CN114085851A

CN114085851A - Gene related to regulating and controlling tobacco flowering time and application

Info

Publication number: CN114085851A
Application number: CN202111450871.XA
Authority: CN
Inventors: 邓乐乐; 杨叶昆; 李雪梅; 徐兴阳; 高茜; 向海英; 杨文武; 米其利; 曾婉俐; 张建铎; 许力; 刘欣; 蒋佳芮
Original assignee: China Tobacco Yunnan Industrial Co Ltd
Current assignee: China Tobacco Yunnan Industrial Co Ltd
Priority date: 2021-12-01
Filing date: 2021-12-01
Publication date: 2022-02-25
Anticipated expiration: 2041-12-01
Also published as: CN114085851B

Abstract

The invention discloses a gene related to tobacco flowering time, which is characterized in that a nucleotide sequence is shown as SEQ ID NO. 1. The invention also discloses the application of the gene related to the tobacco flowering time, and the tobacco flowering time is shortened and the flowering phase is advanced after the gene expression level is reduced.

Description

Gene related to regulating and controlling tobacco flowering time and application

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a gene NtCYP82 related to the regulation and control of tobacco flowering time and application thereof.

Background

The control of the growth period and the flowering time of the tobacco has important significance for tobacco production and realization of rapid breeding. The early flowering of the tobacco can shorten the time required by each generation in the tobacco breeding process, accelerate the breeding process, further shorten the period of breeding new tobacco varieties, and has important significance for realizing rapid breeding.

Plant flowering is an important process for transforming vegetative growth into reproductive growth of plants, and is strictly regulated and controlled by gene networks in plants and external environmental factors in a coordinated manner. With the rapid development of molecular genetics and molecular biology, the regulation and control processes of flowering pathways of arabidopsis, rice and the like are thoroughly researched, and the flowering time of plants and the morphogenesis of flower organs are generally determined by key genes such as SOC1, FT, LFY and the like.

Recent studies have shown that the most critical gene for flowering is the FT gene, which was first found in late-flowering mutants of Arabidopsis [1 ]. Thereafter, a plurality of FT homologous genes were sequentially isolated from plants such as rice, tomato, poplar, apple, corn, wheat, soybean, potato, and the like. The FT gene encodes phosphatidylethanolamine-binding protein [2-3], which promotes flowering in plants by binding lecithin having circadian rhythm variation [4 ]. Only 4 FT homologous genes NtFT1, NtFT2, NtFT3 and NtFT4 have been identified in tobacco at present, all of these 4 genes belonging to the phosphatidylethanolamine-binding protein (PEBP) family.

Shigella et al identified two MADS-box genes, NtMADS1 and NtMADS2, in tobacco, which are transcription factors with wide effects in the plant growth and development process and are particularly important for the flower development process.

It follows that there is less research on flowering-time related genes in tobacco and that flowering pathways and related mechanisms are much behind compared to other crops. Therefore, more genes related to the flowering time of tobacco are identified, and more genes and technical support is provided for optimizing the flowering period of the tobacco and quickly breeding.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provide a novel gene for regulating and controlling the flowering time of tobacco and application thereof, wherein the gene belongs to one of CYP450 gene family members, provides a novel theoretical basis for further clarifying the regulation and control mechanism of the flowering time of the tobacco, and provides a novel genetic material for cultivating tobacco varieties with changed flowering periods.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention discloses a gene related to regulating and controlling the flowering time of tobacco, which has a nucleotide sequence shown in SEQ ID NO.1 and comprises 2568 basic groups, is derived from tobacco (Nicotianatabacacum, named NtCYP82), and belongs to one of members of a CYP450 gene family.

Preferably, the amino acid sequence of the protein coded by the tobacco nicotine transporter-related gene is shown in SEQ ID NO.2 and comprises 463 amino acid residues.

The second aspect of the invention discloses the application of the gene related to the tobacco flowering time, and the tobacco flowering time is shortened and the flowering phase is advanced after the gene expression level is reduced.

Preferably, the gene editing is performed through a CRISPR/Cas 9-mediated gene editing technology, a CRISPR/Cas9 editing vector for knocking out the NtCYP82 gene is constructed, and a tobacco plant with the NtCYP82 gene edited is obtained after genetic transformation.

Preferably, the plant height, the waist leaf length, the waist leaf width, the total leaf number, the stem circumference or the stem leaf included angle of the gene editing tobacco plant in the bud flowering period are all obviously lower than those of a control plant, and the stamen length is shortened.

The regulatory gene is applied to the regulation of the flowering phase of tobacco. After the gene is edited, the NtCYP82 gene knockout-edited tobacco plant is obviously early blossoming compared with a control tobacco plant, and the flowering phase of tobacco can be obviously advanced. Provides a new theoretical basis for further clarifying the flowering time regulation mechanism of tobacco, and provides a new genetic material for cultivating tobacco varieties with changed flowering periods.

The invention also provides the application of the protein or the related biological material in at least one of the following (1) to (7):

(1) the plant height of the tobacco in the bud and flowering period is reduced;

(2) the length of the waist leaves of the tobacco in the bud flowering period is reduced;

(3) reduce the waist leaf width of tobacco in bud and blossom period

(4) Reducing the total leaf number of the tobacco in the bud and blossom period;

(5) the stem circumference of the tobacco in the bud blooming period is reduced;

(6) reducing the included angle of stem leaves of the tobacco in the bud and flowering period;

(7) resulting in the degradation of the stamens of the tobacco and the shortening of the length of the stamens.

The invention has the beneficial effects that:

1. according to the invention, a CRISPR/Cas9 editing vector for knocking out the NtCYP82 gene is constructed through a CRISPR/Cas9 mediated gene editing technology, and a tobacco plant with the NtCYP82 gene knocked out is obtained after editing material creation and molecular detection identification. Compared with a control tobacco plant, the edited tobacco plant obtained by knocking out the NtCYP82 gene obviously shortens the flowering time, and can obviously advance the flowering period of tobacco.

2. The NtCYP82 gene of the invention has pleiotropic effect, which not only has obvious effect on tobacco flowering, but also causes the growth potential of the whole edited tobacco plant population to be slightly worse than that of a control. The plant height, the waist leaf length, the waist leaf width, the total leaf number, the stem circumference and the stem leaf included angle of the edited tobacco plant in the bud flowering period are all obviously lower than those of a control tobacco plant, and the tobacco stamen is degraded and the length of the stamen is shortened.

3. According to the invention, the NtCYP82 gene is knocked out by using a CRISPR/Cas 9-mediated gene editing technology, so that an edited tobacco material with an obviously advanced flowering phase is obtained, a new theoretical basis is provided for further clarifying a tobacco flowering time regulation mechanism, and a new genetic material is provided for cultivating a tobacco variety with a changed flowering phase.

Drawings

FIG. 1 is a statistical plot of flowering time for control (right) and gene-edited tobacco plants (left) from unedited tobacco plants.

FIG. 2 is a statistical chart of plant height, waist leaf length, waist leaf width, total leaf number, stem circumference, and stem leaf included angle of unedited tobacco plant control (right) and gene-edited tobacco plant (left) in the bud flowering period.

FIG. 3 is a graph comparing stamen length of control (right) and gene-edited tobacco plants (left) of unedited tobacco plants.

Detailed Description

The technical solutions of the present invention are described in detail below by examples, and the following examples are only exemplary and can be used only for explaining and explaining the technical solutions of the present invention, but not construed as limiting the technical solutions of the present invention. In the embodiments of the present application, those who do not specify a specific technique or condition, and those who do follow the existing techniques or conditions in the field, and those who do not specify a manufacturer or a material used, are general products that can be obtained by purchasing. The percentage numbers are volume percentages and the ratios are volume ratios unless otherwise specified.

The tobacco variety used in the application is Honghuadajinyuan, a commercialized tobacco variety.

Example 1

This example is described briefly below, mainly with respect to the process of obtaining the tobacco flowering-time related gene NtCYP 82.

The method comprises the following steps of taking cultivated species of tobacco safflower large gold root as an experimental material, extracting total RNA of the tobacco root by using an RNA extraction kit, and carrying out reverse transcription to obtain cDNA for later use:

extracting total RNA of tobacco according to the instruction of the plant RNA extraction kit.

Mu.g of total RNA was extracted from leaves for reverse transcription in the following transcription system:

Total RNA 1μg

Oligo(dT)(10μM) 1.5μL

ddH2O up to 15μL

mixing the above system, placing in PCR, keeping temperature at 70 deg.C for 5min, removing, immediately placing on ice for 5min, and adding the following reagents into the system:

the system is placed in a PCR instrument, and is kept at 42 ℃ for 65min, 65 ℃ for 10min and 4 ℃ and then stored in a refrigerator at-20 ℃ for use.

By a homologous alignment method, referring to the sequence of an arabidopsis gene and the known partial gene sequence of tobacco, the sequence of an amplification primer is designed as follows:

F：5’-ATGGCTGACAATTATGGTCCAGCATTTA-3’；(SEQ ID No.3)

R：5’-TCAACAGCCATAAAGATTGGCATTG-3’；(SEQ ID No.4)

and (3) performing PCR amplification by using the prepared cDNA as a template and the primers:

amplification system (50 μ L):

mixing, centrifuging and performing PCR amplification, wherein the PCR reaction conditions are as follows: 30 cycles of 95 ℃ 10sec, 52 ℃ 30sec, 72 ℃ 2 min; 10min at 72 ℃; hold at 25 ℃.

And purifying and sequencing the amplified product to obtain a gene NtCYP82 sequence related to the tobacco flowering time, wherein the base sequence is shown as SEQ ID No.1 and comprises 2568 bases in total. After the gene sequence is translated, the coded protein sequence is shown as SEQ ID No.2 and comprises 463 amino acid residues in total, and further comparative analysis shows that the protein contains a sequence with high homology and is highly conserved.

Example 2

By utilizing the tobacco flowering time related gene NtCYP82 obtained in example 1, the invention further constructs a CRISPR/Cas9 vector and obtains a gene editing plant by utilizing the leaf disc method for transformation.

The specific 23nt nucleotide sequence (SEQ ID No.5) in the NtCYP82 gene is selected as a guide sequence of CRISPR/Cas9, the sequence fragment is connected with a CRISPR/Cas9 vector (provided by southwest university), transformation and PCR amplification detection are carried out, PCR positive clone is sent to a sequencing company for sequencing confirmation, and finally the CRISPR/Cas9-NtCYP82 editing vector is obtained.

The CRISPR/Cas9-NtCYP82 constructed in the previous step is used for editing vector plasmids, and genetic transformation and tissue culture are carried out by taking a safflower macrogol as an example to obtain plants subjected to knockout and editing of a gene NtCYP82 related to the tobacco flowering time, and related experimental processes are briefly introduced as follows.

Inoculating sterilized tobacco seeds onto MS culture medium, transferring to culture bottle containing MS solid culture medium after 4 cotyledons (15-20d) grow, and culturing at 25 + -1 deg.C under illumination intensity of 30-50 μmol/(m2 s) for 35-40d under illumination time of 16 h/d.

LBA4404 preserved at-80 ℃ is taken out to be electrically transformed into competent Agrobacterium cells, and the cells are frozen and thawed on ice. When competence is just thawed, 2 mu L of CRISPR/Cas9-NtCYP82 editing vector plasmid is added, mixed evenly and placed on ice. And then transferring the uniformly mixed competence into a precooled electric rotor, placing the electric rotor into an electric rotor for conversion, adding 1mL of YEB liquid culture medium after the conversion is finished, mixing with the conversion solution, and then placing the mixture in a shaking table at 28 ℃ and culturing for 1.5-2h at 200 rpm. The cells were centrifuged at 8000rpm to discard the supernatant medium, and then suspended in 200. mu.L of YEB liquid medium, spread on YEB solid medium containing 50mg/L rifampicin, 50mg/L streptomycin and 50mg/L kanamycin, and cultured in 28 ℃ inversion dark for 2-3 d.

Tobacco leaf disks are made into square leaf disks with the side length of 1cm in an ultraclean workbench, and agrobacterium colony containing CRISPR/Cas9-NtCYP82 editing vectors is prepared by MS liquid to form suspension bacterial liquid (OD600 is 0.6-0.8). And soaking and infecting the tobacco leaf discs for 10min by using the suspension agrobacterium liquid. Then, the leaf discs were placed on MS solid medium containing 2.0mg/L NAA +0.5 mg/L6-BA, and were co-cultured at 28 ℃ in the dark for 3 days. Then subculture is carried out, and the subcultured cells are placed on an MS solid culture medium containing 2.0mg/LNAA +0.5 mg/L6-BA +250mg/L Cb +50mg/L Kan under the culture conditions that: culturing at 28 deg.C for 16h/d under illumination with illumination intensity of 30-50 μmol/(m2 s), culturing at 25 deg.C for 8h/d under dark condition, culturing for 45-60d until differentiated bud is formed, and replacing differentiation culture medium every 7-10d for 3-4 times; culturing until a differentiated bud is formed; cutting off callus formed by existing differentiated bud, culturing on MS culture medium containing carbenicillin 500mg/L and kanamycin 50mg/L, culturing for 8-14d when the differentiated bud on callus grows to 2-4cm high and the culture condition is the same as that of differentiated culture; and (2) performing rooting culture on the regenerated plant, cutting a differentiated bud, inserting the cut differentiated bud into an MS culture medium containing 500mg/L carbenicillin and 50mg/L kanamycin to perform rooting culture, wherein the culture condition is consistent with the differentiation culture condition, culturing for 20-30d, performing regeneration and transplantation to a flowerpot, then performing transformation plant leaf sampling, delivering a Huamao gene to perform molecular detection, determining to obtain an NtCYP82 gene editing plant, and then performing seed harvesting to obtain T0 generation editing plant seeds. Carrying out self-copulating propagation on seeds of the T0 generation by 23 times, sampling leaves of a single plant when the plant grows to 5-6 leaves, carrying out molecular detection on the Huada gene, determining to obtain a plant subjected to homozygous editing of the NtCYP82 gene, and then harvesting to obtain T1 generation seeds subjected to homozygous editing of the NtCYP82 gene.

The application of the tobacco flowering phase regulating gene NtCYP82 provided by the invention is to reduce the expression of the NtCYP82 gene in a tobacco plant body, so that the tobacco flowering phase can be advanced. Methods of reducing gene expression or gene silencing commonly used in the art are suitable for use in the present invention.

Example 3

The plants determined to be NtCYP82 gene homozygous knockout by molecular detection in example 2 are used for seed collection to obtain gene homozygous editing materials. And then, carrying out agronomic character investigation at the bud initiation period of the NtCYP82 gene homozygous knockout material, wherein the agronomic character investigation specifically comprises the recording of tobacco flowering time, plant height, waist leaf length, waist leaf width, total leaf number, stem circumference, stem leaf included angle and stamen length.

Selecting tobacco plants in bud flowering period, and recording flowering time, plant height, waist leaf length, waist leaf width, total leaf number, stem girth, stem leaf included angle and stamen length of 60 control sample unedited tobacco plant samples. Selecting tobacco plants in bud flowering period, and recording flowering time, plant height, waist leaf length, waist leaf width, total leaf number, stem circumference, stem leaf included angle and stamen length of 60 tobacco plant samples homozygously edited by NtCYP82 genes.

The results of recording and analyzing the flowering time, plant height, waist leaf length, waist leaf width, total leaf number, stem circumference, stem leaf angle and stamen length of the control unedited tobacco plant and the NtCYP82 gene homozygous edited tobacco plant are shown in FIGS. 1, 2 and 3.

FIG. 1 is a statistical plot of flowering time for control (right) and gene-edited tobacco plants (left) from unedited tobacco plants. As can be seen from fig. 1, the NtCYP82 gene-edited tobacco plants showed shorter flowering times than the control unedited tobacco plants. FIG. 2 is a statistical chart of plant height, waist leaf length, waist leaf width, total leaf number, stem circumference, and stem leaf included angle of unedited tobacco plant control (right) and gene-edited tobacco plant (left) in the bud flowering period. As can be seen from FIG. 2, the plant height, the waist leaf length, the waist leaf width, the total leaf number, the stem circumference and the included angle between the stem and the leaf of the tobacco plant edited by the NtCYP82 gene are all smaller than those of the unedited tobacco plant of the control sample. Comparison of stamen length for unedited tobacco plant control (right) and gene-edited tobacco plants (left). As can be seen from fig. 3, the NtCYP82 gene edited tobacco plants had shorter stamen lengths than the control unedited tobacco plants.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

<110> tobacco industry Limited liability company in Yunnan

<120> gene related to tobacco flowering time regulation and control and application thereof

<130> RIB210585

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2568

<212> DNA

<213> Artificial sequence (NtCYP82)

<400> 1

atggctgaca attatggtcc agcatttagt ataagactag gcagtcgtcg agcatttgtg 60

gttagcagtt gggaaatggc caaagaatgt tttactttga atgacaaagc tttggctagc 120

cgtcccacaa cagttgctgc aaagcatatg ggttatggtt atgcagtttt tgggtttgca 180

ccttatagca ctttctggcg tgagatgagg aaaattgcta tgtatgaact tctttcgaat 240

cgtaggcttg atactcttaa gcatgttcaa gtatcagaag tggagatggg gatccaagag 300

ctgtacaaat tgtgggtcaa caacaactca ggctacaggt aaagctgtca aataggccgg 360

ttgggtcaaa tttgaatcat gacataacgt ttctaaataa cgtgtcatta gtggcgaagc 420

caataatttt acaaagggtg ttcaaggctt aatatatatt tataaaatta ccaaattaaa 480

caaaataatt tttcttttat ctttataaag agaaatggat gcaattatag agagagtaaa 540

tttttgtttg atgggctatg ttcgattatt gacctgtcaa atattttatt ctcaattcaa 600

cggggagatc aattttgtgg tcattacaaa tgtttttttt ttctttttgc attatattgt 660

ccttatcatg aaaatcgcta taaaatataa tataggaaat tggttaataa aatataggag 720

atgttttgta caatttgttt tttctattca aactgtaact gggtctaaga tcatttcctc 780

caattcaaat ccatccgtat atgatggatt ggtcgaatta cacttatttt atgaactaaa 840

tttgacacct ctaactacag gccaatactc gtcgagctca aacattggtt tgaggatttg 900

acattaaacg taatcgttag aatggttgct ggaaagcgtt attttggtgc aggagcaagt 960

tgtgatgatg atgaagctag acgttgccaa aaggcaataa atcaattctt tcatttgatt 1020

ggaatttttg taccatctga tgcttttcca attctggggt ggtttgatat acaagggcat 1080

gaaaaggcta tgaaaagaac agctaaagag cttgattcta tacttgaaag ctggctacaa 1140

gaacaccgag agaagagaag attgagtgag ggtaagaatg aaggtgcaca agatttcatt 1200

gatgttatgt tatcacttca agaagaaggc cgtctctcca attttcaata tgatgctgat 1260

acaagtatca agtccacttg cctggtatgt tcttaaatct tctatgtcat ttttgtttag 1320

aaaaaagcgc ttcgcttatt taaagtcgat tcttccttgc ctttgagaac cggacatatt 1380

gtatcacgat cttatctgaa agtttagatt atagtttctc cactttattt tatgcatctt 1440

agtttgatta gacatgatgt ataaagaaaa cttttgaatc ttgtcttaaa ctacaagtgt 1500

ttatagcata tcaaaaattg ctttttgaat cttgtacatc tcaaacatgc catgtcaatt 1560

aatataaggg aaagtgtcat taagggtaaa atgaaaatct tgaattaaaa acttactaaa 1620

tatagaaaaa taacattctt ttatgaaaca aactaaaaaa aaataagaca cgatgaattg 1680

aaatggatag agtatcaact tttttaagtc tttatattaa taatgaattt ttgcataatc 1740

aatgtaattt aatctctagc agaaaatttc ttgacttatg aattttccat gttattggtt 1800

tacggatata cgcagttacc cttaattaat tttgatatat gatatggtct gatagtataa 1860

atttaagata ataatgtcat tgtataaaag tcaaaccctt tacaatttgt tgtacttaaa 1920

gtattctttt tggtctaggc gcttattctt ggaggcagcg acacaacagc aggaacacta 1980

acatgggcaa tttcattact cctaaataat cctgaaatat tgcgaaaagc acaagaagaa 2040

atagatgttc atgttggtaa agacagacaa gttgacgaat ccgacataca aaacttagcc 2100

tatattcaag ccattattaa agaaacctta cgtttgtacc ctgctggtcc tctcttagga 2160

cctagagaag ccatggacga atgtgaagtt ggtggctata aaattacccc tggaactcgt 2220

ttaattgtaa atgtatggaa aatacaaagg gatccaagaa tttgggaaga tcctgattct 2280

tttaaaccag atagattttt gatgagtaat tcaaatgttg atgtaaaagg ccaagatttt 2340

gagctcattc cgtttggttc tggtagacga tcttgtcctg gcgtatcatt ggcgctacaa 2400

gttcttcatt tgacattggc acgttttctt caggctttcg agttttccaa gcccgtggat 2460

gatcaactta ttgacttgac ggagagccct ggtttaacta tacctaaagc aacaccatta 2520

gatgttctca ttactccgcg cctcaatgcc aatctttatg gctgttga 2568

<210> 2

<211> 463

<212> PRT

<213> Artificial sequence (NtCYP82)

<400> 2

Met Ala Asp Asn Tyr Gly Pro Ala Phe Ser Ile Arg Leu Gly Ser Arg

1 5 10 15

Arg Ala Phe Val Val Ser Ser Trp Glu Met Ala Lys Glu Cys Phe Thr

20 25 30

Leu Asn Asp Lys Ala Leu Ala Ser Arg Pro Thr Thr Val Ala Ala Lys

35 40 45

His Met Gly Tyr Gly Tyr Ala Val Phe Gly Phe Ala Pro Tyr Ser Thr

50 55 60

Phe Trp Arg Glu Met Arg Lys Ile Ala Met Tyr Glu Leu Leu Ser Asn

65 70 75 80

Arg Arg Leu Asp Thr Leu Lys His Val Gln Val Ser Glu Val Glu Met

85 90 95

Gly Ile Gln Glu Leu Tyr Lys Leu Trp Val Asn Asn Asn Ser Gly Tyr

100 105 110

Arg Pro Ile Leu Val Glu Leu Lys His Trp Phe Glu Asp Leu Thr Leu

115 120 125

Asn Val Ile Val Arg Met Val Ala Gly Lys Arg Tyr Phe Gly Ala Gly

130 135 140

Ala Ser Cys Asp Asp Asp Glu Ala Arg Arg Cys Gln Lys Ala Ile Asn

145 150 155 160

Gln Phe Phe His Leu Ile Gly Ile Phe Val Pro Ser Asp Ala Phe Pro

165 170 175

Ile Leu Gly Trp Phe Asp Ile Gln Gly His Glu Lys Ala Met Lys Arg

180 185 190

Thr Ala Lys Glu Leu Asp Ser Ile Leu Glu Ser Trp Leu Gln Glu His

195 200 205

Arg Glu Lys Arg Arg Leu Ser Glu Gly Lys Asn Glu Gly Ala Gln Asp

210 215 220

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

225 230 235 240

Phe Gln Tyr Asp Ala Asp Thr Ser Ile Lys Ser Thr Cys Leu Ala Leu

245 250 255

Ile Leu Gly Gly Ser Asp Thr Thr Ala Gly Thr Leu Thr Trp Ala Ile

260 265 270

Ser Leu Leu Leu Asn Asn Pro Glu Ile Leu Arg Lys Ala Gln Glu Glu

275 280 285

Ile Asp Val His Val Gly Lys Asp Arg Gln Val Asp Glu Ser Asp Ile

290 295 300

Gln Asn Leu Ala Tyr Ile Gln Ala Ile Ile Lys Glu Thr Leu Arg Leu

305 310 315 320

Tyr Pro Ala Gly Pro Leu Leu Gly Pro Arg Glu Ala Met Asp Glu Cys

325 330 335

Glu Val Gly Gly Tyr Lys Ile Thr Pro Gly Thr Arg Leu Ile Val Asn

340 345 350

Val Trp Lys Ile Gln Arg Asp Pro Arg Ile Trp Glu Asp Pro Asp Ser

355 360 365

Phe Lys Pro Asp Arg Phe Leu Met Ser Asn Ser Asn Val Asp Val Lys

370 375 380

Gly Gln Asp Phe Glu Leu Ile Pro Phe Gly Ser Gly Arg Arg Ser Cys

385 390 395 400

Pro Gly Val Ser Leu Ala Leu Gln Val Leu His Leu Thr Leu Ala Arg

405 410 415

Phe Leu Gln Ala Phe Glu Phe Ser Lys Pro Val Asp Asp Gln Leu Ile

420 425 430

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

435 440 445

Asp Val Leu Ile Thr Pro Arg Leu Asn Ala Asn Leu Tyr Gly Cys

450 455 460

<210> 3

<211> 28

<212> DNA

<213> Artificial sequence (NtCYP82)

<400> 3

atggctgaca attatggtcc agcattta 28

<210> 4

<211> 25

<212> DNA

<213> Artificial sequence (NtCYP82)

<400> 4

tcaacagcca taaagattgg cattg 25

<210> 5

<211> 23

<212> DNA

<213> Artificial sequence (NtCYP82)

<400> 5

ttgtgggtca acaacaactc agg 23

Claims

1. A gene related to tobacco flowering time is characterized in that a nucleotide sequence is shown as SEQ ID No. 1.

2. The tobacco flowering-time related gene according to claim 1, wherein the protein is encoded by the tobacco flowering-time related gene.

3. The tobacco flowering-time related gene according to claim 2, wherein the amino acid sequence of the encoded protein is represented by SEQ ID No. 2.

4. Use of a gene involved in flowering-time of tobacco according to any one of claims 1 to 3, wherein the flowering-time of tobacco is shortened and the flowering-time is advanced after the expression level of the gene is decreased.

5. The application of claim 4, wherein the gene editing is performed by a CRISPR/Cas 9-mediated gene editing technology, a CRISPR/Cas9 editing vector for knocking out NtCYP82 gene is constructed, and a tobacco plant with the NtCYP82 gene edited is obtained after genetic transformation.

6. The use of claim 4, wherein the plant height, waist leaf length, waist leaf width, total leaf number, stem circumference or stem and leaf angle of the gene-edited tobacco plant at the bud flowering stage are significantly lower than those of the control plant, and lead to a shorter stamen length.