CN115852019A - Molecular marker for simply, conveniently and efficiently identifying smoke and non-smoke in cigarette making raw materials and application thereof - Google Patents
Molecular marker for simply, conveniently and efficiently identifying smoke and non-smoke in cigarette making raw materials and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker for simply, conveniently and efficiently identifying smoke and non-smoke in a cigarette making raw material and application thereof, and particularly relates to the technical field of molecular biology. The molecular marker is numbered as Ntsp043, and the primer sequences corresponding to the molecular marker Ntsp043 are respectively shown as SEQ ID No.1 and SEQ ID No. 2. The application is that the tobacco genus specific molecular marker is used for carrying out PCR amplification on the genome DNA of the involved tobacco making raw material, and whether a nucleotide sequence of a PCR amplification product with the length of more than 500bp exists is detected, so that whether tobacco materials exist in the involved tobacco making raw material can be accurately judged. The tobacco specific molecular marker disclosed by the invention realizes rapid and effective identification and detection of tobacco and non-tobacco, improves the scientificity, accuracy and authority of identification and detection work of tobacco-making raw materials involved in a case, reduces judicial risks, and provides powerful judicial evidence for counterfeiting and smuggling in monopoly sales work in the tobacco industry.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a molecular marker for simply, conveniently and efficiently identifying smoke and non-smoke in a cigarette making raw material and application thereof.
Background
Most plants in the solanaceae family, especially plants in the nicotiana genus, contain a specific alkaloid, nicotine (nicotine), and thus nicotine is a significant marker for solanaceae plants to distinguish other plants. At present, the chemical inspection method adopted by the identification and detection of the tobacco-making raw materials involved in the case in the counterfeiting and smudging work of the domestic tobacco industry is based on the principle, namely, whether the sample to be detected is a tobacco speciality or not is judged by determining whether the sample involved in the case contains nicotine or not. However, nicotine is not unique to tobacco, and many solanaceae plants also contain nicotine. In addition, due to the restriction of factors such as the sensitivity of instrument detection, the nicotine content level, the detection limit of a detection method and the like, in most flue-cured tobaccos, the content of N-nitrosamine specific to tobaccos is extremely low (ng/g level), and the condition that the N-nitrosamine specific to tobaccos cannot be detected in a flue-cured tobacco sample often occurs, so that the higher misjudgment risk exists when tobacco products involved in the case are identified and detected according to a chemical detection method for detecting the nicotine and N-nitrosamine content in an object to be detected, and the cases of the same kind of cases of corruption are reported, and certain network public opinions and social public opinions are triggered.
Tobacco is a herbaceous economic crop containing a special alkaloid, namely nicotine (nicotine), in the nicotiana of the solanaceae family, and is also the basis of a tobacco-making raw material (tobacco leaf) in the tobacco industry. With the deep development of Chinese-style cigarettes in China, the basic status of cigarette-making raw materials is increasingly prominent, and although the development of the Chinese-style cigarettes has exclusive-selling safe navigation, under the drive of huge benefits, a large number of cases for counterfeiting and smudging the cigarette-making raw materials (namely, whether tobacco materials are detected in case-related raw materials) still remain endless, so that the health and continuous development of the tobacco industry are restricted, and a new challenge is provided for the existing case-related cigarette-making raw material identification and inspection (pre-punishment without smoke inspection) work.
At present, many research reports are reported on the identification and detection of tobacco resources (varieties) on the molecular level, such as the research works of carrying out genetic diversity analysis, fingerprint map construction and the like on the tobacco resources (varieties) by utilizing markers such as RFLP, RAPD, SSR, ISSR, DArT, SNP and the like. In addition, under the condition that the sample to be detected belongs to the tobacco material, the rapid identification research of the varieties of the flue-cured tobacco leaves by utilizing SSR, SCAR and RAPD markers is also reported in a small quantity. However, no research on the identification and detection of the cigarette-making raw materials involved in the scheme on the molecular level is reported abroad. The domestic tobacco industry mainly adopts an organoleptic inspection method and a chemical inspection method aiming at the identification and inspection work of the tobacco raw materials involved in the case, but the two methods have obvious defects: the sensory inspection method mainly depends on the sensory experience and the inspection experience of inspectors on the cigarette making raw materials involved in the case, is easily influenced by the subjective experience and the experience of the inspectors, and has higher probability of misjudgment; the main principle of the chemical test method is to determine whether a sample to be tested contains nicotine or not so as to determine whether the sample is a tobacco speciality, but nicotine is not unique to tobacco, and many non-tobacco (especially solanaceae) plants can also detect nicotine components, so that the chemical test method for detecting the nicotine content in a substance to be tested has higher misjudgment risk.
Chinese patent CN113549704A (patent number: ZL 2021 0558610.3) is a gene sequence which is obtained by the inventor before and has two tobacco-specific molecular marker numbers of Ntsp027 and Ntsp151, and the two sequences are used for rapidly identifying and detecting whether a tobacco-making raw material involved in a case is a tobacco product on a molecular level. Based on the two markers (namely, 2 pairs of primers contain 4 nucleic acid sequences at the upstream and the downstream) and two nucleotide sequence information obtained by PCR amplification, scientific and accurate identification and detection of the tobacco making raw materials related to the case on the molecular level are realized, but the identification and detection steps are relatively complex and tedious, two times of PCR in-vitro amplification are required to be carried out on a sample to be detected respectively, and whether the PCR amplification products of the two times simultaneously meet the size of a target strip or not is detected (namely, the two PCR amplification product nucleotide sequence information must be completely matched to determine that the product belongs to the genus tobacco, and if one of the two PCR amplification product nucleotide sequence information does not meet the target size, the two sequences in the patent are greatly limited, and the time consumption, labor consumption and low efficiency are realized in the actual counterfeiting and smuggling work in the tobacco industry. Therefore, it is particularly important to find a primer which only needs 1 pair of primers to carry out 1 PCR amplification and only needs to detect the existence of PCR products to realize the discrimination and detection of smoke and non-smoke.
Disclosure of Invention
Therefore, the invention provides a molecular marker for simply, efficiently and effectively identifying smoke and non-smoke in a tobacco-making raw material and application thereof, which are used for solving the problems of obvious defects and defects existing in the process of adopting a sensory inspection method and a chemical inspection method for the tobacco-making raw material involved in a case in the current fake-and-smuggling work of the tobacco industry, and effectively making up the limitations of complexity, time consumption and labor consumption of a Chinese patent CN113549704A identification and detection technology on a molecular level, thereby realizing rapid, efficient and accurate molecular identification; the key to realize rapid, efficient and accurate molecular identification needs to scientifically and accurately find the tobacco specific sequence. The invention provides a molecular marker which can simply, quickly, efficiently, scientifically and accurately identify and detect tobacco and non-tobacco materials, and the identification and detection of the tobacco and the non-tobacco materials can be realized only by utilizing the existence of a marked PCR amplification product.
In order to establish a scientific, efficient, simple and easy case-related tobacco raw material identification and inspection method based on the DNA level, the invention utilizes 46 pieces of related gene information known in public databases, such as synthesis, transportation, transformation, metabolism and the like of nicotine in tobacco, and 209 pairs of nicotine-related primers are developed. Meanwhile, the primers are compared with known solanaceae plant genome data and tobacco genome data which are not disclosed in the industry by combining bioinformatics, and candidate tobacco genus specific primers which can be completely compared in all tobacco genome data and have unique results but cannot be compared in other solanaceae plant genomes except tobacco are obtained by screening. And then 108 parts of test materials are utilized to carry out experimental verification on the candidate tobacco genus specific primers, and finally 3 markers with tobacco genus specificity are obtained. The 3 markers respectively belong to 2 structural genes (ADC: array terminator and A662: A622 mRNA for isovola reduction-like protein) and 1 transcription factor (Nt ERF189: nicotiana tabacum ERF189 mRNA for ethylene response factor 189), can quickly and effectively identify and detect smoke and non-smoke, and realizes the identification and detection of the involved smoke-making raw materials based on DNA level for the first time.
In order to achieve the above purpose, the invention provides the following technical scheme:
according to one aspect of the invention, a molecular marker for simply, conveniently and efficiently identifying smoke and non-smoke in a smoke raw material is provided, wherein the molecular marker is numbered Ntsp043; the corresponding primer sequences are as follows:
Ntsp043F：5’-AACAGAACGGGACAATCAGC-3’；SEQ ID No.1；
Ntsp043R：5’-GTTTGGGATGGAACCAAGTG-3’；SEQ ID No.2；
according to another aspect of the invention, the application of the molecular marker for simply, conveniently and efficiently identifying the smoke and the non-smoke in the tobacco raw materials in identifying the nicotiana is provided.
Further, the method comprises the following steps:
firstly, extracting genome DNA of a sample to be detected;
step two, taking the DNA extracted in the step one as a template, and adopting a primer Ntsp043 to perform PCR amplification reaction;
step three, detecting the PCR amplification result in the step two through electrophoresis:
when the primer Ntsp043 is used for carrying out PCR amplification on the reference material, if a PCR amplification product contains a PCR amplification nucleotide fragment, the sample to be detected is a tobacco sample;
if the PCR amplification product does not contain any PCR amplification nucleotide fragment, the sample to be detected is a non-tobacco sample.
The nucleotide fragment of the partial PCR amplification product is more than 500 bp.
Further, the method for extracting the genome DNA of the sample to be detected is a filter column method.
Further, the amplification system in the PCR amplification reaction is 20. Mu.L, which comprises 2.0L of 10 XBuffer, 200M dNTPs,0.5M of upstream and downstream primers, 0.75U of rTaq polymerase, 30-50ng of template DNA, and finally ddH 2 The total amount of O is 20 mu L.
Further, the PCR reaction procedure in the PCR amplification reaction is as follows: pre-denaturation at 95 ℃ for 5min,30 cycles of PCR amplification reaction, extension at 72 ℃ for 5min, and storage at 4 ℃.
Further, the cycling conditions were denaturation 30s at 95 ℃, renaturation 30s at 60 ℃ and extension 30s at 72 ℃.
Further, the 10 XBuffer is 10mM Tris-Cl, pH =8.4,50mM KCl,1.5mM MgCl 2 。
The invention emphasizes primer sequence information of Ntsp043; because the sequence information of the PCR amplification product of Ntsp043 has different and non-unique strip sizes in different materials (varieties) of Nicotiana, the effective PCR amplification nucleotide sequences are all larger than 500bp; whereas in non-nicotiana material no PCR amplification product is obtained (i.e., no PCR amplification product is produced in non-nicotiana material).
The invention has the following advantages:
on the basis of Chinese patent CN113549704A, the invention also discovers a new tobacco specific molecular marker capable of identifying tobacco and non-tobacco, and rapidly identifies and detects whether the tobacco-making raw material involved in the case is a tobacco product on the molecular level; the molecular marker, the primer and the method provided by the invention realize rapid and effective discrimination and detection of tobacco and non-tobacco, are simple and easy to implement and have strong operability, provide an objective, efficient and simple molecular level inspection technology for counterfeiting and smuggling in tobacco industry, further improve the scientificity, accuracy and authority of discrimination and detection of related tobacco making raw materials, reduce judicial risks and provide strong judicial evidence for counterfeiting and smuggling in exclusive sale in industry.
The invention provides a molecular marker which can simply, quickly, efficiently, scientifically and accurately identify and detect tobacco and non-tobacco materials, and the identification and detection of the tobacco and the non-tobacco materials can be realized only by utilizing the existence of a marked PCR amplification product; the method effectively overcomes the limitations of complexity, time consumption and labor consumption of the identification and detection technology of Chinese patent CN113549704A on the molecular level, and realizes the simplicity, rapidness and accuracy of molecular identification of tobacco.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary and that other implementation drawings may be derived from the provided drawings by those of ordinary skill in the art without inventive effort.
The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.
FIG. 1 is the PCR amplification result of Nicotiana specific marker Ntsp043 provided by the invention in 108 test materials; number M001-M091 is a nicotiana material, wherein M001-M083 belongs to the wild tobacco material of 11 groups of 3 nicotiana subgenus, and M084-M091 is 4 different types of cultivated tobacco material; the numbers M092-M108 are non-Nicotiana materials, where M098-M105 are solanaceous materials of 7 genera. The rightmost lane is 500bp DNAmarker, which is respectively 500bp, 600bp, 700bp, 800bp, 900bp and 1000bp from bottom to top;
Detailed Description
The present invention is described in terms of specific embodiments, and other advantages and benefits of the present invention will become apparent to those skilled in the art from the following disclosure. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The embodiment provides a molecular marker for simply, conveniently and efficiently identifying smoke and non-smoke in a tobacco raw material, wherein the number of the nicotiana specific molecular marker is Ntsp043, and in the embodiment, a primer sequence corresponding to the molecular marker Ntsp043 is as follows:
Ntsp043F：5’-AACAGAACGGGACAATCAGC-3’；SEQ ID No.1
Ntsp043R：5’-GTTTGGGATGGAACCAAGTG-3’；SEQ ID No.2
example 2
The embodiment provides an application of molecular markers for simply, conveniently and efficiently identifying smoke and non-smoke in a tobacco raw material, which comprises the following steps:
step one, extracting and purifying gene DNA: the fresh leaves of the test materials and 100mg and 30mg of the leaves subjected to high-temperature enzyme deactivation treatment are respectively placed in a mortar with the diameter of 8cm, liquid nitrogen is poured into the mortar for quick grinding into powder, then the genomic DNA of the test materials is extracted and purified by a filter column method (DNeasy Plant Mini Kit (250) Kit, QIAGEN company), and the operation process and the reagent dosage are carried out according to the Kit instructions.
Step two, a PCR amplification system: the amplification system was 20. Mu.L containing 2.0L of 10 XBuffer (10 mM Tris-Cl, pH =8.4,50mM KCl,1.5mM MgCl 2 ) 200M dNTPs (Takara Biotechnology Co. Ltd., dalian, china), 0.5M upstream and downstream primers (Takara), 0.75U of rTaq polymerase (Takara), 30-50ng template DNA, and finally ddH 2 Supplementing 20uL of O;
the PCR reaction program is: pre-denaturation at 95 ℃ for 5min,30 cycles (denaturation at 95 ℃, renaturation at 60 ℃, extension at 72 ℃ for 30 s), extension at 72 ℃ for 5min, and storage at 4 ℃.
Step three, detecting PCR amplification products: adding 6 × Loading Buffer (1/6 volume) into PCR amplification product, collecting 2.5 μ L of non-denaturing polyacrylamide gel (non-denaturing PAGE,220V,3.5 h) and separating by electrophoresis on DYY-8 type electrophoresis apparatus (Beijing Heishi Co., ltd.), and separating the gel after electrophoresis by referring to children, army, etc [1] The method comprises the steps of fixing, silver staining, rinsing, color development, rinsing and other silver staining detection steps, and finally photographing and film data processing.
When the primer Ntsp043 is used for carrying out PCR amplification on a reference material, if a PCR amplification product contains a PCR amplification nucleotide fragment of more than 500bp, the sample to be detected is a tobacco sample;
if the PCR amplification product does not contain any PCR amplification nucleotide fragment (i.e., no PCR amplification product is generated), the sample to be detected is a non-tobacco sample.
Example 3
In this example, a tobacco-specific molecular marker capable of identifying and detecting the presence of a tobacco material in a tobacco-making raw material involved in a case was obtained by performing experimental verification on 91 parts of a tobacco material and 17 parts of a non-tobacco material using bioinformatics in combination with development markers of gene sequence data related to a nicotine (nicotine) metabolic pathway, tobacco genome data (including unpublished data), and other solanaceae genome data in a public database.
1. Experimental Material
The total of the tested plant materials was 108 parts, including 91 parts of tobacco material (see table 1) and 17 parts of non-tobacco material (see table 2). Among 91 parts of tobacco material, 83 parts of wild tobacco material (seed) belonging to 11 groups of 3 nicotiana, and 8 parts of cultivated tobacco material belonging to 4 different types; the 17 parts of non-tobacco material comprise 1 part of cruciferous material, 2 parts of leguminous and theaceous materials respectively, 4 parts of gramineous material and 8 parts of 7 solanaceous materials. The selection of 108 parts of materials is carried out under the conditions that whether the plant materials contain nicotine or not, main plant materials commonly used in the tobacco making raw materials involved in the case, the verification of developed and marked nicotiana specificity experiments and other factors are fully considered. In addition, in order to meet the drying state of the tobacco raw materials involved in the case, the DNA extraction and purification of the materials are divided into two parts, namely, a fresh leaf tissue piece is divided into two parts, one part is a fresh leaf without any treatment, and the other part is subjected to high-temperature enzyme deactivation treatment in an oven to be in a drying state similar to that of primary flue-cured tobacco leaves.
TABLE 1 91 parts Nicotiana Material details
Note: the numbering of 83 tobacco wild species is counted with PI numbers, i.e. there are wild tobacco species of the same name with different PI numbers.
TABLE 2 information on 17 non-tobacco materials
2. Genome data
A total of 46 genome sequence data related to nicotine (nicotine) synthesis, transport, transformation and metabolism were downloaded from the public database NCBI (National Center for Biotechnology Information; https:// www.ncbi.nlm.nih.gov /) in FASTA file format, wherein 32 structural genes and 14 transcription factors were present, and the detailed Information thereof is shown in Table 3. The genome data of tomato, potato, pepper and the like are downloaded from a solanaceae database (https:// www.sgn.corner.edu /) in a FASTA file format, and the published tobacco genome data is downloaded from https:// www.sgn.corner.edu/organissm/Nicotiana _ tabacum/genome, and meanwhile, the whole genome data and the re-sequencing data of tobacco which are not published in the tobacco industry are combined.
TABLE 3 partial genetic information in the Nicotiana tabacum nicotine (nicotine) metabolic pathway
3. Primer design and validation
Primers were designed using Primer3 (http:// www. Frodo. Wi. Mit. Edu) software for the 46 gene sequences in nicotine metabolic pathways obtained from the download. In principle, 3 pairs of primers are designed for the DNA sequence of each gene, namely, 500bp of nucleic acid sequences are cut at the beginning, the end and the middle of each gene for primer development; if the DNA sequence of a gene is large (3 Kb or more), 2 to 3 pairs of primers can be added to the middle of the gene sequence, and finally, 3 to 6 pairs of primers are designed for each gene. When designing the primer, the setting of the relevant parameters refers to the children's army, etc [2] A method.
The primers obtained by the development are subjected to preliminary analysis of the specificity of the tobacco by using bioinformatics and combining published genome data of tobacco (including data not published in the industry) and genome data of solanaceae plants (except for tobacco). That is, when all the primer sequences developed are aligned to the genome data, the alignment results must satisfy both: a) Primer sequences were aligned completely to the tobacco genomic sequence (Forward & Reverse Ident.%, forward & Reverse Cvg.%: forward and reverse primer sequences are 100% aligned) and the aligned result is unique; b) The primer sequence can not be compared with the genome sequences of other solanaceae plants except for tobacco. Primers satisfying the above 2 conditions are candidate nicotiana species-specific markers.
And performing PCR amplification on 108 tested material genomes by using the candidate nicotiana specific marker, and verifying the nicotiana specific marker according to the result of a PCR amplification product.
PCR amplification and product detection
Extracting and purifying gene DNA: the fresh leaves of the test materials and 100mg and 30mg of the leaves subjected to high-temperature enzyme deactivation treatment are respectively placed in a mortar with the diameter of 8cm, liquid nitrogen is poured into the mortar for quick grinding into powder, then the genomic DNA of the test materials is extracted and purified by a filter column method (DNeasy plant Mini Kit (250) Kit, QIAGEN company), and the operation process and the reagent dosage are carried out according to the Kit instructions.
PCR amplification System: the amplification system was 20. Mu.L, containing 2.0L of 10Buffer (10 mM Tris-Cl, pH =8.4,50mM KCl,1.5mM MgCl) 2 ) 200M dNTPs (Takara Biotechnology Co. Ltd., dalian, china), 0.5M upstream and downstream primers (Takara), 0.75U of rTaq polymerase (Takara), 30-50ng template DNA, and finally 20uL supplemented with ddH 2O. The PCR reaction program is: pre-denaturation at 95 ℃ for 5min,30 cycles (denaturation at 95 ℃, renaturation at 60 ℃, extension at 72 ℃ for 30 s), extension at 72 ℃ for 5min, and storage at 4 ℃.
And (3) detecting a PCR amplification product: adding 6 × Loading Buffer (1/6 volume) into PCR amplification product, collecting 2.5 μ L, electrophoretically separating with 8% non-denaturing polyacrylamide gel (non-denaturing PAGE,220V,3.5 h) in DYY-8 type electrophoresis apparatus (Beijing Liuyi Dai Co., ltd.), and referring to gel after electrophoresis such as Chin Zhi Jun et al [1] The method comprises the steps of fixing, silver staining, rinsing, color development, rinsing and other silver staining detection steps, and finally photographing and film data processing.
4. Results and analysis
1. Nicotiana specific marker assay
Based on the full-length DNA sequences of 46 genes related to tobacco nicotine synthesis, transport, transformation, metabolism and the like, primers 209 pairs meeting the design requirements are jointly developed, and each gene is developed to obtain 3-6 pairs of primers, and 4.5 (209/46) pairs on average. The 209 pairs of primer sequences are compared with solanaceae plant genome data (including tobacco) in a public database and unpublished tobacco genome data in the tobacco industry (the genome data sequenced from the beginning, such as Honghuadajinyuan, yunyan 87, beiinhart 1000-1, yunyan No.1, yellow tobacco G366 and the like, and 369 parts of 10-15 multiplied genome re-sequencing data of tobacco core resources), and finally 5 pairs of candidate nicotiana specific primers are obtained. From this, it was found that the yield of the candidate nicotiana-specific primers was extremely low, and was only about 2.39% (5/209).
2. Nicotiana-specific marker validation
The candidate tobacco genus specific primers obtained by the preliminary screening are experimentally verified by using 108 parts of materials to be tested, and finally 1 pair of primers with tobacco genus specificity (the primer information is shown in table 4) is obtained, namely, the primers can obtain effective PCR amplification products of more than 500bp in 91 parts of tobacco materials, but no PCR amplification products are obtained in 17 parts of non-tobacco materials. The primer Ntsp043 has PCR amplification products of more than 500bp in 91 parts of tobacco materials and no PCR amplification products in 17 parts of non-tobacco materials, but the PCR amplification products (bands) in 91 parts of tobacco materials are not unique, and the sizes of the bands amplified in different tobacco materials are different (see figure 1). In a word, the primer can amplify an effective PCR product strip of more than 500bp in 91 parts of tobacco materials, and no PCR amplification product is detected in 17 parts of non-tobacco materials, so the primer has the specificity of tobacco, and the aim of scientifically and efficiently identifying and testing the markers of tobacco and non-tobacco plant materials is fulfilled.
TABLE 4 Nicotiana-specific marker information
The invention focuses on the identification and detection technology of the tobacco-making raw materials involved in the case on the genome DNA level, utilizes bioinformatics and combines the nicotine metabolic pathway related gene sequence data in a public database and solanaceae (including unpublished tobacco) genome data to develop molecular markers, obtains 1 molecular marker with tobacco genus specificity through reasonable selection and experimental verification of plant materials, realizes quick and effective identification and detection of tobacco and non-tobacco, improves the scientificity, accuracy and authority of the identification and detection work of the tobacco-making raw materials involved in the case, reduces the risk of justice, and provides powerful justice evidence for counterfeiting and prividing for trade monopoly.
[1] Development and application of tobacco microsatellite markers in junior army [ D ] Zhejiang university, 2012, pp.
[2] The SSR locus analysis of ordinary tobacco and ancestral genome thereof [ J ] is the agricultural science of China, 2015,48 (11): 2108-2117.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (8)
1. A molecular marker for simply, conveniently and efficiently identifying smoke and non-smoke in a tobacco raw material is characterized in that the molecular marker is numbered as Ntsp043, and a primer sequence corresponding to the molecular marker Ntsp043 is as follows:
Ntsp043F：5’-AACAGAACGGGACAATCAGC-3’；SEQ ID No.1
Ntsp043R：5’-GTTTGGGATGGAACCAAGTG-3’；SEQ ID No.2。
2. the application of the molecular marker for simply, efficiently and effectively identifying the smoke and the non-smoke in the tobacco raw materials in the identification of the nicotiana in the claim 1.
3. The use according to claim 2, comprising:
step one, extracting genome DNA of a sample to be detected;
step two, taking the DNA extracted in the step one as a template, and adopting a primer Ntsp043 to carry out PCR amplification reaction;
step three, detecting the PCR amplification result in the step two through electrophoresis:
when the primer Ntsp043 is used for carrying out PCR amplification on a material to be detected, if a PCR amplification product contains a PCR amplification nucleotide fragment of more than 500bp, the sample to be detected is a tobacco sample;
and if the PCR amplification does not exist in the product during the PCR amplification, the sample to be detected is a non-tobacco sample.
4. The use of claim 3, wherein the method for extracting the genomic DNA of the sample to be tested is a filter column method.
5. The use of claim 4, wherein the amplification system in the PCR amplification reaction is 20 μ L, which comprises 2.0L of 10 xBuffer, 200M dNTPs,0.5M upstream and downstream primers, 0.75U rTaq polymerase, 30-50ng template DNA, and ddH 2 The total amount of O is 20 mu L.
6. The use of claim 5, wherein the PCR reaction program in the PCR amplification reaction is: pre-denaturation at 95 ℃ for 5min,30 cycles of PCR amplification reaction, extension at 72 ℃ for 5min, and storage at 4 ℃.
7. Use according to claim 6, wherein said cycling conditions are denaturation at 95 ℃ for 30s, renaturation at 60 ℃ for 30s and extension at 72 ℃ for 30s.
8. The use according to claim 7, wherein said 10 XBuffer is 10mM Tris-Cl, pH =8.4,50mM KCl,1.5mM MgCl 2 。
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| 张敏等: "烟叶烟碱含量与腐胺 Ｎ－甲基转移酶基因表达间的相关性", 贵州农业科学, vol. 45, no. 9, pages 1 * |
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