CN102796821A - Method of detecting relative level of tobacco nicotine conversion - Google Patents

Abstract

The invention discloses a method for detecting the relative level of tobacco nicotine conversion, which comprises: (1) collecting the leaves of the same variety of tobacco at different stages or different parts or different varieties of tobacco; (2) extracting the total RNA of the leaves respectively, reversing Record and synthesize cDNA; (3) design specific primers for genes related to nicotine conversion, use the cDNA as a template, and use the specific primers to perform real-time fluorescent quantitative PCR to obtain respective Ct values; (4) according to the Ct Value, using the standard curve to calculate the template amount of nicotine conversion-related genes in each cDNA, to determine the relative level of nicotine conversion between different stages or different parts of the same variety of tobacco or leaves of different varieties of tobacco. The method disclosed by the invention can not only monitor the relative level of tobacco nicotine conversion in real time, but also has high accuracy and strong sensitivity, and can eliminate bad tobacco transformed strains in time.

Description

A method for detecting the relative level of nicotine conversion in tobacco

technical field

The invention belongs to the technical field of molecular biology, in particular to a method for detecting the relative level of tobacco nicotine conversion.

Background technique

Alkaloids are a class of secondary metabolites composed of small molecule nitrogen-containing compounds that widely exist in plants. At present, more than 12,000 alkaloids have been isolated and identified in the world. Alkaloids have a special status in tobacco and its products. It is not only an important quality factor of tobacco, but also defines the characteristics of tobacco as a commodity. Nicotine, nornicotine, anatabine, and anatabine are the four main alkaloids in tobacco. People smoke tobacco mainly to inhale nicotine (nicotine), also known as nicotine. Ordinary tobacco belongs to the nicotine accumulation type, and the nicotine content accounts for 90-95% of the total alkaloid content, and the other three types only account for 5%-10% of the total alkaloid content, and the nornicotine content generally does not exceed 50% of the total alkaloid content. 3.5%.

Common tobacco is a homozygous double recessive genotype (ctctcscs), which does not have the ability to demethylate nicotine. However, in the population of cultivated tobacco plants, some plants have the ability to demethylate nicotine due to genetic mutations, resulting in a significant decrease in nicotine content, and a corresponding increase in nornicotine content, which can even reach more than 95%. The trait of converting nicotine to nornicotine is called nicotine conversion, and the tobacco strain with the ability to convert nicotine is called a converter.

The increase of nornicotine content will directly affect the quality of flavor and taste of tobacco leaves, resulting in the phenomenon of "sakura red" and poor flavor of flue-cured tobacco. At the same time, because nornicotine is prone to biochemical reactions such as oxidation, nitrosation, and acylation during tobacco leaf modulation and aging after modulation, many harmful components are formed, such as myosmine and acylated nornicotine (acylnornicotines). And nitroso nornicotine (NNN), etc., so the increase of nornicotine content also has adverse effects on human health.

Controlling lower levels of nornicotine is not only because of its potential carcinogenicity to the human body as a precursor of many harmful components, but also because nornicotine itself has negative effects on human health. Nornicotine can lead to abnormal glycosylation of proteins, and can also covalently react with many commonly used steroid drugs (such as prednisone), thereby changing the efficacy and toxicity of these drugs.

Therefore, timely elimination of transformed strains in tobacco populations to control lower levels of nornicotine content has become one of the hot issues of international tobacco industry and academic circles.

The current method for detecting the nicotine conversion level of tobacco is mainly to detect its alkaloid components and content by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS), and then calculate the nicotine conversion level. Although the method has good stability and operability, it can usually only analyze mature tobacco leaves or processed tobacco leaves after harvesting, and requires certain treatment after harvesting, which is time-consuming and cannot be used throughout the growth period. Dynamically understand the nicotine conversion levels of different varieties of tobacco leaves and different populations of the same variety, so that it is impossible to eliminate bad transformed strains in a timely manner.

Contents of the invention

The invention provides a method for detecting the relative level of nicotine transformation in tobacco, which can be used to simply and quickly detect the relative level of nicotine transformation in different stages or different parts of the same variety of tobacco or between leaves of different kinds of tobacco.

A method for detecting the relative level of tobacco nicotine conversion, comprising the steps of:

(1) Collect leaves of the same variety of tobacco at different periods or different parts or different varieties of tobacco;

(2) extracting the total RNA of the leaves respectively, reverse transcription and synthesizing cDNA;

(3) Design specific primers for genes related to nicotine conversion, use the cDNA as a template, and use the specific primers to perform real-time fluorescent quantitative PCR to obtain respective Ct values;

(4) According to the Ct value, use the standard curve to calculate the template amount of nicotine conversion-related genes in each cDNA, and determine the relative level of nicotine conversion between the leaves of the same variety of tobacco in different periods or different parts or different varieties of tobacco;

The specific primers are:

Upstream primer: 5'-ACGTGATCCTAAACTCTGGTCTG-3';

Downstream primer: 5'-GCCTGCACCTTCCTTCATG-3';

The nicotine conversion-related gene is the nicotine demethylase gene CYP82E4, and its full-length cDNA nucleotide sequence is shown in SEQ ID NO.1.

When detecting the relative levels of nicotine transformation in different parts of the same tobacco variety or leaves of different tobacco varieties, mature leaves are generally selected.

The nicotine demethylase gene CYP82E4 is a member of the CYP82E2 gene family and mediates the conversion of nicotine to nornicotine. The transcription level of this gene family, especially the nicotine demethylase gene CYP82E4, in tobacco transformed strains is generally higher than that of non-transformed strains. Using RNAi interference technology to inhibit the expression of this gene can effectively reduce the high nornicotine content in tobacco. The proportion of transformants. Therefore, the gene design method can accurately and sensitively detect the relative level of tobacco nicotine conversion.

What the real-time fluorescent quantitative PCR of the present invention amplifies is not the complete sequence of nicotine conversion-related genes, but only a certain fragment thereof, and its base sequence is shown in SEQ ID No.4. During the reverse transcription process, the volume and concentration of RNA taken by each group are the same, and the expression level of the gene can be obtained by calculating the template amount (concentration) of the gene related to nicotine conversion.

The real-time fluorescence quantitative PCR adopts SYBR Green I fluorescence mosaic method.

The amplification system of the real-time fluorescent quantitative PCR is 20 μL, including: Takara 2×SYBR Premix EX Taq 10 μL, 10 μM forward and reverse primers 0.5 μL each, cDNA template 2 μL, sterilized distilled water 7 μL.

The reaction program of the real-time fluorescent quantitative PCR is: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 50s, 35 cycles; extension at 72°C for 5 minutes.

The preparation method of described standard curve is:

(1) Prepare a group of standard substances with gradient distribution;

(2) Using the standard as a template, using the primers, perform real-time fluorescent quantitative PCR, and record the Ct value;

(3) Draw a standard curve.

The standard product is a recombinant vector suspension containing the full-length cDNA nucleotide sequence of the nicotine demethylase gene CYP82E4, or a transgenic host cell suspension containing the recombinant vector.

The preparation method of described standard substance is:

(1) Extract tobacco leaf total RNA and reverse transcribe it into cDNA;

(2) Using the cDNA as a template, perform PCR amplification to obtain the target fragment;

(3) Link the target fragment into a T vector to obtain a recombinant vector.

The primers used in the PCR amplification are:

The upstream primer is: 5'-ATGCTTTCTCCCATAGAAGCC-3';

The downstream primer is: 5'-TTAATAAAGCTCAGGTGCCAG-3'.

The quality of the total RNA is crucial, and high-quality kits or reagents should be used for extraction, such as Qiagen RNeasy Plant Mini kit.

Since RNA is easily degraded and impurities are difficult to remove, the purity of the extracted RNA should be determined. The OD ₂₆₀ /OD ₂₈₀ ratio of pure RNA is 2.0. If the ratio is low, it indicates that there is protein or phenol contamination; if the ratio is too high, it indicates that the RNA has been degraded to varying degrees. Generally, RNA with an OD ₂₆₀ /OD ₂₈₀ ratio of 1.8-2.0 is available.

PCR amplification of the full-length cDNA of the nicotine demethylase gene CYP82E4 is used to construct a recombinant vector; and real-time fluorescent quantitative PCR is to detect the expression level of the gene, so full-sequence amplification is not required during amplification, but The selected primers should have better specificity.

The present invention adopts the SYBR Green I fluorescence mosaic method to carry out real-time fluorescent quantitative PCR, which is based on the following advantages: no selectivity to DNA templates, applicable to any DNA; no need to design complex probes, easy to use; very sensitive; low price.

However, it is precisely because SYBR Green I has no selectivity for DNA templates that it is easy to combine with non-specific double-stranded DNA, resulting in false positives. Therefore, after the real-time fluorescent quantitative PCR reaction is completed, a melting curve analysis is required to determine whether the amplified product is the target fragment. If the curve has only a single peak, and the position of the peak is the annealing temperature, there will be no non-specific fluorescence, and the quantification will be accurate; if there are miscellaneous peaks or the position of the peak is not the annealing temperature, there will be non-specific fluorescence of other products, and the quantification will be inaccurate.

In real-time fluorescent quantitative PCR, log x ₀ (template amount) has a linear relationship with the Ct value, and a standard curve can be made through the standard substance of the known template amount, and the corresponding template amount can be calculated according to the Ct value of each sample, and then Each sample was analyzed for relative levels of nicotine conversion.

Compared with prior art, the beneficial effect of the present invention is:

(1) The SYBR Green I fluorescence mosaic method adopted by the present invention to detect the relative level of tobacco nicotine conversion has high accuracy and strong sensitivity; The relative level of nicotine conversion of the leaves of the same variety of tobacco at different stages or different parts or different varieties of tobacco has a greater advantage.

(2) The method of the present invention has less sampling amount, compared with the existing detection methods (GC method and GC-MS method), it will not cause obvious damage to the sampling tobacco strain, and it is easy to operate, accurate and reproducible good.

(3) Existing detection methods can only analyze the nicotine conversion level after the tobacco leaves are harvested, but the present invention can monitor the relative levels of nicotine conversion of different tobacco leaf samples in real time throughout the growth period, avoiding the existing The time limit of the detection method has opened up a new and effective way for researchers and growers to eliminate bad transformed strains in time.

Description of drawings

Fig. 1 is the gel electrophoresis detection result of the full-length routine PCR of the nicotine demethylase gene CYP82E4 cDNA. Among them, M is Takara DL2000 DNA Marker, and 1 is the full-length amplification product of nicotine demethylase gene CYP82E4 cDNA.

Fig. 2 is the gel electrophoresis detection result of conventional PCR verification of recombinant plasmid standard construction. Among them, M is Takara DL2000 DNA Marker, and 1-6 are target fragments in different positive clones.

Fig. 3 is the amplification curve of the real-time fluorescent quantitative PCR of the recombinant plasmid standard product with different dilution gradients.

Fig. 4 is the standard curve of real-time fluorescent quantitative PCR of recombinant plasmid standard products with different dilution gradients. Wherein, the abscissa represents the logarithmic value of the number of templates, and the ordinate represents the Ct value.

Fig. 5A is the melting curve of the real-time fluorescence quantitative PCR of the recombinant plasmid standard product with different dilution gradients.

Fig. 5B shows the melting peaks of real-time fluorescence quantitative PCR of recombinant plasmid standards with different dilution gradients.

Fig. 6 is the gel electrophoresis detection result of the routine PCR of the recombinant plasmid standard product of different dilution gradients. Among them, M is Takara 100bp DNA Marker, 1-7 are 1×10 ⁵ , 1×10 ⁶ , 1×10 ⁷ , 1×10 ⁸ , 1×10 ⁹ , 1×10 ¹⁰ , 1×10 ¹¹ copies/ mL of recombinant plasmid standard.

Detailed ways

Example 1 Preparation of Standards for Detecting Relative Levels of Tobacco Nicotine Conversion

The following only takes the recombinant plasmid containing the full-length cDNA of the nicotine demethylase gene CYP82E4 as an example to illustrate its effect on detecting the relative level of tobacco nicotine conversion. All other plasmids containing the full-length cDNA of the nicotine demethylase gene CYP82E4 Or the recombinant vector of partial nucleotide sequence or the transgenic host cell containing the recombinant vector has the same effect on detecting the relative level of tobacco nicotine conversion as the standard product.

(1) Sampling of tobacco leaves

Using Burley tobacco B37 as the material, select 2g of the middle leaves at the mature stage, wrap the fresh leaf samples with tinfoil paper and mark them, put them in an ice box filled with liquid nitrogen for storage, and send them to the laboratory for further analysis. work in one step.

(2) Total RNA extraction, content determination and reverse transcription reaction

Extraction of total RNA: Take 200 mg of the tobacco leaf sample in the above-mentioned tin foil paper, and use the RNeasy Plant Mini kit of Qiagen Company to extract its total RNA. The specific steps are as follows:

Grind the tobacco leaves with liquid nitrogen, transfer to an Eppendorf tube before the liquid nitrogen volatilizes and add 450 μL of RLT extraction buffer, shake and incubate at 56°C for 3 minutes; transfer the extract to a purple column, centrifuge at 10,000g for 2 minutes, and transfer the filtrate to a new Eppendorf tube , add 225μL absolute ethanol and mix well; transfer the filtrate to the pink column, centrifuge at 8000g for 15s, discard the filtrate; add 700μL RW1 to the pink column, centrifuge at 8000g for 15s, discard the filtrate; add 500μL RPE to the pink column, centrifuge at 8000g for 15s , discard the filtrate, repeat once, and centrifuge the empty tube for 2 min; insert the pink column into a new Eppendorf tube, add 30 μL of RNase-free water, and centrifuge at 10,000 g for 1 min; store the extracted RNA sample at -80 °C.

Determination of total RNA content: Use the built-in RNA determination program of the nucleic acid protein analyzer to read the RNA content as 345.8 μg/mL, and the OD ₂₆₀ /OD ₂₈₀ value is 1.933, indicating that the purity of the total RNA is good, and it is ready for use.

Reverse transcription reaction: The mRNA in the total RNA was reverse-transcribed into cDNA using AMV reverse transcriptase from Takara Company, and diluted 10 times for later use. Specific steps are as follows:

The reaction system is 20 μL, including: 2 μL of total RNA, 1 μL of 50 pmol/μL Oligo(dT)18 primer, 2.5 μL of 10 mM dNTP Mixture, 20 U of RNase Inhibitor, 10 U of AMV reverse transcriptase, 4 μL of 5×AMV Buffer, and constant volume of DEPC H ₂ O to 20 μL. Leave it at room temperature for 10 minutes, move it into a 42°C constant temperature bath for 1.5 hours, and cool it in ice water for 2 minutes. The obtained cDNA was diluted 10 times before use.

(3) Preparation of standard products

Using the above cDNA as a template, the following primers were used for conventional PCR amplification of the full-length cDNA sequence of the nicotine demethylase gene CYP82E4, and the primers were:

Forward primer sequence: 5'-ATGCTTTCTCCCATAGAAGCC-3';

Reverse primer sequence: 5'-TTAATAAAGCTCAGGTGCCAG-3'.

The concrete steps of described routine PCR amplification are:

1) Cloning and purification of the target gene

The PCR reaction system is 50 μL, including: 5U/μL Takara LATaq 0.5 μL, 2.5mMdNTP 8 μL, LA Taq Buffer (Mg ²⁺ Free) 5 μL, 25mM MgCl ₂ 5 μL, 20 μM forward and reverse primers 1 μL, cDNA template 1 μL, sterilized 28.5 μL of distilled water.

The PCR reaction program was: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 90 s, 30 cycles; extension at 72°C for 7 min.

5 μL of PCR products were taken for 1% agarose gel electrophoresis, and the electrophoresis results are shown in Figure 1. The result showed that the size of the amplified product was about 1600bp, indicating that the amplified product was the target fragment. The remaining 45 μL PCR product was recovered by 1% agarose gel electrophoresis, and the target fragment was recovered and purified using the Promega Gel Recovery Kit (No. A9281). After recovery and purification, 5 μL was taken for 1% agarose gel electrophoresis. The results showed that the recovery and purification effect was better, and it could be used for T-A or other double-digested clones.

2) T-A cloning technology

a. Reaction: Ligate the recovered and purified target fragment with the Takara pMD18-T simple Vector carrier. The connection system is 10 μL, including: 2 μL of the target fragment, 1 μL of the pMD18-T simple Vector carrier, 5 μL of high-efficiency linking solution Solution I, and 2 μL of dH ₂ O ; Ligate overnight at 4°C.

b. Transformation of competent cells: Add 10 μL of the ligation product to 100 μL DH5α competent cells, and place on ice for 30 minutes; heat at 42°C for 45 seconds, and then place in ice for 1 minute; add 890 μL of LB medium, shake at 37°C for 60 minutes; Culture overnight on LB medium containing X-Gal, IPTG and Amp to form a single colony.

3) Identification of positive clones

a. Verification of positive clones: Pick 16 white single colonies from the plated culture medium after transformation and transfer them to test tubes containing 5mL LB (Amp+) liquid medium, and shake overnight at 37°C. Take 1 μL of bacterial liquid as a template, and use the PCR system and program in step 1) to carry out PCR amplification to verify whether the target band of about 1600 bp has been transferred into the plasmid. The verification result is shown in Figure 2. The bacterium solution that was proved to be a positive clone by PCR was sent for sequencing, and the sequencing result was 1554bp, and the blast comparison result proved that it was a positive recombinant plasmid, and the recombinant plasmid and the nicotine demethylase gene CYP82E4 mRNA sequence in GenBank (GenBank accession number : DQ205656.1; DQ131885.1; DQ131886.1) have more than 99% homology, indicating that the plasmid standard was successfully constructed. The nucleotide sequence carried in the recombinant plasmid is shown in SEQ ID NO.1.

b. Plasmid extraction: Select the bacterial solution containing the target fragment, and use the improved SDS alkaline lysis method AxyPrep plasmid DNA kit from Axygen Company to extract plasmid DNA. The specific process is as follows:

Take 3mL of bacterial solution, centrifuge at 12000g for 1min, discard the supernatant; add 250μL S1 buffer to resuspend the bacterial pellet; add 250μL S2 buffer, mix gently and thoroughly to fully lyse the bacteria; add 350μL S3 buffer, mix gently Evenly, centrifuge at 12000g for 10min; transfer the supernatant to the preparation tube, centrifuge at 12000g for 1min, discard the filtrate; add 500μL W1 buffer, centrifuge at 12000g for 1min, discard the filtrate; add 700μL W2 buffer, centrifuge at 12000g for 1min, discard the filtrate; Add 80 μL of deionized water to the tube, centrifuge at 12,000 g for 1 min; store at -20°C for later use.

(4) Recombinant plasmid standard detection

1) Concentration determination and copy number calculation of recombinant plasmid

Take 2 μL of the purified plasmid standard, and use a nucleic acid protein analyzer to measure its concentration to be 94.5 μg/mL, and the ratio of OD ₂₆₀ /OD ₂₈₀ is 1.888, indicating that the purity of the plasmid standard is relatively good. The copy number can be calculated according to the concentration of the plasmid standard, and the calculation formula is as follows:

DNA copy number = (DNA mass/DNA molar mass) × 6.02 × 10 ²³

1bp in double-stranded DNA=660Da, then the molar mass of DNA=660Da×(2692+1554)bp. Substituting the above formula, the copy number of the plasmid standard was obtained as 2.030×10 ¹³ copies/mL.

2) Real-time fluorescence quantitative PCR detects the scope of recombinant plasmid standard products

The recombinant plasmid standard was diluted 10 times, and each dilution gradient was 1×10 ¹¹ , 1×10 ¹⁰ ... 1×10 ¹ copy/mL. Utilize the following specific primers for real-time fluorescent quantitative PCR detection, the nucleotide sequence of the primers is:

The forward primer sequence is: 5'-ACGTGATCCTAAACTCTGGTCTG-3';

The reverse primer sequence is: 5'-GCCTGCACCTTCCTTCATG-3'.

The SYBR Green I fluorescence mosaic method was used for real-time fluorescent quantitative PCR detection. The reaction system was 20 μL, including: Takara 2×SYBR Premix EX Taq 10 μL, 10 μM forward and reverse primers 0.5 μL each, plasmid standard 2 μL, and sterilized distilled water 7 μL.

The real-time fluorescence quantitative PCR reaction program was: 94°C pre-denaturation for 3 minutes; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 50 s, 35 cycles; 72°C extension for 5 min; 55-95°C slow heating to generate a melting curve.

The amplification curve is shown in Figure 3, and it can be seen from the figure that the real-time fluorescent quantitative PCR detection range of the recombinant plasmid standard is 1×10 ⁵ -1×10 ¹¹ copies/mL.

3) Drawing of real-time fluorescent quantitative PCR detection standard curve

Take known 7 times diluted recombinant plasmid standard (1×10 ¹¹ , 1×10 ¹⁰ , 1×10 ⁹ , 1×10 ⁸ , 1×10 ⁷ , 1×10 ⁶ , 1×10 ⁵ copies /mL) to draw the standard curve for real-time fluorescent quantitative PCR detection. Each dilution gradient was repeated 3 times to perform a real-time fluorescent quantitative PCR reaction. The reaction system and procedures were the same as those in steps (4)-2) of Example 1. The standard curve is shown in Figure 4. The plasmid standard product is in the range of 1×10 ⁵ -1×10 ¹⁰ copies/mL. R ² = 0.9937, indicating that the system has good linearity; the slope is -4.4208, indicating the amplification efficiency of the system higher. The results in the figure are the average of 3 repeated experiments.

4) Real-time fluorescence quantitative PCR detects the melting curve of the standard curve

The method for determining the melting curve of the recombinant plasmid standard is the same as step (4)-2). The measurement results are shown in Figures 5A and 5B, the Tm value of the specific reaction was 81.5±0.5°C, and the curve was a single peak, indicating that there was no non-specific fluorescence and the quantification was accurate.

5) The scope of routine PCR detection of recombinant plasmid standards

Routine PCR detection was performed on the recombinant plasmid standard with a dilution gradient of 1×10 ⁵ -1×10 ¹¹ copies/mL.

The conventional PCR reaction system is 50 μL, of which, 5U/μL Takara LA Taq 0.5 μL, 2.5 mM dNTP 8 μL, LA Taq Buffer (Mg ²⁺ Free) 5 μL, 25 mM MgCl ₂ 5 μL, 20 μM forward and reverse primers 1 μL each, recombinant plasmid standard product 1 μL, sterilized distilled water 28.5 μL.

The routine PCR reaction program is: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 90 s, 30 cycles; 72°C extension for 7 min.

Take 5 μL of PCR product for 1% agarose gel electrophoresis. The electrophoresis result is shown in Figure 6. The size of the amplified product is between 200-300bp, and the sequencing result is 238bp. After comparison by blast, it is proved that it is a recombinant plasmid standard item Part of the nucleotide fragment in the fragment, the sequence of the nucleotide fragment is shown in SEQ ID NO.4.

Example 2 Detecting the Relative Levels of Burley Tobacco Wild Type and Mutant Nicotine Conversion

Using Burley tobacco B37 and the high nicotine conversion mutant HNC-1 as materials, the primers were used to detect the relative level of nicotine conversion, and the specific steps were as follows:

(1) Sampling of tobacco leaves

Using Burley tobacco B37 and the high-nicotine conversion mutant HNC-1 as materials, select 2 g of the middle leaves of the cluster stage, flourishing stage, and mature stage, respectively, and wrap the fresh leaf samples with tin foil and mark them. Quickly put it in an ice box filled with liquid nitrogen for storage, and quickly send it to the laboratory for the next step.

(2) Total RNA extraction, content determination and reverse transcription reaction

Extraction of total RNA: Take 200 mg of the tobacco leaf sample in the above-mentioned tin foil paper, and use the RNeasy Plant Mini kit of Qiagen Company to extract its total RNA. The specific steps are as follows:

Grind the tobacco leaves with liquid nitrogen, transfer to an Eppendorf tube before the liquid nitrogen volatilizes and add 450 μL of RLT extraction buffer, shake and incubate at 56°C for 3 minutes; transfer the extract to a purple column, centrifuge at 10,000g for 2 minutes, and transfer the filtrate to a new Eppendorf tube. Add 225μL absolute ethanol and mix well; transfer the filtrate to the pink column, centrifuge at 8000g for 15s, discard the filtrate; add 700μL RW1 to the pink column, centrifuge at 8000g for 15s, discard the filtrate; add 500μL RPE to the pink column, centrifuge at 8000g for 15s, Discard the filtrate, repeat once, and centrifuge the empty tube for 2 min; insert the pink column into a new Eppendorf tube, add 30 μL of RNase-free water, and centrifuge at 10,000 g for 1 min; store the extracted total RNA sample at -80 °C.

Determination of total RNA content: the total RNA was diluted 60 times with RNase-free treated water, and the RNA content was read with the built-in RNA determination program of the nucleic acid protein analyzer (Table 2).

Table 2 RNA content of Burley tobacco samples

Variety period RNA content (μg/mL) B37 Reunion period 801.6 B37 Long term 294.4 B37 Maturity 340.0 HNC-1 Reunion period 372.0 HNC-1 Long term 153.6 HNC-1 Maturity 200.8

Reverse transcription reaction: Dilute the RNA concentration of the 6 samples to 100 μg/mL, use AMV reverse transcriptase from Takara Company, reverse-transcribe the mRNA in the total RNA of the sample into cDNA, dilute it by 10 times and prepare it for later use, specific steps as follows:

The reaction system is 20μL, including: 100μg/mL total RNA 2μL, 50pmol/μL Oligo(dT)18primer 1μL, 10mM dNTP Mixture 2.5μL, RNase Inhibitor 20U, AMV reverse transcriptase 10U, 5×AMV Buffer 4μL, DEPC H ₂ O Dilute to 20 μL. Leave it at room temperature for 10 minutes, move it into a 42°C constant temperature bath for 1.5 hours, and cool it in ice water for 2 minutes. The obtained cDNA was diluted 10 times before use.

(3) Real-time fluorescent quantitative PCR detection

Using the diluted cDNA of the sample to be tested as a template, the relative level of nicotine conversion was analyzed by real-time fluorescent quantitative PCR. The reaction system and procedure are the same as those in Step (4)-2) of Example 1. Record the Ct value of each sample, calculate the corresponding template amount through the standard curve of the recombinant plasmid standard, and then compare the relative level of nicotine conversion of each sample. The test results are shown in Table 3. The Ct value and template amount are repeated three times. average of.

Table 3 Burley tobacco sample Ct value and corresponding template amount

Variety period Ct value Amount of template (copy/mL) B37 Reunion period 30.91 8.15×10 ⁵ B37 Long term 31.44 6.18×10 ⁵ B37 Maturity 29.88 1.40×10 ⁶ HNC-1 Reunion period 31.14 7.25×10 ⁵

HNC-1 Long term 28.40 3.01×10 ⁶ HNC-1 Maturity 23.90 3.13×10 ⁷

(4) Validation of traditional detection methods

In order to verify the accuracy of the method of the present invention, the inventors also used traditional detection methods to verify the batch of samples, measured the alkaloid content of each sample, and then detected the nicotine conversion level. The analysis method refers to the tobacco industry standard YC/T383-2010, Determination of nicotine, nornicotine, anonicotine, mesmin and pseudobasine in tobacco and tobacco products-gas chromatography-mass spectrometry. Nicotine conversion rate (%)=100×nornicotine content/(nornicotine content+nicotine content).

The nicotine conversion rate of each sample is shown in Table 4. The results showed that the high nicotine transformant strain HNC-1 had a higher level of nicotine conversion, especially in mature leaves, which was significantly higher than that of wild-type B37, which was consistent with the expression trend of nicotine demethylase gene CYP82E4. It can be seen that the method provided by the present invention has a high degree of accuracy, which is completely consistent with the actual situation.

Table 4 Nicotine conversion rate of Burley tobacco samples

Variety period Nicotine conversion rate (%) B37 Reunion period 2.79 B37 Long term 2.19 B37 maturity 3.36 HNC-1 Reunion period 2.35 HNC-1 Long term 11.58 HNC-1 maturity 69.68

Example 3 Detection of relative levels of nicotine conversion in flue-cured tobacco at different developmental stages

Using the flue-cured tobacco variety Honghua Dajinyuan as the material, the method was used to detect the relative levels of nicotine conversion in different developmental stages (group tree stage, flourishing stage, lower mature stage, middle mature stage and upper mature stage). The specific steps are as follows:

(1) Sampling of tobacco leaves:

Using Dajinyuan safflower as the material, select 2g each of the leaves of the group tree stage, the flourishing stage, the lower mature stage, the middle mature stage and the upper mature stage. Store in an ice box filled with liquid nitrogen and quickly send to the laboratory for the next step.

(2) Total RNA extraction, content determination and reverse transcription reaction

Extraction of total RNA: Take 200 mg of the tobacco leaf samples of Honghua Dajinyuan in each developmental stage in the above-mentioned tinfoil, and use the RNeasy Plant Mini kit of Qiagen Company to extract its total RNA. The specific steps are as follows:

Grind the tobacco leaves with liquid nitrogen, transfer to an Eppendorf tube before the liquid nitrogen volatilizes and add 450 μL of RLT extraction buffer, shake and incubate at 56°C for 3 minutes; transfer the extract to a purple column, centrifuge at 10,000g for 2 minutes, and transfer the filtrate to a new Eppendorf tube. Add 225μL absolute ethanol and mix well; transfer the filtrate to the pink column, centrifuge at 8000g for 15s, discard the filtrate; add 700μL RW1 to the pink column, centrifuge at 8000g for 15s, discard the filtrate; add 500μL RPE to the pink column, centrifuge at 8000g for 15s, Discard the filtrate, repeat once, and centrifuge the empty tube for 2 min; insert the pink column into a new Eppendorf tube, add 30 μL of RNase-free water, and centrifuge at 10,000 g for 1 min; store the extracted total RNA sample at -80 °C.

Determination of total RNA content: the total RNA was diluted 60 times with RNase-free treated water, and the RNA content was read with the built-in RNA determination program of the nucleic acid protein analyzer (Table 5).

Table 5 RNA content of samples of Honghua Dajinyuan

Variety period RNA content (μg/mL) safflower gold dollar Reunion period 664.1 safflower gold dollar Long term 482.4 safflower gold dollar Lower mature stage 312.5 safflower gold dollar Middle mature stage 201.3 safflower gold dollar Upper mature stage 140.4

Reverse transcription reaction: Dilute the RNA concentration of 5 samples to 100 μg/mL, then use Takara’s AMV reverse transcriptase to reverse-transcribe the mRNA in the total RNA of the sample into cDNA, dilute it by 10 times and prepare it for later use. Proceed as follows:

The reaction system is 20μL, including: 100μg/mL total RNA 2μL, 50pmol/μL Oligo(dT)18primer 1μL, 10mM dNTP Mixture 2.5μL, RNase Inhibitor 20U, AMV reverse transcriptase 10U, 5×AMV Buffer 4μL, DEPC H ₂ O Dilute to 20 μL. Leave it at room temperature for 10 minutes, move it into a 42°C constant temperature bath for 1.5 hours, and cool it in ice water for 2 minutes. The obtained cDNA was diluted 10 times before use.

(3) Real-time fluorescent quantitative PCR detection

Using the diluted cDNA of the sample to be tested as a template, real-time fluorescent quantitative PCR was used to detect the relative level of nicotine conversion. The reaction system and procedures are the same as those in Step (4)-2) of Example 1. Record the Ct value of each sample, calculate the corresponding template amount through the standard curve of the recombinant plasmid standard, and then compare the relative level of nicotine conversion. The test results are shown in Table 6. The Ct value and template amount are repeated three times. average value.

Table 6 Ct value and corresponding template amount of Honghua Dajinyuan sample

Variety period Ct value Amount of template (copy/mL) safflower gold dollar Reunion period 34.20 1.47×10 ⁵ safflower gold dollar Long term 34.37 1.35×10 ⁵ safflower gold dollar Lower mature stage 33.91 1.70×10 ⁵ safflower gold dollar Middle mature stage 32.94 2.83×10 ⁵ safflower gold dollar Upper mature stage 32.77 3.09×10 ⁵

(4) Validation of traditional detection methods

In order to verify the accuracy of the method of the present invention, the inventors also used traditional detection methods to verify the batch of samples, measured the alkaloid content of each sample, and then calculated the nicotine conversion level. The analysis method refers to the tobacco industry standard YC/T383-2010, Determination of nicotine, nornicotine, anonicotine, mesmin and pseudobasine in tobacco and tobacco products-gas chromatography-mass spectrometry. Nicotine conversion rate (%)=100×nornicotine content/(nornicotine content+nicotine content).

The nicotine conversion rate of each sample is shown in Table 7. The results showed that the nicotine conversion rate of Dajinyuan leaves gradually increased during the developmental period, and reached the highest at the upper mature stage, which was consistent with the expression trend of the nicotine demethylase gene CYP82E4. It can be seen that the method provided by the present invention has a high degree of accuracy, which is completely consistent with the actual situation.

Table 7 Nicotine conversion rate of Burley tobacco samples

Variety period Nicotine conversion rate (%) safflower gold dollar Reunion period 0.64 safflower gold dollar Long term 0.60 safflower gold dollar Lower mature stage 0.71 safflower gold dollar Middle mature stage 1.06 safflower big gold dollar Upper mature stage 1.46

Example 4 Detecting the Relative Levels of Nicotine Conversion of Different Varieties of Flue-cured Tobacco

Using the flue-cured tobacco varieties Zhongyan 100 and K326 as materials, the method was used to detect the relative level of nicotine conversion in the mature leaves of the tobacco, and the specific steps were as follows:

(1) Sampling of tobacco leaves:

Using China Tobacco 100 and K326 as materials, select 2g of mature leaves respectively, wrap and label the fresh leaf samples with tinfoil paper, put them in an ice box filled with liquid nitrogen for storage, and quickly send them to the experiment room for further work.

(2) Total RNA extraction, content determination and reverse transcription reaction

Extraction of total RNA: Take 200 mg of China Tobacco 100 and K326 tobacco leaf samples in the above-mentioned foil paper, and use Qiagen’s RNeasy Plant Mini kit to extract the total RNA. The specific steps are as follows:

Grind the tobacco leaves with liquid nitrogen, transfer to an Eppendorf tube before the liquid nitrogen volatilizes and add 450 μL of RLT extraction buffer, shake and incubate at 56°C for 3 minutes; transfer the extract to a purple column, centrifuge at 10,000g for 2 minutes, and transfer the filtrate to a new Eppendorf tube. Add 225μL absolute ethanol and mix well; transfer the filtrate to the pink column, centrifuge at 8000g for 15s, discard the filtrate; add 700μL RW1 to the pink column, centrifuge at 8000g for 15s, discard the filtrate; add 500μL RPE to the pink column, centrifuge at 8000g for 15s, Discard the filtrate, repeat once, and centrifuge the empty tube for 2 min; insert the pink column into a new Eppendorf tube, add 30 μL of RNase-free water, and centrifuge at 10,000 g for 1 min; store the extracted total RNA sample at -80 °C.

Determination of total RNA content: the total RNA was diluted 60 times with RNase-free treated water, and the RNA content was read with the built-in RNA determination program of the nucleic acid protein analyzer (Table 8).

Table 8 RNA content of flue-cured tobacco samples of different varieties

Variety period RNA content (μg/mL) China Tobacco 100 Maturity 170.8 K326 Maturity 312.4

Reverse transcription reaction: Dilute the RNA concentration of the two samples to 100 μg/mL, then use Takara’s AMV reverse transcriptase to reverse-transcribe the mRNA in the total RNA of the sample into cDNA, dilute it by 10 times and prepare it for later use. Proceed as follows:

The reaction system is 20μL, including: 100μg/mL total RNA 2μL, 50pmol/μL Oligo(dT)18primer 1μL, 10mM dNTP Mixture 2.5μL, RNase Inhibitor 20U, AMV reverse transcriptase 10U, 5×AMV Buffer 4μL, DEPC H ₂ O Dilute to 20 μL. Leave it at room temperature for 10 minutes, move it into a 42°C constant temperature bath for 1.5 hours, and cool it in ice water for 2 minutes. The obtained cDNA was diluted 10 times before use.

(3) Real-time fluorescent quantitative PCR detection

Using the diluted cDNA of the sample to be tested as a template, real-time fluorescent quantitative PCR was used to analyze the relative level of nicotine conversion. The reaction system and procedures are the same as those in Step (4)-2) of Example 1. Record the Ct value of each sample, calculate its corresponding value through the standard curve of the recombinant plasmid standard, and then compare the relative level of nicotine conversion. The test results are shown in Table 9, and the Ct value and template amount are the average values of three repetitions.

Table 9 Ct values and corresponding template amounts of flue-cured tobacco samples of different varieties

Variety period Ct value Amount of template (copy/mL) China Tobacco 100 Maturity 30.61 9.53×10 ⁵ K326 Maturity 31.95 4.74×10 ⁵

(4) Validation of traditional detection methods

In order to verify the accuracy of the method of the present invention, the inventors also used traditional detection methods to verify the batch of samples, measured the alkaloid content of each sample, and then calculated the nicotine conversion level. The analysis method refers to the tobacco industry standard YC/T383-2010, Determination of nicotine, nornicotine, anonicotine, mesmin and pseudobasine in tobacco and tobacco products-gas chromatography-mass spectrometry. Nicotine conversion rate (%)=100×nornicotine content/(nornicotine content+nicotine content).

The nicotine conversion rate of each sample is shown in Table 10. The results showed that the nicotine conversion rate of Zhongyan 100 leaves was higher than that of K326 leaves, which was consistent with the expression trend of nicotine demethylase gene CYP82E4. It can be seen that the method provided by the present invention has a high degree of accuracy, which is completely consistent with the actual situation.

Table 10 Nicotine conversion rate of flue-cured tobacco samples of different varieties

Variety period Nicotine conversion rate (%) China Tobacco 100 Maturity 3.21 K326 Maturity 2.65

Claims (6)

1. A method for detecting the relative level of tobacco nicotine conversion, characterized in that, comprising the steps: (1) Collect leaves of the same variety of tobacco at different periods or different parts or different varieties of tobacco; (2) extracting the total RNA of the leaves respectively, reverse transcription and synthesizing cDNA; (3) Design specific primers for genes related to nicotine conversion, use the cDNA as a template, and use the specific primers to perform real-time fluorescent quantitative PCR to obtain respective Ct values; (4) According to the Ct value, use the standard curve to calculate the template amount of nicotine conversion-related genes in each cDNA, and determine the relative level of nicotine conversion between the leaves of the same variety of tobacco in different periods or different parts or different varieties of tobacco; The specific primers are: Upstream primer: 5'-ACGTGATCCTAAACTCTGGTCTG-3'; Downstream primer: 5'-GCCTGCACCTTCCTTCATG-3'; The nicotine conversion-related gene is the nicotine demethylase gene CYP82E4, and its full-length cDNA nucleotide sequence is shown in SEQ ID NO.1. 2. the method for claim 1, is characterized in that, described real-time fluorescence quantitative PCR adopts SYBR Green I fluorescence mosaic method. 3. The method according to claim 1, wherein the amplification system of the real-time fluorescent quantitative PCR is 20 μL, wherein: 10 μL of Takara 2×SYBR Premix EX Taq, 0.5 μL of 10 μM forward and reverse primers, cDNA template 2 μL, 7 μL of sterilized distilled water. 4. The method according to claim 1, wherein the reaction program of the real-time fluorescent quantitative PCR is: 94°C pre-denaturation for 3 minutes; 94°C denaturation for 30s, 55°C annealing for 30s, 72°C extension for 50s, 35 cycles ; 72°C extension for 5 min. 5. method as claimed in claim 1, is characterized in that, the preparation method of described standard curve is: (1) Prepare a group of standard substances with gradient distribution; (2) take described standard product as template, utilize the primer described in claim 1, carry out real-time fluorescence quantitative PCR, record Ct value; (3) Draw a standard curve; The standard product is a recombinant vector suspension containing the full-length cDNA nucleotide sequence of the nicotine demethylase gene CYP82E4, or a transgenic host cell suspension containing the recombinant vector. 6. the method as claimed in claim 5 is characterized in that, the preparation method of described recombinant vector is: (1) extracting tobacco leaf total RNA, and reverse transcription into cDNA; (2) Using the cDNA as a template, perform PCR amplification to obtain the target fragment; (3) Link the target fragment into a T vector to obtain a recombinant vector. The primers used in the PCR amplification are: The upstream primer is: 5'-ATGCTTTCTCCCATAGAAGCC-3'; The downstream primer is: 5'-TTAATAAAGCTCAGGTGCCAG-3'.

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