Tobacco ntmyc2 allelic mutant and application thereof

Abstract

This invention discloses tobacco NtMYC2a Allelic mutants and their applications belong to the field of genetic engineering technology. The tobacco provided by this invention... NtMYC2a Gene allelic variant mutant, nucleotide sequence as shown in SEQ ID NO:2, derived from wild-type tobacco NtMYC2a A T deletion at nucleotide position 1239 of the gene results in the wild-type sequence shown in SEQ ID NO:1. This single-base deletion triggers a frameshift mutation, causing premature termination of translation at amino acid position 430, encoding the amino acid sequence shown in SEQ ID NO:4. Experiments have confirmed that the total alkaloid content in tobacco leaves carrying this mutant is significantly reduced, with nicotine being the predominant alkaloid; reducing total alkaloids equates to reducing nicotine content. This mutant is genetically stable and exhibits excellent alkaloid-reducing effects, making it suitable for constructing recombinant vectors, transforming host cells, and cultivating tobacco plants with low total alkaloid content. It can also be used to develop molecular diagnostic kits for trait screening, demonstrating significant application value in tobacco quality improvement and low-alkaloid breeding.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to tobacco. NtMYC2 Allelic mutants and their applications. Background Technology

[0002] MYC2 Transcription factors belong to the conserved bHLH (basic helix-loop-helix) family in plants, and play a central role in plant growth and development, environmental response, and secondary metabolism regulation. MYC2 Its most well-known function is integrating environmental and hormonal signals, particularly participating in the jasmonic acid (JA) signaling pathway, where it is responsible for regulating plant growth, development, resistance responses, and the accumulation of secondary metabolites (Pauwels, L., et al. (2009). Jasmonate-inducible gene: What does it mean? Trends Plant Sci, 14, 87-91.; Dombrecht, B., et al. (2007). MYC2 differentially modulates diverse jasmonate-dependent functions in Arabidopsis Plant Cell, 19, 2225-2245.). In Arabidopsis thaliana ( Arabidopsis thaliana In tobacco, AtMYC2 participates in regulating root elongation, leaf senescence, and the accumulation of various secondary metabolites. Its mechanisms include directly activating the promoter regions of downstream target genes to directly regulate their transcriptional levels, and indirectly regulating them by forming an interaction network with other transcription factor families. Nicotiana tabacum In *L.*, two homologous genes of MYC2, NtMYC2a and NtMYC2b, have been shown to be key transcription factors regulating nicotine biosynthesis, directly involved in tobacco pest and disease defense mechanisms and the growth and development of phenotypic traits (Zhang, Z., et al. (2012). Tobacco transcription factors NtMYC2a and NtMYC2b form nuclear complexes with the NtJAZ1 repressor and regulate multiple jasmonate-inducible steps in nicotine biosynthesis. *Molecular Plant*, 5, 73-84.).

[0003] In cultivated tobacco, the total alkaloids mainly include nicotine, nornicotine, neonicotine, and pseudoeatidine. Nicotine is the most abundant component, typically accounting for 90%–95% of the total alkaloids. The remaining alkaloids, such as nornicotine, pseudoeatidine, and neonicotine, are present in relatively low amounts, accounting for only a small proportion. Nicotine biosynthesis in tobacco, as a classic model of plant secondary metabolism regulation, can be divided into two precursor pathways: the pyrrolidine ring originates from ornithine and arginine metabolism, and the pyridine ring originates from nicotinic acid or nicotinamide metabolism (Hibi, N., et al. (1994). Gene expression in tobacco low-nicotine mutants. The Plant Cell, 6, 723–735.). Key enzymes in the synthesis pathway include putrescine N-methyltransferase (N-methyltransferase). PMT ), catalyzing the conversion of putrescine to N-methylputrescine, is the rate-limiting step in nicotine biosynthesis; A622 participates in the coupling of the pyridine ring and the pyrrolidine ring to form nicotine precursors; nicotinic acid phosphoribosyltransferase (NPT) QPT These enzymes catalyze the formation of pyridine ring precursors. The expression of these enzymes is regulated by the JA signaling pathway. When tobacco plants are subjected to mechanical damage, insect feeding, or exogenous JA treatment, the nicotine biosynthesis genes in the plant are induced to express (Shoji, T., Kajikawa, M., & Hashimoto, T. (2010). Clustered transcription factor genes regulate nicotine biosynthesis in tobacco. Plant Cell, 22, 3390-3409.).

[0004] NtMYC2aThe gene NtMYC2a plays a key regulatory role in the tobacco JA signaling pathway. Under JA induction, NtMYC2a activates the promoter region of a key nicotine biosynthetic enzyme gene to regulate its transcription. Specifically, the NtMYC2a protein possesses a DNA-binding domain of the bHLH transcription factor family and a JAZ protein-protein interaction domain, allowing it to directly bind to the G-box sequence (CACGTG) in the promoter of downstream target genes. Simultaneously, it is negatively regulated by JAZ repressor proteins (Kazan, K. and Manners, JM (2013). MYC2: the master in action. Mol Plant, 6, 686-703.; Pauwels, L., et al. (2009). Jasmonate-inducible gene: What does it mean? Trends Plant Sci, 14, 87-91.). In the unstimulated state, JAZ proteins inhibit NtMYC2a's transcriptional activity by binding to it; however, under JA signaling, SCF... COI1 The complex mediates the ubiquitination and degradation of JAZ proteins, thereby releasing NtMYC2a to activate the expression of downstream target genes. Furthermore, NtMYC2a can interact with… AP2 / ERF The transcription factor NtERF189 protein works synergistically to activate the expression of nicotine biosynthesis enzyme genes. On the other hand, NtMYC2a can also directly bind to... NtERF189 The gene promoter region is enhanced, thereby forming a positive feedback regulatory loop that ensures efficient initiation of nicotine biosynthesis under stress or JA-induced conditions (Shoji, T. and Hashimoto, T. (2011). Tobacco MYC2 regulates jasmonate-inducible nicotine biosynthesis genes directly and by way of the NIC2 -Locus ERF genes, Plant Cell Physiol, 52,1117-1130.).

[0005] right NtMYC2a Verification of transcription factor function is mainly achieved through RNA interference (RNAi) and gene editing methods. NtMYC2a -RNAi strains showed a significant decrease in nicotine content, while PMT , QPT , A622The transcriptional levels of key genes such as Tobacco MYC2 are reduced (Shoji, T. and Hashimoto, T. (2011). Tobacco MYC2 regulates jasmonate-inducible nicotine biosynthesis genes directly and by way of the NIC2 -Locus ERF Genes, Plant Cell Physiol, 52, 1117-1130.). In gene editing mediated by CRISPR / Cas9... NtMYC2a In knockout lines, nicotine accumulation in tobacco leaves was significantly reduced, with some studies showing a decrease of approximately 80% in leaf nicotine content (Sui, X., et al. (2020)). NtMYC2a These results indicate that... (The text abruptly ends here, likely due to an incomplete translation or a missing section.) NtMYC2a Not only is it essential in the transcriptional regulation of nicotine biosynthesis, but it also has a rate-limiting effect on nicotine accumulation, providing an effective target for the metabolic engineering of low-nicotine tobacco.

[0006] NtMYC2a Natural allelic variation is the molecular basis for the diversity of nicotine content in tobacco. Analysis of tobacco germplasm resources with low nicotine content revealed that... NtMYC2a Coding regions and promoters can contain various structural variations such as single nucleotide polymorphisms (SNPs) and small deletions / insertions (indels). For example, tobacco germplasm TI313 carries... NtMYC2a A five-base deletion allelic variant leads to protein truncation and reduced transcriptional activity (Burner, N., et al. (2022). Analyses of diverse low alkaloid tobacco germplasm identify naturally occurring nucleotide variability contributing to reduced leaf nicotine accumulation, Molecular Breeding, 42, 4.). Tobacco plants carrying this variant showed a significant decrease in nicotine accumulation in tobacco leaves, indicating... NtMYC2aNatural variations in tobacco have significant physiological implications for secondary metabolism (Shoji, T., Kajikawa, M., & Hashimoto, T. (2010). Clustered transcription factor genes regulate nicotine biosynthesis in tobacco. Plant Cell, 22, 3390-3409.). Besides TI313, variations from wild and cultivated tobacco species... NtMYC2a Natural variations have also been reported. For example, in the two ancestral tobacco species, forest tobacco (Nicotiana spp.) N. sylvestris ) and fluffy tobacco ( N. tomentosiformis Multiple bHLH domain amino acid substitution alleles were found in the genome, potentially affecting DNA binding affinity and transcriptional activity. These variations are associated with nicotine accumulation levels, suggesting that natural selection plays a role in regulating secondary metabolism (Kazan, K. and Manners, JM (2013). MYC2: themaster in action. Mol Plant, 6, 686-703.). Furthermore, related patents utilize... NtMYC2a Allelic variation can be used to regulate nicotine content, for example, by screening tobacco germplasm resources with high and low nicotine content. NtMYC2a A single nucleotide sequence shift from A to G can result in a significant difference in nicotine accumulation in tobacco (Xie, H., et al. (2025). Molecular marker nicotine associated SNP 1 for identifying high or low nicotine content of tobacco and its kit as well as use thereof. US Patent, US12215394B2.). Therefore, NtMYC2a Natural variations not only have scientific research value but also play an important role in industrial applications. Besides coding region variations, NtMYC2a Promoter polymorphism also plays an important regulatory role in nicotine biosynthesis. Differences in the G-box and other cis-regulatory elements within the promoter affect... NtMYC2a The response to JA signals alters the induction amplitude of downstream genes. Promoter haplotypes in different cultivars and... NtMYC2aExpression levels are correlated with nicotine content, indicating that transcriptional regulation and coding sequence variations jointly determine metabolite levels (Shoji, T. and Hashimoto, T. (2011). Tobacco MYC2 regulates jasmonate-inducible nicotine biosynthesis genes directly and by way of the NIC2 -Locus ERF genes, Plant Cell Physiol, 52,1117-1130.; Dombrecht, B., etal. (2007). MYC2 differentially modulates diverse jasmonate-dependent functions in Arabidopsis (Plant Cell, 19, 2225-2245). These findings highlight that the regulation of nicotine biosynthesis is a complex trait involving the combined effects of gene sequences and transcriptional network architecture. Given... NtMYC2a Allelic variation holds significant promise for applications in tobacco breeding and metabolic engineering. Therefore, combining gene editing and information on natural variant sites allows for the design of tobacco lines with precisely controllable nicotine levels. For example, the 5-base deletion allelic variation of TI313 or similar low-activity alleles can be introduced into commercially cultivated varieties to breed low-nicotine lines; while selecting high-activity alleles... NtMYC2a Alleles and NtERF189 Overexpression of this transcription factor can increase the alkaloid content of specialty tobaccos, demonstrating its dual value in public health and industrial applications.

[0007] In summary, NtMYC2a It is a core transcriptional regulator of nicotine biosynthesis in tobacco, playing a pivotal role in the JA signaling pathway and transcriptional metabolic regulatory network. By integrating functional analysis and natural variation studies, this research not only provides a theoretical foundation for basic research on tobacco secondary metabolism but also offers actionable strategies for developing low-nicotine and high-functionality tobacco varieties. However, the disclosed technologies in the prior art... NtMYC2a Natural allelic variants are limited in type, resulting in generally low nicotine-lowering effects. Furthermore, most allelic mutations involve multiple base deletions or point mutations, leading to poor stability and limited breeding value. Therefore, there is an urgent need to discover new nicotine-lowering varieties with stronger effects, genetic stability, and ease of breeding application. NtMYC2a Natural allelic variations provide key genetic resources for breeding low-nicotine tobacco. Summary of the Invention

[0008] The purpose of this invention is to provide tobacco. NtMYC2This study describes an allelic mutant and its application. This mutant significantly reduces the total alkaloid (nicotine) content in tobacco leaves, exhibiting genetic stability and high safety. It also provides the encoded protein, recombinant vector, host cell, low-total-alkaloid tobacco plants, and their applications, offering core gene resources and technical support for large-scale breeding of low-total-alkaloid tobacco.

[0009] In a first aspect, the present invention provides a tobacco NtMYC2a Gene allelic variant mutants, the tobacco NtMYC2a The nucleotide sequence of the gene allelic variant mutant is shown in SEQ ID NO:2, derived from wild-type tobacco. NtMYC2a The nucleotide sequence of the wild-type tobacco NtMYC2a gene is formed by the deletion of nucleotide T at position 1239. The nucleotide sequence is shown in SEQ ID NO:1.

[0010] Preferably, the tobacco NtMYC2a The amino acid sequence encoded by the gene allelic variant mutant is shown in SEQ ID NO:4.

[0011] Preferably, the mutant is derived from the wild type. NtMYC2a The deletion of nucleotide T at position 1239 of the gene triggers a frameshift mutation, leading to premature termination of translation.

[0012] Specifically, compared to the tobacco shown in SEQ ID NO:3 NtMYC2a The amino acid sequence encoded by the gene resulted in a frameshift mutation after amino acid position 412 (proline P) and a termination mutation at position 430. The tobacco contained therein... NtMYC2a Tobacco leaves containing allelic mutants and tobacco NtMYC2a The alkaloid content is lower in tobacco leaves compared to those with genetically modified varieties; wherein, the alkaloids are total alkaloids.

[0013] Secondly, the present invention provides a recombinant carrier comprising the aforementioned tobacco. NtMYC2a Gene allelic variant mutant.

[0014] Thirdly, the present invention provides a host cell into which the recombinant vector is introduced, or into which the tobacco is integrated into the genome. NtMYC2a Gene allelic variant mutant.

[0015] Fourthly, the present invention provides a tobacco plant with low total alkaloids, the genome of which contains the tobacco described above. NtMYC2a Gene allelic variant mutant.

[0016] Fifthly, the present invention provides the aforementioned tobacco. NtMYC2a Application of gene allelic mutants in the cultivation of tobacco with low total alkaloids.

[0017] Preferably, in the application described, the total alkaloid content of tobacco leaves containing the mutant is significantly reduced.

[0018] Specifically, in the above applications, the tobacco is included. NtMYC2a Tobacco leaves containing allelic mutants and tobacco NtMYC2a The alkaloid content in tobacco leaves with the gene was significantly lower than that in tobacco leaves with the gene.

[0019] The alkaloids mentioned are total alkaloids.

[0020] In a sixth aspect, the present invention provides a kit for detecting low total alkaloids in tobacco, comprising primer pairs that specifically recognize the mutant of claim 1, the nucleotide sequences of which are shown in SEQ ID NO:5 and SEQ ID NO:6.

[0021] The present invention has the following advantages: 1. This invention includes the tobacco described. NtMYC2a Tobacco leaves containing allelic mutants and tobacco NtMYC2a Compared to tobacco leaves containing the mutant of this invention, the average total alkaloid content of tobacco leaves containing the mutant of this invention is 0.28%, while that of wild-type Yunyan 87 (YN87) is 1.57%, representing a reduction of 82.17% in total alkaloid content, reaching the level of gene editing knockout. Therefore, the tobacco of this invention... NtMYC2a Allelic mutants can significantly reduce the total alkaloid content in tobacco leaves and can be used to create tobacco materials with reduced nicotine content.

[0022] 2. The mutants of this invention are allelic variations naturally occurring in tobacco, without the integration of exogenous genes, thus avoiding the off-target risks of gene editing and the biosafety controversies of genetically modified organisms. They meet the requirements of green breeding and are easy to pass variety approval and promotion.

[0023] 3. The mutants of this invention can be rapidly introduced into mainstream cultivated varieties through conventional hybridization breeding without the need for complex gene editing operations, resulting in a short breeding cycle and low cost; the accompanying detection kit can achieve early and accurate screening of low nicotine traits, improving breeding efficiency.

[0024] 4. The mutant of this invention loses translation by premature termination. NtMYC2a It functions normally, inhibiting nicotine synthesis at the source with stable effects; it can be used to cultivate different types of tobacco varieties such as low-nicotine flue-cured tobacco, air-cured tobacco, and burley tobacco, with broad application prospects. Attached Figure Description

[0025] Figure 1 For the present invention NtMYC2a In wild-type tobacco and NtMYC2a Sequencing results in tobacco with allelic variant mutants; Figure 2 Tobacco of the present invention NtMYC2a Gene allelic variant mutants and wild-type tobacco NtMYC2a The results of gene coding sequence alignment; where the gene represents tobacco. NtMYC2a The coding sequence of gene allelic variant mutants. NtMYC2a Indicates wild-type tobacco NtMYC2a The coding sequence of a gene; Figure 3 This is the amino acid sequence alignment result of the protein encoded by the allelic mutant of the tobacco NtMYC2a gene in this invention and the protein encoded by the wild-type tobacco NtMYC2a gene; among which 429 amino acid sequences are from tobacco. NtMYC2a The amino acid sequence encoded by gene allelic variant mutants. NtMYC2a Indicates wild-type tobacco NtMYC2a The 659-amino acid sequence encoded by the gene; Figure 4 This invention includes NtMYC2a Tobacco with allelic variants of the gene and wild-type tobacco NtMYC2a The total alkaloid content (%) of the middle tobacco leaves after topping. * This indicates a highly significant difference (P < 0.05). Wild-type tobacco NtMYC2a and allelic variant mutant tobacco NtMYC2a The total alkaloid content of tobacco leaves. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0028] This invention provides a tobacco method for reducing the alkaloid content in tobacco leaves. NtMYC2a A single nucleotide mutant of the gene. The tobacco... NtMYC2a The nucleotide sequence of the gene allelic variant mutant is shown in SEQ ID NO:2.

[0029] like Figure 2 As shown, the nucleotide sequence is similar to that of wild-type tobacco as shown in SEQ ID NO:1. NtMYC2a Compared to genes, tobacco NtMYC2aThe allelic variant mutant has a deletion of nucleotide T at position 1239.

[0030] like Figure 3 As shown, compared with wild tobacco NtMYC2a Compared to the amino acid sequence encoded by the gene (SEQ ID NO:3), tobacco NtMYC2a The amino acid sequence encoded by the gene allelic mutant undergoes a frameshift mutation after amino acid 412 (proline P) and a termination mutation at 430 (SEQ ID NO:4).

[0031] Wild tobacco NtMYC2a The coding sequence of the gene (1980 bp) is as follows: Wild tobacco NtMYC2a The coding sequence (1290 bp) of the gene allelic variant mutant is as follows: Wild tobacco NtMYC2a The amino acid sequence encoded by the gene (659 aa) is as follows: - (SEQ ID NO:3) tobacco NtMYC2a The amino acid sequence (429 aa) encoded by the gene allelic variant mutant is as follows: MTDYRIPTMTNIWSNTTSDDNMMEAFLSSDPSSFWPGTTTTPRSSVSPAPAPVTGIAGDPLKSMPYFNQESLQQRLQTLIDGARKGWTYAIFWQSSVVDFASPSVL GWGDGYYKGEEDKNKRKTASFSPDFITEQAHRKKVLRELNSLISGTQTGGENDAVDEEVTDTEWFFLISMTQSFVNGSGLPGLAMYSSSPIWVTGTERLAVSHCERARQ AQGFGLQTIVCIPSANGVVELGSTELIFQTADLMNKVKVLFNFNIDMGATTGSGSGSCAIQAEPDPSALWLTDPASSVVEVKDSSNTVPSRNTSKQLVFGNENSENGN QNSQQTQGFFTRELNFSEYGFDGSNTRYGNGNANSSRSCKPESGEILNFGDSTKRSACSANGSLFSGQSQFGPGPAEENKNKNKKRSPHQEEATMKESFHLFRV-(SEQ ID NO:4) Example 1 This embodiment uses tobacco germplasm resources with different nicotine contents and employs TILLING technology to screen for mutants related to transcriptional regulatory genes involved in nicotine biosynthesis, obtaining mutants containing the above-mentioned... NtMYC2a Tobacco with gene allelic variant mutants.

[0032] 1. Screening for natural mutants of the target gene Using TILLING technology to screen for transcriptional regulatory genes of tobacco nicotine. NtMYC2a Natural allelic mutants were obtained. Tobacco germplasm resources with different nicotine contents were sown in a greenhouse. Once the seedlings reached the seedling stage, individual leaves were harvested, and genomic DNA was extracted using a DNA extraction kit (QIAGEN, 69106) according to the kit's instructions. The obtained DNA samples were arranged sequentially. 2 μl of each DNA sample was taken and its concentration was determined using a 16-channel Tecan Infinite M200 instrument. All DNA samples were then diluted to 40 ng / μl, and eight samples were pooled to create an 8-fold DNA pool for tilling analysis.

[0033] Designed using Primer 3 online software NtMYC2a Gene-specific TILLING analysis primers NtMYC2a -F / NtMYC2a -R, whose nucleotide sequence is as follows: NtMYC2a-F: 5'- GATTTGATGGAAGTAATACTCGGTA-3' (SEQ ID NO: 5); NtMYC2a -R:5'-AGATTCGATTTGGTTCCTCAAGTCC-3' (SEQ ID NO:6).

[0034] The target fragment amplified by the above primers is 623 bp (wildtype) or 622 bp (mutant). The above DNA sample was amplified according to the following reaction system and procedure.

[0035]

[0036] The reaction program was as follows: 95℃ for 3 min; (94℃ for 30 s, 62℃ for 30 s, -1℃ / cycle, 72℃ for 1 min) for 7 cycles; (94℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min) for 40 cycles; 72℃ for 5 min; 99℃ for 10 min; (70℃ for 20 s, -0.3℃ / cycle) for 70 cycles; and stored at 4℃.

[0037] After the reaction, the amplification products were analyzed by capillary electrophoresis to screen for nicotine synthesis transcriptional regulatory genes. NtMYC2a Tobacco germplasm resources with low nicotine content due to nucleotide mutations were obtained and sequenced for verification. The results are as follows: Figure 1 As stated above.

[0038] Obtain a mutant, whose NtMYC2a The coding sequence (CDS) of the gene contains a T deletion at nucleotide position 1239. Figure 2 This causes a frameshift mutation in the amino acid sequence encoded by the gene, ultimately leading to premature termination of the gene's translation. Figure 3 These low-nicotine germplasm resources were grown under greenhouse conditions, and nucleotide sequencing was used to identify those containing homozygous... NtMYC2a Tobacco nicotine mutants with allelic gene variation were self-crossed and propagated.

[0039] The tobacco shown NtMYC2a The mutation is caused by a frameshift mutation resulting from the deletion of nucleotide T at position 1239 of the gene, which leads to premature termination of the gene's translation.

[0040] Example 2 This invention utilizes SNP markers to identify tobacco materials with low total alkaloid content. Determination of total alkaloid content in tobacco leaves According to standard YC / T 160 The total alkaloid content of tobacco materials was tested in 2002. The selected tobacco materials were wild-type tobacco (Yunyan 87) in its prime growing season and those obtained in Example 1 containing homozygous alkaloids. NtMYC2a The allelic mutant (FCT12) was used as the treatment target. At least five wild-type tobacco plants (Yunyan 87) and plants containing homozygous allelic variants were collected. NtMYC2a Allelic mutant tobacco plants were topped during the budding stage. Fourteen days after topping, wild-type tobacco Yunyan 87 and plants containing homozygous alleles were harvested. NtMYC2a The middle leaf of the gene allelic mutant was dried and ground into powder.

[0041] Tobacco samples were extracted with a 5% acetic acid aqueous solution. The total alkaloids (calculated as nicotine) in the extract were reacted with p-aminobenzenesulfonic acid and cyanogen chloride, which was produced by the online reaction of potassium cyanide and chloramine T. The reaction products were measured at 460 nm using a colorimeter.

[0042] Required instruments and equipment for measurement: continuous flow analyzer (API, USA; SEALAA3, Germany; ALLIANCE, France).

[0043] Prepare reagents: Brij 35 solution (polyethoxylated lauryl ether): Add 5 drops of 22% Brij 35 to water and stir well.

[0044] Buffer solution A: Weigh 2.35g sodium chloride (NaCl) and 7.60g sodium borate (Na2B4O7·10H2O), dissolve in water, then transfer to a 1L volumetric flask, add 1mL Brij 35, and dilute to 1L with distilled water. Filter with qualitative filter paper before use.

[0045] Buffer solution B: Weigh 26g disodium hydrogen phosphate (Na2HPO4), 10.4g citric acid [COH(COOH)(CH2COOH)2·H2O], and 7g p-aminobenzenesulfonic acid (NH2C6H4SO3H). Dissolve in water, then transfer to a 1L volumetric flask. Add 1mL Brij 35 and dilute to 1L with distilled water. Filter with qualitative filter paper before use.

[0046] Chloramine T solution (N chlorine 4 Sodium methylbenzylthioamide [CH3C6H4SO2N(Na)Cl·3H2O]: Dissolve 8.65g of chloramine T in water, then transfer to a 500mL volumetric flask and dilute to the mark with water. Filter with qualitative filter paper before use.

[0047] 0.22mol / L NaOH buffer: 8.8g NaOH, 26.0g Na2HPO4, 10.4g C6H8O7·H2O (citric acid monohydrate), dissolved in water and brought to a final volume of 1000mL.

[0048] p-Aminobenzenesulfonic acid buffer: Weigh 7g of C6H7NO3S (p-aminobenzenesulfonic acid), 26.0g of Na2HPO4, and 10.4g of C6H8O7·H2O (citric acid monohydrate), dissolve in water and bring the volume to 1000mL.

[0049] Chloramine T: Weigh 1.2g of chloramine T, dissolve it in pure water and bring the volume to 100mL, then store it in a brown reagent bottle.

[0050] Potassium cyanide: 0.4g KCN, dissolved in pure water and brought to a final volume of 100mL. NaCO3 solution: 10g NaCO3, dissolved in distilled water and brought to a final volume of 1000mL.

[0051] Analytical procedure: Weigh 0.3g of smoke sample into a 150mL Erlenmeyer flask or plastic bottle (accurate to 0.0001g); add 50mL of 5% acetic acid solution and stopper the bottle; extract on a regular shaker for 30min at 170r / min; filter through filter paper and transfer to the instrument. (If the concentration of the sample solution exceeds the concentration range of the working standard solution, it should be diluted).

[0052] Calculation and expression of results: The total alkaloid content (total plant alkaloids) on a dry basis is calculated using the following formula: In the formula: C – Instrumental observation value of total alkaloids in the sample solution, in mg / mL; V – Volume of the extract, in mL; m — mass of the sample, in mg; W – Moisture content of the sample, in percentage.

[0053] The average of the two measurements was used as the result, with the result accurate to 0.01%.

[0054] Measurements showed that, under greenhouse growing conditions, the average total alkaloid content of the middle leaves of wild-type tobacco Yunyan 87 (YN87) after topping was 1.57%; while the corresponding content of homozygous alkaloids was... NtMYC2a The average total alkaloid content of tobacco leaves from the FCT12 allelic mutant was 0.28%, containing... NtMYC2a Compared with wild-type tobacco, the total alkaloid content in the leaves of the allelic mutant was reduced by 82.17%, which was significant (P < 0.05) (Table 1 and ). Figure 4 ).

[0055] Table 1 Wild-type tobacco and its contents NtMYC2a Total alkaloid content in allelic mutant tobacco leaves

[0056] The table " * "" indicates a significant difference (P < 0.05).

[0057] In summary, the present invention provides a tobacco NtMYC2a The gene allelic variant mutant, whose nucleotide sequence is shown in SEQ ID NO:2, is a tobacco species whose nucleotide sequence is shown in SEQ ID NO:1. NtMYC2a The gene was obtained by a frameshift mutation caused by the deletion of nucleotide T at position 1239, leading to premature termination of translation. This tobacco... NtMYC2a Allelic mutants can significantly reduce the total alkaloid content in tobacco leaves and can be used to create tobacco materials with reduced nicotine content.

[0058] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A type of tobacco NtMYC2a Gene allelic variant mutants, characterized by, The tobacco NtMYC2a The nucleotide sequence of the gene allelic variant mutant is shown in SEQ ID NO:2, derived from wild-type tobacco. NtMYC2a The nucleotide sequence of the wild-type tobacco NtMYC2a gene is formed by the deletion of nucleotide T at position 1239. The nucleotide sequence is shown in SEQ ID NO:

1.

2. The tobacco according to claim 1 NtMYC2a Gene allelic variant mutants, characterized by, The tobacco NtMYC2a The amino acid sequence encoded by the gene allelic variant mutant is shown in SEQ ID NO:

4.

3. The tobacco according to claim 1 NtMYC2a Gene allelic variant mutants, characterized by, The mutant is derived from the wild type. NtMYC2a The deletion of nucleotide T at position 1239 of the gene triggers a frameshift mutation, leading to premature termination of translation.

4. A recombinant vector, characterized in that, Tobacco comprising the tobacco of claim 1 NtMYC2a Gene allelic variant mutant.

5. A host cell, characterized in that, The recombinant vector of claim 4 is introduced, or the tobacco of claim 1 is integrated into the genome. NtMYC2a Gene allelic variant mutant.

6. A tobacco plant with low total alkaloids, characterized in that, The genome contains the tobacco as described in claim 1 NtMYC2a Gene allelic variant mutant.

7. The tobacco according to any one of claims 1 to 3 NtMYC2a Application of gene allelic mutants in the cultivation of tobacco with low total alkaloids.

8. The application according to claim 7, characterized in that, The total alkaloid content in tobacco leaves containing the mutant was significantly reduced.

9. A kit for detecting low total alkaloids in tobacco, characterized in that, The primer pair comprises a primer pair that specifically recognizes the mutant of claim 1, the nucleotide sequences of which are shown in SEQ ID NO:5 and SEQ ID NO:6.

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