10000g; Extract good total RNA sample and put-80 ℃ of preservations.
Total rna content is measured: total RNA reads the content (table 2) of RNA with 60 times of RNase-free treating water dilutions with the built-in RNA mensuration program of nucleic acid-protein determinator.
Table 2 burley tobaccos sample rna content
| Kind | Period | Rna content (μ g/mL) |
| B37 | Group's phase | 801.6 |
| B37 | Prosperous long-term | 294.4 |
| B37 | Ripening stage | 340.0 |
| HNC-1 | Group's phase | 372.0 |
| HNC-1 | Prosperous long-term | 153.6 |
| HNC-1 | Ripening stage | 200.8 |
Reverse transcription reaction: the RNA concentration of 6 samples all being diluted to 100 μ g/mL, adopting the AMV ThermoScript II of Takara company, is cDNA with the mRNA reverse transcription among the total RNA of sample, and subsequent use after diluting 10 times, concrete steps are following:
Reaction system is 20 μ L, wherein: the total RNA2 μ of 100 μ g/mL L, 50pmol/ μ L Oligo (dT) 18primer 1 μ L, 10mM dNTP Mixture 2.5 μ L, RNase Inhibitor 20U, AMV ThermoScript II 10U, 5 * AMV Buffer, 4 μ L, DEPC H
2O is settled to 20 μ L.Room temperature held 10min moves in 42 ℃ of thermostatic baths and is incubated 1.5h, and cooling 2min gets final product in frozen water.With subsequent use after 10 times of the cDNA that the obtains dilutions.
(3) real-time fluorescence quantitative PCR detects
CDNA with after the testing sample dilution is a template, carries out its nicotine of real-time fluorescence quantitative PCR analysis and transforms level relatively.Reaction system and program are with embodiment 1 step (4)-2).Write down the Ct value of each sample, through its corresponding template amount of typical curve calculating of recombinant plasmid standard substance, and then relatively the nicotine of each sample transforms level relatively, and test-results is as shown in table 3, and Ct value and template amount are 3 multiple MVs.
Table 3 burley tobaccos sample Ct value and corresponding templates amount
| Kind | Period | The Ct value | The template amount (copy/mL) |
| B37 | Group's phase | 30.91 | 8.15×10 5 |
| B37 | Prosperous long-term | 31.44 | 6.18×10 5 |
| B37 | Ripening stage | 29.88 | 1.40×10 6 |
| HNC-1 | Group's phase | 31.14 | 7.25×10 5 |
| ?HNC-1 | Prosperous long-term | 28.40 | 3.01×10 6 |
| ?HNC-1 | Ripening stage | 23.90 | 3.13×10 7 |
(4) traditional detection method checking
In order to verify the accuracy of the inventive method, the inventor adopts traditional detection method to verify to these lot sample article simultaneously, measures the alkaloid of each sample, and then detects its nicotine level of conversion.Analytical procedure is with reference to tobacco industry standard YC/T383-2010, the mensuration-GC-MS of tobacco and tobacco product nicotine, nornicotine, neonicotine, myosmine and neonicotine.Nicotine conversion rate (%)=100 * nornicotine content/(nornicotine content+nicotine content).
Each sample nicotine conversion rate is seen table 4.The result shows that high-nicotine transformant HNC-1 has higher nicotine level of conversion, particularly ripening stage blade, is significantly higher than the level of wild-type B37, and is consistent with the expression trend of nicotine demethylase gene C YP82E4.This shows that method provided by the present invention has the accuracy of height, fits like a glove with practical situation.
Table 4 burley tobaccos sample nicotine conversion rate
| Kind | Period | Nicotine conversion rate (%) |
| B37 | Group's phase | 2.79 |
| B37 | Prosperous long-term | 2.19 |
| B37 | Ripening stage | 3.36 |
| HNC-1 | Group's phase | 2.35 |
| HNC-1 | Prosperous long-term | 11.58 |
| HNC-1 | Ripening stage | 69.68 |
With the big gold dollar of flue-cured tobacco cultivars safflower is material, adopts said method to detect its different developmental phases (group's phase, prosperous long-term, bottom ripening stage, middle part ripening stage and top ripening stage) nicotine and transforms level relatively, and concrete steps are following:
(1) tobacco leaf sampling:
With the big gold dollar of safflower is material; Choose each 2g of blade in its group's phase, prosperous long-term, bottom ripening stage, middle part ripening stage and top ripening stage respectively; The bright leaf sample of getting is wrapped and mark with masking foil; Put into the ice chest that liquid nitrogen is housed rapidly and preserve, be sent to the laboratory fast and carry out further work.
(2) total RNA extraction, assay and reverse transcription reaction
Total RNA extracts: get each the developmental stage tobacco sample 200mg of the big gold dollar of safflower in the above-mentioned masking foil, adopt the RNeasy Plant Mini kit of Qiagen company to extract its total RNA, concrete steps are following:
It is levigate that tobacco leaf is added liquid nitrogen, changes the Eppendorf pipe before the liquid nitrogen volatilization over to and add 450 μ L RLT to extract damping fluid, and the back 56 ℃ of temperature of vibrating are bathed 3min; Change extracting solution over to the purple post, the centrifugal 2min of 10000g, filtrating changes new Eppendorf pipe over to, adds 225 μ L absolute ethyl alcohol mixings; Filtrating changes pink post again over to, and the centrifugal 15s of 8000g abandons filtrating; Add 700 μ L RW1 in pink post, the centrifugal 15s of 8000g abandons filtrating; Add 500 μ L RPE in pink post, the centrifugal 15s of 8000g abandons filtrating, repeats once the centrifugal 2min of blank pipe; Pink post is inserted on the new Eppendorf pipe, and adding 30 μ L does not have RNase water, the centrifugal 1min of 10000g; Extract good total RNA sample and put-80 ℃ of preservations.
Total rna content is measured: total RNA reads the content (table 5) of RNA with 60 times of RNase-free treating water dilutions with the built-in RNA mensuration program of nucleic acid-protein determinator.
The big gold dollar sample of table 5 safflower rna content
| Kind | Period | Rna content (μ g/mL) |
| The big gold dollar of safflower | Group's phase | 664.1 |
| The big gold dollar of safflower | Prosperous long-term | 482.4 |
| The big gold dollar of safflower | The bottom ripening stage | 312.5 |
| The big gold dollar of safflower | The middle part ripening stage | 201.3 |
| The big gold dollar of safflower | The top ripening stage | 140.4 |
Reverse transcription reaction: the RNA concentration of 5 samples all being diluted to 100 μ g/mL, adopting the AMV ThermoScript II of Takara company then, is cDNA with the mRNA reverse transcription among the total RNA of sample, and subsequent use after diluting 10 times, concrete steps are following:
Reaction system is 20 μ L, wherein: the total RNA2 μ of 100 μ g/mL L, 50pmol/ μ L Oligo (dT) 18primer 1 μ L, 10mM dNTP Mixture 2.5 μ L, RNase Inhibitor 20U, AMV ThermoScript II 10U, 5 * AMV Buffer, 4 μ L, DEPC H
2O is settled to 20 μ L.Room temperature held 10min moves in 42 ℃ of thermostatic baths and is incubated 1.5h, and cooling 2min gets final product in frozen water.With subsequent use after 10 times of the cDNA that the obtains dilutions.
(3) real-time fluorescence quantitative PCR detects
CDNA with after the testing sample dilution is a template, carries out its nicotine of real-time fluorescence quantitative PCR detection and transforms level relatively.Reaction system and program are with embodiment 1 step (4)-2).Write down the Ct value of each sample, through its corresponding template amount of typical curve calculating of recombinant plasmid standard substance, and then relatively its nicotine transforms level relatively, and test-results is as shown in table 6, and Ct value and template amount are multiple MV 3 times.
The big gold dollar sample of table 6 safflower Ct value and corresponding templates amount
| Kind | Period | The Ct value | The template amount (copy/mL) |
| The big gold dollar of safflower | Group's phase | 34.20 | 1.47×10 5 |
| The big gold dollar of safflower | Prosperous long-term | 34.37 | 1.35×10 5 |
| The big gold dollar of safflower | The bottom ripening stage | 33.91 | 1.70×10 5 |
| The big gold dollar of safflower | The middle part ripening stage | 32.94 | 2.83×10 5 |
| The big gold dollar of safflower | The top ripening stage | 32.77 | 3.09×10 5 |
(4) traditional detection method checking
In order to verify the accuracy of the inventive method, the inventor adopts traditional detection method to verify to these lot sample article simultaneously, to its alkaloid of each sample determination, and then calculates its nicotine level of conversion.Analytical procedure is with reference to tobacco industry standard YC/T383-2010, the mensuration-GC-MS of tobacco and tobacco product nicotine, nornicotine, neonicotine, myosmine and neonicotine.Nicotine conversion rate (%)=100 * nornicotine content/(nornicotine content+nicotine content).
Each sample nicotine conversion rate is as shown in table 7.The result shows that the big gold dollar blade of safflower is along with the developmental stage nicotine conversion rate increases gradually, and the ripening stage reaches the highest to top, and this expression trend with nicotine demethylase gene C YP82E4 is consistent.This shows that method provided by the present invention has the accuracy of height, fits like a glove with practical situation.
Table 7 burley tobaccos sample nicotine conversion rate
| Kind | Period | Nicotine conversion rate (%) |
| The big gold dollar of safflower | Group's phase | 0.64 |
| The big gold dollar of safflower | Prosperous long-term | 0.60 |
| The big gold dollar of safflower | The bottom ripening stage | 0.71 |
| The big gold dollar of safflower | The middle part ripening stage | 1.06 |
| The big gold dollar of safflower | The top ripening stage | 1.46 |
Is material with cigarette in the flue-cured tobacco cultivars 100 with K326, utilizes said method to detect its ripening stage blade nicotine and transforms level relatively, and concrete steps are following:
(1) tobacco leaf sampling:
Is material with middle cigarette 100 with K326, chooses each 2g of blade in its ripening stage respectively, and the bright leaf sample of getting is wrapped and mark with masking foil, puts into the ice chest that liquid nitrogen is housed rapidly and preserves, and is sent to the laboratory fast and carries out further work.
(2) total RNA extraction, assay and reverse transcription reaction
Total RNA extracts: get middle cigarette 100 and K326 tobacco sample 200mg in the above-mentioned masking foil, adopt the RNeasy Plant Mini kit of Qiagen company to extract its total RNA, concrete steps are following:
It is levigate that tobacco leaf is added liquid nitrogen, changes the Eppendorf pipe before the liquid nitrogen volatilization over to and add 450 μ L RLT to extract damping fluid, and the back 56 ℃ of temperature of vibrating are bathed 3min; Change extracting solution over to the purple post, the centrifugal 2min of 10000g, filtrating changes new Eppendorf pipe over to, adds 225 μ L absolute ethyl alcohol mixings; Filtrating changes pink post again over to, and the centrifugal 15s of 8000g abandons filtrating; Add 700 μ L RW1 in pink post, the centrifugal 15s of 8000g abandons filtrating; Add 500 μ L RPE in pink post, the centrifugal 15s of 8000g abandons filtrating, repeats once the centrifugal 2min of blank pipe; Pink post is inserted on the new Eppendorf pipe, and adding 30 μ L does not have RNase water, the centrifugal 1min of 10000g; Extract good total RNA sample and put-80 ℃ of preservations.
Total rna content is measured: total RNA reads the content (table 8) of RNA with 60 times of RNase-free treating water dilutions with the built-in RNA mensuration program of nucleic acid-protein determinator.
Table 8 different varieties flue-cured tobacco sample rna content
| Kind | Period | Rna content (μ g/mL) |
| Middle cigarette 100 | Ripening stage | 170.8 |
| ?K326 | Ripening stage | 312.4 |
Reverse transcription reaction: the RNA concentration of 2 samples all being diluted to 100 μ g/mL, adopting the AMV ThermoScript II of Takara company then, is cDNA with the mRNA reverse transcription among the total RNA of sample, and subsequent use after diluting 10 times, concrete steps are following:
Reaction system is 20 μ L, wherein: the total RNA2 μ of 100 μ g/mL L, 50pmol/ μ L Oligo (dT) 18primer 1 μ L, 10mM dNTP Mixture 2.5 μ L, RNase Inhibitor 20U, AMV ThermoScript II 10U, 5 * AMV Buffer, 4 μ L, DEPC H
2O is settled to 20 μ L.Room temperature held 10min moves in 42 ℃ of thermostatic baths and is incubated 1.5h, and cooling 2min gets final product in frozen water.With subsequent use after 10 times of the cDNA that the obtains dilutions.
(3) real-time fluorescence quantitative PCR detects
CDNA with after the testing sample dilution is a template, carries out its nicotine of real-time fluorescence quantitative PCR analysis and transforms level relatively.Reaction system and program are with embodiment 1 step (4)-2).Write down the Ct value of each sample, the typical curve through the recombinant plasmid standard substance calculates that it is corresponding, and then relatively its nicotine transforms level relatively, and test-results is as shown in table 9, and Ct value and template amount are 3 multiple MVs.
Table 9 different varieties flue-cured tobacco sample Ct value and corresponding templates amount
| Kind | Period | The Ct value | The template amount (copy/mL) |
| Middle cigarette 100 | Ripening stage | 30.61 | 9.53×10 5 |
| ?K326 | Ripening stage | 31.95 | 4.74×10 5 |
(4) traditional detection method checking
In order to verify the accuracy of the inventive method, the inventor adopts traditional detection method to verify to these lot sample article simultaneously, to its alkaloid of each sample determination, and then calculates its nicotine level of conversion.Analytical procedure is with reference to tobacco industry standard YC/T383-2010, the mensuration-GC-MS of tobacco and tobacco product nicotine, nornicotine, neonicotine, myosmine and neonicotine.Nicotine conversion rate (%)=100 * nornicotine content/(nornicotine content+nicotine content).
Each sample nicotine conversion rate is as shown in table 10.The result shows that middle cigarette 100 blade nicotine conversion rates are higher than the K326 blade, and are consistent with the expression trend of nicotine demethylase gene C YP82E4.This shows that method provided by the present invention has the accuracy of height, fits like a glove with practical situation.
Table 10 different varieties flue-cured tobacco sample nicotine conversion rate
| Kind | Period | Nicotine conversion rate (%) |
| Middle cigarette 100 | Ripening stage | 3.21 |
| ?K326 | Ripening stage | 2.65 |
Claims (6)
1. one kind is detected tobacco smoke alkaloid and transforms the method for level relatively, it is characterized in that, comprises the steps:
(1) collects the grow tobacco blade of different times or different sites or different varieties tobacco of same article;
(2) extract total RNA of said blade respectively, rt synthesizes cDNA;
(3) transforming the genes involved design specific primers to nicotine, is template with said cDNA, utilizes said Auele Specific Primer to carry out real-time fluorescence quantitative PCR, obtains Ct value separately;
(4) according to said Ct value, utilize typical curve to calculate the template amount that nicotine among each cDNA transforms genes involved, judge that the nicotine that same article grow tobacco between the blade of different times or different sites or different varieties tobacco transforms level relatively;
Said Auele Specific Primer is:
Upstream primer: 5 '-ACGTGATCCTAAACTCTGGTCTG-3 ';
Downstream primer: 5 '-GCCTGCACCTTCCTTCATG-3 ';
It is nicotine demethylase gene C YP82E4 that said nicotine transforms genes involved, and its full-length cDNA nucleotide sequence is shown in SEQ ID NO.1.
2. the method for claim 1 is characterized in that, said real-time fluorescence quantitative PCR adopts the chimeric method of SYBR Green I fluorescence.
3. the method for claim 1; It is characterized in that; The amplification system of said real-time fluorescence quantitative PCR is 20 μ L, wherein: Takara 2 * SYBR Premix EX Taq 10 μ L, each 0.5 μ L of the forward and reverse primer of 10 μ M, cDNA template 2 μ L, sterile purified water 7 μ L.
4. the method for claim 1 is characterized in that, the response procedures of said real-time fluorescence quantitative PCR is: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 50s, 35 circulations; 72 ℃ are extended 5min.
5. the method for claim 1 is characterized in that, the preparation method of said typical curve is:
(1) standard substance of one group of concentration in gradient distribution of preparation;
(2) be template with said standard substance, utilize the described primer of claim 1, carry out real-time fluorescence quantitative PCR, record Ct value;
(3) drafting obtains typical curve;
Said standard substance are the recombinant vectors suspension that contains nicotine demethylase gene C YP82E4 full-length cDNA nucleotide sequence, or contain the genetically modified host cell suspension of said recombinant vectors.
6. method as claimed in claim 5 is characterized in that, the preparation method of said recombinant vectors is:
(1) extract the total RNA of tobacco leaf, and reverse transcription is cDNA;
(2) be template with said cDNA, carry out pcr amplification and obtain the purpose fragment;
(3) said purpose fragment is connected into the T carrier, obtains recombinant vectors.
The employed primer of said pcr amplification is:
Upstream primer is: 5 '-ATGCTTTCTCCCATAGAAGCC-3 ';
Downstream primer is: 5 '-TTAATAAAGCTCAGGTGCCAG-3 '.
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| CN104946781A (en) * | 2015-07-16 | 2015-09-30 | 湖北省烟草科学研究院 | Method for early-stage screening of high-nicotine and low-nicotine tobacco converter plants |
| CN106577771A (en) * | 2016-12-01 | 2017-04-26 | 湖北中烟工业有限责任公司 | Method for preparing inhibitor for reducing tobacco nicotine synthesis by double-stranded RNA interference technology and application thereof |
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| CN113584211A (en) * | 2021-08-09 | 2021-11-02 | 云南省烟草农业科学研究院 | Method for screening cinnabar plants based on CYP82E4 gene expression level |
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| CN103589779A (en) * | 2012-12-11 | 2014-02-19 | 湛江师范学院 | Fluorescent quantification PCR method for determining eucalyptus embryogenic and non-embryogenic calluses |
| CN103589779B (en) * | 2012-12-11 | 2016-08-17 | 湛江师范学院 | A kind of fluorescence quantifying PCR method judging tail alpine ash embryo and non embryogenic callus |
| CN104946781A (en) * | 2015-07-16 | 2015-09-30 | 湖北省烟草科学研究院 | Method for early-stage screening of high-nicotine and low-nicotine tobacco converter plants |
| CN104946781B (en) * | 2015-07-16 | 2018-06-01 | 湖北省烟草科学研究院 | A kind of high method with low nicotine transformant of early screening tobacco |
| CN106577771A (en) * | 2016-12-01 | 2017-04-26 | 湖北中烟工业有限责任公司 | Method for preparing inhibitor for reducing tobacco nicotine synthesis by double-stranded RNA interference technology and application thereof |
| CN110839367A (en) * | 2019-11-20 | 2020-02-28 | 云南省烟草农业科学研究院 | Method for screening cinnabar tobacco seeds based on nicotine quantitative conversion rate and gene expression |
| CN113584211A (en) * | 2021-08-09 | 2021-11-02 | 云南省烟草农业科学研究院 | Method for screening cinnabar plants based on CYP82E4 gene expression level |
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