CN113174395B - Mutant gene DHQ-SDH1 related to smoke phenol release amount - Google Patents

Abstract

The invention belongs to the technical field of tobacco biology, and particularly relates to a plurality of mutant gene sequences related to the release amount of phenol in smoke. Mutant gene related to phenol release amount of smokeDHQ‑SDH1Is tobacco dehydroquinic acid dehydrogenase/shikimic acid dehydrogenase mutant gene. In the present application, the inventor treats tobacco byDHQ‑SDH1、TyrAFurther research on the genes shows that the two genes are highly related to the content of the released tyrosine and the released phenol in the tobacco. Based on the discovery, the method further combines the Tilling screening technology, carries out protein tertiary structure prediction by analyzing and predicting sense mutation generated by the encoded protein, and combines actual field experiment verification at the same time, and the result shows that the tyrosine content and the smoke phenol release amount in the tobacco are obviously reduced after the two genes are mutated. Based on the research results, certain theoretical basis and technical basis can be established for the cultivation of new varieties of tobacco leaves with low phenol content.

Description

Mutant gene DHQ-SDH1 related to smoke phenol release amount

Technical Field

The invention belongs to the technical field of tobacco biology, and particularly relates to a plurality of mutant gene sequences related to the release amount of phenol in smoke.

Background

Phenol, one of the main harmful components in smoke, is rapidly distributed in all tissues after being inhaled along with the smoke, has remarkable mucosal permeability, and can cause tissue necrosis and erosion and shedding. Therefore, the control of phenol content in smoke is important from the viewpoint of reducing the smoking hazard of tobacco.

The research shows that tobacco leaf protein, free tyrosine (Tyr), chlorogenic acid (CGA) and the like are the main precursors of smoke phenol. Tyr (tyrosine) as source of phenolic group in plant bodyMost are stored as proteins in roots, stems and leaves. The existing research shows that the precursor of Tyr in the plant can generate free Tyr under the action of arogenate dehydrogenase (TyrA). On the other hand, Tyr is one of Aromatic Amino Acids (AAA), and is closely related to the shikimic acid pathway of AAA biosynthesis in plants. In the shikimic acid pathway, Dehydroquinate Dehydrogenase (DHQ) and shikimic acid dehydrogenase (SDH) can be fused to form bifunctional enzyme (DHQ-SDH), and further participate in catalyzing the generation of shikimic acid. Research finds that the tobaccoDHQ-SDH1The series of RNAi tobacco lines of (a) exhibits different phenotypes; when the silencing degree is 40-60%, the downstream aromatic metabolites are slightly reduced, and the tobacco does not show a remarkable phenotype; while the degree of silencing is 60-85%, the downstream aromatic metabolites are significantly reduced and the tobacco shows a slightly dwarf phenotype (Ding L et al. Functional analysis of the developmental biofunctional tobacco 3-dehydrogenate dehydrogenase/shikimate dehydrogenase in transgenic tobacco plants. Journal of Experimental Botany, 2007). Further research shows that the number of the corns is 4ZmTyrAThe expression patterns of the gene members, 4 members, are different, whereinZmTyrA1The mutation(s) in (e) results in a decrease in the amino acid content of the aromatic amino acid (Holding DR et al. Identification and characterization of the mail aromatic dehydrogenase gene family. Journal of experimental botanic, 2010). This indicates that it is desirable to have,DHQ-SDH1andTyrAit is suitable as a target gene for genetic engineering modification. Therefore, based on these biological studies, if the content of the tobacco-related precursor can be controlled by related biotechnology, it is possible to effectively and fundamentally reduce the release amount of phenol in smoke.

Disclosure of Invention

Based on TILLING (Targeted Induced Local losses IN genes, directionally Induced gene mutation) technology, the application aims to provide a plurality of mutated gene sequences related to phenol precursors, thereby laying a certain technical foundation for breeding new varieties of related low-phenol release tobacco.

The technical scheme provided by the application is detailed as follows.

Mutant gene related to phenol release amount of smokeDHQ-SDH1The mutant geneDHQ-SDH1The mutant gene of tobacco dehydroquinate dehydrogenase/shikimate dehydrogenase has a base sequence shown in SEQ ID No.1, wherein the specific nucleic acid fragment is 24-1530 base;

the tobacco dehydroquinic acid dehydrogenase/shikimic acid dehydrogenase mutant gene (DHQ-SDH1) The amino acid sequence of the encoded tobacco DHQ-SHD1 protein is shown in SEQ ID NO.2, and the encoded tobacco DHQ-SHD1 protein consists of 515 amino acid residues, wherein amino acids 8-510 are conserved 3-dehydroquinic acid dehydratase/shikimate dehydrogenase structural domains.

The tobacco dehydroquinic acid dehydrogenase/shikimic acid dehydrogenase mutant geneDHQ-SDH1Specific CDS sequences and corresponding amino acid sequence mutation sites compared to wild type are referenced below:

A262G→K88E，G286A→E96K，G295A→E99K，G298A→V100I，G307A→E103K，G317A→C106Y，G445A→G149R，C637T→P213S，C647T→P216L，A863G→D288G。

mutant gene related to phenol release amount of smokeTyrAThe mutant geneTyrAThe gene is a tobacco aroate dehydrogenase gene, the base sequence of which is shown as SEQ ID NO.3, wherein the specific nucleic acid segment is 1-708 bases;

the tobacco aronic acid dehydrogenase geneTyrAThe amino acid sequence of the encoded tobacco TyrA protein is shown in SEQ ID NO.4 and consists of 243 amino acid residues, wherein the amino acids from 1 to 236 are NADB _ Rossmann superfamily conserved domains.

The tobacco aronic acid dehydrogenase geneTyrACompared with wild type, the CDS sequence and the mutation site of the corresponding amino acid sequence are referred to as follows: G118A → E40K, G185A → C62Y, C217T → L73F, G229A → E77K, C404T → S135L, C485T → T162I.

The tobacco dehydroquinic acid dehydrogenase/shikimic acid dehydrogenase mutant gene (DHQ-SDH1) Or the tobacco DHQ-SHD1 protein has tyrosine content in tobacco leaf or the phenol release amount in smokeThe application in regulation and control, the protein DHQ-SHD1 of tobacco is related to the tyrosine content and the release amount of phenol in smoke in plant leaves, and after the expression amount of the protein is reduced, the tyrosine content and the release amount of phenol in smoke in leaves are obviously reduced; by regulating tobacco using biotechnological methodsDHQ-SDH1The gene expression level, and further the expression level of the DHQ-SHD1 protein of the tobacco can be regulated, and the tyrosine content and the smoke phenol release amount in the tobacco leaves can be regulated and controlled.

The tobacco aroate dehydrogenase mutant gene or tobaccoTyrAIn the application of the protein in regulating and controlling the tyrosine substance content or the smoke phenol release amount of the tobacco leaves, the TyrA protein of the tobacco is related to the tyrosine content and the smoke phenol release amount of the plant leaves, and after the expression amount of the protein is reduced, the tyrosine content and the smoke phenol release amount of the leaves are obviously reduced; by regulating tobacco using biotechnological methodsTyrAThe gene expression level, and further the tobacco TyrA protein expression level can be regulated, and the tyrosine substance content and the smoke phenol release amount in the tobacco leaves can be regulated and controlled.

Using the tobacco dehydroquinate dehydrogenase/shikimate dehydrogenase mutant gene (DHQ-SDH1) The method for cultivating a new variety of tobacco comprises constructing a tobacco product containing a gene having a sequence of mutations, a gene transfer technique, a transient expression technique or a genome editing techniqueDHQ-SDH1The virus-induced silencing vector, RNAi interference vector, overexpression vector or genome editing vector of the gene are used for transforming tobacco and screening to obtain a new tobacco variety with reduced tyrosine content and smoke phenol release amount.

Using said tobacco aroate dehydrogenase mutant gene (a)TyrA) The method for cultivating a new variety of tobacco comprises constructing a tobacco product containing a gene having a sequence of mutations, a gene transfer technique, a transient expression technique or a genome editing techniqueTyrAThe virus-induced silencing vector, RNAi interference vector, overexpression vector or genome editing vector of the gene are used for transforming tobacco and screening to obtain a new tobacco variety with reduced tyrosine content and smoke phenol release amount.

Tobaccotyra/dhq-sdh1Method for breeding new tobacco variety with double mutants by using mutant genesTyrATobacco and tobacco containing mutant geneDHQ-SDH1The tobacco is selected as parent and obtained by genetic hybridizationtyra/dhq-sdh1A double mutant homozygote;

the mutant geneTyrAIn this mutant, compared with the wild typeTyrAThe 217 th base of the gene is mutated C → T, the corresponding 73 rd amino acid is mutated L → F;

the mutant geneDHQ-SDH1In this mutant, compared with the wild typeDHQ-SDH1The 863 th base of the gene is mutated A → G and the corresponding 288 th amino acid is mutated D → G;

specifically, the method comprises the following steps:

simultaneously seeding tobaccotyraAnddhq-sdh1mutants and cultivation;

genetic hybridization is carried out at the flowering stage, and specifically, the hybridization: to be provided withdhq-sdh1Using the mutant as female parent, removing excessive bud, selecting unopened bud, removing stamen, and collectingtyraPollen of mutant and applying it todhq-sdh1The stigmas of the pistils of the mutants are put on the stigmas and then bagged for reserving seeds; after the hybridized seeds are mature, collecting the seeds;

preferably, the homozygosity is ensured by means of a multi-generation seeding screen, in particular:

about 30 days after F1 generation, DNA of tobacco was extracted and sequenced, and the DNA contained thereinTyrAAndDHQ-SDH1reserving the mutant;

about 30 days after F2 generation, DNA of tobacco was extracted and sequenced, and the DNA contained thereinTyrAAndDHQ-SDH1reserving the mutant;

about 30 days after F3 sowing, extracting DNA of tobacco, sequencing, and determining the seed reservation of F2 progenytyra/dhq-sdh1Double mutation as the finaltyra/dhq-sdh1Double mutant homozygotes.

It has been shown that tobaccoDHQ-SDH1The gene family comprises a plurality of family members which are expressed in a plurality of plant organs, but the functions of different members are different, and the expression quantity of part of genes has certain correlation with the phenotypic change of plants (Zhanglin, and the like, the analysis of common tobacco DHD-SDH gene family, tobacco science and technology, 2018; Ding L et al, Funct)An ionic analysis of the addressing biofunctional tobacaco enzyme 3-hydrolysis dehydrogenase/shikimate dehydrogenase in transgenic tobacaco plants, Journal of Experimental Botany, 2007). The number and expression pattern of TyrA proteins vary from species to species (Rippet P et al, Purification and kinetic analysis of the two recombinant expression of the microorganisms of Arabidopsis thaliana, European Journal of biological chemistry, 2002; Holding DR et al, Identification and characterization of the microbial expression, Journal of experimental biology, 2010).

Based on the existing research, the inventor in the application carries out the research on the tobaccoDHQ-SDH1、TyrAFurther research on the genes shows that the two genes are highly related to the content of the released tyrosine and the released phenol in the tobacco. Based on the discovery, the prediction of the tertiary structure of the protein is carried out by further combining with the Tilling screening technology and analyzing and predicting the sense mutation generated by the encoded protein, and the results are verified by combining with the actual field experiment, so that the tyrosine content and the smoke phenol release amount in the tobacco are obviously reduced after the two genes are mutated. Based on the research results, a certain theoretical basis and technical basis can be established for the cultivation of new varieties of tobacco leaves with low phenol content.

Drawings

FIG. 1 shows the Tyr content results in mutant tobacco leaves;

FIG. 2 is the results of total protein content in mutant tobacco leaves;

FIG. 3 shows the result of maximum photochemical efficiency of the mutant tobacco leaves;

FIG. 4 is a result of the release amount of phenolic substances in smoke of a cigarette sample prepared from a mutant tobacco leaf;

FIG. 5 shows the Tyr content results in double mutant tobacco leaves;

FIG. 6 is a result of the release amount of phenolic substances in smoke of a cigarette sample prepared by using double mutant tobacco leaves.

Detailed Description

The technical solution of the present application is further explained with reference to the following examples.

Example 1

It should be noted that, since all the related mutant sequences were obtained by screening based on the EMS mutant library by the Tilling technique, the construction and screening process of the related mutant library in this example are summarized as follows.

(I) EMS mutant library construction

EMS treatment is carried out on the ordinary tobacco Yunyan 87 seeds (provided by tobacco agricultural science research institute in Yunnan province) by referring to conventional operation, the treated seeds are used as M1 generation mutant seeds for field planting, and M2 generation seeds are harvested by bagging and selfing.

Finally, about 2200 parts of EMS mutant plants of M2 generation are harvested after field planting, and the collected leaves are subjected to genome DNA extraction. During determination, all sample DNA concentrations are diluted to 40 ng/. mu.L, each 8 DNA samples are mixed to form an 8-time mixing pool, and the mixing pool is stored in a 96-well plate for subsequent mutant screening. Finally, a DNA library containing 1842M 2 generation Yunnan tobacco 87 mutant was established.

(II) Tilling screening

Based on the existing studies, respectively related to Tyr (tyrosine) synthesisDHQ-SDH1The gene,TyrAAnd (3) taking the gene as a target gene, and carrying out Tilling screening on the EMS mutant library constructed in the step (I).

(1) Designing primers and carrying out PCR amplification

To is directed atDHQ-SDH1When a gene is selected, the DNA sequence of the gene is too long, and the gene is segmented. Based on the existingDHQ-SDH1Specific primers were designed for the GS1 sequence (823 bp, containing the second and third exons) and the GS2 sequence (947 bp, containing the fourth exon) of the gene.

DHQ-SDH1GS1 sequence of gene:

AAAATCTTACTGATTGGGAATTTGTTGACATTTTACTCCAATTACTGAAAATGGTTCTCTTCGATTTTGTGATCCAATCTGCCAAAAAGTGAGGCGAATTGACCAAAAGCTTGAACCTTTGCGAAATCTTGCTGATTGAGAATTTGGGGTTTGTTTTCTCAGGCCAAATTCGCCGAGATATTCTTGCAGAGAAAACTGGAAGAAAACATGCTTGCAAGTTCTGAAATTGGCTGTTGAATTGGACGTCGAGTTCGTTGAAGTTGACCGCGAGGTTAGTCCAACTGATTTTCTCAAATTACTCTCTAATTGATACAGTTTTATTGAAGCTAAATAAATCAAAATAATCAAAAAAGAATCTTGCTTAAGAGAGTAAATCATTGTACAAGTTTCCTCTTTTATGTTTATATGATGATTTAACAATGATAGCAGGTTTACTATCATTTTTATCAGGTTATATATTTTTTACATAATCAGCTTATAGAAATTAAACTCTTTGCAACTTTATAGGTTGCTTGCGATGAGGTCATCTGTGAATTAATGACCAAACGATCGAACTGCAAGATAATTGCCTCCAGTCATGTGAATGGTGGAAATCCTACAAAAGAGAGACTTTGTAATTTAATTGCAAACCTGCAGTCAACAGGAGCAGATATCATCAAATTAGTGATTGATGTAGCTTATATTACAGATGTTGCACCAGTTTTTCATATGCTTACACATTGTCAGGTATTTCTTTTCTTGCTTTAACTTGCACTTTTTCCTTGCAAAAACACACCCTCAACTTGCTTTTAGCTTTACACCTATTGATAAATTTGAGGTGTTC

DHQ-SDH1GS2 sequence of gene:

TAGCTTTACACCTATTGATAAATTTGAGGTGTTCAGTATTTATATTCTCACTACTGCGTTCGTTGCAATTTTAGTGATCATTCACATATATAGTTATCATCCGTGTAAAAGATACTGGGTTTTCTTGAACCCGTGGAAACTACATTGCATCCCCTCTGATGGTATACTTGCTCAATTCAACTTACCGACAAGAAGAGAGAAAAGGGGTCTGCTTTAAAATCTTTCTACGCGAACTGCTGTTGATATACTTAATTTTTCTGCGATGAAGTGTTCATTAGAAAATACTCTCATATTTTCTTCCATATATATGGCCTCTTATTTGTATCTGCAGCCATCTCTTGGGCTTATATGGTTGTAAAGAAAATCTTTTAATGCTAGTTTCCTTGTGAGTAGTGATAAGTTTGTTGAACTTCTCTTTTTAAAACTGAGAATGAGGTACTTTTTTGCCTCATTATAAGGTCTCTATGCAGGTGCCTCTAATTGCCAGGGCAGCAGGAGATAGAGGTCTTATAAGCCAACTATTGGGTCCAAAATATGGTGCTTTCTTTGTTTGTGGATCTTTAGGAGGCAAATCCAACCCTGGCTTGCCAGCTTTGACTAGCATTAAAGACGTTTATAAACTGGAATATGTGAACCAAGATACTAGAGTTTTTGGCGTAATCTCTAATCCTGTTGGCCATAGCAAGGGCCCTCTTCTGCACAACCCTGCCTTTAGACATACAGGATACAATGGAATATATGTGCCTCTACTAGTTGATAATATCAAGGAATTTTTTCGGGTCTTCTCATGCAATGACTATGCTGGTTTTAGGTATGCTCCTTATGTACAAGCTAAAATTGTTCTGTGCCGCATACTGATGAATAACTGCCAGATTGTATTAGGAATATCTAAAGATTCCTACTCGTCAAGCATAAATGTATGCCATATATAAGCAACACTCATCTTA

the specific primer design is as follows:

a forward primer:

DHQ1_Tilling_F：5′- AAAATCTTACTGATTGGGAATTTGTTG-3′，

DHQ2_Tilling_F：5′- TAGCTTTACACCTATTGATAAATTTGAGG-3′；

reverse primer:

DHQ1_Tilling_R：5′- GAACACCTCAAATTTATCAATAGGTGT-3′，

DHQ2_Tilling_R：5′- TAAGATGAGTGTTGCTTATATATGGC-3′。

to is directed atTyrAWhen the gene is selected, the gene is based onTyrAThe specific primer is designed according to the partial DNA sequence GS1 (976 bp, containing second and third exons);

TyrAGS1 sequence of gene:

GAATGAACAACTTTTCCAGCTAATGACTGCTTATGTTCATTTATTCACTTGCAGGGATATGGGTGCATTTCTTGAATCAGACAATGAGGTTATTATAATTAGCACGTCGATACTGTCTCTATCACGAGTTGTAGAGTCCATACCATTCCATTGTCTCAAGCGGCCTACACTTTTCGTTGATGTACTCTCAGTTAAAGAACACCCAAAAGATGTCCTTTTGCGAGTATGCAATTCACACCAACATTATTAATTTCAAGACTTTTGACTTCTGCAGCTTCACATACGTTTGTATGTTGAACAGTAATATGTCTAACAACGGTTGCATACTATGTGTTCGAAAGATATTGCCCGAGGAGTGCGACTTGCTGTGTACTCACCCAATGTTTGGACCACAAAGTGGAAAAGATGGATGGACTGATTTGACTTTTATGTACGACATGATTCGAATTAGAGATAAATCTCTGTGTTCCAGTTTTCTGCAAATATTCTCAAGTGAGGTAAGAAGTTCAAATATGCCCAAAGTTGTGTTCGTCAATGTTATTACTTTCTCATTGATTTTGGCTAGCTAGACTAGCATGGTTCATTCTATTACTAGCTCCTTGAGCTGAAATTCTCCTGTATTGACTGGAAAACTGAGGAAAGAGAACCCCATATCTGAGTATTGCACGAATCTTCTGTTGCAGGGGTGCAAAATGCTGGAAATGACTTGTGAAGAGCATGACAAATTGGCTGCTCGAAGTCAATTTCTGACTCACACAATTGGCAGGTAACTTGTGTCACCTACAACTGTAGAAAGAGGTCATCACAATTACCAACTACTATTAATCTTCTTCACCTAACAGGATCTTATCCGAAATGGAGGTTGAACCCACCCCCATAGACACGAAGGGATTTCAGAAACTTGTTCAAGTGGTAAATACAACATTGACTTGTACCTTTTCAAGATTATACTTGTAGCAAATTAAGCGTGTTTTTC

the forward primer is

TyrA_Tilling_F：5′- GAATGAACAACTTTTCCAGCTAATGAC-3′，

The reverse primer is

TyrA_Tilling_R：5′-GAAAAACACGCTTAATTTGCTACAAGTA-3′。

And (3) during PCR amplification, taking DNA extracted from the mutant library sample in the step (I) as a template, and respectively carrying out PCR amplification by using the primer pairs.

During PCR amplification, a 10 mu L amplification system is designed as follows:

DNA template, 0.5 μ L (about 20 ng);

2MM dNTPs，2µL；

KOD FX polymerase (TOYOBO Co.), 0.5. mu.L;

a forward primer of 1 muL;

reverse primer, 1 μ L;

2× KOD FX buffer，5μL。

amplification conditions: 2min at 95 ℃; extending at 98 deg.C, 10s, 64 deg.C, 30s, and 68 deg.C for 1min, 4 cycles, and sequentially reducing annealing temperature by 1 deg.C; at 98 deg.C, 10s, 60 deg.C, 30s, 68 deg.C, 1min, 45 cycles; at 68 deg.C for 5 min; keeping at 4 ℃.

(2) CELI digestion and electrophoresis analysis

Subsequently, CELI enzyme digestion is respectively carried out on the PCR amplification products, and a 6-mu-L enzyme digestion system is designed as follows:

CEL I (Takara Co., Ltd.), 0.2. mu.L;

10× CEL1 buffer，1.2 μL；

PCR product, 2. mu.L;

sterile water, 2.6 μ L.

The cleavage products were subjected to capillary electrophoresis (Advanced Analytical Technologies, USA)) for analysis.

(3) Mutation site sequencing verification and protein function prediction

And (3) screening and determining target EMS mutant individuals based on the enzyme digestion analysis result in the step (2).

Aiming at the target EMS mutant individuals determined by preliminary screening, extracting the genomic DNA of the target tobacco mutant leaf by using a DNA extraction kit (Shanghai Biotechnology); using the extracted genomic DNA as a template, amplifying with DHQ1_ Tilling _ F/R, DHQ2_ Tilling _ F/R, TyrA _ Tilling _ F/R primer pairs, recovering PCR products, and performing sequencing analysis (see conventional procedures in the prior art for related operations).

Furthermore, the on-line tool PROVEAN Protein (http:// Provean. jcvi. org/seq _ sumit. php) was used to perform the prediction analysis of gene function of the sequenced mutant sequence. When analyzed, a score of less than-2.5 indicates that the mutation at that site affects its protein function, and conversely, that the mutation does not affect much.

When screening is carried out based on the Tilling technology, 10 mutants of the Yunyan 87 are obtained by primary screening in a Yunyan 87 mutant libraryDHQ-SDH1Gene mutationType (b). The specific mutation sites, function prediction, scores, and the like are shown in Table 1 below. Among them, mutant M330 underwent E99K and G149R mutations, and M283 underwent P213S mutation.

TABLE 1 screening based on the Tilling techniqueDHQ-SDH1Mutation status of Gene mutant

。

When screening is carried out based on the Tilling technology, 6 mutants of the Yunyan 87 are obtained by screening in a Yunyan 87 mutant libraryTyrAThe type of gene mutation. The specific mutation sites, function prediction, scores, and the like are shown in Table 2 below. Among them, mutant M837 underwent E40K mutation.

TABLE 2 screening based on the Tilling techniqueTyrAMutation status of Gene mutant

。

Sequencing results show that the M330 mutant strainDHQ-SDH1The GS1 sequence of the gene is underlined (257 sites and 642 sites) and comprises 823 bases, and the sequence is as follows:

AAAATCTTACTGATTGGGAATTTGTTGACATTTTACTCCAATTACTGAAAATGGTTCTCTTCGATTTTGTGATCCAATCTGCCAAAAAGTGAGGCGAATTGACCAAAAGCTTGAACCTTTGCGAAATCTTGCTGATTGAGAATTTGGGGTTTGTTTTCTCAGGCCAAATTCGCCGAGATATTCTTGCAGAGAAAACTGGAAGAAAACATGCTTGCAAGTTCTGAAATTGGCTGTTGAATTGGACGTCGAGTTCGTTAAAGTTGACCGCGAGGTTAGTCCAACTGATTTTCTCAAATTACTCTCTAATTGATACAGTTTTATTGAAGCTAAATAAATCAAAATAATCAAAAAAGAATCTTGCTTAAGAGAGTAAATCATTGTACAAGTTTCCTCTTTTATGTTTATATGATGATTTAACAATGATAGCAGGTTTACTATCATTTTTATCAGGTTATATATTTTTTACATAATCAGCTTATAGAAATTAAACTCTTTGCAACTTTATAGGTTGCTTGCGATGAGGTCATCTGTGAATTAATGACCAAACGATCGAACTGCAAGATAATTGCCTCCAGTCATGTGAATGGTGGAAATCCTACAAAAGAGAGACTTTGTAATTTAATTGCAAACCTGCAGTCAACAAGAGCAGATATCATCAAATTAGTGATTGATGTAGCTTATATTACAGATGTTGCACCAGTTTTTCATATGCTTACACATTGTCAGGTATTTCTTTTCTTGCTTTAACTTGCACTTTTTCCTTGCAAAAACACACCCTCAACTTGCTTTTAGCTTTACACCTATTGATAAATTTGAGGTGTT。

sequencing results show that the M283 mutant strainDHQ-SDH1The GS2 sequence of the gene comprises 947 bases, and the underlined (579 site) is the mutated base, which is specifically as follows:

TAGCTTTACACCTATTGATAAATTTGAGGTGTTCAGTATTTATATTCTCACTACTGCGTTCGTTGCAATTTTAGTGATCATTCACATATATAGTTATCATCCGTGTAAAAGATACTGGGTTTTCTTGAACCCGTGGAAACTACATTGCATCCCCTCTGATGGTATACTTGCTCAATTCAACTTACCGACAAGAAGAGAGAAAAGGGGTCTGCTTTAAAATCTTTCTACGCGAACTGCTGTTGATATACTTAATTTTTCTGCGATGAAGTGTTCATTAGAAAATACTCTCATATTTTCTTCCATATATATGGCCTCTTATTTGTATCTGCAGCCATCTCTTGGGCTTATATGGTTGTAAAGAAAATCTTTTAATGCTAGTTTCCTTGTGAGTAGTGATAAGTTTGTTGAACTTCTCTTTTTAAAACTGAGAATGAGGTACTTTTTTGCCTCATTATAAGGTCTCTATGCAGGTGCCTCTAATTGCCAGGGCAGCAGGAGATAGAGGTCTTATAAGCCAACTATTGGGTCCAAAATATGGTGCTTTCTTTGTTTGTGGATCTTTAGGAGGCAAATCCAACTCTGGCTTGCCAGCTTTGACTAGCATTAAAGACGTTTATAAACTGGAATATGTGAACCAAGATACTAGAGTTTTTGGCGTAATCTCTAATCCTGTTGGCCATAGCAAGGGCCCTCTTCTGCACAACCCTGCCTTTAGACATACAGGATACAATGGAATATATGTGCCTCTACTAGTTGATAATATCAAGGAATTTTTTCGGGTCTTCTCATGCAATGACTATGCTGGTTTTAGGTATGCTCCTTATGTACAAGCTAAAATTGTTCTGTGCCGCATACTGATGAATAACTGCCAGATTGTATTAGGAATATCTAAAGATTCCTACTCGTCAAGCATAAATGTATGCCATATATAAGCAACACTCATCTTA。

sequencing results show that the M837 mutant strainTyrAThe GS1 sequence of the gene comprises 976 bases, and the underlined (86 sites) is the mutated base, which is as follows:

GAATGAACAACTTTTCCAGCTAATGACTGCTTATGTTCATTTATTCACTTGCAGGGATATGGGTGCATTTCTTGAATCAGACAATAAGGTTATTATAATTAGCACGTCGATACTGTCTCTATCACGAGTTGTAGAGTCCATACCATTCCATTGTCTCAAGCGGCCTACACTTTTCGTTGATGTACTCTCAGTTAAAGAACACCCAAAAGATGTCCTTTTGCGAGTATGCAATTCACACCAACATTATTAATTTCAAGACTTTTGACTTCTGCAGCTTCACATACGTTTGTATGTTGAACAGTAATATGTCTAACAACGGTTGCATACTATGTGTTCGAAAGATATTGCCCGAGGAGTGCGACTTGCTGTGTACTCACCCAATGTTTGGACCACAAAGTGGAAAAGATGGATGGACTGATTTGACTTTTATGTACGACATGATTCGAATTAGAGATAAATCTCTGTGTTCCAGTTTTCTGCAAATATTCTCAAGTGAGGTAAGAAGTTCAAATATGCCCAAAGTTGTGTTCGTCAATGTTATTACTTTCTCATTGATTTTGGCTAGCTAGACTAGCATGGTTCATTCTATTACTAGCTCCTTGAGCTGAAATTCTCCTGTATTGACTGGAAAACTGAGGAAAGAGAACCCCATATCTGAGTATTGCACGAATCTTCTGTTGCAGGGGTGCAAAATGCTGGAAATGACTTGTGAAGAGCATGACAAATTGGCTGCTCGAAGTCAATTTCTGACTCACACAATTGGCAGGTAACTTGTGTCACCTACAACTGTAGAAAGAGGTCATCACAATTACCAACTACTATTAATCTTCTTCACCTAACAGGATCTTATCCGAAATGGAGGTTGAACCCACCCCCATAGACACGAAGGGATTTCAGAAACTTGTTCAAGTGGTAAATACAACATTGACTTGTACCTTTTCAAGATTATACTTGTAGCAAATTAAGCGTGTTTTTC。

based on the sequencing result of the sense mutation information and the known gene sequence information, the sequence shown in SEQ ID No. 1-4 is finally obtainedDHQ-SDH1Gene, gene,TyrAGenes and their corresponding coding amino acid sequences. For specific coding sequence (i.e., CDS) sequence information, reference may also be made to the following.

Mutant genesDHQ-SDH1The coding sequence of (SEQ ID NO.1, 1548 bp):

ATGGGTTTCAAACAAGACCTTTTAGTGTACACAACATTAGAATGTGAAAGCTTGTCTGAAATGGCAGCTTGTATGCAGAAAGCAAAAGAAGAAGGAGCAGATCTAGTGGAACTTTGCATTGACTCTTTAACTTTCACACACATTTCAGAAGTTGAACACCTTCTCAAACAGAGGACTTTACCCTCCATCGTTTCTTTCAGGCCAAATTCGCCGAGATATTCTTGCAGAGAAAACTGGAAGAAAACATGCTTGCAAGTTCTGGAATTGGCTGTTGAATTGGACGTCAAGTTTGTTAAAATTGACCGCAAGGTTGCTTATGATGAGGTCATCTGTGAATTAATGACCAAACGATCGAACTGCAAGATAATTGCCTCTAGTCATGTGAATGGTGGAAATCCTACAAAAGAGAGACTTTGTAATTTAATTGCAAACCTGCAATCAACAAGAGCAGATATCATCAAATTAGTGATTGATGTAGCTTATATTACAGATGTTGCACCAGTTTTTCATATGCTTACACATTGTCAGGTGCCTCTAATTGCCAGGGCAGCAGGAGATAAAGGTCTTATAAGCCAACTATTAGGTCCAAAATATGGTGCTTTCTTTGTTTGTGGATCTTTAGGAGGCAAATCCAACTCTGGCTTGCTAGCTTTGACTAGCATTAAAGACGTTTATAAACTGGAATATGTGAACCAAGATACTAGAGTTTTTGGCGTAATCTCTAATCCTATTGGCCATAGCAAGGGCCCTCTACTGCACAACCCTGCCTTTAGACATACAGGATACAATGGAATATATGTGCCTCTACTAGTTGATAATATCAAGGAATTTTTTCGGGTCTTCTCATGCAATGACTATGCTGGTTTTAGTGTTGGACTCCCACATAAGGAAGCAGCAGTACGGTGCTGTGATGAAGTAGATCCACTTGCTAAGTCTATAGGAGCTGTTAACACAATTATAAGGAGACCTTCTGATGGCAAGCTCATTGGTTACAATACAGATTGTGAGGCTTGTGCGACGGCAATTGAGGATGCACTTAGAGAGAGACAAAAGACCAATGGCCATGCATCAAATGTTTCTCCAATTGCTGGAAAATTGTTCGTATTAGTTGGAGCAGGTGGTGCTGGGAGAGCTATTGCTTTTGGTGTCAAAAGTAGAGGGGCAAGGGTTGTAATATTTAACCGCAAATACGAGAGAGCAAAAGCTCTGGCCGCAGCAGTATCTGGTGAAGCCTTGCCATATGAACAACTAAACGATTTCTGCCCTGAGAAGGGAATGATTCTTGCAAATGCTTCTGCTGTAGGCATGCAGCCAAGGACAGATCAAACTCCTATTTCCAAGGAGGCCTTGAGATCATATGAGCTAGTATTTGATGCAGTTTACACACCTAGAAACACGCGGCTATTGCAGGAGGCTACAGAGGTTGGAGCTGCAGTGGTGGGTGGGGTTGAGATGTTCGTCAGGCAGGCAATTGGACAGTTCAAATTGTTCACCAATGGATTAGCACCAGAAGACTTTATGCGGAGGATTGTATATGAGCAATTTTGA。

mutant DHQ-SHD1 protein (SEQ ID NO.2, 515 amino acids)

MGFKQDLLVYTTLECESLSEMAACMQKAKEEGADLVELCIDSLTFTHISEVEHLLKQRTLPSIVSFRPNSPRYSCRENWKKTCLQVLELAVELDVKFVKIDRKVAYDEVICELMTKRSNCKIIASSHVNGGNPTKERLCNLIANLQSTRADIIKLVIDVAYITDVAPVFHMLTHCQVPLIARAAGDKGLISQLLGPKYGAFFVCGSLGGKSNSGLLALTSIKDVYKLEYVNQDTRVFGVISNPIGHSKGPLLHNPAFRHTGYNGIYVPLLVDNIKEFFRVFSCNDYAGFSVGLPHKEAAVRCCDEVDPLAKSIGAVNTIIRRPSDGKLIGYNTDCEACATAIEDALRERQKTNGHASNVSPIAGKLFVLVGAGGAGRAIAFGVKSRGARVVIFNRKYERAKALAAAVSGEALPYEQLNDFCPEKGMILANASAVGMQPRTDQTPISKEALRSYELVFDAVYTPRNTRLLQEATEVGAAVVGGVEMFVRQAIGQFKLFTNGLAPEDFMRRIVYEQF。

Mutant genesTyrAThe coding sequence of (SEQ ID NO.3, 732 bp):

ATGATAAAACAAGGCCATATAATCAGGGCTACTTCTAGATCAGATTATTCAGACTTGTGCGCAAATTTGGGTATCCTATTCTTCAGGGATATGGGTGCATTTCTTGAATCAGACAATAAGATTATTATAATTAGCACGTCGATACTGTCTCTATCACGAGTTGTAGAGTCCATACCATTCCATTATCTCAAGCGGCCTACACTTTTCATTGATGTATTCTCAGTTAAAAAACATCCAAAAGATGTCCTTTTGCGAATATTGCCTGAGGAATGCGACTTGCTGTGTACTCACCCAATGTTTGGACCACAAAGTGGAAAAGATGGATGGACTGATTTGACTTTTATGTACGACATGATTCGAATTAGAAATAAATCTCTGTGTTCCAGTTTTCTGCAAATATTTTTAAGTGAGGGGTGCAAAATGCTGGAAATGACTTGTGAAGAGCATGATAAATTGGCTGCTCGAAGTCAATTTCTGACTCACATAATTGGCAGGATCTTATCCGAAATGGAGGTTGAACCCACCCCCATAGACACGAAGGGATTTCAGAAACTTGTTCAAGTGAAGGAGAGCTCAGTTAGAGATAGTTTTGATCTATTCAGCGGGCTATTCATACACAATAGGTTTGCCAGGCAACAGATGAAAAATTTAGAAGTAGCAGTGGAGAAAACTAAACAGAAGCTTGAAGAGAGGTCGAAGGAGCTGCAGGATCCTATCATATCTAAGTTCTAG。

mutant TyrA protein (SEQ ID No.4, 243 amino acids):

MIKQGHIIRATSRSDYSDLCANLGILFFRDMGAFLESDNKIIIISTSILSLSRVVESIPFHYLKRPTLFIDVFSVKKHPKDVLLRILPEECDLLCTHPMFGPQSGKDGWTDLTFMYDMIRIRNKSLCSSFLQIFLSEGCKMLEMTCEEHDKLAARSQFLTHIIGRILSEMEVEPTPIDTKGFQKLVQVKESSVRDSFDLFSGLFIHNRFARQQMKNLEVAVEKTKQKLEERSKELQDPIISKF。

example 2

Based on the screening and analysis in example 1, the specific phenotypic change of related mutants is further determined. The mutant pure hybrid plants are further subjected to greenhouse planting and field planting, the tyrosine content of tobacco leaves and the release amount of phenol in smoke are measured, and the specific experimental process is briefly described as follows.

(I) detection of tyrosine content

M4 generation homozygous mutant strain mutant M837 (LH 2), M330 (LH 5), M283 (LH 10) and control material Yunyan 87 (LH 1) are planted in the culture room of the national tobacco gene research center (the temperature is 25 +/-2 ℃, the light is 16 h/the dark is 8 h).

And after the 5 th true leaf grows out, taking down the third true leaf and freeze-drying. Accurately weighing 50mg of lyophilized tobacco powder, placing into 1.5ml EP tube, accurately measuring to 0.0001g, accurately adding 1.6mg/ml leucine brain phenolphthalein methanol solution 1ml, placing in shaking table at 30 deg.C and 200rpm, and oscillating for 30 min; after centrifugation at 14000rpm for 10min, 600. mu.L of the supernatant was carefully collected and assayed by LC-MS (Agilent, 1290-. Detection conditions are as follows:

a chromatographic column: waters BEH Amide, 2.1 × 100mm,1.7 μm;

mobile phase A: acetonitrile solution containing 0.1% formic acid; mobile phase B: an aqueous solution containing 0.1% formic acid;

column temperature: 30 ℃; flow rate: 0.3 mL/min; sample introduction volume: 10 mu L of the solution;

gradient:

0min, mobile phase A5%, mobile phase B95%;

5min, mobile phase A25%, mobile phase B75%;

6min, mobile phase A30% and mobile phase B70%;

7min, mobile phase A25% and mobile phase B75%;

8min, 20% of mobile phase A and 80% of mobile phase B;

10min, mobile phase A5%, and mobile phase B95%.

The results of the tyrosine content detection of the mutant tobacco leaves (figure 1) show that compared with the control Yunyan 87, the free tyrosine content in the mutant strains LH2, LH5 and LH10 is greatly reduced by 61.37 percent, 76.78 percent and 81.71 percent respectively. The results show that the smoke is 87DHQ-SDH1Genes orTyrAMutation of the gene can obviously reduce the content of Tyr.

(II) detection of protein content

In the greenhouse, after the 5 th true leaf was grown, the third true leaf was removed and the protein content was measured using BCA protein concentration assay kit (Sevin Innovation Biotech Co., Ltd.) according to the kit instructions.

The results of leaf protein content assays on mutant strains showed that the protein content in LH2, LH5 and LH10 decreased by about 7.39%, 8.699% and 21.74%, respectively (fig. 2). This indicates that the cloud 87 wasDHQ-SDH1Genes andTyrAmutation of the gene can effectively reduce the content of leaf protein, probably by inhibiting the synthesis of free Tyr to influence the synthesis of protein content, because Tyr is one of indispensable amino acid components of protein synthesis.

(III) analysis of photo-biological Properties

In the greenhouse, after the 5 th true leaf grows out, the change of the photosynthetic physiological index of the homozygous mutant after being exposed for 4 hours in the sun is detected by using Imaging-PAM (Whole leaf fluorescence Imaging system) of the Germany WALZ company.

The results of measurements of the photosynthetic physiology of the leaves of the mutant lines showed that the maximum photochemical efficiency (Fv/Fm) of photosynthetic system II of the plants remained essentially the same as the control, both before and after normal growth and intense light treatment (FIG. 3). This indicates that although the leaf protein and Tyr content are significantly reduced, the photosynthetic efficiency of the leaf is not significantly interfered, indicating that the site-directed mutation of the gene does not significantly interfere with the normal growth and development of the plant.

(IV) detection of release amount of smoke phenolic substances

In an experimental field located in the royal town of Xiang county, Henan, M4 generation homozygous lines LH5, LH10 and LH2 corresponding to mutants M330, M283 and M837 and a control material Yunyan 87 (LH 1) are planted, wherein each material has 3 repeated cells, and each cell is planted with 20 tobacco plants. The plant spacing is 50 cm, and the row spacing is 120 cm. Topping is carried out when the central flower of the first tobacco plant blooms 50%, and the number of leaves left on each plant is about 22. The field management measures and the baking are carried out according to the local high-quality tobacco leaf cultivation and baking technical regulations.

After the middle leaves of the Yunyan cigarette 87 and the mutant material are mature and picked, the tobacco leaves are baked according to a local high-quality tobacco baking process, the baked tobacco leaves are balanced and then cut into shreds, and the tobacco shred samples are filled into empty cigarettes by using a cigarette beater to prepare sample cigarettes.

Further, after the sample cigarettes are put into a constant temperature and humidity room (temperature is 22 ℃, humidity is 60% RH) to be balanced for 48 hours, cigarettes with weight deviation within the range of +/-20 mg are screened as test cigarettes.

The cigarettes were smoked using an automatic smoking machine (SM 450, CERULEAN, uk), 4 cigarettes were drawn for each sample, the glass fiber filter with the total particulate matter of the mainstream smoke trapped therein was folded and placed in a 200mL conical flask with a stopper, 50mL of 1% acetic acid solution (volume fraction) was added accurately, ultrasonic extraction was carried out at room temperature for 20min, and the flask was allowed to stand for 5 min. 2ml of the extract was filtered through a 0.45 μm aqueous filter. Detection was performed using a high performance liquid chromatograph (WATERS, USA, E2695). Detection conditions are as follows:

mobile phase A: 1% acetic acid solution; and (3) mobile phase B: acetic acid-acetonitrile-water (1: 30: 69);

column temperature: 30 ℃; flow rate: 1 mL/min; sample introduction volume: 10 mu L of the solution;

gradient:

0min, mobile phase A80%, and mobile phase B20%;

40min, mobile phase A0% and mobile phase B100%.

For is toDHQ-SDH1The results of the measurement of phenol release from mutant cigarettes (fig. 4) show that the phenol release of each mutant cigarette sample is significantly reduced compared to the control cigarette sample. Wherein the LH10 has the largest reduction amplitude, and the reduction rate is 48.99%; LH5 and LH2 decreased by 24.09% and 11.89%, respectively.

The results of testing the release amounts of other 5 phenols in the smoke of the mutant material are shown in fig. 4, and compared with the wild type, the release amounts of phenols in the smoke of the mutant test cigarette are increased except for the release amount of p-benzenediol, and the release amounts of other phenols, such as the release amounts of m-benzenediol, o-benzenediol, p-cresol and o-cresol, are reduced to different degrees. The content of o-dihydroxybenzene in the 5 phenols is the highest, the reduction amplitude is greatly increased than that of the o-dihydroxybenzene, so that the smoke phenolic substance release amount of the mutant material is reduced, and the LH10 is reduced maximally.

Example 3

Based on the embodiment 1 and the embodiment 2, the inventor further constructs tobacco by means of genetic hybridization based on the single mutant determined by screening and in order to further reduce the phenol content in the smoketyra/dhq-sdh1The specific construction process for the double mutant is briefly described below.

It should be explained that, in order to avoid the occurrence of the condition of over-change of tobacco quality and even death after mutation accumulation, the inventors adopted other mutant materials for further research and discussion in the process of constructing the double mutant. Specifically, the method comprises the following steps:

male parent: with selected homozygous mutant tobaccotyra(M846) material as male parent in the mutantTyrAThe 217 th base of the gene is mutated C → T, the corresponding 73 rd amino acid is mutated L → F;

female parent: with selected homozygous mutant tobaccoDHQ-SDH1(M398) material as female parent, in the mutantDHQ- SDH1The 863 th base of the gene is mutated A → G and the corresponding 288 th amino acid is mutated D → G.

The tobacco M846 and M398 mutants were sown simultaneously, and approximately 90 days after sowing, genetic crossing was performed at the time of flowering. Removing redundant buds by taking the M398 mutant as a female parent, selecting unopened buds, removing stamens, collecting pollen of the M846 mutant, smearing the pollen on stigmas of pistils of the M398 mutant, and bagging for reserving seeds. And (4) harvesting the hybrid seeds after the hybrid seeds are mature.

About 30 days after F1 generation, DNA of tobacco was extracted and sequenced, and the DNA contained thereinTyrAAndDHQ-SDH1reserving seeds of two gene mutations;

about 30 days after F2 generation, DNA of tobacco was extracted and sequenced, and the DNA contained thereinTyrAAndDHQ-SDH1seed reservation for two gene mutations;

about 30 days after F3 sowing, extracting DNA of tobacco, sequencing, and determining the seed reservation of F2 progenyTyrAAndDHQ-SDH1mutation of both genes and harvesting.

When sequencing identification is carried out on the basis of the PCR amplification product, primers DHQ2_ Tilling _ F/R and TyrA _ Tilling _ F/R are respectively used for carrying out PCR amplification, 1% agarose gel electrophoresis is carried out on the PCR amplification product, then gel cutting recovery is carried out, the gel is connected to a T carrier, and then the product is sent to Huada gene company for sequencing.

Further, referring to the related planting process in example 2, F4 generation double mutants were planted, and the tyrosine content and phenol release amount of the tobacco leaves were measured.

The result of the tyrosine content detection of the double mutant tobacco leaves (fig. 5) shows that the content of free tyrosine in the mutant strain LH14 is greatly reduced by 85.4% compared with the control Yunyan 87.

The measurement result of the double mutant phenol release amount (fig. 6) shows that the mutant LH14 cigarette sample has significantly reduced phenol release amount compared with the control cigarette sample, wherein the reduction rate of phenol release amount is 49.8%.

SEQUENCE LISTING

<110> Zhengzhou tobacco institute of China tobacco general Co

<120> mutant gene DHQ-SDH1 related to smoke phenol release amount

<130> none

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1548

<212> DNA

<213> Nicotiana tabacum

<400> 1

atgggtttca aacaagacct tttagtgtac acaacattag aatgtgaaag cttgtctgaa 60

atggcagctt gtatgcagaa agcaaaagaa gaaggagcag atctagtgga actttgcatt 120

gactctttaa ctttcacaca catttcagaa gttgaacacc ttctcaaaca gaggacttta 180

ccctccatcg tttctttcag gccaaattcg ccgagatatt cttgcagaga aaactggaag 240

aaaacatgct tgcaagttct ggaattggct gttgaattgg acgtcaagtt tgttaaaatt 300

gaccgcaagg ttgcttatga tgaggtcatc tgtgaattaa tgaccaaacg atcgaactgc 360

aagataattg cctctagtca tgtgaatggt ggaaatccta caaaagagag actttgtaat 420

ttaattgcaa acctgcaatc aacaagagca gatatcatca aattagtgat tgatgtagct 480

tatattacag atgttgcacc agtttttcat atgcttacac attgtcaggt gcctctaatt 540

gccagggcag caggagataa aggtcttata agccaactat taggtccaaa atatggtgct 600

ttctttgttt gtggatcttt aggaggcaaa tccaactctg gcttgctagc tttgactagc 660

attaaagacg tttataaact ggaatatgtg aaccaagata ctagagtttt tggcgtaatc 720

tctaatccta ttggccatag caagggccct ctactgcaca accctgcctt tagacataca 780

ggatacaatg gaatatatgt gcctctacta gttgataata tcaaggaatt ttttcgggtc 840

ttctcatgca atgactatgc tggttttagt gttggactcc cacataagga agcagcagta 900

cggtgctgtg atgaagtaga tccacttgct aagtctatag gagctgttaa cacaattata 960

aggagacctt ctgatggcaa gctcattggt tacaatacag attgtgaggc ttgtgcgacg 1020

gcaattgagg atgcacttag agagagacaa aagaccaatg gccatgcatc aaatgtttct 1080

ccaattgctg gaaaattgtt cgtattagtt ggagcaggtg gtgctgggag agctattgct 1140

tttggtgtca aaagtagagg ggcaagggtt gtaatattta accgcaaata cgagagagca 1200

aaagctctgg ccgcagcagt atctggtgaa gccttgccat atgaacaact aaacgatttc 1260

tgccctgaga agggaatgat tcttgcaaat gcttctgctg taggcatgca gccaaggaca 1320

gatcaaactc ctatttccaa ggaggccttg agatcatatg agctagtatt tgatgcagtt 1380

tacacaccta gaaacacgcg gctattgcag gaggctacag aggttggagc tgcagtggtg 1440

ggtggggttg agatgttcgt caggcaggca attggacagt tcaaattgtt caccaatgga 1500

ttagcaccag aagactttat gcggaggatt gtatatgagc aattttga 1548

<210> 2

<211> 515

<212> PRT

<213> Nicotiana tabacum

<400> 2

Met Gly Phe Lys Gln Asp Leu Leu Val Tyr Thr Thr Leu Glu Cys Glu

1 5 10 15

Ser Leu Ser Glu Met Ala Ala Cys Met Gln Lys Ala Lys Glu Glu Gly

20 25 30

Ala Asp Leu Val Glu Leu Cys Ile Asp Ser Leu Thr Phe Thr His Ile

35 40 45

Ser Glu Val Glu His Leu Leu Lys Gln Arg Thr Leu Pro Ser Ile Val

50 55 60

Ser Phe Arg Pro Asn Ser Pro Arg Tyr Ser Cys Arg Glu Asn Trp Lys

65 70 75 80

Lys Thr Cys Leu Gln Val Leu Glu Leu Ala Val Glu Leu Asp Val Lys

85 90 95

Phe Val Lys Ile Asp Arg Lys Val Ala Tyr Asp Glu Val Ile Cys Glu

100 105 110

Leu Met Thr Lys Arg Ser Asn Cys Lys Ile Ile Ala Ser Ser His Val

115 120 125

Asn Gly Gly Asn Pro Thr Lys Glu Arg Leu Cys Asn Leu Ile Ala Asn

130 135 140

Leu Gln Ser Thr Arg Ala Asp Ile Ile Lys Leu Val Ile Asp Val Ala

145 150 155 160

Tyr Ile Thr Asp Val Ala Pro Val Phe His Met Leu Thr His Cys Gln

165 170 175

Val Pro Leu Ile Ala Arg Ala Ala Gly Asp Lys Gly Leu Ile Ser Gln

180 185 190

Leu Leu Gly Pro Lys Tyr Gly Ala Phe Phe Val Cys Gly Ser Leu Gly

195 200 205

Gly Lys Ser Asn Ser Gly Leu Leu Ala Leu Thr Ser Ile Lys Asp Val

210 215 220

Tyr Lys Leu Glu Tyr Val Asn Gln Asp Thr Arg Val Phe Gly Val Ile

225 230 235 240

Ser Asn Pro Ile Gly His Ser Lys Gly Pro Leu Leu His Asn Pro Ala

245 250 255

Phe Arg His Thr Gly Tyr Asn Gly Ile Tyr Val Pro Leu Leu Val Asp

260 265 270

Asn Ile Lys Glu Phe Phe Arg Val Phe Ser Cys Asn Asp Tyr Ala Gly

275 280 285

Phe Ser Val Gly Leu Pro His Lys Glu Ala Ala Val Arg Cys Cys Asp

290 295 300

Glu Val Asp Pro Leu Ala Lys Ser Ile Gly Ala Val Asn Thr Ile Ile

305 310 315 320

Arg Arg Pro Ser Asp Gly Lys Leu Ile Gly Tyr Asn Thr Asp Cys Glu

325 330 335

Ala Cys Ala Thr Ala Ile Glu Asp Ala Leu Arg Glu Arg Gln Lys Thr

340 345 350

Asn Gly His Ala Ser Asn Val Ser Pro Ile Ala Gly Lys Leu Phe Val

355 360 365

Leu Val Gly Ala Gly Gly Ala Gly Arg Ala Ile Ala Phe Gly Val Lys

370 375 380

Ser Arg Gly Ala Arg Val Val Ile Phe Asn Arg Lys Tyr Glu Arg Ala

385 390 395 400

Lys Ala Leu Ala Ala Ala Val Ser Gly Glu Ala Leu Pro Tyr Glu Gln

405 410 415

Leu Asn Asp Phe Cys Pro Glu Lys Gly Met Ile Leu Ala Asn Ala Ser

420 425 430

Ala Val Gly Met Gln Pro Arg Thr Asp Gln Thr Pro Ile Ser Lys Glu

435 440 445

Ala Leu Arg Ser Tyr Glu Leu Val Phe Asp Ala Val Tyr Thr Pro Arg

450 455 460

Asn Thr Arg Leu Leu Gln Glu Ala Thr Glu Val Gly Ala Ala Val Val

465 470 475 480

Gly Gly Val Glu Met Phe Val Arg Gln Ala Ile Gly Gln Phe Lys Leu

485 490 495

Phe Thr Asn Gly Leu Ala Pro Glu Asp Phe Met Arg Arg Ile Val Tyr

500 505 510

Glu Gln Phe

515

<210> 3

<211> 732

<212> DNA

<213> Nicotiana tabacum

<400> 3

atgataaaac aaggccatat aatcagggct acttctagat cagattattc agacttgtgc 60

gcaaatttgg gtatcctatt cttcagggat atgggtgcat ttcttgaatc agacaataag 120

attattataa ttagcacgtc gatactgtct ctatcacgag ttgtagagtc cataccattc 180

cattatctca agcggcctac acttttcatt gatgtattct cagttaaaaa acatccaaaa 240

gatgtccttt tgcgaatatt gcctgaggaa tgcgacttgc tgtgtactca cccaatgttt 300

ggaccacaaa gtggaaaaga tggatggact gatttgactt ttatgtacga catgattcga 360

attagaaata aatctctgtg ttccagtttt ctgcaaatat ttttaagtga ggggtgcaaa 420

atgctggaaa tgacttgtga agagcatgat aaattggctg ctcgaagtca atttctgact 480

cacataattg gcaggatctt atccgaaatg gaggttgaac ccacccccat agacacgaag 540

ggatttcaga aacttgttca agtgaaggag agctcagtta gagatagttt tgatctattc 600

agcgggctat tcatacacaa taggtttgcc aggcaacaga tgaaaaattt agaagtagca 660

gtggagaaaa ctaaacagaa gcttgaagag aggtcgaagg agctgcagga tcctatcata 720

tctaagttct ag 732

<210> 4

<211> 243

<212> PRT

<213> Nicotiana tabacum

<400> 4

Met Ile Lys Gln Gly His Ile Ile Arg Ala Thr Ser Arg Ser Asp Tyr

1 5 10 15

Ser Asp Leu Cys Ala Asn Leu Gly Ile Leu Phe Phe Arg Asp Met Gly

20 25 30

Ala Phe Leu Glu Ser Asp Asn Lys Ile Ile Ile Ile Ser Thr Ser Ile

35 40 45

Leu Ser Leu Ser Arg Val Val Glu Ser Ile Pro Phe His Tyr Leu Lys

50 55 60

Arg Pro Thr Leu Phe Ile Asp Val Phe Ser Val Lys Lys His Pro Lys

65 70 75 80

Asp Val Leu Leu Arg Ile Leu Pro Glu Glu Cys Asp Leu Leu Cys Thr

85 90 95

His Pro Met Phe Gly Pro Gln Ser Gly Lys Asp Gly Trp Thr Asp Leu

100 105 110

Thr Phe Met Tyr Asp Met Ile Arg Ile Arg Asn Lys Ser Leu Cys Ser

115 120 125

Ser Phe Leu Gln Ile Phe Leu Ser Glu Gly Cys Lys Met Leu Glu Met

130 135 140

Thr Cys Glu Glu His Asp Lys Leu Ala Ala Arg Ser Gln Phe Leu Thr

145 150 155 160

His Ile Ile Gly Arg Ile Leu Ser Glu Met Glu Val Glu Pro Thr Pro

165 170 175

Ile Asp Thr Lys Gly Phe Gln Lys Leu Val Gln Val Lys Glu Ser Ser

180 185 190

Val Arg Asp Ser Phe Asp Leu Phe Ser Gly Leu Phe Ile His Asn Arg

195 200 205

Phe Ala Arg Gln Gln Met Lys Asn Leu Glu Val Ala Val Glu Lys Thr

210 215 220

Lys Gln Lys Leu Glu Glu Arg Ser Lys Glu Leu Gln Asp Pro Ile Ile

225 230 235 240

Ser Lys Phe

Claims (5)

1. Mutant gene related to phenol release amount of smokeDHQ-SDH1Characterized in that the mutant geneDHQ-SDH1A mutant gene for tobacco dehydroquinate dehydrogenase/shikimate dehydrogenase; compared withDHQ-SDH1The gene wild type CDS, the sense mutation site and the mutation type are as follows: G445A → G149R and G295A → E99K, or C637T → P213S;

the wild typeDHQ-SDH1The CDS sequence of the gene is as follows:

ATGGGTTTCAAACAAGACCTTTTAGTGTACACAACATTAGAATGTGAAAGCTTGTCTGAAATGGCAGCTTGTATGCAGAAAGCAAAAGAAGAAGGAGCAGATCTAGTGGAACTTTGCATTGACTCTTTAACTTTCACACACATTTCAGAAGTTGAACACCTTCTCAAACAGAGGACTTTACCCTCCATCGTTTCTTTCAGGCCAAATTCGCCGAGATATTCTTGCAGAGAAAACTGGAAGAAAACATGCTTGCAAGTTCTGAAATTGGCTGTTGAATTGGACGTCGAGTTTGTTGAAGTTGACCGCGAGGTTGCTTGTGATGAGGTCATCTGTGAATTAATGACCAAACGATCGAACTGCAAGATAATTGCCTCTAGTCATGTGAATGGTGGAAATCCTACAAAAGAGAGACTTTGTAATTTAATTGCAAACCTGCAATCAACAGGAGCAGATATCATCAAATTAGTGATTGATGTAGCTTATATTACAGATGTTGCACCAGTTTTTCATATGCTTACACATTGTCAGGTGCCTCTAATTGCCAGGGCAGCAGGAGATAAAGGTCTTATAAGCCAACTATTAGGTCCAAAATATGGTGCTTTCTTTGTTTGTGGATCTTTAGGAGGCAAATCCAACCCTGGCTTGCCAGCTTTGACTAGCATTAAAGACGTTTATAAACTGGAATATGTGAACCAAGATACTAGAGTTTTTGGCGTAATCTCTAATCCTATTGGCCATAGCAAGGGCCCTCTACTGCACAACCCTGCCTTTAGACATACAGGATACAATGGAATATATGTGCCTCTACTAGTTGATAATATCAAGGAATTTTTTCGGGTCTTCTCATGCAATGACTATGCTGATTTTAGTGTTGGACTCCCACATAAGGAAGCAGCAGTACGGTGCTGTGATGAAGTAGATCCACTTGCTAAGTCTATAGGAGCTGTTAACACAATTATAAGGAGACCTTCTGATGGCAAGCTCATTGGTTACAATACAGATTGTGAGGCTTGTGCGACGGCAATTGAGGATGCACTTAGAGAGAGACAAAAGACCAATGGCCATGCATCAAATGTTTCTCCAATTGCTGGAAAATTGTTCGTATTAGTTGGAGCAGGTGGTGCTGGGAGAGCTATTGCTTTTGGTGTCAAAAGTAGAGGGGCAAGGGTTGTAATATTTAACCGCAAATACGAGAGAGCAAAAGCTCTGGCCGCAGCAGTATCTGGTGAAGCCTTGCCATATGAACAACTAAACGATTTCTGCCCTGAGAAGGGAATGATTCTTGCAAATGCTTCTGCTGTAGGCATGCAGCCAAGGACAGATCAAACTCCTATTTCCAAGGAGGCCTTGAGATCATATGAGCTAGTATTTGATGCAGTTTACACACCTAGAAACACGCGGCTATTGCAGGAGGCTACAGAGGTTGGAGCTGCAGTGGTGGGTGGGGTTGAGATGTTCGTCAGGCAGGCAATTGGACAGTTCAAATTGTTCACCAATGGATTAGCACCAGAAGACTTTATGCGGAGGATTGTATATGAGCAATTTTGA。

2. the mutant gene according to claim 1DHQ-SDH1The encoded tobacco DHQ-SHD1 mutant protein is characterized in that compared with the wild-type DHQ-SHD1 protein, the sense mutation site and the mutation type are as follows: G445A → G149R and G295A → E99K, or C637T → P213S;

the amino acid sequence of the wild DHQ-SHD1 protein is as follows:

MGFKQDLLVYTTLECESLSEMAACMQKAKEEGADLVELCIDSLTFTHISEVEHLLKQRTLPSIVSFRPNSPRYSCRENWKKTCLQVLKLAVELDVEFVEVDREVACDEVICELMTKRSNCKIIASSHVNGGNPTKERLCNLIANLQSTGADIIKLVIDVAYITDVAPVFHMLTHCQVPLIARAAGDKGLISQLLGPKYGAFFVCGSLGGKSNPGLPALTSIKDVYKLEYVNQDTRVFGVISNPIGHSKGPLLHNPAFRHTGYNGIYVPLLVDNIKEFFRVFSCNDYADFSVGLPHKEAAVRCCDEVDPLAKSIGAVNTIIRRPSDGKLIGYNTDCEACATAIEDALRERQKTNGHASNVSPIAGKLFVLVGAGGAGRAIAFGVKSRGARVVIFNRKYERAKALAAAVSGEALPYEQLNDFCPEKGMILANASAVGMQPRTDQTPISKEALRSYELVFDAVYTPRNTRLLQEATEVGAAVVGGVEMFVRQAIGQFKLFTNGLAPEDFMRRIVYEQF。

3. the process of claim 1Gene variantsDHQ-SDH1The application of the protein in the regulation and control of the tyrosine substance content or the smoke phenol release amount of tobacco leaves is characterized in that the regulation and control is to wild typeDHQ-SDH1The CDS of the gene is mutated at a corresponding sense mutation site, and is used for reducing the content of tyrosine substances in the tobacco leaves and the release amount of phenol in the tobacco leaves.

4. The use of the tobacco DHQ-SHD1 mutein of claim 2 for the modulation of tobacco leaf tyrosine content or smoke phenol release, wherein the modulation is a mutation of the sense mutation site corresponding to wild-type DHQ-SHD1 protein for down-regulating the tyrosine content in tobacco leaves and for down-regulating the phenol release in smoke.

5. Tobaccotyra/dhq-sdh1The method for cultivating the tobacco variety with double mutants is characterized in that the tobacco variety contains mutant genesTyrATobacco and tobacco containing mutant geneDHQ-SDH1The tobacco is selected as parent and obtained by genetic hybridizationtyra/dhq-sdh1Double mutant homozygotes;

and (3) during genetic hybridization: to be provided withdhq-sdh1The mutant is the female parent, andtyrathe mutant is a male parent;

the mutant geneTyrACompared withTyrAGene wild type CDS, in mutantTyrAThe 217 th base of the gene is mutated C → T, the corresponding 73 rd amino acid is mutated L → F;

the wild typeTyrAThe CDS sequence of the gene is as follows:

ATGATAAAACAAGGCCATATAATCAGGGCTACTTCTAGATCAGATTATTCAGACTTGTGCGCAAATTTGGGTATCCTATTCTTCAGGGATATGGGTGCATTTCTTGAATCAGACAATGAGATTATTATAATTAGCACGTCGATACTGTCTCTATCACGAGTTGTAGAGTCCATACCATTCCATTGTCTCAAGCGGCCTACACTTTTCATTGATGTACTCTCAGTTAAAGAACATCCAAAAGATGTCCTTTTGCGAATATTGCCTGAGGAATGCGACTTGCTGTGTACTCACCCAATGTTTGGACCACAAAGTGGAAAAGATGGATGGACTGATTTGACTTTTATGTACGACATGATTCGAATTAGAAATAAATCTCTGTGTTCCAGTTTTCTGCAAATATTTTCAAGTGAGGGGTGCAAAATGCTGGAAATGACTTGTGAAGAGCATGATAAATTGGCTGCTCGAAGTCAATTTCTGACTCACACAATTGGCAGGATCTTATCCGAAATGGAGGTTGAACCCACCCCCATAGACACGAAGGGATTTCAGAAACTTGTTCAAGTGAAGGAGAGCTCAGTTAGAGATAGTTTTGATCTATTCAGCGGGCTATTCATACACAATAGGTTTGCCAGGCAACAGATGAAAAATTTAGAAGTAGCAGTGGAGAAAACTAAACAGAAGCTTGAAGAGAGGTCGAAGGAGCTGCAGGATCCTATCATATCTAAGTTCTAG；

the mutant geneDHQ-SDH1Compared with wild typeDHQ-SDH1CDS sequence of Gene, in mutantDHQ-SDH1The 863 th base of the gene is mutated A → G and the corresponding 288 th amino acid is mutated D → G;

the wild typeDHQ-SDH1The CDS sequence of the gene is as follows:

ATGGGTTTCAAACAAGACCTTTTAGTGTACACAACATTAGAATGTGAAAGCTTGTCTGAAATGGCAGCTTGTATGCAGAAAGCAAAAGAAGAAGGAGCAGATCTAGTGGAACTTTGCATTGACTCTTTAACTTTCACACACATTTCAGAAGTTGAACACCTTCTCAAACAGAGGACTTTACCCTCCATCGTTTCTTTCAGGCCAAATTCGCCGAGATATTCTTGCAGAGAAAACTGGAAGAAAACATGCTTGCAAGTTCTGAAATTGGCTGTTGAATTGGACGTCGAGTTTGTTGAAGTTGACCGCGAGGTTGCTTGTGATGAGGTCATCTGTGAATTAATGACCAAACGATCGAACTGCAAGATAATTGCCTCTAGTCATGTGAATGGTGGAAATCCTACAAAAGAGAGACTTTGTAATTTAATTGCAAACCTGCAATCAACAGGAGCAGATATCATCAAATTAGTGATTGATGTAGCTTATATTACAGATGTTGCACCAGTTTTTCATATGCTTACACATTGTCAGGTGCCTCTAATTGCCAGGGCAGCAGGAGATAAAGGTCTTATAAGCCAACTATTAGGTCCAAAATATGGTGCTTTCTTTGTTTGTGGATCTTTAGGAGGCAAATCCAACCCTGGCTTGCCAGCTTTGACTAGCATTAAAGACGTTTATAAACTGGAATATGTGAACCAAGATACTAGAGTTTTTGGCGTAATCTCTAATCCTATTGGCCATAGCAAGGGCCCTCTACTGCACAACCCTGCCTTTAGACATACAGGATACAATGGAATATATGTGCCTCTACTAGTTGATAATATCAAGGAATTTTTTCGGGTCTTCTCATGCAATGACTATGCTGATTTTAGTGTTGGACTCCCACATAAGGAAGCAGCAGTACGGTGCTGTGATGAAGTAGATCCACTTGCTAAGTCTATAGGAGCTGTTAACACAATTATAAGGAGACCTTCTGATGGCAAGCTCATTGGTTACAATACAGATTGTGAGGCTTGTGCGACGGCAATTGAGGATGCACTTAGAGAGAGACAAAAGACCAATGGCCATGCATCAAATGTTTCTCCAATTGCTGGAAAATTGTTCGTATTAGTTGGAGCAGGTGGTGCTGGGAGAGCTATTGCTTTTGGTGTCAAAAGTAGAGGGGCAAGGGTTGTAATATTTAACCGCAAATACGAGAGAGCAAAAGCTCTGGCCGCAGCAGTATCTGGTGAAGCCTTGCCATATGAACAACTAAACGATTTCTGCCCTGAGAAGGGAATGATTCTTGCAAATGCTTCTGCTGTAGGCATGCAGCCAAGGACAGATCAAACTCCTATTTCCAAGGAGGCCTTGAGATCATATGAGCTAGTATTTGATGCAGTTTACACACCTAGAAACACGCGGCTATTGCAGGAGGCTACAGAGGTTGGAGCTGCAGTGGTGGGTGGGGTTGAGATGTTCGTCAGGCAGGCAATTGGACAGTTCAAATTGTTCACCAATGGATTAGCACCAGAAGACTTTATGCGGAGGATTGTATATGAGCAATTTTGA。

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