CN111004808B

CN111004808B - Tobacco protein NtVHA-a1 and application thereof

Info

Publication number: CN111004808B
Application number: CN201911317395.7A
Authority: CN
Inventors: 郑庆霞; 徐国云; 翟妞; 周会娜; 刘萍萍; 张慧; 陈千思; 王晨; 卢鹏; 金立锋; 曹培健
Original assignee: Zhengzhou Tobacco Research Institute of CNTC
Current assignee: Zhengzhou Tobacco Research Institute of CNTC
Priority date: 2019-12-19
Filing date: 2019-12-19
Publication date: 2022-06-10
Anticipated expiration: 2039-12-19
Also published as: CN111004808A

Abstract

The invention belongs to the field of tobacco genetic engineering, and particularly relates to a tobacco protein NtVHA-a1 and an application patent application thereof. The gene consists of 2454 bases, wherein the specific nucleic acid fragment is 1042-1371 base, and the specific base sequence is shown as SEQ ID NO. 1. The amino acid sequence of the tobacco protein NtVHA-a1 is shown in SEQ ID NO.2 and consists of 817 amino acid residues. In the process of researching the related regulatory gene of the content of the biogenic amines in the tobacco, the inventor discovers that the related regulatory gene is highly related to the content of the biogenic amines in the tobacco, particularly the content of spermine through researching a specific tobacco protein NtVHA-a 1. In the process of further gene function verification, after the gene is silenced, the spermine content in the tobacco is obviously reduced. Based on the characteristic, a certain technical basis can be established for the quality control of tobacco and the cultivation of new tobacco varieties and a certain reference can be provided.

Description

Tobacco protein NtVHA-a1 and application thereof

Technical Field

The invention belongs to the field of tobacco genetic engineering, and particularly relates to a tobacco protein NtVHA-a1 and an application patent application thereof.

Background

Tobacco (A)Nicotiana tabacum L.) Belonging to the family Solanaceae (Solanaceae) Genus Nicotiana (Nicotiana) Annual herbaceous plants. Tobacco is originally produced in America, and is introduced into China through a plurality of routes, so that the China becomes the country with the largest tobacco planting area and the highest yield by virtue of the superior geographical environment conditions of the China. China is also the largest tobacco producing country and consuming country in the world, and the yield and sales volume accounts for one third of the world, so that the tobacco planting and product processing industry occupies a very important position in the development of national economy.

Biogenic amines are a general term for a class of nitrogen-containing low molecular weight basic organic compounds having biological activity, which are precursors for the synthesis of hormones, nucleotides and proteins by organisms themselves and can be regarded as low molecular weight alkaloids formed by substituting 1 to 3 hydrogen atoms in an ammonia molecule with an alkyl or aryl group. Spermine (Spermine), a polyamine substance containing two amino groups and two imino groups, is produced in plants by a series of enzymatic reactions of butanediamine and S-adenosylmethionine. As a pure natural plant active substance in biogenic amine, spermine widely exists in plant kingdom and has the physiological functions of regulating the growth and development of plants, delaying the senescence of plant cells and the like. When plants are stressed by adversity, polyamines in the plants can directly eliminate active oxygen through a bifidus reaction, and can reduce the accumulation of the active oxygen by improving the activity of antioxidant enzyme.

In view of the important effect of the biogenic amine substances on the growth of tobacco leaves, the deep analysis of the metabolic pathways of the biogenic amine substances is obviously of great technical significance. With the deepening of tobacco genetic engineering, the deep research and development of coding genes related to biogenic amines in tobacco can lay a good technical foundation for the regulation and control of tobacco quality and the cultivation of new tobacco varieties.

Disclosure of Invention

Based on the research of the related regulation genes of the content of the biological amine substances in the tobacco, the invention aims to provide the tobacco protein NtVHA-a1 gene and the application thereof in the regulation of the content of the biological amine substances in the tobacco, thereby laying a certain foundation for the development of the work of tobacco leaf quality regulation, tobacco resistance research, new variety cultivation and the like.

The technical solution adopted in the present application is detailed as follows.

Coding gene of tobacco protein NtVHA-a1NtVHA-a1Consists of 2454 bases, wherein the specific nucleic acid segment is 1042-1371 base, and the specific base sequence is shown as SEQ ID NO. 1.

The coding geneNtVHA-a1The method is applied to regulation and control of leaf biogenic amine substances, and the content of biogenic amine substances in tobacco leaves is regulated and controlled by regulating the expression level of tobacco NtVHA-a1 genes by using a gene silencing technology or a gene overexpression method, wherein the biogenic amine substances are mainly spermine.

The coding geneNtVHA-a1The PCR amplification preparation method comprises the following steps:

(1) extracting (for example, taking tobacco K326 leaves as a sample) genome, and carrying out reverse transcription to obtain cDNA for later use;

(2) designing a primer for PCR amplification, and carrying out PCR amplification, wherein the specific primer sequence is designed as follows:

NtVHA-a1-F：5’- TTTGCGAAGACCAAGATACA - 3’，

NtVHA-a1-R：5’- GCTGCCTAGTTTCTGAGAGC - 3’。

The amino acid sequence of the tobacco protein NtVHA-a1 is shown in SEQ ID NO.2 and consists of 817 amino acid residues.

The tobacco protein NtVHA-a1 is applied to regulation and control of the content of biological amines in leaves, the protein is related to the content of the biological amines in plant leaves, and after the expression content of the protein is reduced, the spermine content in the leaves is obviously reduced.

Using the coding geneNtVHA-a1The method for cultivating the new variety of tobacco comprises constructing the tobacco containing gene by a transgenic technology, a transient expression technology or a genome editing technologyNtVHA-a1Transforming tobacco by using a virus-induced silencing vector, an RNAi interference vector, an overexpression vector or a genome editing vector of the gene, and screening to obtain a new tobacco variety with variable spermine content;

specific examples thereof include: gene silencing by Virus Induction (VIGS)Technique of, interferenceNtVHA-a1The expression of the gene causes it to be silenced,NtVHA-a1the spermine content in the gene silencing plant is obviously reduced, and then a new plant variety with reduced spermine content is obtained.

In other words, a method for breeding a new variety of tobacco with low biogenic amine content utilizes the technology of virus-induced gene silencing (VIGS) to interfere withNtVHA-a1The expression of the gene causes it to be silenced,NtVHA-a1the content of biogenic amine, especially spermine, in the plants of the new tobacco variety with gene silencing is obviously reduced.

In the process of researching the related regulatory gene of the content of the biogenic amines in the tobacco, the inventor finds that the related regulatory gene is highly related to the content of the biogenic amines in the tobacco, particularly the content of spermine through the research on specific tobacco protein NtVHA-a 1. In the process of further gene function verification, after the gene is silenced, the content of spermine in the tobacco is obviously reduced. Based on the characteristic, a certain technical basis can be established for the quality control of tobacco and the cultivation of new tobacco varieties and a certain reference can be provided.

Drawings

FIG. 1 shows the comparison of control plants,NtVHA-a1relative expression of the gene in the gene-silenced plant;

FIG. 2 is a comparison of spermine content in virus-induced gene-silenced tobacco leaves and control tobacco leaves.

Detailed Description

The present application is further illustrated by the following examples, which are intended to provide a brief summary of the experimental background information in the examples given below, before describing the examples.

Biological material:

the Nicotiana benthamiana, a common tobacco material, is planted in a Zhenzhou tobacco institute planting base in the following examples, seedling is grown in a seedling raising pot, seedling division is carried out two weeks after germination, the seedling is planted in a plastic pot (10 cm multiplied by 10 cm), and daily fertilizer and water management and the like are carried out under the dark condition of 16h light/8 h at the temperature of 22 ℃;

The VIGS vector used in the following examples is a viral vector (TRV) derived from tobacco rattle virus, specifically TRV2 (a commonly used vector) having a Kanna selection marker and a 35S promoter, and TRV2 having multiple cloning sites such as EcoR I and BamH I, and can be used for carrying and transforming a foreign gene;

experimental reagent:

LB liquid medium, 1L content contains: 10 g of bacto peptone (bacteriological peptone); 10 g sodium chloride (NaCl); 5g yeast extract (yeast extract), autoclaving;

YEB liquid culture medium, 1L content contains: 5g beef extract (beef extract); 5g bacterial peptone (bacteriological peptone); 5g sucrose (sucrose); 1 g yeast extract (yeast extract); 2 mL of 1M magnesium sulfate (MgSO 4), autoclaved;

1M 2- (N-morpholine) ethanesulfonic acid (MES) stock: ddH₂Dissolving O, filtering, sterilizing, and storing at-20 ℃ for later use;

200 mM Acetosyringone (Acetosyringone, As) stock solution: dissolving Dimethyl Sulfoxide (DSMO), and storing at-20 deg.C;

MMA（100 mL）：1 mL（1 M）MgCl2；1 mL（1 M,pH5.6）MES；75 μL（200 mM）As。

example 1

This example relates to tobaccoNtVHA-a1The construction of gene cloning and silencing vectors is briefly described below.

(1) TobaccoNtVHA-a1Cloning of genes

Tobacco genome and related methods based on prophaseNtVHA-a1Gene research, selecting specific coding sequence as target segment, designing primer sequence for PCR amplification as follows:

NtVHA-a1-F：5’- TTTGCGAAGACCAAGATACA - 3’，

NtVHA-a1-R：5’- GCTGCCTAGTTTCTGAGAGC - 3’；

using cDNA of tobacco K326 leaf as template, carrying out PCR amplification to obtainNtVHA-a1A gene;

the PCR amplification procedure was: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 30s, and complete extension at 72 ℃ for 5min after 34 cycles;

and carrying out agarose gel electrophoresis detection on the PCR amplification product, and recovering the electrophoresis product for later use.

(2) Construction of recombinant TRV 2-NtVHA-a 1 vector

Carrying out EcoRI and BamHI double enzyme digestion on the PCR amplification product in the step (1), simultaneously carrying out EcoRI and BamHI double enzyme digestion on an empty vector TRV2, respectively recovering enzyme digestion products, and utilizing T4 DNA ligase to carry out ligation;

transforming the ligation product into escherichia coli competent DH5 alpha, coating the transformation product on LB solid culture medium containing 50mg/L Kan after the transformation operation is finished, and culturing at 37 ℃ overnight;

and selecting positive single colonies, amplifying, and then further performing PCR identification, and ensuring that a correctly constructed recombinant vector TRV 2-NtVHA-a 1 is obtained by combining sequencing verification.

Note that, tobaccoNtVHA-a1The gene comprises 2454 bases, the base sequence is shown as SEQ ID NO.1, and the specific base sequence is as follows:

ATGGAGTACATAGACAACATGCCGCCAATGGATCTGATGCGCTCGGAGAAGATGACTTTCGTTCAGCTTATTATTCCTTTTGAGTCCGCTCATCGTGCTGTCACTTACCTTGGCCAACTCGGACTTCTCCAATTCCGTGATCTAAATGCCGAAAAGAGCCCTTTCCAGAGAACATTTGTAAACCAGGTAAAAAGATGCGCCGAGATGTCAAGAAAACTACGATATTTCAAAGATCAGATACACAAAGCCGGTCTATTGCCTCCTCCTCTTCCTGCTTCCCAACCTGATATTGAATTAGAAGAATTGGAGATACAACTGGCAGAGCATGAACATGAGTTGATTGAAATGAATGCTAATAGTGAGAAATTGCGGCAATCATATAATGAGCTGCTTGAGTTTAAGATGGTATTGCAAAAGGCTAGTGGCTTCCTTGTTTCAAGTAGTCATACCACTGATCAGGAAACAGAACTGGTTGAAAATGTGTACTCCAATGATAACCATGCTGATACAGCATCATTACTTGAGCAGGAGATGCGTTCAGAACTGTCAAATCAGTCTGGAGTTAGATTTATGAGTGGCATTATCTGCAAGTCCAAGGTTGTTCAATTTGAAAGAATGCTGTTTCGTGCTACAAGGGGTAATATGCTTTTCAATCAGGCAGTTGCTGATGATGAGATACTGGATCCTTCCTCAAACGAAATGGTTGAGAAAGTAGTCTTTGTAGTATTCTTTTCAGGTGAGCAGGCGAGAACAAAAATACTGAAAATATGTGAGGCATTTGGTGCAAATTGCTATCCTGTTCCTGAAGACACGACAAAGAGAAGGCAGATAACTCAAGAAGTTTTGTTTCGGCTATCTGAATTAGAGACCACTCTGGATGCTGGACTGCGCCATAGAGATAAGGCTTTGACCTCCATAGGGTATCACCTTACAAAATGGATAAACATGGTTAAAACACAAAAGGCAGTGTATGACACGTTAAATATGCTAAATTTCGATGTTACAAAGAAGTGCCTTGTGGGTGAGGGCTGGTGTCCAATATTTGCGAAGACCAAGATACAAGAGGCTTTGCAGCGTGGAACATTTGATAGCAGTTCACAAGTGGGCATTATATTTCATGTGATGGATGCTGTGGAGTCACCTCCAACATACTTTAGGACAAACAGTTTCACAAATGCATTTCAAGAAATTGTTGATGCATATGGTGTTGCTAAATACCAGGAGGCAAATCCAGCTGTTTATACCATTGTTACATTTCCTTTCCTTTTTGCTGTGATGTTTGGGGACTGGGGTCATGGAATCTGCTTGCTGTTGGGAGCATTAGTTCTTATCGCAAGGGAAAGCAAACTTAGCTCTCAGAAACTAGGCAGCTTCATGGAGATGCTCTTTGGGGGTCGCTATGTACTCCTGTTGATGTCAATATTTTCAATTTACTGTGGCTTGATATATAATGAATTCTTCTCAGTTCCCTTTCACATATTTGGTGACTCAGCGTACAAATGCCGAGATGCTACATGCAGTGATGCACGGACAGTTGGTTTAGTAAAATACAATGATCCATATCCATTTGGTGTGGACCCAAGCTGGAGAGGCAGCCGTTCAGAGCTTCCTTTCTTGAATTCTCTTAAGATGAAGATGTCTATTTTGTTGGGTGTGGCCCAGATGAACCTCGGAATTATTTTAAGTTATTTCAATGCACGTTTCTTCAGCAGCTCAATTGATATTAAGTATCAGTTTATTCCACAAATAATCTTTCTCAACAGCCTCTTTGGATACCTTTCTCTTCTTATTCTTGTCAAATGGTGCACTGGTTCTCAAGCAGATCTATATCATGTTATGATTTATATGTTCTTAAGTCCTTTTGAGGCTCTTGGTGAAAATAGGTTGTTCTGGGGCCAGAGTGTGCTTCAGGTAATATTGCTGCTTTTGGCACTTGTTGCTGTCCCGTGGATGCTCTTCCCAAAACCTTTTATTTTGAAAAGTCTTCATATGGAGAGATTTCAAGGTCGTACATACGGGATACTTGGAACCTCCGAGATGGTTATTGACGAAGAACCAGGCTCTGCCAGTCAGCATGCTGAGGAGTTTAACTTTAGTGAGGTTTTTGTGCACCAAATGATACACTCTATTGAATTTGTTCTTGGTGCTGTTTCAAACACTGCATCGTATCTTCGGCTTTGGGCTTTGAGTTTGGCTCACTCTGAATTATCTACTGTGTTCTATGAGAAAGTTCTCCTCCTTGCCTGGGGGTATGATAACATCGTCATTCGGCTAGTGGGGCTGGCAGTTTTTGCCTTTGCGACAACTTTTATCCTACTTATGATGGAGACTCTTAGTGCATTCCTTCATGCATTGCGTCTTCATTGGGTGGAATTCCAGAACAAGTTTTATCATGGGGATGGTTATAAGTTCAAGCCATTCTCCTTTGCCTTGTTAGCAGATGATGACGATTAG。

The tobacco protein NtVHA-a1 comprises 817 amino acids, the amino acid sequence is shown as SEQ ID NO.2, and the specific amino acid sequence is as follows:

MEYIDNMPPMDLMRSEKMTFVQLIIPFESAHRAVTYLGQLGLLQFRDLNAEKSPFQRTFVNQVKRCAEMSRKLRYFKDQIHKAGLLPPPLPASQPDIELEELEIQLAEHEHELIEMNANSEKLRQSYNELLEFKMVLQKASGFLVSSSHTTDQETELVENVYSNDNHADTASLLEQEMRSELSNQSGVRFMSGIICKSKVVQFERMLFRATRGNMLFNQAVADDEILDPSSNEMVEKVVFVVFFSGEQARTKILKICEAFGANCYPVPEDTTKRRQITQEVLFRLSELETTLDAGLRHRDKALTSIGYHLTKWINMVKTQKAVYDTLNMLNFDVTKKCLVGEGWCPIFAKTKIQEALQRGTFDSSSQVGIIFHVMDAVESPPTYFRTNSFTNAFQEIVDAYGVAKYQEANPAVYTIVTFPFLFAVMFGDWGHGICLLLGALVLIARESKLSSQKLGSFMEMLFGGRYVLLLMSIFSIYCGLIYNEFFSVPFHIFGDSAYKCRDATCSDARTVGLVKYNDPYPFGVDPSWRGSRSELPFLNSLKMKMSILLGVAQMNLGIILSYFNARFFSSSIDIKYQFIPQIIFLNSLFGYLSLLILVKWCTGSQADLYHVMIYMFLSPFEALGENRLFWGQSVLQVILLLLALVAVPWMLFPKPFILKSLHMERFQGRTYGILGTSEMVIDEEPGSASQHAEEFNFSEVFVHQMIHSIEFVLGAVSNTASYLRLWALSLAHSELSTVFYEKVLLLAWGYDNIVIRLVGLAVFAFATTFILLMMETLSAFLHALRLHWVEFQNKFYHGDGYKFKPFSFALLADDDD。

example 2

Based on example 1, by using the agrobacterium-mediated VIGS technology, the inventors further transformed tobacco plants with the constructed recombinant TRV2-NtVHA-a1 vector, and performed verification analysis on the phenotypic change of the relevant plants, and the specific experimental process is summarized as follows.

(1) Transformation of Agrobacterium

It should be noted that, referring to the operation of example 1 and the prior art, the inventors prepared TRV2-PDS recombinant vector as a control, and the specific transformation process was:

positive cloning plasmids of TRV2 (vector control), TRV2-PDS (VIGS efficiency control) and TRV2-NtVHA-a1 were transformed into Agrobacterium GV3101 competent cells by electric shock transformation, cultured and selected using YEB plates containing 50mg/L Kan and 50mg/L Rif, and cultured at 28 ℃ for 2 days in an inverted manner, and then screened for Agrobacterium carrying the gene of interest by colony PCR.

(2) Preparation of a bacterial solution for transfection

Culturing the positive agrobacterium clones obtained by screening in the step (1) in 5 mL of YEB liquid culture medium (containing 50mg/L Kan and 50mg/L Rif) at 28 ℃ and 250 rpm overnight;

50uL of the overnight culture was inoculated into 50 mL of YEB liquid medium (containing 50 mg/L Kan), and cultured to OD₆₀₀= 1.0-1.5, centrifuging at 4000g for 5 min, collecting the cells, resuspending with MMA (1 mL (1M) MgCl 2; 1 mL (1M, pH 5.6) MES; 75. mu.L (200 mM) As), and adjusting OD₆₀₀= about 1.0; finally, the mixture is placed at room temperature for about 3 hours and then used as a bacterial liquid for transfection.

(3) Transient transformation

And (3) taking 3-4 w (week) of seedling-age Nicotiana benthamiana leaves as an experimental material, injecting the bacterial liquid for transfection prepared in the step (2) into the tobacco leaves by using a1 mL-specification injector, continuously culturing the injected tobacco in an artificial incubator, and observing the phenotypic change.

Further by qRT-PCRNtVHA-a1The gene expression condition is detected, and the result is shown in figure 1, and the expression quantity of NtVHA-a1 in the infected plant of TRV 2-NtVHA-a 1 is obviously reduced.

Further, the inventors examined the content of spermine in the experimental group (TRV 2-NtVHA-a 1-impregnated plants) and the control group (TRV 2-GFP impregnated plants), and the results are shown in FIG. 2:

as can be seen from the results of FIG. 2, the spermine content in the experimental group is significantly reduced compared with that in the control group, which further indicates that the content of the biogenic amine component in the tobacco leaves can be regulated and controlled by silencing the NtVHA-a1 gene, and further a certain technical basis can be laid for tobacco breeding and quality regulation and control.

SEQUENCE LISTING

<110> Zhengzhou tobacco institute of China tobacco general Co

<120> tobacco protein NtVHA-a1 and application thereof

<130> none

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 2454

<212> DNA

<213> Nicotiana tabacum

<400> 1

atggagtaca tagacaacat gccgccaatg gatctgatgc gctcggagaa gatgactttc 60

gttcagctta ttattccttt tgagtccgct catcgtgctg tcacttacct tggccaactc 120

ggacttctcc aattccgtga tctaaatgcc gaaaagagcc ctttccagag aacatttgta 180

aaccaggtaa aaagatgcgc cgagatgtca agaaaactac gatatttcaa agatcagata 240

cacaaagccg gtctattgcc tcctcctctt cctgcttccc aacctgatat tgaattagaa 300

gaattggaga tacaactggc agagcatgaa catgagttga ttgaaatgaa tgctaatagt 360

gagaaattgc ggcaatcata taatgagctg cttgagttta agatggtatt gcaaaaggct 420

agtggcttcc ttgtttcaag tagtcatacc actgatcagg aaacagaact ggttgaaaat 480

gtgtactcca atgataacca tgctgataca gcatcattac ttgagcagga gatgcgttca 540

gaactgtcaa atcagtctgg agttagattt atgagtggca ttatctgcaa gtccaaggtt 600

gttcaatttg aaagaatgct gtttcgtgct acaaggggta atatgctttt caatcaggca 660

gttgctgatg atgagatact ggatccttcc tcaaacgaaa tggttgagaa agtagtcttt 720

gtagtattct tttcaggtga gcaggcgaga acaaaaatac tgaaaatatg tgaggcattt 780

ggtgcaaatt gctatcctgt tcctgaagac acgacaaaga gaaggcagat aactcaagaa 840

gttttgtttc ggctatctga attagagacc actctggatg ctggactgcg ccatagagat 900

aaggctttga cctccatagg gtatcacctt acaaaatgga taaacatggt taaaacacaa 960

aaggcagtgt atgacacgtt aaatatgcta aatttcgatg ttacaaagaa gtgccttgtg 1020

ggtgagggct ggtgtccaat atttgcgaag accaagatac aagaggcttt gcagcgtgga 1080

acatttgata gcagttcaca agtgggcatt atatttcatg tgatggatgc tgtggagtca 1140

cctccaacat actttaggac aaacagtttc acaaatgcat ttcaagaaat tgttgatgca 1200

tatggtgttg ctaaatacca ggaggcaaat ccagctgttt ataccattgt tacatttcct 1260

ttcctttttg ctgtgatgtt tggggactgg ggtcatggaa tctgcttgct gttgggagca 1320

ttagttctta tcgcaaggga aagcaaactt agctctcaga aactaggcag cttcatggag 1380

atgctctttg ggggtcgcta tgtactcctg ttgatgtcaa tattttcaat ttactgtggc 1440

ttgatatata atgaattctt ctcagttccc tttcacatat ttggtgactc agcgtacaaa 1500

tgccgagatg ctacatgcag tgatgcacgg acagttggtt tagtaaaata caatgatcca 1560

tatccatttg gtgtggaccc aagctggaga ggcagccgtt cagagcttcc tttcttgaat 1620

tctcttaaga tgaagatgtc tattttgttg ggtgtggccc agatgaacct cggaattatt 1680

ttaagttatt tcaatgcacg tttcttcagc agctcaattg atattaagta tcagtttatt 1740

ccacaaataa tctttctcaa cagcctcttt ggataccttt ctcttcttat tcttgtcaaa 1800

tggtgcactg gttctcaagc agatctatat catgttatga tttatatgtt cttaagtcct 1860

tttgaggctc ttggtgaaaa taggttgttc tggggccaga gtgtgcttca ggtaatattg 1920

ctgcttttgg cacttgttgc tgtcccgtgg atgctcttcc caaaaccttt tattttgaaa 1980

agtcttcata tggagagatt tcaaggtcgt acatacggga tacttggaac ctccgagatg 2040

gttattgacg aagaaccagg ctctgccagt cagcatgctg aggagtttaa ctttagtgag 2100

gtttttgtgc accaaatgat acactctatt gaatttgttc ttggtgctgt ttcaaacact 2160

gcatcgtatc ttcggctttg ggctttgagt ttggctcact ctgaattatc tactgtgttc 2220

tatgagaaag ttctcctcct tgcctggggg tatgataaca tcgtcattcg gctagtgggg 2280

ctggcagttt ttgcctttgc gacaactttt atcctactta tgatggagac tcttagtgca 2340

ttccttcatg cattgcgtct tcattgggtg gaattccaga acaagtttta tcatggggat 2400

ggttataagt tcaagccatt ctcctttgcc ttgttagcag atgatgacga ttag 2454

<210> 2

<211> 817

<212> PRT

<213> Nicotiana tabacum

<400> 2

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1 5 10 15

Lys Met Thr Phe Val Gln Leu Ile Ile Pro Phe Glu Ser Ala His Arg

20 25 30

Ala Val Thr Tyr Leu Gly Gln Leu Gly Leu Leu Gln Phe Arg Asp Leu

35 40 45

Asn Ala Glu Lys Ser Pro Phe Gln Arg Thr Phe Val Asn Gln Val Lys

50 55 60

Arg Cys Ala Glu Met Ser Arg Lys Leu Arg Tyr Phe Lys Asp Gln Ile

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His Lys Ala Gly Leu Leu Pro Pro Pro Leu Pro Ala Ser Gln Pro Asp

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Ile Glu Leu Glu Glu Leu Glu Ile Gln Leu Ala Glu His Glu His Glu

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Leu Ile Glu Met Asn Ala Asn Ser Glu Lys Leu Arg Gln Ser Tyr Asn

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Glu Leu Leu Glu Phe Lys Met Val Leu Gln Lys Ala Ser Gly Phe Leu

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Val Ser Ser Ser His Thr Thr Asp Gln Glu Thr Glu Leu Val Glu Asn

145 150 155 160

Val Tyr Ser Asn Asp Asn His Ala Asp Thr Ala Ser Leu Leu Glu Gln

165 170 175

Glu Met Arg Ser Glu Leu Ser Asn Gln Ser Gly Val Arg Phe Met Ser

180 185 190

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

195 200 205

Arg Ala Thr Arg Gly Asn Met Leu Phe Asn Gln Ala Val Ala Asp Asp

210 215 220

Glu Ile Leu Asp Pro Ser Ser Asn Glu Met Val Glu Lys Val Val Phe

225 230 235 240

Val Val Phe Phe Ser Gly Glu Gln Ala Arg Thr Lys Ile Leu Lys Ile

245 250 255

Cys Glu Ala Phe Gly Ala Asn Cys Tyr Pro Val Pro Glu Asp Thr Thr

260 265 270

Lys Arg Arg Gln Ile Thr Gln Glu Val Leu Phe Arg Leu Ser Glu Leu

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Glu Thr Thr Leu Asp Ala Gly Leu Arg His Arg Asp Lys Ala Leu Thr

290 295 300

Ser Ile Gly Tyr His Leu Thr Lys Trp Ile Asn Met Val Lys Thr Gln

305 310 315 320

Lys Ala Val Tyr Asp Thr Leu Asn Met Leu Asn Phe Asp Val Thr Lys

325 330 335

Lys Cys Leu Val Gly Glu Gly Trp Cys Pro Ile Phe Ala Lys Thr Lys

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Ile Gln Glu Ala Leu Gln Arg Gly Thr Phe Asp Ser Ser Ser Gln Val

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Gly Ile Ile Phe His Val Met Asp Ala Val Glu Ser Pro Pro Thr Tyr

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Phe Arg Thr Asn Ser Phe Thr Asn Ala Phe Gln Glu Ile Val Asp Ala

385 390 395 400

Tyr Gly Val Ala Lys Tyr Gln Glu Ala Asn Pro Ala Val Tyr Thr Ile

405 410 415

Val Thr Phe Pro Phe Leu Phe Ala Val Met Phe Gly Asp Trp Gly His

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Gly Ile Cys Leu Leu Leu Gly Ala Leu Val Leu Ile Ala Arg Glu Ser

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

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Gly Arg Tyr Val Leu Leu Leu Met Ser Ile Phe Ser Ile Tyr Cys Gly

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Leu Ile Tyr Asn Glu Phe Phe Ser Val Pro Phe His Ile Phe Gly Asp

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Ser Ala Tyr Lys Cys Arg Asp Ala Thr Cys Ser Asp Ala Arg Thr Val

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Gly Leu Val Lys Tyr Asn Asp Pro Tyr Pro Phe Gly Val Asp Pro Ser

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Trp Arg Gly Ser Arg Ser Glu Leu Pro Phe Leu Asn Ser Leu Lys Met

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Lys Met Ser Ile Leu Leu Gly Val Ala Gln Met Asn Leu Gly Ile Ile

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Leu Ser Tyr Phe Asn Ala Arg Phe Phe Ser Ser Ser Ile Asp Ile Lys

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Tyr Gln Phe Ile Pro Gln Ile Ile Phe Leu Asn Ser Leu Phe Gly Tyr

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Leu Ser Leu Leu Ile Leu Val Lys Trp Cys Thr Gly Ser Gln Ala Asp

595 600 605

Leu Tyr His Val Met Ile Tyr Met Phe Leu Ser Pro Phe Glu Ala Leu

610 615 620

Gly Glu Asn Arg Leu Phe Trp Gly Gln Ser Val Leu Gln Val Ile Leu

625 630 635 640

Leu Leu Leu Ala Leu Val Ala Val Pro Trp Met Leu Phe Pro Lys Pro

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Phe Ile Leu Lys Ser Leu His Met Glu Arg Phe Gln Gly Arg Thr Tyr

660 665 670

Gly Ile Leu Gly Thr Ser Glu Met Val Ile Asp Glu Glu Pro Gly Ser

675 680 685

Ala Ser Gln His Ala Glu Glu Phe Asn Phe Ser Glu Val Phe Val His

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Gln Met Ile His Ser Ile Glu Phe Val Leu Gly Ala Val Ser Asn Thr

705 710 715 720

Ala Ser Tyr Leu Arg Leu Trp Ala Leu Ser Leu Ala His Ser Glu Leu

725 730 735

Ser Thr Val Phe Tyr Glu Lys Val Leu Leu Leu Ala Trp Gly Tyr Asp

740 745 750

Asn Ile Val Ile Arg Leu Val Gly Leu Ala Val Phe Ala Phe Ala Thr

755 760 765

Thr Phe Ile Leu Leu Met Met Glu Thr Leu Ser Ala Phe Leu His Ala

770 775 780

Leu Arg Leu His Trp Val Glu Phe Gln Asn Lys Phe Tyr His Gly Asp

785 790 795 800

Gly Tyr Lys Phe Lys Pro Phe Ser Phe Ala Leu Leu Ala Asp Asp Asp

805 810 815

Asp

Claims

1. Coding gene of tobacco protein NtVHA-a1NtVHA-a1The application of the gene silencing technology in the regulation and control of the biological amine substances in the tobacco leaves is characterized in that the content of the biological amine substances in the tobacco leaves is reduced by reducing the expression level of the NtVHA-a1 gene of the tobacco;

the biogenic amine substance is spermine;

the coding gene of the tobacco protein NtVHA-a1NtVHA-a1The base sequence is shown in SEQ ID NO. 1.

2. The application of the tobacco protein NtVHA-a1 in the regulation and control of the content of the biological amine substances in the tobacco leaves is characterized in that the protein is related to the content of the biological amine substances in the tobacco leaves, and the content of the biological amine substances in the tobacco leaves is obviously reduced after the expression content of the protein is reduced;

the biogenic amine substance is spermine;

the amino acid sequence of the tobacco protein NtVHA-a1 is shown in SEQ ID NO. 2.

3. Coding gene of tobacco protein NtVHA-a1NtVHA-a1The method for breeding a new variety of tobacco is characterized in that the technology of virus-induced gene silencing VIGS is utilized to construct a tobacco variety containing VIGSNtVHA-a1The virus-induced silencing vector of the gene is used for transforming tobacco and screening to obtain a new tobacco variety with the reduced spermine content;

The describedNtVHA-a1The base sequence of the gene is shown in SEQ ID NO. 1.

4. The method of breeding a new variety of tobacco as claimed in claim 3, wherein the method comprisesNtVHA-a1PCR amplification preparation in the course of virus-induced silencing of geneNtVHA-a1When the gene is used, the following steps are carried out:

(1) extracting genome and reverse transcribing into cDNA for later use;

NtVHA-a1-F：5’- TTTGCGAAGACCAAGATACA - 3’，

NtVHA-a1-R：5’- GCTGCCTAGTTTCTGAGAGC - 3’。