CN114085851B - Gene for regulating and controlling tobacco flowering time and application thereof - Google Patents
Gene for regulating and controlling tobacco flowering time and application thereof Download PDFInfo
- Publication number
- CN114085851B CN114085851B CN202111450871.XA CN202111450871A CN114085851B CN 114085851 B CN114085851 B CN 114085851B CN 202111450871 A CN202111450871 A CN 202111450871A CN 114085851 B CN114085851 B CN 114085851B
- Authority
- CN
- China
- Prior art keywords
- gene
- tobacco
- flowering time
- ntcyp82
- plant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
- C12N9/0081—Cholesterol monooxygenase (cytochrome P 450scc)(1.14.15.6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
- C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Abstract
The invention discloses a tobacco flowering time related gene which is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1. The invention also discloses application of the gene related to the flowering time of the tobacco, and after the expression quantity of the gene is reduced, the flowering time of the tobacco is shortened, and the flowering period is advanced.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a gene NtCYP82 related to regulation and control of tobacco flowering time and application thereof.
Background
The regulation and control of the tobacco growth period and flowering time have important significance for tobacco leaf production and rapid breeding. The tobacco early flowers can shorten the time required by each generation in the tobacco breeding process, quicken the breeding process, further shorten the period of breeding new tobacco varieties, and have important significance for realizing rapid breeding.
Flowering of plants is an important process for transforming plants from vegetative growth to reproductive growth, and is strictly regulated and controlled by the cooperation of plant internal gene networks and external environmental factors. Along with the rapid development of molecular genetics and molecular biology, the regulation and control process of flowering pathways of arabidopsis, rice and the like is thoroughly researched, and the whole body is formed by determining the flowering time of plants and the morphology of flowers 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, and at the earliest is 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, etc. The FT gene encodes phosphatidylethanolamine-binding protein [2-3], which promotes plant flowering by binding to lecithin with circadian variation [4]. Only 4 FT homologous genes NtFT1, ntFT2, ntFT3 and NtFT4 are currently identified in tobacco, all 4 belonging to the phosphatidylethanolamine-binding protein (PEBP) family.
Xie He and the like identify two MADS-box genes in tobacco, and NtMADS1 and NtMADS2 genes, which are transcription factors with wide functions in the plant growth and development process, and are particularly important for the flower development process.
It follows that flowering time related genes in tobacco are less studied and flowering pathways and related mechanisms are less studied than in other crops. Therefore, the identification of more genes related to flowering time of tobacco provides more genes and technical support for optimizing flowering time of tobacco and rapid breeding.
Disclosure of Invention
The invention aims to solve the defects of the prior art, provides a novel gene for regulating and controlling the flowering time of tobacco and application thereof, wherein the gene belongs to one member of CYP450 gene families, provides a novel theoretical basis for further elucidating the regulating and controlling mechanism of the flowering time of the tobacco, and provides novel genetic materials for cultivating tobacco varieties with changed flowering time.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the first aspect of the invention discloses a gene for regulating and controlling flowering time of tobacco, the nucleotide sequence of the gene is shown as SEQ ID NO.1, the gene comprises 2568 bases, the gene is derived from tobacco (Nicotianatabacum and named NtCYP 82), and the gene belongs to one member of CYP450 gene families.
Preferably, the amino acid sequence of the coding protein of the tobacco nicotine transporter related gene is shown as SEQ ID NO.2 and comprises 463 amino acid residues.
The invention also discloses the application of the gene related to the flowering time of the tobacco, and the flowering time of the tobacco is shortened and the flowering period is advanced after the expression level of the gene is reduced.
Preferably, the gene editing is performed by using a CRISPR/Cas9 mediated gene editing technology, a CRISPR/Cas9 editing vector for knocking out the NtCYP82 gene is constructed, and a tobacco plant edited by the NtCYP82 gene is obtained after genetic transformation.
Preferably, the plant height, waist leaf length, waist leaf width, total leaf number, stem circumference or stem leaf included angle of the gene-edited tobacco plant in the bud bloom stage are all significantly lower than those of the control plant, and the stamen length is shortened.
The application of the regulatory gene in regulating the flowering phase of tobacco. After the gene is edited, the NtCYP82 gene knockout edited tobacco plant is obviously early-flowering compared with a control tobacco plant, and the tobacco flowering period can be obviously advanced. Provides a new theoretical basis for further elucidating the flowering time regulation mechanism of the tobacco and provides a new genetic material for cultivating tobacco varieties with changed flowering time.
The invention also provides the use of the protein or the related biological material in at least one of the following (1) - (7):
(1) Reducing the plant height of tobacco in bud bloom stage;
(2) Reducing the waist leaf length of tobacco in bud bloom stage;
(3) Reducing the waist leaf width of tobacco in bud bloom stage
(4) Reducing the total leaf number of tobacco in bud bloom stage;
(5) Reducing the stem circumference of tobacco in bud bloom stage;
(6) Reducing the included angles of stems and leaves of tobacco in the bud bloom stage;
(7) Resulting in tobacco stamen degradation and shorter stamen length.
The beneficial effects of the invention are as follows:
1. the CRISPR/Cas9 editing vector for knocking out the NtCYP82 gene is constructed by a CRISPR/Cas9 mediated gene editing technology, and the tobacco plant with the knocked-out NtCYP82 gene is obtained after editing material creation and molecular detection and identification. Compared with a control tobacco plant, the obtained edited tobacco plant with the NtCYP82 gene knocked-out obviously shortens the flowering time, and can obviously advance the tobacco flowering period.
2. The NtCYP82 gene has multiple effects, has obvious influence on tobacco flowering, and also causes the growth vigor of the whole edited tobacco plant population to be slightly worse than that of a control. The plant height, waist leaf length, waist leaf width, total leaf number, stem circumference and stem leaf included angle of the edited tobacco plant in the bud bloom stage are all extremely lower than those of a control tobacco plant, and the tobacco stamen are degraded and the length of the stamen is shortened.
3. The invention uses CRISPR/Cas9 mediated gene editing technology to knock out the NtCYP82 gene to obtain edited tobacco materials with obviously advanced flowering phase, which provides a new theoretical basis for further elucidating the flowering time regulation mechanism of tobacco and provides new genetic materials for cultivating tobacco varieties with changed flowering phase.
Drawings
FIG. 1 is a statistical plot of flowering time for control (right) and gene-edited tobacco plants (left) for unedited tobacco plants.
FIG. 2 is a statistical plot of plant height, waist leaf length, waist leaf width, total leaf number, stem circumference, and stem leaf angle data for an unedited tobacco plant control (right) and a genetically-edited tobacco plant (left) at the bud bloom stage.
FIG. 3 is a graph comparing stamen length of unedited tobacco plant control (right) with that of genetically-edited tobacco plant (left).
Detailed Description
The following examples are given by way of illustration only and are not to be construed as limiting the scope of the invention. In the examples of the present application, where no specific technique or condition is noted, and where the materials or equipment used are not noted to the manufacturer, they are conventional products available for purchase, according to the state of the art or condition. Unless otherwise indicated, the percentages are by volume and the proportions are by volume.
The tobacco variety used in the present application is Honghuadajinyuan, a commercial tobacco variety.
Example 1
This example is mainly described below in terms of the process of obtaining the gene NtCYP82 related to the flowering time of tobacco.
The method comprises the steps of (1) taking a cultivar tobacco safflower large Jin Yuangen as an experimental material, extracting total RNA of tobacco roots 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.
1 μg total RNA extracted from leaf for reverse transcription was as follows:
Total RNA 1μg
Oligo(dT)(10μM) 1.5μL
ddH2O up to 15μL
mixing the above systems, placing in PCR, maintaining at 70deg.C for 5min, removing, immediately placing on ice for 5min, and adding the following reagents:
placing the above system into a PCR instrument, holding at 65min at 65deg.C, 10min at 65deg.C, and holding at 4deg.C, and storing in a refrigerator at-20deg.C.
By a homology comparison method, referring to the sequence of the Arabidopsis gene and the sequence of the known tobacco part gene, the amplification primer sequence is designed as follows:
F：5’-ATGGCTGACAATTATGGTCCAGCATTTA-3’；(SEQ ID No.3)
R：5’-TCAACAGCCATAAAGATTGGCATTG-3’；(SEQ ID No.4)
PCR amplification was performed using the cDNA prepared as described above as a template and the above primers:
amplification system (50 μl):
and (3) carrying out PCR amplification after uniformly mixing and centrifuging, wherein the PCR reaction conditions are as follows: 30 cycles of 95℃10sec,52℃30sec,72℃2 min; 72 ℃ for 10min; hold at 25 ℃.
And (3) purifying and sequencing the amplified product to obtain a gene NtCYP82 sequence related to the flowering time of the tobacco, 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 contains 463 amino acid residues, and further comparative analysis shows that the protein contains a sequence with high homology and is highly conserved.
Example 2
The invention further constructs a CRISPR/Cas9 vector by using the gene NtCYP82 related to the flowering time of tobacco obtained in the example 1, and obtains a gene editing plant by using a leaf disc method for transformation.
The 23nt nucleotide sequence (SEQ ID No. 5) which is more specific in the NtCYP82 gene is selected as a CRISPR/Cas9 guide sequence, and the sequence fragment is connected with a CRISPR/Cas9 carrier (provided by southwest university), transformed and PCR amplified detected, and the PCR positive clone is sent to a sequencing company for sequencing confirmation, so that the CRISPR/Cas9-NtCYP82 editing carrier is finally obtained.
The CRISPR/Cas9-NtCYP82 constructed in the last step is utilized to edit vector plasmids, and genetic transformation and tissue culture are carried out by taking safflower Dajinyuan as an example to obtain a plant with the gene NtCYP82 related to the flowering time of tobacco subjected to knockout editing, and the related experimental process is briefly described as follows.
And (3) after the surfaces of the tobacco seeds are disinfected, dibbling the tobacco seeds on an MS culture medium, growing until 4 cotyledons (15-20 d) are grown, transferring the cotyledons into a culture bottle containing an MS solid culture medium, and continuously culturing for 35-40d at the temperature of 25+/-1 ℃ under the condition that the illumination intensity is 30-50 mu mol/(m 2 s) and the illumination time is 16h/d for standby.
LBA4404 stored at-80℃was removed and competent Agrobacterium cells were electrotransformed and frozen and thawed on ice. When the competence was just thawed, 2 μl of CRISPR/Cas9-NtCYP 82-edited vector plasmid was added, mixed well and placed on ice. Transferring the uniformly mixed competence into a precooled electric rotating cup, placing the electric rotating cup into an electric rotating instrument for conversion, adding 1mL of YEB liquid culture medium and conversion liquid for mixing after conversion is finished, and placing the mixture into a shaking table at 28 ℃ for culturing at 200rpm for 1.5-2h. The cells were centrifuged at 8000rpm, the supernatant medium was discarded, and then 200. Mu.L of YEB liquid medium was used to suspend the cells, which were spread on YEB solid medium containing 50mg/L rifampicin, 50mg/L streptomycin and 50mg/L kanamycin, and inverted dark culture was performed at 28℃for 2-3d.
Square leaf discs with side length of 1cm were made in an ultra clean bench, and agrobacterium colonies containing CRISPR/Cas9-NtCYP82 editing vector were prepared as suspension (od600=0.6-0.8) with MS liquid. And soaking and infecting tobacco leaf discs for 10min by using suspension agrobacterium liquid. Then, the leaf discs were placed on MS solid medium containing 2.0mg/L NAA+0.5 mg/L6-BA, at 28℃in the dark, and co-cultured for 3d. Then carrying out secondary culture, and placing on an MS solid culture medium containing 2.0mg/LNAA+0.5 mg/L6-BA+250 mg/L Cb+50mg/L Kan, wherein the culture conditions are as follows: culturing at 28deg.C for 16h/d with light intensity of 30-50 μmol/(m2.s), culturing at 25deg.C in dark for 8h/d, culturing for 45-60d until differentiation bud forms, and changing differentiation culture medium for 3-4 times every 7-10 d; culturing until differentiation buds are formed; cutting off the callus formed by the existing differentiation buds, placing the callus on an MS culture medium containing 500mg/L carbenicillin and 50mg/L kanamycin for culture, and culturing for 8-14 days when the differentiation buds on the callus grow to 2-4cm high under the condition consistent with the differentiation culture condition; rooting and culturing regenerated plants, cutting off differentiated buds, inserting the cut off differentiated buds into an MS culture medium containing 500mg/L of carbenicillin and 50mg/L of kanamycin for rooting and culturing, wherein the culture conditions are consistent with the differentiation culture conditions, culturing for 20-30d, regenerating and transplanting the cut off differentiated buds to a flowerpot for culturing, sampling leaves of the transformed plants, carrying out molecular detection on the leaves of the transformed plants, determining to obtain NtCYP82 gene edited plants, and then harvesting to obtain T0 generation edited plant seeds. And (3) carrying out selfing homozygous propagation on the T0 generation seeds according to 23 times, sampling the leaves of a single plant when the plants grow to 5-6 leaves, carrying out molecular detection on the large gene, determining to obtain plants subjected to homozygous editing of the NtCYP82 genes, and then carrying out seed collection to obtain T1 generation seeds subjected to homozygous editing of the NtCYP82 genes.
The application of the tobacco flowering phase regulating gene NtCYP82 provided by the invention is that the expression of the NtCYP82 gene is reduced 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
And (3) carrying out seed collection by using the plant which is determined to be homozygous knockout of the NtCYP82 gene by molecular detection in the embodiment 2 to obtain the homozygous editing material of the gene. And then, carrying out agronomic character investigation in the bud bloom stage of the NtCYP82 gene homozygous knockout material, wherein the agronomic character investigation specifically comprises the recording of the flowering time, plant height, waist leaf length, waist leaf width, total leaf number, stem circumference, stem leaf included angle and stamen length of the tobacco.
And selecting tobacco plants in the bud bloom stage, and recording the flowering time, plant height, waist leaf length, waist leaf width, total leaf number, stem circumference, stem leaf included angle and stamen length of the sample of the 60 control unedited tobacco plants. And selecting tobacco plants in the bud bloom stage, and recording the flowering time, plant height, waist leaf length, waist leaf width, total leaf number, stem circumference, stem and leaf included angle and stamen length of a tobacco plant sample obtained by homozygous editing of 60 NtCYP82 genes.
The results of the record analysis of flowering time, plant height, waist leaf length, waist leaf width, total leaf number, stem circumference, stem leaf included 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) for unedited tobacco plants. As can be seen from fig. 1, the flowering time of the NtCYP82 gene-edited tobacco plants was shorter than that of the control-like unedited tobacco plants. FIG. 2 is a statistical plot of plant height, waist leaf length, waist leaf width, total leaf number, stem circumference, and stem leaf angle data for an unedited tobacco plant control (right) and a genetically-edited tobacco plant (left) at the bud bloom stage. As can be seen from fig. 2, the plant height, waist leaf length, waist leaf width, total leaf number, stem circumference and stem leaf included angle of the NtCYP82 gene edited tobacco plant are all smaller than those of the unedited tobacco plant of the control sample. The stamen length of the unedited tobacco plants (left) versus the control (right) of unedited tobacco plants (right). As can be seen from fig. 3, the stamen length of the NtCYP82 gene-edited tobacco plants was shorter than that of the control-like unedited tobacco plants.
The foregoing has shown and described the basic principles, principal 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, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
/>
Sequence listing
<110> Yunnan Zhongyan industry Limited liability company
<120> gene for regulating and controlling flowering time of tobacco and use thereof
<130> RIB210585
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2568
<212> DNA
<213> Artificial sequence (NtCYP 82)
<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 (NtCYP 82)
<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 (NtCYP 82)
<400> 3
atggctgaca attatggtcc agcattta 28
<210> 4
<211> 25
<212> DNA
<213> Artificial sequence (NtCYP 82)
<400> 4
tcaacagcca taaagattgg cattg 25
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence (NtCYP 82)
<400> 5
ttgtgggtca acaacaactc agg 23
Claims (3)
1. The application of the tobacco flowering time related gene is characterized in that the nucleotide sequence of the tobacco flowering time related gene is shown as SEQ ID NO. 1; the amino acid sequence of the coding protein of the tobacco flowering time related gene is shown as SEQ ID NO. 2; after the gene expression quantity is reduced, the flowering time of tobacco is shortened, and the flowering period is advanced; the gene is the NtCYP82 gene.
2. The use according to claim 1, wherein the reduction of gene expression is by gene editing by CRISPR/Cas9 mediated gene editing technique, a CRISPR/Cas9 editing vector for knocking out the NtCYP82 gene is constructed, and a tobacco plant edited by the NtCYP82 gene is obtained after genetic transformation.
3. The use according to claim 2, wherein the genetically edited tobacco plant has a significantly lower plant height, waist leaf length, waist leaf width, total leaf count, stem circumference or stem leaf angle than the control plant during the bud bloom stage, and results in a shorter stamen length.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111450871.XA CN114085851B (en) | 2021-12-01 | 2021-12-01 | Gene for regulating and controlling tobacco flowering time and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111450871.XA CN114085851B (en) | 2021-12-01 | 2021-12-01 | Gene for regulating and controlling tobacco flowering time and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114085851A CN114085851A (en) | 2022-02-25 |
| CN114085851B true CN114085851B (en) | 2023-08-04 |
Family
ID=80305954
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111450871.XA Active CN114085851B (en) | 2021-12-01 | 2021-12-01 | Gene for regulating and controlling tobacco flowering time and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114085851B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045609A (en) * | 2012-11-16 | 2013-04-17 | 云南省烟草农业科学研究院 | Complementary DNA (cDNA) sequence of tobacco NtFT1 genes and transient expression thereof for inducing tobacco early blossoming |
| CN107653253A (en) * | 2017-11-28 | 2018-02-02 | 云南省烟草农业科学研究院 | One kind regulation and control Tobacco Flowering period NtMADS2 genes and its cloning process and application |
| CN110819639A (en) * | 2019-12-19 | 2020-02-21 | 中国烟草总公司郑州烟草研究院 | Tobacco low-temperature early-flowering related gene NtDUF599 and application thereof |
| CN111620936A (en) * | 2020-07-01 | 2020-09-04 | 北京林业大学 | Protein related to plant flowering time and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8426677B2 (en) * | 2008-06-16 | 2013-04-23 | Academia Sinica | Method of controlling plant growth and architecture by controlling expression of gibberellin 2-oxidase |
-
2021
- 2021-12-01 CN CN202111450871.XA patent/CN114085851B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045609A (en) * | 2012-11-16 | 2013-04-17 | 云南省烟草农业科学研究院 | Complementary DNA (cDNA) sequence of tobacco NtFT1 genes and transient expression thereof for inducing tobacco early blossoming |
| CN107653253A (en) * | 2017-11-28 | 2018-02-02 | 云南省烟草农业科学研究院 | One kind regulation and control Tobacco Flowering period NtMADS2 genes and its cloning process and application |
| CN110819639A (en) * | 2019-12-19 | 2020-02-21 | 中国烟草总公司郑州烟草研究院 | Tobacco low-temperature early-flowering related gene NtDUF599 and application thereof |
| CN111620936A (en) * | 2020-07-01 | 2020-09-04 | 北京林业大学 | Protein related to plant flowering time and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| XP_016458765;Nicotiana tabacum;《GENBANK》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114085851A (en) | 2022-02-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Boss et al. | A grapevine TFL1 homologue can delay flowering and alter floral development when overexpressed in heterologous species | |
| CN114369599B (en) | Long-chain non-coding RNA gene for increasing rice yield and application thereof | |
| CN113583099B (en) | Method for cultivating alfalfa male sterile line and corresponding maintainer line and related biological material thereof | |
| CN112680461B (en) | Male sterile gene ZmPHD11 and application thereof in creating male sterile line of corn | |
| CN112921051B (en) | Method for creating male sterile breeding quality of watermelons through gene editing technology | |
| CN114438105B (en) | Tobacco NtMLO6-1 gene and knockout method and application thereof | |
| CN114085851B (en) | Gene for regulating and controlling tobacco flowering time and application thereof | |
| CN113980978B (en) | Tobacco nicotine transporter related gene and application thereof | |
| Swinnen et al. | KIX8 and KIX9 are conserved repressors of organ size in the asterid species tomato | |
| CN114507688A (en) | Application of tobacco NtCNGC4 gene in preparation of tobacco mutant material for regulating plant height and leaf amino acid content | |
| CN117024541A (en) | Tobacco iron redox protein gene Ntab0899580 and application thereof | |
| CN110964728B (en) | Gene ED1 for controlling rape plant height and application thereof | |
| CN114085845B (en) | Tobacco nicotine metabolism related gene and application thereof | |
| CN110592106A (en) | Molecular marker Lb14-3-3c gene and application thereof | |
| CN114807081B (en) | Tobacco glutathione transfer related gene and application thereof | |
| CN114540368B (en) | Application and method of tobacco amino acid transporter related gene NtLHT1 | |
| CN116144698A (en) | Application of tobacco alcohol dehydrogenase related gene NtADH1 in regulation and control of tobacco waist leaf width | |
| CN114106121B (en) | FvGR3 protein, and coding gene and application thereof | |
| CN115960189B (en) | Application of shinyleaf yellowhorn protein and encoding gene thereof in improving anthocyanin content in plant petals | |
| CN115960849B (en) | Rice heading stage related gene OsJMJ718 and application of encoding protein thereof | |
| CN117603325A (en) | Tobacco Ntab0167450 gene and application of encoding protein thereof in regulating and controlling polyphenol in tobacco leaves | |
| CN114591979B (en) | Tobacco ubiquitin conjugated enzyme NtE gene and application thereof | |
| CN116555301B (en) | SlMETS1 gene and application thereof in regulation and control of tomato growth and development | |
| CN113652434B (en) | Gorgon fruit DNA molecule with function of promoting rice grain enlargement and application thereof | |
| CN117126883A (en) | Application of tobacco biotin synthetase gene NtBIO3-BIO1 in regulation and control of tobacco waist leaf length, waist leaf width and polyphenol content |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |