WO2023082780A1 - 用于鉴别烟草尼古丁含量高低的分子标记Nicotine Associated SNP 1及其试剂盒与应用 - Google Patents

用于鉴别烟草尼古丁含量高低的分子标记Nicotine Associated SNP 1及其试剂盒与应用 Download PDF

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WO2023082780A1
WO2023082780A1 PCT/CN2022/115610 CN2022115610W WO2023082780A1 WO 2023082780 A1 WO2023082780 A1 WO 2023082780A1 CN 2022115610 W CN2022115610 W CN 2022115610W WO 2023082780 A1 WO2023082780 A1 WO 2023082780A1
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tobacco
nicotine
snp
molecular marker
seq
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PCT/CN2022/115610
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English (en)
French (fr)
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谢贺
白戈
李勇
杨爱国
逄涛
杨大海
肖炳光
李永平
任民
费明亮
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云南省烟草农业科学研究院
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

Definitions

  • the invention belongs to the technical field of molecular biology, and in particular relates to a molecular marker Nicotine Associated SNP 1 for identifying tobacco nicotine content, a kit and application thereof.
  • Nicotine has a strong physiological stimulating effect on the human body and is the material basis for the commercial use of tobacco. Many of the world's top tobacco companies such as Philip Morris, Imperial Tobacco, Japan Tobacco, British American Tobacco and other companies have invested heavily in research on the metabolic pathways and regulatory mechanisms of tobacco nicotine.
  • Nicotine is the most important secondary metabolite of cultivated tobacco (Nicotiana tabacum). As one of the most well-known alkaloids, nicotine content can directly determine the quality of tobacco leaves and is the most economically valuable part of tobacco. In most tobacco varieties, alkaloids account for 2%-4% of the dry weight, while nicotine accounts for 90%-95% of the total content of alkaloids.
  • Arginine decarboxylase catalyzes the decarboxylation of arginine to form putrescine
  • ODC ornithine decarboxylase
  • Putrescine obtains the methyl group provided by S-adenosylmethionine (SAM, S-adenosyl-L-methionine) under the action of putrescine N-methyltransferase (PMT, putrescine-N-methyltransferase) to form N-methyl putrescine Amine, which is a reaction that depends on the activity of S-adenosylmethionine synthase (SAMS, S-adenosylmethionine synthase).
  • SAM S-adenosylmethionine
  • PMT putrescine-N-methyltransferase
  • N-methylputrescine is catalyzed by N-methylputrescine oxidase (MPO, N-methylputrescine oxidase) to form 4-methylaminobutyl ether, and through self-cyclization to form N-methyl- ⁇ 1-pyrroline cation , followed by a condensation reaction with a niacin derivative providing a pyridine ring moiety to form nicotine.
  • MPO N-methylputrescine oxidase
  • the pyridine ring part of nicotine is provided by nicotinic acid, and its precursor is quinolinic acid synthesized from aspartic acid.
  • Quinolinic acid forms nicotinamide adenine dinucleotide (NAD) under the catalysis of quinolinate phosphoribosyltransferase (QPRT, quinolinate phosphoribosyltransferase), and then generates nicotinic acid through the pyridine nucleotide cycle pathway.
  • NAD nicotinamide adenine dinucleotide
  • QPRT quinolinate phosphoribosyltransferase
  • Phytohormones are one of the basic ways to regulate the biosynthesis of secondary metabolites.
  • To analyze the regulation mechanism of hormone signals on the biosynthesis of secondary metabolites is one of the focuses of plant science research.
  • Plant hormone jasmonates (Jas) have been proved to be widely involved in the metabolic regulation of plant secondary metabolites, and JA has a significant induction effect on nicotine biosynthesis.
  • the jasmonic acid derivative JA-Ile binds to the jasmonic acid receptor COI1 to degrade the ubiquitination of the negative regulator JAZ protein, thereby releasing downstream transcriptional activators and activating the plant's jasmonic acid response.
  • Tobacco jasmonic acid pathway regulators COI1 and JAZ proteins have been shown to be regulators of nicotine synthesis.
  • Recent studies have also identified some transcription factors that regulate nicotine synthesis, such as homologous genes of ERF transcription factor family members JAP1, ERF32 and ORC1, bHLH transcription factor family members bHLH1/2 and MYC2, etc. These ERF transcription factors and bHLH transcription factors Factors can also affect nicotine metabolism through mutual regulation.
  • the object of the present invention is to provide a molecular marker Nicotine Associated SNP 1 (nicas1 for short) for identifying the level of tobacco nicotine content and its kit and application.
  • the molecular marker Nicotine Associated SNP 1 used to identify the level of tobacco nicotine is characterized in that it is located at the SNP site Nitab4.5_0002539:95304A/ at No. 0002539 genome fragment No. 95304 of the tobacco genome version Nitab v4.5 Genome Scaffolds Edwards2017 g.
  • the specific primers of the molecular marker Nicotine Associated SNP 1 used to identify the level of tobacco nicotine content are shown in SEQ ID NO.1 and 2.
  • the kit for identifying the level of tobacco nicotine is characterized in that it comprises: molecular marker Nicotine Associated SNP 1; said molecular marker Nicotine Associated SNP 1 is genome fragment No. 0002539 located in tobacco genome version Nitab v4.5 Genome Scaffolds Edwards2017 The SNP site at position 95304 Nitab4.5_0002539:95304A/G.
  • the kit for identifying the level of tobacco nicotine also includes: specific primers for the molecular marker Nicotine Associated SNP 1;
  • the specific primers of the molecular marker Nicotine Associated SNP 1 are shown in SEQ ID NO.1 and 2;
  • the kit further includes: reagents for PCR, reagents for sequencing, and/or reagents for KASP genotyping detection;
  • the reagents for PCR include: dNTPs, Taq enzyme, PCR buffer, ddH 2 O;
  • the reagents for sequencing include: Tris-HCl, agarose, EB;
  • the reagents for the KASP genotyping detection include: KASP Master mix.
  • a method for identifying tobacco nicotine content characterized in that the tobacco material to be tested is screened using the molecular marker Nicotine Associated SNP 1; the molecular marker Nicotine Associated SNP 1 is located in the genome of tobacco genome version Nitab v4.5 Genome Scaffolds Edwards2017 No. 0002539 The SNP site Nitab4.5_0002539:95304A/G at the 95304th position of the fragment.
  • the method for identifying the level of tobacco nicotine comprises: using the specific primer of the molecular marker Nicotine Associated SNP 1 to perform PCR amplification on the DNA of the tobacco material to be tested; the specific primer of the molecular marker Nicotine Associated SNP 1 As shown in SEQ ID NO.1 and 2;
  • the method for identifying tobacco nicotine content further includes: performing sequencing or KASP genotyping detection on the PCR amplification product;
  • the sequencing results or KASP detection results show that the SNP site genotype is GG corresponding to the tobacco material to be tested is a tobacco material with low nicotine content;
  • Sequencing results or KASP detection results show that the SNP site genotype is AA and the tobacco material to be tested is a tobacco material with high nicotine content;
  • the reaction system of the PCR amplification comprises:
  • the reaction program of PCR amplification includes: 98°C for 5min; 98°C for 30s, 58°C for 30s, and 72°C for 30s as a cycle, a total of 35 cycles; 72°C for 5min;
  • the DNA of the tobacco material to be tested is extracted from leaves, seeds, roots, stems, flowers or fruits of tobacco.
  • a method for breeding tobacco varieties with high nicotine content characterized in that the tobacco material with high nicotine content is selected from the tobacco materials to be tested by using the method for identifying high or low tobacco nicotine content.
  • the selected tobacco material with high nicotine content is used as the female parent or male parent, and the tobacco material to be improved is used as the male parent or female parent, and the F1 generation is obtained by crossing the two;
  • the F1 generation plants are used as parents to perform self-crossing to obtain the F2 generation population, and the F2 generation population plants are backcrossed with the selected tobacco material with high or low nicotine content or the tobacco material to be improved.
  • a method for activating a gene promoter related to tobacco nicotine synthesis pathway characterized in that, overexpressing a gene containing an SNP site; the SNP site is a molecular marker Nicotine Associated SNP 1 for identifying the level of tobacco nicotine content SNP site; the molecular marker Nicotine Associated SNP 1 is the SNP site Nitab4.5_0002539:95304A/G located at the 95304th position of the genome fragment No. 0002539 of the tobacco genome version Nitab v4.5 Genome Scaffolds Edwards2017.
  • the base of the SNP site in the gene containing the SNP site is A or G;
  • primers such as SEQ ID NO.7 and SEQ ID NO.8 are used to amplify the DNA of tobacco materials with high nicotine content or low nicotine content to obtain the gene sequence containing the SNP site,
  • the DNA of the tobacco material is amplified using primers such as SEQ ID NO.3 and SEQ ID NO.4, or SEQ ID NO.5 and SEQ ID NO.6 to obtain the genes related to the tobacco nicotine synthesis pathway.
  • primers such as SEQ ID NO.3 and SEQ ID NO.4, or SEQ ID NO.5 and SEQ ID NO.6 to obtain the genes related to the tobacco nicotine synthesis pathway.
  • Promoter region sequence SEQ ID NO.3 and SEQ ID NO.4, or SEQ ID NO.5 and SEQ ID NO.6
  • the sequence of the gene containing the SNP site is connected to the overexpression vector, and the sequence of the gene related to the tobacco nicotine synthesis pathway is connected to the expression vector;
  • the overexpression vector connected to the sequence of the gene containing the SNP site and the expression vector connected to the sequence of the gene related to the tobacco nicotine synthesis pathway are transformed into Agrobacterium and then transfected into tobacco;
  • the nicotine synthesis pathway-related gene promoters are selected from the promoters of the following genes: NtPMT2 and/or NtQPT2;
  • the overexpression vector is pB2GW7 overexpression vector; the expression vector is pGreen0800 fluorescent expression vector.
  • the SNP site is a molecular marker Nicotine used to identify the level of nicotine in tobacco The SNP site of Associated SNP 1;
  • the molecular marker Nicotine Associated SNP 1 is the SNP site Nitab4.5_0002539:95304A/G located at the 95304th position of the genome fragment No. 0002539 of the tobacco genome version Nitab v4.5 Genome Scaffolds Edwards2017.
  • the base of the SNP site in the gene containing the SNP site is A or G;
  • primers shown in SEQ ID NO.9 and SEQ ID NO.10 to amplify the gene sequence related to promoting nicotine synthesis;
  • the gene fragment containing the SNP site is amplified with primers as shown in SEQ ID NO.11 and SEQ ID NO.12;
  • the amplified nicotine synthesis-related gene sequence and the gene fragment containing the SNP site are respectively connected to an expression vector for the co-expression;
  • the expression vector connected with the gene sequence related to promoting nicotine synthesis and the expression vector connected with the gene fragment containing the SNP site are jointly transformed into tobacco;
  • the gene related to promoting nicotine synthesis is NtMED25, and the expression vector is pCAMBIA1300-cLUC.
  • the present invention provides a SNP (Single Nucleotide Polymorphism) site Nicotine Associated SNP 1 associated with tobacco nicotine content traits, characterized in that: the position of the SNP site is tobacco genome version Nitab v4.5 Genome Scaffolds Edwards 2017 No. 0002539 At position 95304 of the genome fragment, there is an A/G SNP site.
  • SNP Single Nucleotide Polymorphism
  • the SNP Single Nucleotide Polymorphism site associated with the tobacco nicotine content trait.
  • the method for GWAS analysis of SNP (single nucleotide polymorphism) sites associated with tobacco nicotine content traits is characterized in that it includes the following steps: 1) using senteion software to detect population SNPs, and the detection has obtained 47140188 SNP positions in total point; 2) After the SNPs were filtered by vcftools software with the conditions of Miss0.5, Het0.2, and maf0.05, a total of 6,957,682 high-quality SNP sites were obtained for subsequent analysis 3) BreakDancer and CNVnator standards were used Analysis process SVs analysis of multiple tobacco natural populations; 4) Genome-wide association analysis of tobacco nicotine content trait phenotype data by using the mixed linear model method according to the population structure and kinship analysis.
  • SNP single nucleotide polymorphism
  • SNP single nucleotide polymorphism
  • the SNP (single nucleotide polymorphism) site associated with tobacco nicotine content traits is applied in high and low nicotine content tobacco breeding.
  • a SNP (single nucleotide polymorphism) locus associated with tobacco nicotine content traits is located at the SNP locus Nitab4.5_0002539 at position 95304 of the genome fragment No. 0002539 of the tobacco genome version Nitab v4.5 Genome Scaffolds Edwards2017 :95304A/G.
  • SNP Single Nucleotide Polymorphism
  • SNP Single Nucleotide Polymorphism
  • a SNP (Single Nucleotide Polymorphism) locus associated with tobacco nicotine content traits is applied in high and low nicotine content tobacco breeding.
  • the present invention has the advantages that: the present invention identifies the SNP (single nucleotide polymorphism) site associated with tobacco nicotine content traits, clarifies the new function of NtMYC2a and provides a method for application, and lays the foundation for cultivating tobacco with different nicotine content.
  • SNP-based GWAS analysis the SNP (single nucleotide polymorphism) loci and genes that control tobacco nicotine content traits were identified, and laid the foundation for molecular marker-assisted breeding and aggregation breeding to improve cotton fiber yield.
  • the patent of the present invention uses the Genome wide association study (GWAS) method to identify SNP sites that mainly affect tobacco nicotine content from 339 tobacco materials, so that high nicotine content can be cultivated by conventional methods of hybridization and then backcrossing or low-nicotine tobacco varieties. It can be used to provide genetic resources for cultivating tobacco with different nicotine content, and can also provide marker information for tobacco molecular marker-assisted breeding, and accelerate the progress of tobacco nicotine content breeding.
  • the invention can directly enter the tobacco breeding process without transgenic methods.
  • the present invention provides a SNP (Single Nucleotide Polymorphism) site associated with tobacco nicotine content and a GWAS analysis method and its application.
  • the SNP site is located in the tobacco genome version Nitab v4.5 Genome Scaffolds Edwards2017 No. 0002539 Genome Fragment No. Position 95304 is a SNP site with a base change of A/G.
  • This site is an A/G SNP at position 95304 of the genome fragment No. 0002539 of the tobacco genome version Nitab v4.5 Genome Scaffolds Edwards 2017.
  • the present invention uses SNP-based GWAS analysis to identify the key site for controlling tobacco nicotine content as the SNP site Nitab4.5_0002539:95304A/G at position 95304 of the genome fragment No. 0002539 of the tobacco genome version Nitab v4.5 Genome Scaffolds Edwards 2017.
  • the present invention adopts Genome wide association study (GWAS) method to identify the SNP sites that mainly affect the tobacco nicotine content from 339 tobacco materials, so the conventional method of hybridization and then backcrossing can be used to breed high nicotine content or Tobacco varieties with low nicotine content. It can be used to provide genetic resources for cultivating tobacco with different nicotine content, and can also provide marker information for tobacco molecular marker-assisted breeding, and accelerate the progress of tobacco nicotine content breeding.
  • the invention can directly enter the tobacco breeding process without transgenic methods.
  • FIG. 1 is a linkage disequilibrium diagram of Chr8 chromosome SNP in Experimental Example 1 of the present invention.
  • Fig. 2 is a histogram of the fluorescent value of the expression of the promoter fragment of the nicotine synthesis-related enzyme gene when the gene of the SNP site of the molecular marker of the present invention is overexpressed in Experimental Example 3 of the present invention; the left figure is the expression of the promoter fragment of the nicotine synthesis-related enzyme NtQPT2 gene Among them, the label QPT2 on the abscissa refers to the fluorescence detection value of the Agrobacterium-transformed tobacco that has been transferred into the NtQPT2Pgreen0800 carrier and the pB2GW7 empty vector, and NtMYC2a_A+QTP2 refers to the fluorescence detection value of the Agrobacterium-transformed tobacco that has been transferred into the NtMYC2a_A carrier and the NtQTP2Pgreen0800 carrier.
  • the label QPT2 on the abscissa refers to the fluorescence detection value of the Agrobacterium-transformed tobacco that has been transferred into the Nt
  • NtMYC2a_G+QTP2 refers to the fluorescence detection value of the Agrobacterium transformed tobacco transformed into the NtMYC2a_G vector and NtQTP2Pgreen0800 vector; the right figure shows the fluorescence value expressed by the promoter fragment of the nicotine synthesis-related enzyme NtPMT2 gene.
  • Fig. 3 is the fluorescent photo of the gene fragment where the SNP site of the molecular marker of the present invention is co-expressed in tobacco leaves in experimental example 4 of the present invention and the NtMED25 gene, and the left side of the leaf is AA. Fluorescence results, the right side of the leaf is the fluorescence result of the genotype of the SNP site co-expressing the molecular marker of the present invention as GG.
  • the 339 tobacco materials used in Experimental Example 1 of the present invention and the 17 tobacco materials used in Experimental Example 2 are all tobacco germplasm resources owned by the applicant unit, and the applicant promises that they can be distributed to the public within 20 years from the filing date of the present invention For verifying the effect of the present invention.
  • the Honghua Dajinyuan used in Experimental Examples 3 and 4, and the Yunyan 87 used in Experimental Examples 3 and 4 are well-known and commonly used tobacco varieties, which are commercially available.
  • the Nicotiana benthamiana plant used in Experimental Example 3 is a common tobacco resource for testing that exists worldwide and is commercially available.
  • the Agrobacterium used in Experimental Example 3 is commercially available.
  • This group of examples provides a molecular marker Nicotine Associated SNP 1 (nicas1 for short) for identifying the level of tobacco nicotine. All the examples in this group have the following common features: the molecular marker nicas1 is located at the SNP site Nitab4.5_0002539:95304 A/G at position 95304 of the genome fragment No. 0002539 of the tobacco genome version Nitab v4.5 Genome Scaffolds Edwards 2017. The molecular marker nicas1 for identifying tobacco nicotine content according to the present invention is the SNP site Nitab4.5_0002539:95304 A/G located at the 95304th position of the genome fragment No. 0002539 of the tobacco genome version Nitab v4.5 Genome Scaffolds Edwards 2017.
  • the SNP site corresponds to the same site on the two versions of the genome, the nucleotide is G or A, and it has been confirmed by experiments that the site of tobacco with high nicotine content is A, and the genome of tobacco with low nicotine content
  • the site is G, and the molecular marker can be used to accurately screen out tobacco varieties with high and low nicotine content, and confirm their genotype and phenotype.
  • the tobacco genome version of the present invention is the Nitab v4.5 Genome Scaffolds Edwards2017 version genome.
  • the primer sequence of the molecular marker nicas1 for identifying the level of tobacco nicotine content can be amplified as shown in SEQ ID NO.1 and SEQ ID NO.2:
  • SNPs can be distinguished by using the above-mentioned primers through the KASP labeling method, but those skilled in the art can also use other detection methods using the SNP of the present invention, such as caps labeling methods, etc., and he can design other detection methods suitable for different detection methods according to the SNP site. available primers.
  • the 2nd group embodiment, the test kit for identifying the level of tobacco nicotine content of the present invention is a test kit for identifying the level of tobacco nicotine content of the present invention.
  • the embodiment of this group provides a kit for identifying the level of tobacco nicotine content. All the embodiments of this group have the following common features: the kit includes: molecular marker nicas1; the molecular marker nicas1 is located at the SNP position at the 95304th position of the genome fragment No. 0002539 of the tobacco genome version Nitab v4.5 Genome Scaffolds Edwards 2017 Point Nitab4.5_0002539:95304A/G.
  • the nucleotide sequence of the molecular marker Nicotine Associated SNP 1 is shown in SEQ ID NO.1;
  • the kit also includes: specific primers for the molecular marker Nicotine Associated SNP 1; preferably, the specific primers for the molecular marker Nicotine Associated SNP 1 are shown in SEQ ID NO.1 and 2;
  • the kit further includes: reagents for PCR, reagents for sequencing, and/or reagents for KASP genotyping detection;
  • the reagents for PCR include: dNTPs, Taq enzyme, PCR buffer, ddH 2 O;
  • the reagents for sequencing include: Tris-HCl, agarose, EB;
  • the reagents for the KASP genotyping detection include: KASP Master mix.
  • This group of examples provides a method for identifying the level of tobacco nicotine content.
  • This group of embodiments has the following common features: the tobacco material to be tested is screened using the molecular marker nicas1; the molecular marker nicas1 is located at the SNP site Nitab4 at position 95304 of the genome fragment No. 0002539 of the tobacco genome version Nitab v4.5 Genome Scaffolds Edwards 2017 .5_0002539:95304A/G.
  • the method for identifying tobacco nicotine content includes: using the specific primers of the molecular marker nicas1 to perform PCR amplification on the DNA of the tobacco material to be tested; the specificity of the molecular marker nicas1 Primers are shown in SEQ ID NO.1 and 2;
  • the method for identifying tobacco nicotine content further includes: performing sequencing or KASP genotyping detection on the PCR amplification product;
  • the sequencing results or KASP detection results show that the SNP site genotype is GG corresponding to the tobacco material to be tested is a tobacco material with low nicotine content;
  • Sequencing results or KASP detection results show that the SNP site genotype is AA and the tobacco material to be tested is a tobacco material with high nicotine content;
  • reaction system and reaction program of the PCR amplification are shown in Table 1 below:
  • the DNA of the tobacco material to be tested is extracted from leaves, seeds, roots, stems, flowers or fruits of tobacco.
  • KASP is a well-known technology in the art. Those skilled in the art can make routine selection and adjustment according to the specific conditions in the actual test and with reference to conventional technical means in the art. For example, you can purchase "KASP Master mix” and refer to the reaction recorded in its product manual Conditions, select appropriate parameters and adjust appropriate reaction conditions according to the fragment size of the PCR amplification product obtained in the present invention.
  • KASP and DNA sequencing are well-known technologies in the art, and they are also mature commercial technologies in the market. Those skilled in the art can perform PCR amplification according to the primer sequences recorded in the present invention, and send the amplified products to paid KASP and DNA sequencing.
  • the company determines the fragment size or sequence of the PCR amplification product, so as to obtain the fragment size or specific sequence of the PCR amplification product, and judge whether the tobacco variety corresponding to the template is a high-nicotine content type according to whether the SNP site is AA or GG Or low nicotine.
  • PCR amplification products can also use other conventional methods in the art to detect PCR amplification products, for example, methods such as competitive allele-specific PCR amplification, electrophoresis, and DNA sequencing can be used to obtain the sequence of the PCR amplification product, and then obtain It is known whether the tobacco material to be tested is specifically low-nicotine-content tobacco or high-nicotine-content tobacco.
  • a Kluster Caller genotyper can be used to detect the sequence of the PCR product.
  • the Kluster Caller genotyping instrument is a common commercially available instrument in this field, and its operation method can refer to the product manual of the instrument for operation.
  • the fourth group of embodiments, the breeding method of tobacco varieties with high nicotine content of the present invention is the breeding method of tobacco varieties with high nicotine content of the present invention
  • the examples of this group provide a method for breeding tobacco varieties with high nicotine content. All the embodiments of this group have the following common features: use a method for identifying the level of tobacco nicotine content provided by the third group of embodiments to screen out tobacco materials with high nicotine content from the tobacco materials to be tested.
  • Those skilled in the art can use the method for identifying tobacco nicotine content to screen tobacco materials at any stage of tobacco plant growth, and obtain tobacco materials with high or low nicotine content.
  • the selected tobacco material with high nicotine content or low nicotine content is used as the female parent or male parent, and the tobacco material to be improved is used as the male parent or female parent, and the F1 generation is obtained by crossing the two ;
  • the F1 generation plants are used as parents to self-cross to obtain the F2 generation, and then the F2 generation plants are used for backcrossing with the selected tobacco material with high nicotine content or low nicotine content or the tobacco material to be improved.
  • the fifth group of embodiments a method of activating the promoters of genes related to the tobacco nicotine synthesis pathway of the present invention
  • This group of examples provides a method for activating the promoters of genes related to tobacco nicotine synthesis pathway.
  • the method has the following common features: overexpression of the gene containing the SNP site; the SNP site is a SNP site of the molecular marker nicas1 used to identify the level of tobacco nicotine content;
  • the molecular marker nicas1 is the SNP site Nitab4.5_0002539:95304A/G located at position 95304 of the genome fragment No. 0002539 of the tobacco genome version Nitab v4.5 Genome Scaffolds Edwards 2017.
  • the base of the SNP site in the gene containing the SNP site is A or G;
  • primers such as SEQ ID NO.7 and SEQ ID NO.8 are used to amplify the DNA of tobacco materials with high nicotine content or low nicotine content to obtain the gene sequence containing the SNP site,
  • the DNA of the tobacco material is amplified to obtain tobacco nicotine synthesis Sequences of pathway-related genes;
  • sequence of the gene containing the SNP site is connected to the overexpression vector, and the sequence of the gene related to the tobacco nicotine synthesis pathway is connected to the expression vector;
  • the overexpression vector connected with the sequence of the gene containing the SNP site and the expression vector connected with the sequence of the gene related to the tobacco nicotine synthesis pathway are transformed into Agrobacterium and then transfected into tobacco;
  • the nicotine synthesis pathway-related genes are selected from: NtPMT2 and/or NtQPT2;
  • the overexpression vector is pB2GW7 overexpression vector; the expression vector is pGreen0800 fluorescent expression vector.
  • the examples of this group provide a method for enhancing the interaction with genes related to promoting nicotine synthesis. All the examples in this group have the following common features: co-expressing genes related to promoting nicotine synthesis and gene fragments containing SNP sites; the SNP sites are SNPs of nicas1, a molecular marker used to identify the level of tobacco nicotine content site; the molecular marker nicas1 is the SNP site Nitab4.5_0002539:95304A/G located at the 95304th position of the genome fragment No. 0002539 of the tobacco genome version Nitab v4.5 Genome Scaffolds Edwards 2017.
  • the base of the SNP site in the gene containing the SNP site is A or G;
  • primers shown in SEQ ID NO.9 and SEQ ID NO.10 to amplify the gene sequence related to promoting nicotine synthesis;
  • the gene fragment containing the SNP site is amplified with primers as shown in SEQ ID NO.11 and SEQ ID NO.12;
  • the amplified nicotine synthesis-related gene sequence and the gene fragment containing the SNP site are respectively connected to an expression vector for the co-expression;
  • the expression vector connected with the gene sequence related to promoting nicotine synthesis and the expression vector connected with the gene fragment containing the SNP site are jointly transformed into tobacco;
  • the gene related to promoting nicotine synthesis is NtMED25, and the expression vector is pCAMBIA1300-cLUC.
  • the interaction effect with the NtMED25 gene can be significantly improved.
  • a SNP (Single Nucleotide Polymorphism) site associated with tobacco nicotine content traits the position of the SNP site is the SNP site Nitab4 located at the 95304th position of the genome fragment No. 0002539 of the tobacco genome version Nitab v4.5 Genome Scaffolds Edwards 2017 .5_0002539:95304A/G. , the site is located in the 259bp coding region of the gene NtMYC2a.
  • a method of SNP (single nucleotide polymorphism) site GWAS analysis associated with tobacco nicotine content traits comprising the following steps:
  • the senteion software was used to detect the population SNP, and a total of 47140188 SNP sites were obtained;
  • the genome-wide association analysis was performed on the phenotype data of tobacco nicotine content traits by using the mixed linear model method.
  • the present invention uses SNP to type the phenotypic traits of 339 GWAS seeds, and finds that among the tobacco materials, there are 53 materials with genotype "GG” and 286 materials with "AA”.
  • the SNP site at position 95304 is also located in an exon of the MYC2a transcription factor protein (CASP) gene (Ga08G0117), which is an A-G SNP variation, and the corresponding amino acid variation is Glutamic acid-lysine (Lysin) showed that the SNP variation of NtMYC2a may be the key variation that determines the content of nicotine.
  • CASP transcription factor protein
  • the nicotine content of the tobacco material in the test is tested.
  • the selected tobacco materials were non-transgenic tobacco plants and transgenic tobacco plants with similar developmental phenotypes in the flourishing period as the treatment objects, and the wild-type tobacco K326 was used as the control. Take the upper, middle and lower leaves of 5 non-transgenic tobacco plants and transgenic tobacco plants. For the other group, five non-transgenic tobacco plants and transgenic tobacco plants were subjected to topping treatment, and then the upper, middle and lower leaves of the non-transgenic tobacco plants and the transgenic tobacco plants were collected.
  • the tobacco sample was extracted with 5% acetic acid aqueous solution, and the total plant alkaloids (calculated as nicotine) in the extract reacted with p-sulfanilic acid and cyanogen chloride, which was produced by the online reaction of potassium cyanide and chloramine T.
  • the reaction product was measured with a colorimeter at 460 nm.
  • Brij 35 solution polyethoxylauryl ether: add 5 drops of 22% Brij35 to water and stir well.
  • Buffer solution A Weigh 2.35g sodium chloride (NaCl), 7.60g sodium borate (Na 2a4 O 3 10H 2 O), dissolve in water, then transfer to a 1L volumetric flask, add 1mL Brij 35, dilute with distilled water to 1L. Filter with qualitative filter paper before use.
  • Buffer solution B weigh 26g disodium hydrogen phosphate (Na 2 HPO 4 ), 10.4g citric acid [COH(COOH)(CH 2 COOH) 2 ⁇ H 2 O], 7g p-aminobenzenesulfonic acid (NH 2 C 6 H 4 SO 3 H), dissolved in water, then transferred to a 1L volumetric flask, added 1mL Brij 35, and diluted to 1L with distilled water. Filter with qualitative filter paper before use.
  • Chloramine T solution N-chloro-4-methylbenzenesulfonamide sodium salt [CH 3 C 6 H 4 SO 2 N(Na)Cl ⁇ 3H 2 O]: take 8.65g of chloramine T, dissolve it in water, Then transfer to a 500mL volumetric flask, and dilute to the mark with water. Filter with qualitative filter paper before use.
  • NaOH buffer solution 8.8g NaOH, 26.0g Na 2 HPO 4 , 10.4g C 6 H 8 O 7 ⁇ H 2 O (citric acid monohydrate), dissolve in water and make up to 1000mL.
  • 4-aminobenzenesulfonic acid buffer Weigh 7g of C 6 H 7 NO 3 S (4-aminobenzenesulfonic acid), 26.0g of Na 2 HPO 4 , 10.4g of C 6 H 8 O 7 ⁇ H 2 O (citric acid monohydrate) g, dissolved in water and adjusted to 1000mL.
  • Chloramine T Weigh 1.2g of chloramine T, dissolve it in pure water to 100mL, and store it in a brown reagent bottle.
  • Potassium cyanide KCN 0.4g, dissolved in pure water to 100mL.
  • NaCO 3 solution 10g NaCO 3 , dissolved in distilled water and adjusted to 1000mL.
  • the content of total plant alkaloids in dry basis is obtained by the following formula:
  • V - the volume of the extract, in mL
  • W moisture content of the sample, in %.
  • the average value of the two determinations was taken as the measurement result, and the result was accurate to 0.01%.
  • the tobacco material with a nicotine content > 20 mg/g is a tobacco material with a high nicotine content of genotype AA;
  • the tobacco material with a nicotine content of ⁇ 20 mg/g is a tobacco material with a low nicotine content of genotype GG.
  • the molecular markers of the present invention are used to verify and identify the other 17 tobacco materials to be tested as shown in Table 2 below:
  • the molecular marker nicas1 and the identification method of the present invention Detected by the molecular marker nicas1 and the identification method of the present invention, among the 17 tobacco materials to be tested, a total of 17 were of genotype and phenotype, of which 9 were of the AA genotype and 9 were of the GG genotype. 8 tobacco materials, the SNP marker and method of the present invention identify tobacco materials with high or low nicotine content, and the accuracy rate is as high as 100%.
  • PMT2_Pgreen_0800_R primer cgctctagaactagtggatccTTTCAAAAATTAAACTAAAC (SEQ ID NO.4) was cloned to obtain the promoter fragment of tobacco NtPMT2 gene, and then the fragment was cloned into pGreen0800 fluorescent vector.
  • QPT2_Pgreen_0800_F primer gtcgacggtatcgataagcttGAAACTATAAATAGCTAAG (SEQ ID NO.5) and QPT2_Pgreen_0800_R primer cgctctagaactagtggatccGGTTTATTTTCTTGGGGCT (SEQ ID NO.6) to clone the promoter sequence of the NtQPT2 gene and then clone it into pGreen 0800 Carrier.
  • the c The DNA was amplified to obtain the gene sequences of NtMYC2a of different genotypes respectively, wherein The SNP site of Honghua Dajinyuan is A, and the SNP site genotype of Yunyan 87 is G.
  • the two fragments were cloned into the PDONR-Zeo vector using geteway's BP reaction.
  • the sequence fragment was finally introduced into the pB2GW7 overexpression vector by LR reaction. These vectors were then transformed into Agrobacterium by transformation method.
  • the 14-day-old Nicotiana benthamiana plant leaves were injected by the method of transient transformation, and then the injected Nicotiana benthamiana plant leaves were stored in the dark for 24 hours, and then restored to culture under light for 48 hours.
  • NtMYC2a_A significantly activates tobacco NtPMT2 and NtQPT2 promoters than NtMYC2a_G (as shown in Figure 2). It has been reported that these two genes, NtPMT2 and NtQPT2, are two key enzymes in the nicotine synthesis pathway.
  • NtMED25 can promote the synthesis of nicotine.
  • This experimental example verified the influence of different genotypes of the SNP site of the present invention on the binding strength of NtMED25, and confirmed that the AA genotype of the SNP site of the present invention can be more strongly combined than the GG genotype. Interaction with NtMED25 gene, which significantly affects the content of nicotine.
  • NtMED25F AAGGTACCatgtgtaaaaatgcgttgggagctg (SEQ ID NO.9)
  • NtMED25R AAGTCGACgggcatgtttggcagtcctcgtgaa (SEQ ID NO.10) clone to obtain NtMED25 full-length sequence, then clone the sequence into 1300nLUC vector, use NtMYC 2F: TTGGTACCatgacggactatagaataccaacgatgacta (SEQ ID NO.11 ), and NtMYC2R: AAGTCGACtcgcgattcagcaattctggatgtcaatgat (SEQ ID NO.12) was cloned to obtain two fragments of NtMYC2A/G and then respectively ligated into the pCAMBIA1300-cLUC vector to obtain NtMYC2A/G 1300-cLUC expression vector and NtMED25
  • the NtMYC2A/G 1300-cLUC expression vector and the NtMED25 1300-cLUC expression vector were co-transformed into Nicotiana benthamiana leaves, and the fluorescence intensity was observed three days later.
  • the results are shown in Figure 3, and the results show that the AA-type SNP fragment has a stronger interaction with NtMED25, and the fluorescence is brighter.
  • GG-type SNP fragments interacted weaker with NtMED25 and had weaker fluorescence.
  • the left side of the picture is the AA type SNP interaction, and the right side is the GG type SNP interaction.
  • the present invention combined the high-nicotine material Honghua Dajinyuan (nicotine content of 21.4 mg/g, genotype AA) with the low-nicotine material Yunyan 87 (nicotine The content is 9.4mg/g, and the genotype is GG) for crossing, and plant the harvested F1 seeds. The genotype and nicotine content of the F2 population and F1 individuals were detected.
  • the genotype detection method is the KASP marker method, and the detection primers are MYC2a-F1 GTATCAAGAATCAAACAGATCTGAATTGATTTGTCT (SEQ ID NO.1), MYC2a-R1 TGATAAAGTCAGGCGAAAACGA (SEQ ID NO.2).

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Abstract

本发明属于分子生物学技术领域，所述分子标记Nicotine Associated SNP 1为位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017的第0002539号基因组片段第95304位的SNP位点Nitab4.5_0002539:95304 A/G。本发明可鉴别筛选尼古丁含量高低的烟草种质资源，筛选得到的烟草材料可不经转基因方法，直接用于选育新的烟草品种，同时SNP位点所在基因序列还可激活尼古丁合成途径关键酶基因的启动子。

Description

用于鉴别烟草尼古丁含量高低的分子标记Nicotine Associated SNP 1及其试剂盒与应用 技术领域
本发明属于分子生物学技术领域，尤其涉及一种用于鉴别烟草尼古丁含量高低的分子标记Nicotine Associated SNP 1及其试剂盒与应用。
背景技术
研究烟草尼古丁的代谢调控是非常有意义的一项工作，通过基因调控可以提供不同尼古丁含量的烟草品种，为烟草商业生产个性化烟碱烟草产品提供原材料。尼古丁对人体有很强的生理刺激作用，是烟草商业性使用的物质基础。许多世界顶级烟草公司如菲利普莫里斯，帝国烟草、日本烟草、英美烟草等公司都投入巨资对烟草尼古丁的代谢途径、调控机制进行研究。
烟碱(Nicotine)是栽培烟草(Nicotiana tabacum)最为重要的次生代谢产物，尼古丁作为一种最为知名的生物碱，其含量的多少可以直接决定烟叶品质，是烟草中最具有经济价值的部分。在大多数的烟草品种中生物碱占干重的2％-4％，而烟碱占生物碱总含量的90％-95％。
烟草生长受到昆虫、病原菌侵害或受到胁迫时,烟株通过诱导产生烟碱抵御侵害,因此烟碱是一种烟草自身的保护性代谢物,对自身的正常生长发育提供保护。在工业生产中烟碱含量过高或过低都会导致烟叶化学成分含量不协调,降低可用性,不利于卷烟的生产。因此研究烟草的烟碱合成与代谢调控对我国烟草工业以及农业生产的发展具有重要意义。
烟碱生物合成途径已经部分解析，烟草中的腐胺可以用于合成烟碱吡咯烷环。精氨酸脱羧酶(ADC,arginine decarboxylase)催化精氨酸脱羧生成腐氨,或由鸟氨酸脱羧酶(ODC,ornithine decarboxylase)催化鸟氨酸脱羧形成腐氨。腐胺在腐胺N-甲基转移酶(PMT,putrescine-N-methyltransferase)作用下获得由S-腺苷蛋氨酸(SAM,S-adenosyl-L-methionine)提供的甲基形成N-甲基腐胺,这是一个依赖S-腺苷蛋氨酸合成酶(SAMS,S-adenosylmethionine synthase)活性的反应。N-甲基腐胺在N-甲基腐胺氧化酶(MPO,N-methylputrescine oxidase)催化下形成4-甲氨基丁醚,并通过自身环化形成N-甲基-△1-吡咯啉阳离子,随后与提供吡啶环部分的烟酸衍生物发生缩合反应形成烟碱。
烟碱吡啶环部分由烟酸提供,其前体是由天冬氨酸合成的喹啉酸。喹啉酸在喹啉酸磷酸核糖转移酶(QPRT,quinolinate phosphoribosyltransferase)催化下形成烟酰胺腺嘌呤二核苷酸(NAD),然后经由吡啶核苷酸循环途径生成烟酸。
近期有关烟碱吡咯烷环部分和吡啶环部分缩合反应的研究表明,NADPH依赖性还原酶的PIP家族成员类异黄酮还原酶基因A622及其同源基因参与了这一过程。
植物激素是调控次生代谢产物生物合成基本方式之一，解析激素信号对次生代谢产物生物合成的调控机制是植物科学研究的重点之一。植物激素茉莉酸类化合物(Jasmonates,JAs)已被证明广泛参与植物次生代谢物质的代谢调控，JA对烟碱生物合成具有显著的诱导作用。
在茉莉酸存在时,茉莉酸衍生物JA-Ile与茉莉酸受体COI1相结合使负调控因子JAZ蛋白的泛素化降解,从而释放下游转录激活因子并激活植物的茉莉酸应答。烟草的茉莉酸途径调控因子COI1和JAZ蛋白都已被证明是烟碱合成调控因子。近期研究还鉴定了一些调控烟碱合成的转录因子,如ERF转录因子家族成员JAP1、ERF32及ORC1的同源基因等,bHLH转录因子家族成员bHLH1/2和MYC2等,这些ERF转录因子和bHLH转录因子还可以通过彼此间的相互调控影响烟碱代谢过程。
目前本领域尚不存在利用SNP位点鉴别烟草尼古丁含量高低的分子标记。
发明内容
基于本领域的上述空白，本发明的目的在于提供一种用于鉴别烟草尼古丁含量高低的分子标记Nicotine Associated SNP 1(简称nicas1)及其试剂盒与应用。
为达到上述目的，本发明采用的技术方案是：
用于鉴别烟草尼古丁含量高低的分子标记Nicotine Associated SNP 1，其特征在于，为位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017的第0002539号基因组片段第95304位的SNP位点Nitab4.5_0002539:95304A/G。
所述用于鉴别烟草尼古丁含量高低的分子标记Nicotine Associated SNP 1的特异性引物如SEQ ID NO.1和2所示。
用于鉴别烟草尼古丁含量高低的试剂盒，其特征在于，包括：分子标记Nicotine Associated SNP 1；所 述分子标记Nicotine Associated SNP 1为位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017的第0002539号基因组片段第95304位的SNP位点Nitab4.5_0002539:95304A/G。
所述的用于鉴别烟草尼古丁含量高低的试剂盒还包括：分子标记Nicotine Associated SNP 1的特异性引物；
优选地，所述分子标记Nicotine Associated SNP 1的特异性引物如SEQ ID NO.1和2所示；
优选地，所述试剂盒还包括：用于PCR的试剂、用于测序的试剂、和/或，用于KASP基因分型检测的试剂；
优选地，所述用于PCR的试剂包括：dNTPs、Taq酶、PCR缓冲液、ddH 2O；
优选地，所述用于测序的试剂包括：Tris-HCl、琼脂糖、EB；
优选地，所述KASP基因分型检测的试剂包括：KASP Master mix。
一种鉴别烟草尼古丁含量高低的方法，其特征在于，采用分子标记Nicotine Associated SNP 1对待测烟草材料进行筛选；所述分子标记Nicotine Associated SNP 1位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017第0002539基因组片段第95304位的SNP位点Nitab4.5_0002539:95304A/G。
所述的一种鉴别烟草尼古丁含量高低的方法包括：采用所述分子标记Nicotine Associated SNP 1的特异性引物对待测烟草材料的DNA进行PCR扩增；所述分子标记Nicotine Associated SNP 1的特异性引物如SEQ ID NO.1和2所示；
优选地，所述一种鉴别烟草尼古丁含量高低的方法还包括：所述PCR扩增产物进行测序或KASP基因分型检测；
优选地，测序结果或KASP检测结果显示SNP位点基因型为GG对应的待测烟草材料为尼古丁含量低的烟草材料；
测序结果或KASP检测结果显示SNP位点基因型为AA对应的待测烟草材料为尼古丁含量高的烟草材料；
优选地，所述PCR扩增的反应体系包括：
模板0.02μL/μL、正向引物0.02μL/μL、反向引物0.02μL/μL、5×buffer 0.2μL/μL、dNTP mixture 0.02μL/μL、DNA聚合酶0.02μL/μL、其余为水；
PCR扩增的反应程序包括：98℃5min；以98℃30s、58℃30s、72℃30s为1个循环，共35个循环；72℃5min；
优选地，所述待测烟草材料的DNA提取自烟草的叶片、种子、根、茎、花、或果实。
一种高尼古丁含量的烟草品种的选育方法，其特征在于，采用所述一种鉴别烟草尼古丁含量高低的方法从待测烟草材料筛选出高尼古丁含量的烟草材料。
以筛选出的高尼古丁含量的烟草材料做为母本或父本、以待改良的烟草材料做为父本或母本，将二者杂交得到F1代；
优选地，再以F1代植株为亲本进行自交获得F2代群体，以F2代群体植株与筛选出的高尼古丁含量或低尼古丁含量的烟草材料或待改良的烟草材料进行回交。
一种激活烟草尼古丁合成途径相关基因启动子的方法，其特征在于，过表达含有SNP位点的基因；所述SNP位点为一种用于鉴别烟草尼古丁含量高低的分子标记Nicotine Associated SNP 1的SNP位点；所述分子标记Nicotine Associated SNP 1为位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017第0002539号基因组片段第95304位的SNP位点Nitab4.5_0002539:95304A/G。
所述含有SNP位点的基因中SNP位点碱基为A或G；
优选地，采用如SEQ ID NO.7和SEQ ID NO.8所示的引物对高尼古丁含量或低尼古丁含量的烟草材料的DNA进行扩增获得含有SNP位点的基因序列，
优选地，采用如SEQ ID NO.3和SEQ ID NO.4、或，SEQ ID NO.5和SEQ ID NO.6所示的引物对烟草材料的DNA进行扩增获得烟草尼古丁合成途径相关基因的启动子区域序列；
优选地，将含有SNP位点的基因的序列连接过表达载体、将烟草尼古丁合成途径相关基因的序列连接表达载体；
优选地，将连接了含有SNP位点的基因的序列的过表达载体和连接了烟草尼古丁合成途径相关基因的 序列的表达载体转化农杆菌再转染烟草；
优选地，所述尼古丁合成途径相关基因启动子选自下述基因的启动子：NtPMT2和/或NtQPT2；
优选地，所述过表达载体为pB2GW7过表达载体；所述表达载体为pGreen0800荧光表达载体。
一种增强与促尼古丁合成相关基因互作的方法，将促尼古丁合成相关基因与含有SNP位点的基因片段共表达；所述SNP位点为一种用于鉴别烟草尼古丁含量高低的分子标记Nicotine Associated SNP 1的SNP位点；所述分子标记Nicotine Associated SNP 1为位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017第0002539号基因组片段第95304位的SNP位点Nitab4.5_0002539:95304A/G。
优选地，所述含SNP位点的基因中SNP位点碱基为A或G；
优选地，用如SEQ ID NO.9和SEQ ID NO.10所示的引物扩增促尼古丁合成相关基因序列；
优选地，用如SEQ ID NO.11和SEQ ID NO.12所示的引物扩增含SNP位点的基因片段；
优选地，将扩增得到的促尼古丁合成相关基因序列和含SNP位点的基因片段分别连接至表达载体中进行所述共表达；
优选地，将连接有促尼古丁合成相关基因序列的表达载体和连接有含SNP位点的基因片段的表达载体共同转化烟草；
优选地，所述促尼古丁合成相关基因为NtMED25，所述表达载体为pCAMBIA1300-cLUC。
本发明提供一种与烟草尼古丁含量性状关联的SNP(Single Nucleotide Polymorphism)位点Nicotine Associated SNP 1，其特征在于：所述SNP位点的位置为烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017第0002539号基因组片段第95304位，存在一个A/G的SNP位点。
所述的与烟草尼古丁含量性状关联的SNP(Single Nucleotide Polymorphism)位点。
所述的与烟草尼古丁含量性状关联的SNP(single nucleotide polymorphism)位点GWAS分析的方法，其特征在于，包括以下步骤：1)采用senteion软件进行群体SNP的检测，检测共获得了47140188个SNP位点；2)SNP经过vcftools软件，采用条件为Miss0.5、Het0.2、maf0.05的过滤后，最后共获得了6,957,682个高质量的SNP位点用于后续分析3)采用BreakDancer和CNVnator标准分析流程对多份烟草自然群体进行SVs分析；4)根据群体结构和亲缘关系分析，利用混合线性模型方法，对烟草尼古丁含量性状表型数据进行全基因组关联分析。
所述的与烟草尼古丁含量多少相关联的SNP(single nucleotide polymorphism)位点在烟草尼古丁含量多少的早期预测和筛选中的应用。
所述的与烟草尼古丁含量性状关联的SNP(single nucleotide polymorphism)位点在烟草分子标记辅助育种中的应用。
所述与烟草尼古丁含量性状关联的SNP(single nucleotide polymorphism)位点在高，低尼古丁含量烟草育种中进行应用。
一种与烟草尼古丁含量性状关联的SNP(single nucleotide polymorphism)位点，所述SNP位点为位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017第0002539号基因组片段第95304位的SNP位点Nitab4.5_0002539:95304A/G。
一种烟草尼古丁含量多少相关联的SNP(Single Nucleotide Polymorphism)位点在烟草尼古丁含量多少的早期预测和筛选中的应用。
一种烟草尼古丁含量性状关联的SNP(Single Nucleotide Polymorphism)位点在烟草分子标记辅助育种中的应用。
一种烟草尼古丁含量性状关联的SNP(Single Nucleotide Polymorphism)位点在高，低尼古丁含量烟草育种中进行应用。
本发明具有的优点是：本发明鉴定到烟草尼古丁含量性状关联的SNP(single nucleotide polymorphism)位点，明确了NtMYC2a的新功能并为应用提供方法，为培育不同尼古丁含量的烟草奠定基础，本发明利用基于SNP的GWAS分析，鉴定到控制烟草尼古丁含量性状关联的SNP(single nucleotide polymorphism)位点以及基因，同时为分子标记辅助育种以及聚合育种提高棉纤维产量奠定基础。
本发明专利采用全基因组关联分析(Genome wide association study,GWAS)方法从339份烟草材料中鉴定出主效影响烟草尼古丁含量的SNP位点，因此可以通过杂交然后回交的常规方法培育高尼古丁含量或 低尼古丁含量的烟草品种。可以用来为培育不同尼古丁含量的烟草提供基因资源，同时也可为烟草分子标记辅助育种提供标记信息，加速烟草尼古丁含量育种的进展。该发明可以不通过转基因方法直接进入烟草育种环节。
本发明提供一种与烟草烟碱含量相关联的SNP(Single Nucleotide Polymorphism)位点与GWAS分析方法及应用，所述SNP位点位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017第0002539号基因组片段第95304位，是一个碱基变化为A/G的SNP位点。
该位点为烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017第0002539号基因组片段第95304位的一个A/G的SNP。本发明利用基于SNP的GWAS分析，鉴定到控制烟草尼古丁含量的关键位点为烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017第0002539号基因组片段第95304位的SNP位点Nitab4.5_0002539:95304A/G。
通过鉴定该SNP有助于理解NtMYC2a生物学功能，同时为分子标记辅助育种以及聚合育种改变烟草尼古丁含量奠定基础。
本发明采用全基因组关联分析(Genome wide association study,GWAS)方法从339份烟草材料中鉴定出主效影响烟草尼古丁含量的SNP位点，因此可以通过杂交然后回交的常规方法培育高尼古丁含量或低尼古丁含量的烟草品种。可以用来为培育不同尼古丁含量的烟草提供基因资源，同时也可为烟草分子标记辅助育种提供标记信息，加速烟草尼古丁含量育种的进展。该发明可以不通过转基因方法直接进入烟草育种环节。
附图说明
图1为本发明实验例1的Chr8号染色体SNP连锁不平衡图。
图2为本发明实验例3过表达本发明分子标记的SNP位点所在基因时尼古丁合成相关酶基因启动子片段表达的荧光值柱状图；左图为尼古丁合成相关酶NtQPT2基因的启动子片段表达的荧光值，其中，横坐标上的标记QPT2指转入了NtQPT2Pgreen0800载体和pB2GW7空载体的农杆菌转化烟草的荧光检测值、NtMYC2a_A+QTP2指转入了NtMYC2a_A载体和NtQTP2Pgreen0800载体的农杆菌转化烟草的荧光检测值、NtMYC2a_G+QTP2指转入了NtMYC2a_G载体和NtQTP2Pgreen0800载体的农杆菌转化烟草的荧光检测值；右图为尼古丁合成相关酶NtPMT2基因的启动子片段表达的荧光值，其中，横坐标上的标记PMT2指转入了NtPMT2Pgreen0800载体和pB2GW7空载体的农杆菌转化烟草的荧光检测值、NtMYC2a_A+PMT2指转入了NtMYC2a_A载体和Nt PMT2Pgreen0800载体的农杆菌转化烟草的荧光检测值、NtMYC2a_G+PMT2指转入了NtMYC2a_G载体和Nt PMT2Pgreen0800载体的农杆菌转化烟草的荧光检测值。
图3为本发明实验例4在烟草叶片中共表达本发明分子标记的SNP位点所在基因片段与NtMED25基因的荧光照片，叶片左侧为共表达本发明分子标记的SNP位点基因型为AA的荧光结果，叶片右侧为共表达本发明分子标记的SNP位点基因型为GG的荧光结果。
具体实施方式
下面结合具体实施例对本发明的内容做进一步详细描述，但并不以此限制本发明的保护范围。
生物材料的来源
本发明的实验例1使用的339份烟草材料以及实验例2使用的17份烟草材料均为申请人单位自有的烟草种质资源，申请人承诺自本发明申请日起20年内可向公众发放用于验证本发明的效果。
实验例3、4使用的红花大金元、实验例3、4使用的云烟87为公知公用的烟草品种，可商购获得。实验例3使用的本氏烟植株为世界范围内普遍存在的试验用共用烟草资源，可商购获得。
实验例3使用的农杆菌可商购获得。
第1组实施例、本发明的分子标记
本组实施例提供一种用于鉴别烟草尼古丁含量高低的分子标记Nicotine Associated SNP 1(简称nicas1)。本组所有的实施例都具备如下共同特征：所述分子标记nicas1为位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017第0002539号基因组片段第95304位的SNP位点Nitab4.5_0002539:95304 A/G。本发明所述的用于鉴别烟草尼古丁含量高低的分子标记nicas1为位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017第0002539号基因组片段第95304位的SNP位点Nitab4.5_0002539:95304 A/G。该SNP位点在此两个版本的基因组上对应同一个位点，的核苷酸为G或A，并经实验证实尼古丁含 量高的烟草的该位点为A，尼古丁含量低的烟草的基因组该位点为G，并且可用该分子标记准确筛选出尼古丁含量高低的烟草品种，并确认其基因型与表型。本发明的烟草基因组版本为Nitab v4.5 Genome Scaffolds Edwards2017版本基因组。
在具体的实施例中，可扩增所述用于鉴别烟草尼古丁含量高低的分子标记nicas1的引物序列如SEQ ID NO.1和SEQ ID NO.2所示：
MYC2a-F1 GTATCAAGAATCAAACAGATCTGAATTGATTTGTCT(SEQ ID NO.1)
MYC2a-R1 TGATAAAGTCAGGCGAAAACGA(SEQ ID NO.2)
采用上述引物通过KASP标记方法对就可以区分SNP，但是本领域技术人员利用本发明的SNP还可以采用其它检测手段，例如caps标记方法等，他可根据SNP位点设计出其它适用于不同检测手段的可用的引物。
第2组实施例、本发明的用于鉴别烟草尼古丁含量高低的试剂盒
本组实施例提供用于鉴别烟草尼古丁含量高低的试剂盒。本组所有的实施例都具备如下共同特征：所述试剂盒包括：分子标记nicas1；所述分子标记nicas1为位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017第0002539号基因组片段第95304位的SNP位点Nitab4.5_0002539:95304A/G。在一些实施例中，所述分子标记Nicotine Associated SNP 1的核苷酸序列如SEQ ID NO.1所示；
优选地，所述试剂盒还包括：分子标记Nicotine Associated SNP 1的特异性引物；优选地，所述分子标记Nicotine Associated SNP 1的特异性引物如SEQ ID NO.1和2所示；
优选地，所述试剂盒还包括：用于PCR的试剂、用于测序的试剂、和/或，用于KASP基因分型检测的试剂；
优选地，所述用于PCR的试剂包括：dNTPs、Taq酶、PCR缓冲液、ddH 2O；
优选地，所述用于测序的试剂包括：Tris-HCl、琼脂糖、EB；
优选地，所述KASP基因分型检测的试剂包括：KASP Master mix。
第3组实施例、本发明的鉴别烟草尼古丁含量高低的方法
本组实施例提供一种鉴别烟草尼古丁含量高低的方法。本组实施例具备如下共同特征：采用分子标记nicas1对待测烟草材料进行筛选；所述分子标记nicas1为位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017第0002539号基因组片段第95304位的SNP位点Nitab4.5_0002539:95304A/G。在一些具体的实施例中，所述的一种鉴别烟草尼古丁含量高低的方法包括：采用所述分子标记nicas1的特异性引物对待测烟草材料的DNA进行PCR扩增；所述分子标记nicas1的特异性引物如SEQ ID NO.1和2所示；
优选地，所述一种鉴别烟草尼古丁含量高低的方法还包括：所述PCR扩增产物进行测序或KASP基因分型检测；
优选地，测序结果或KASP检测结果显示SNP位点基因型为GG对应的待测烟草材料为尼古丁含量低的烟草材料；
测序结果或KASP检测结果显示SNP位点基因型为AA对应的待测烟草材料为尼古丁含量高的烟草材料；
优选地，所述PCR扩增的反应体系及反应程序如下表1所示：
表1
Figure PCTCN2022115610-appb-000001
优选地，所述待测烟草材料的DNA提取自烟草的叶片、种子、根、茎、花、或果实。
KASP是本领域公知技术，本领域技术人员可根据实际试验中的具体情况，参照本领域常规技术手段进行常规选择和调整的，例如，可购买“KASP Master mix”并参照其产品说明书记载的反应条件，根据本发明所得的PCR扩增产物的片段大小选择合适的参数、调整合适的反应条件。
KASP、DNA测序均为本领域公知技术，也是目前市场上成熟的商品化技术，本领域技术人员可根据本发明记载的引物序列进行PCR扩增，将扩增产物送至有偿进行KASP、DNA测序的公司进行PCR扩增产物的片段大小确定或序列确定，从而得出PCR扩增产物的片段大小或具体序列，根据SNP位点是AA还是GG来判断模板所对应的烟草品种是尼古丁高含量型还是尼古丁低含量型。
本领域技术人员也可采用本领域其它常规手段检测PCR扩增产物，例如，可采用竞争性等位基因特异性PCR扩增、电泳、DNA测序等方法均可获知PCR扩增产物序列，进而得知待测烟草材料具体是尼古丁低含量型烟草还是尼古丁高含量型烟草。
在另一些实施例中，可采用Kluster Caller基因分型仪，检测PCR产物序列。
Kluster Caller基因分型仪是本领域常见的可商购获得的仪器，其操作方法可参考该仪器的产品说明书进行操作。
第4组实施例、本发明高尼古丁含量的烟草品种的选育方法
本组实施例提供一种高尼古丁含量的烟草品种的选育方法。本组所有的实施例都具备如下共同特征：采用第3组实施例所一项所提供的一种鉴别烟草尼古丁含量高低的方法从待测烟草材料筛选出高尼古丁含量的烟草材料。
本领域技术人员可在烟草植株生长的任意阶段采用所述鉴别烟草尼古丁含量高低的方法对烟草材料进行筛选，并得到高尼古丁含量或低尼古丁含量的烟草材料。
在进一步的实施例中，以筛选出的高尼古丁含量或低尼古丁含量的烟草材料做为母本或父本、以待改良的烟草材料做为父本或母本，将二者杂交得到F1代；
优选地，再以F1代植株为亲本自交获得F2代，再用F2代植株与筛选出的高尼古丁含量的烟草材料或低尼古丁含量的烟草材料或待改良的烟草材料进行回交。
第5组实施例、本发明的一种激活烟草尼古丁合成途径相关基因启动子的方法
本组实施例提供一种激活烟草尼古丁合成途径相关基因启动子的方法。在本组所有的实施例中，所述方法具备如下共同特征：过表达含有SNP位点的基因；所述SNP位点为一种用于鉴别烟草尼古丁含量高低的分子标记nicas1的SNP位点；所述分子标记nicas1为位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017第0002539号基因组片段第95304位的SNP位点Nitab4.5_0002539:95304A/G。在一些实施例中，所述含有SNP位点的基因中SNP位点碱基为A或G；
在具体的实施例中，采用如SEQ ID NO.7和SEQ ID NO.8所示的引物对高尼古丁含量或低尼古丁含量的烟草材料的DNA进行扩增获得含有SNP位点的基因序列，
在优选的实施例中，采用如SEQ ID NO.3和SEQ ID NO.4、或，SEQ ID NO.5和SEQ ID NO.6所示的引物对烟草材料的DNA进行扩增获得烟草尼古丁合成途径相关基因的序列；
在另一些实施例中，将含有SNP位点的基因的序列连接过表达载体、将烟草尼古丁合成途径相关基因的序列连接表达载体；
在进一步的实施例中，将连接了含有SNP位点的基因的序列的过表达载体和连接了烟草尼古丁合成途径相关基因的序列的表达载体转化农杆菌再转染烟草；
在具体的实施例中，所述尼古丁合成途径相关基因选自：NtPMT2和/或NtQPT2；
在优选的实施例中，所述过表达载体为pB2GW7过表达载体；所述表达载体为pGreen0800荧光表达载体。
第6组实施例、本发明增强基因互作的方法
本组实施例提供一种增强与促尼古丁合成相关基因互作的方法。本组所有的实施例都具备如下共同特征：将促尼古丁合成相关基因与含有SNP位点的基因片段共表达；所述SNP位点为一种用于鉴别烟草尼古丁含量高低的分子标记nicas1的SNP位点；所述分子标记nicas1为位于烟草基因组版本Nitab v4.5Genome Scaffolds Edwards2017第0002539号基因组片段第95304位的SNP位点Nitab4.5_0002539:95304A/G。
在一些实施例中，所述含SNP位点的基因中SNP位点碱基为A或G；
优选地，用如SEQ ID NO.9和SEQ ID NO.10所示的引物扩增促尼古丁合成相关基因序列；
优选地，用如SEQ ID NO.11和SEQ ID NO.12所示的引物扩增含SNP位点的基因片段；
优选地，将扩增得到的促尼古丁合成相关基因序列和含SNP位点的基因片段分别连接至表达载体中进行所述共表达；
优选地，将连接有促尼古丁合成相关基因序列的表达载体和连接有含SNP位点的基因片段的表达载体共同转化烟草；
优选地，所述促尼古丁合成相关基因为NtMED25，所述表达载体为pCAMBIA1300-cLUC。
本发明SNP位点AA基因型相比GG基因型可显著提高与NtMED25基因的互作效应。
实验例1：本发明分子标记的获得
一种与烟草尼古丁含量性状关联的SNP(Single Nucleotide Polymorphism)位点，所述SNP位点的位置为位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017第0002539号基因组片段第95304位的SNP位点Nitab4.5_0002539:95304A/G。，该位点位于基因NtMYC2a编码区域259bp。
一种与烟草尼古丁含量性状关联的SNP(single nucleotide polymorphism)位点GWAS分析的方法，包括以下步骤：
1.采用senteion软件进行群体SNP的检测，检测共获得了47140188个SNP位点；
2.SNP经过vcftools软件，采用条件为Miss0.5、Het0.2、maf0.05的过滤后，最后共获得了6,957,682个高质量的SNP位点用于后续分析；
3.采用BreakDancer和CNVnator标准分析流程对多份烟草自然群体进行SVs分析；
4.根据群体结构和亲缘关系分析，利用混合线性模型方法，对烟草尼古丁含量性状表型数据进行全基因组关联分析。进行全基因组关联分析时，所有被测性状与SNP位点关联的显著性阈值用以下公式进行评估，P＝0.05/n，n为被检测SNP个数。
本发明利用SNP对339份GWAS种子表型性状进行分型发现烟草材料中基因型为“GG”的有53个材料、“AA”的有286个材料。
本发明对烟草基因组约900kb的GWAS区间进行了详细的分析，发现这段区域共包含12个基因(*、**、**)和2个明显的LD blocks(表2、图1)，并发性一个与烟碱含量性状相关联的强信号SNP(-logP＝11.41,Nitab4.5_0002539:95304A/G。)，该位点为位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017第0002539号基因组片段第95304位的SNP位点，同时也位于一个编码MYC2a转录因子蛋白(CASP)基因(Ga08G0117)的外显子中，为一个A-G SNP变异，相应氨基酸变异为谷氨酸(Glutamic acid)-赖氨酸(Lysin)说明NtMYC2a的SNP变异可能是决定烟碱含量的关键变异。
实验例2：本发明SNP标记鉴别尼古丁含量高低的烟草材料
测定烟叶中尼古丁含量
依据标准YC/T 160-2002检测中烟草材料的尼古丁含量。选取的烟草材料为旺长期，发育表型相近的非转基因烟株及转基因烟株为处理对象，以野生型烟草K326为对照。取5株非转基因烟株及转基因烟株的上部、中部及下部叶片。对于另一组，对5株非转基因烟株及转基因烟株进行打顶处理，然后采取非转基因烟株及转基因烟株的上部、中部及下部叶片。
用5％乙酸水溶液萃取烟草样品，萃取液中的总植物碱(以烟碱计)与对氨基苯磺酸和氯化氰反应，氯化氰由氰化钾和氯胺T在线反应产生。反应产物用比色计在460nm测定。
主要仪器设备：连续流动分析仪(美国API)(德国SEAL AA3)(法国ALLIANCE)。
配置试剂：
Brij 35溶液(聚乙氧基月桂醚)：水中加5滴22％Brij35，搅拌均匀。
缓冲溶液A：称取2.35g氯化钠(NaCl)，7.60g硼酸钠(Na 2a4O 3·10H 2O)，用水溶解，然后转入1L容量瓶中，加入1mL Brij 35，用蒸馏水稀释至1L。使用前用定性滤纸过滤。
缓冲溶液B：称26g磷酸氢二钠(Na 2HPO 4)，10.4g柠檬酸[COH(COOH)(CH 2COOH) 2·H 2O]，7g对氨基苯磺酸(NH 2C 6H 4SO 3H)，用水溶解，然后转入1L容量瓶中，加入1mL Brij 35，用蒸馏水稀释至1L。使用前用定性滤纸过滤。
氯胺T溶液(N-氯-4-甲基苯硫酰胺钠盐)[CH 3C 6H 4SO 2N(Na)Cl·3H 2O]：取8.65g氯胺T，溶于水中，然后转入500mL的容量瓶中，用水定容至刻度。使用前用定性滤纸过滤。
0.22mol/L NaOH缓冲液：NaOH 8.8g，Na 2HPO 4 26.0g，C 6H 8O 7·H 2O(一水柠檬酸)10.4g，用水溶解并定容至1000mL。
对氨基苯磺酸缓冲液：称取C 6H 7NO 3S(对氨基苯磺酸)7g，Na 2HPO 4 26.0g，C 6H 8O 7·H 2O(一水柠檬酸)10.4g，用水溶解并定容至1000mL。
氯胺T：称取氯胺T 1.2g，用纯水溶解定容至100mL，用棕色试剂瓶保存。
氰化钾：KCN 0.4g，用纯水溶解定容至100mL。
NaCO 3溶液：10g NaCO 3，蒸馏水溶解并定容至1000mL。
分析步骤：称取0.3g烟样于150mL三角瓶或塑料瓶中(精确至0.0001g)；加入50mL 5％乙酸溶液盖上塞子；在普通摇床上振荡萃取30min，转速控制170r/min，用滤纸过滤上机。(如样品液浓度超出工作标准液的浓度范围，则应稀释)。
结果的计算与表述：
以干基计的总植物碱的含量，由下面公式得出：
[根据细则91更正 15.09.2022]　
Figure WO-DOC-FIGURE-1
式中：
C——样品液总植物碱的仪器观测值，单位为mg/mL；
V——萃取液的体积，单位为mL；
m——试料的质量，单位为mg；
W——试样的水分含量，单位为％。
以两次测定的平均值作为测定结果，结果精确至0.01％。
在本文中，尼古丁含量＞20mg/g的烟草材料为基因型为AA的尼古丁高含量的烟草材料；
尼古丁含量≤20mg/g的烟草材料为基因型为GG的尼古丁低含量的烟草材料。
采用本发明的分子标记对另外17份待测烟草材料进行验证并鉴别结果如下表2所示：
表2
SNP基因型 品种 烟碱含量(mg/g)
AA TI 516 31
AA TI 401 27
AA TI 383 21.5
AA TI 1457 27.5
AA TN90 24
AA Burley 21 22.5
AA TI 319 22.5
AA TI 179 21.1
AA HondDa 21.4
GG TI 245 17.5
GG TW 7 2.5
GG TI 857 13.5
GG SC 72 13
GG K 399 18.5
GG YanYan97 10.25
GG YunYan87 9.4
GG YunYan02 9.1
经本发明的分子标记nicas1及鉴定方法进行检测，在待测的17份待测烟草材料中，基因型与表型相符的一共17份，其中AA基因型的烟草材料9份，GG基因型的烟草材料8份，本发明的SNP标记及方法鉴别尼古丁含量高低的烟草材料，准确率高达100％。
实验例3：过表达SNP位点所在基因激活尼古丁合成相关酶基因启动子
采用PMT2_Pgreen_0800_F引物gtcgacggtatcgataagcttAGTATTCAAGGTATCTAAC(SEQ ID NO.3)，
PMT2_Pgreen_0800_R引物cgctctagaactagtggatccTTTCAAAATTAAACTAAAC(SEQ ID NO.4)克隆获得烟草NtPMT2基因的启动子片段，然后将该片段克隆到pGreen0800荧光载体中。采用QPT2_Pgreen_0800_F引物gtcgacggtatcgataagcttGAAACTATAAATAGCTAAG(SEQ ID NO.5)以及QPT2_Pgreen_0800_R引物cgctctagaactagtggatccGGTTTATTTTCTTGGGGCT(SEQ ID NO.6)克隆获得NtQPT2基因的启动子序列然后再次克隆到pGreen0800载体中。
采用NtMYC2a_F引物GGGGACAAGTTTGTACAAAAAAGCAGGCTGCATGACGGATTATAGAATACCAAC(SEQ ID NO.7)以及NtMYC2a_R引物GGGGACCACTTTGTACAAGAAAGCTGGGTCTCATCGCGATTCAGCAATTCTGGATG(SEQ ID NO.8)分别以红花大金元及云烟87的cDNA进行扩增，分别获得不同基因型的NtMYC2a的基因序列，其中红花大金元的SNP位点为A，云烟87的SNP位点基因型为G。采用geteway的BP反应将两条片段克隆到PDONR-Zeo载体中。采用LR反应将序列片段最终导入到pB2GW7过表达载体中。之后采用化转法将这些载体转入到农杆菌中。挑取农杆菌单克隆在2ml抗性液体培养基中过夜培养，第二天将2ml菌液放入50ml抗性培养基培养，待到OD值为0.6时收获农杆菌沉淀。
按下表3的以下组合配置农杆菌注射液：
表3
NtPMT2 Pgreen0800载体+pB2GW7空载体
NtQPT2 Pgreen0800载体+pB2GW7空载体
NtMYC2a_A载体+NtPMT Pgreen0800载体
NtMYC2a_G载体+NtPMT Pgreen0800载体
NtMYC2a_A载体+NtQTP2 Pgreen0800载体
NtMYC2a_G载体+NtQPT2 Pgreen0800载体
采用瞬时转化的方法注射14天大的本氏烟植株叶片，之后将注射后的本氏烟避光保存24小时，之后恢复在光下培养48小时。将所取的材料放入1.5mL的EP管中，并向其中加入2粒直径为0.5cm的钢珠，超低温环境下，60Hz，振荡破碎30sec；向其粉末中加入100μL PLB裂解液，涡旋震荡10sec，使其快速溶解，提取总蛋白；4℃，13000rpm离心10sec，
吸取上清8μL加入到另外EP管中，加入40μL LARⅡ，轻轻混匀，荧光分光光度计第1次检测；检测完成后再其混合液中加入40μL Stop&GloReagent，轻轻混匀，再次进行荧光检测；记录其数值，对其进行比对计算发现NtMYC2a_A比NtMYC2a_G显著的激活烟草NtPMT2和NtQPT2启动子(如图2)。已有报道证实NtPMT2和NtQPT2这两个基因是尼古丁合成途径的2个关键酶，这两个关键酶基因的启动子的激活与尼古丁合成及含量呈显著正相关，换言之，本领域技术人员可以预期尼古丁含量随着这两个关键酶基因的启动子的激活而升高。因此本实验例进一步证明，本发明的SNP位点所在基因的过表达可激活与尼古丁合成正相关的2个关键酶，且SNP位点为A时的激活程度显著高于位点为G时的激活程度(图2)。
实验例4：本发明分子标记增强与NtMED25互作效应
NtMED25能够促进尼古丁的合成，本实验例验证了本发明的SNP位点不同基因型对NtMED25结合强度的影响，并证实本发明的SNP位点AA基因型相比GG基因型能够更强地结合并互作NtMED25基因，从而显著地影响尼古丁的含量。用引物NtMED25F：AAGGTACCatgtgtaaaaatgcgttgggagctg(SEQ ID NO.9)，NtMED25R:AAGTCGACgggcatgtttggcagtcctcgtgaa(SEQ ID NO.10)克隆获得NtMED25全长序列，然后将该序列克隆到1300nLUC载体中，用NtMYC2F：TTGGTACCatgacggactatagaataccaacgatgacta(SEQ ID NO.11)，及NtMYC2R：AAGTCGACtcgcgattcagcaattctggatgtcaatgat(SEQ ID NO.12)克隆获得两个NtMYC2A/G两个片段然后分别连接至pCAMBIA1300-cLUC载体中获得NtMYC2A/G 1300-cLUC表达载体和NtMED251300-cLUC表达载体。将NtMYC2A/G 1300-cLUC表达载体和NtMED25 1300-cLUC表达载体两个载体共同转化本氏烟叶片，三天后观测荧光强度。结果如图3所示，结果表明AA型SNP片段与NtMED25互作更强，荧光更亮。与之相反，GG型SNP片段与NtMED25互作更弱，荧光弱。图片左边为AA型SNP互作情况，右边为GG型SNP互作情况。
实验例5：SNP位点在育种上应用
为了进一步确认该SNP位点在育种中的用处，本发明将高烟碱材料红花大金元(烟碱含量为21.4mg/g，基因型为AA)与低烟碱材料云烟87(烟碱含量为9.4mg/g，基因型为GG)进行杂交，种植收获到的F1种子。检测F2群体及F1个体的基因型及烟碱含量。检测基因型的方法为KASP标记方法，检测引物为MYC2a-F1 GTATCAAGAATCAAACAGATCTGAATTGATTTGTCT(SEQ ID NO.1)，MYC2a-R1 TGATAAAGTCAGGCGAAAACGA(SEQ ID NO.2)。
F2群体中GG基因型为54株，尼古丁含量均高于20mg/g。实验结果表明，用本发明的SNP位点检测F2群体的基因型和表型，准确率仍然达到100％。
Figure PCTCN2022115610-appb-000003
Figure PCTCN2022115610-appb-000004
Figure PCTCN2022115610-appb-000005
Figure PCTCN2022115610-appb-000006
Figure PCTCN2022115610-appb-000007

Claims (12)

  1. 用于鉴别烟草尼古丁含量高低的分子标记Nicotine Associated SNP 1，其特征在于，为位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017的第0002539号基因组片段第95304位的SNP位点Nitab4.5_0002539:95304 A/G。
  2. 根据权利要求1所述的用于鉴别烟草尼古丁含量高低的分子标记Nicotine Associated SNP 1，其特征在于，所述用于鉴别烟草尼古丁含量高低的分子标记Nicotine Associated SNP 1的特异性引物如SEQ ID NO.1和SEQ ID NO.2所示。
  3. 用于鉴别烟草尼古丁含量高低的试剂盒，其特征在于，包括：分子标记Nicotine Associated SNP 1；所述分子标记Nicotine Associated SNP 1为位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017的第0002539号基因组片段第95304位的SNP位点Nitab4.5_0002539:95304 A/G。
  4. 根据权利要求3所述的用于鉴别烟草尼古丁含量高低的试剂盒，其特征在于，还包括：分子标记Nicotine Associated SNP 1的特异性引物；和/或，所述分子标记Nicotine Associated SNP 1的特异性引物如SEQ ID NO.1和SEQ ID NO.2所示；
    和/或，所述试剂盒还包括：用于PCR的试剂、用于测序的试剂、和/或，用于KASP基因分型检测的试剂；
    和/或，所述用于PCR的试剂包括：dNTPs、Taq酶、PCR缓冲液、ddH 2O；
    和/或，所述用于测序的试剂包括：Tris-HCl、琼脂糖、EB；
    和/或，所述KASP基因分型检测的试剂包括：KASP Master mix。
  5. 一种鉴别烟草尼古丁含量高低的方法，其特征在于，采用分子标记Nicotine Associated SNP 1对待测烟草材料进行筛选；所述分子标记Nicotine Associated SNP 1位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017第0002539基因组片段第95304位的SNP位点Nitab4.5_0002539:95304 A/G。
  6. 根据权利要求5所述的一种鉴别烟草尼古丁含量高低的方法，其特征在于，包括：采用所述分子标记Nicotine Associated SNP 1的特异性引物对待测烟草材料的DNA进行PCR扩增；所述分子标记Nicotine Associated SNP 1的特异性引物如SEQ ID NO.1和SEQ ID NO.2所示；
    和/或，所述一种鉴别烟草尼古丁含量高低的方法还包括：所述PCR扩增产物进行测序或KASP基因分型检测；
    和/或，测序结果或KASP检测结果显示SNP位点基因型为GG对应的待测烟草材料为尼古丁含量低的烟草材料；
    测序结果或KASP检测结果显示SNP位点基因型为AA对应的待测烟草材料为尼古丁含量高的烟草材料；
    和/或，所述PCR扩增的反应体系包括：
    模板0.02μL/μL、正向引物0.02μL/μL、反向引物0.02μL/μL、5×buffer 0.2μL/μL、dNTP mixture 0.02μL/μL、DNA聚合酶0.02μL/μL、其余为水；
    PCR扩增的反应程序包括：98℃ 5min；以98℃ 30s、58℃ 30s、72℃ 30s为1个循环，共35个循环；72℃ 5min；
    和/或，所述待测烟草材料的DNA提取自烟草的叶片、种子、根、茎、花、或果实。
  7. 一种高尼古丁含量的烟草品种的选育方法，其特征在于，采用权利要求1或2所述的用于鉴别烟草尼古丁含量高低的分子标记Nicotine Associated SNP 1、和/或，权利要求3或4所述的用于鉴别烟草尼古丁含量高低的试剂盒、和/或，权利要求5或6所述一种鉴别烟草尼古丁含量高低的方法从待测烟草材料筛选出高尼古丁含量的烟草材料。
  8. 根据权利要求7所述的一种高尼古丁含量的烟草品种的选育方法，其特征在于，以筛选出的高尼古丁含量的烟草材料做为母本或父本、以待改良的烟草材料做为父本或母本，将二者杂交得到F1代；
    和/或，再以F1代植株为亲本进行自交获得F2代群体，以F2代群体植株与筛选出的高尼古丁含量或低尼古丁含量的烟草材料或待改良的烟草材料进行回交；
    和/或，再通过权利要求5或6所述一种鉴别烟草尼古丁含量高低的方法、和/或，权利要求1或2所述的用于鉴别烟草尼古丁含量高低的分子标记Nicotine Associated SNP 1、和/或，权利要求3或4所述的用于鉴别烟草尼古丁含量高低的试剂盒从回交群体中筛选获得高尼古丁含量或低尼古丁含量的烟草品种。
  9. 一种激活烟草尼古丁合成途径相关基因启动子的方法，其特征在于，过表达含有SNP位点的基因；所述SNP位点为一种用于鉴别烟草尼古丁含量高低的分子标记Nicotine Associated SNP 1的SNP位点；所述分子标记Nicotine Associated SNP 1为位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017第0002539号基因组片段第95304位的SNP位点Nitab4.5_0002539:95304 A/G。
  10. 根据权利要求9所述的一种激活烟草尼古丁合成途径相关基因启动子的方法，其特征在于，所述含有SNP位点的基因中SNP位点碱基为A或G；
    和/或，采用如SEQ ID NO.7和SEQ ID NO.8所示的引物对高尼古丁含量或低尼古丁含量的烟草材料的DNA进行扩增获得含有SNP位点的基因序列，
    和/或，采用如SEQ ID NO.3和SEQ ID NO.4、或，SEQ ID NO.5和SEQ ID NO.6所示的引物对烟草材料的DNA进行扩增获得烟草尼古丁合成途径相关基因的启动子区域序列；
    和/或，将含有SNP位点的基因的序列连接过表达载体、将烟草尼古丁合成途径相关基因的序列连接表达载体；
    和/或，将连接了含有SNP位点的基因的序列的过表达载体和连接了烟草尼古丁合成途径相关基因的序列的表达载体转化农杆菌再转染烟草；
    和/或，所述尼古丁合成途径相关基因的启动子选自下述基因的启动子：NtPMT2和/或NtQPT2；
    和/或，所述过表达载体为pB2GW7过表达载体；所述表达载体为pGreen0800荧光表达载体。
  11. 一种增强与促尼古丁合成相关基因互作的方法，其特征在于，将促尼古丁合成相关基因与含有SNP位点的基因片段共表达；所述SNP位点为一种用于鉴别烟草尼古丁含量高低的分子标记Nicotine Associated SNP 1的SNP位点；所述分子标记Nicotine Associated SNP 1为位于烟草基因组版本Nitab v4.5 Genome Scaffolds Edwards2017第0002539号基因组片段第95304位的SNP位点Nitab4.5_0002539:95304 A/G。
  12. 根据权利要求11所述的一种增强与促尼古丁合成相关基因互作的方法，其特征在于，所述含SNP位点的基因中SNP位点碱基为A或G；
    和/或，用如SEQ ID NO.9和SEQ ID NO.10所示的引物扩增促尼古丁合成相关基因序列；
    和/或，用如SEQ ID NO.11和SEQ ID NO.12所示的引物扩增含SNP位点的基因片段；
    和/或，将扩增得到的促尼古丁合成相关基因序列和含SNP位点的基因片段分别连接至表达载体中进行所述共表达；
    和/或，将连接有促尼古丁合成相关基因序列的表达载体和连接有含SNP位点的基因片段的表达载体共同转化烟草；
    和/或，所述促尼古丁合成相关基因为NtMED25，所述表达载体为pCAMBIA1300-cLUC。
PCT/CN2022/115610 2021-11-10 2022-08-29 用于鉴别烟草尼古丁含量高低的分子标记Nicotine Associated SNP 1及其试剂盒与应用 WO2023082780A1 (zh)

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