CN106480067A - The old and feeble application of Nicotiana tabacum L. NtNAC096 Gene Handling Nicotiana tabacum L. - Google Patents

Abstract

The invention provides an application of tobacco NtNAC096 gene to control tobacco senescence, belongs to the field of tobacco biological control, and can significantly regulate tobacco senescence by using tobacco NtNAC096 gene. The genomic DNA sequence of the tobacco NtNAC096 gene is 1203bp in length, consisting of 3 exons and 2 introns. The above application specifically includes the following steps: (1) tobacco NtNAC096 gene cloning and sequence analysis; (2) expression analysis of the NtNAC096 gene; (3) functional analysis of the tobacco NtNAC096 gene by constructing a tobacco NtNAC096 gene overexpression vector; (4) Using CRISPR/Cas9 gene editing technology to design the target site of tobacco NtNAC096 gene, construct NtNAC096 gene knockout vector; carry out genetic transformation of tobacco varieties, and obtain tobacco plants with delayed aging.

Description

Application of Tobacco NtNAC096 Gene in Controlling Tobacco Senescence

technical field

The invention relates to the field of tobacco aging control, in particular to an application of tobacco NtNAC096 gene to control tobacco aging.

Background technique

Leaf senescence is the last stage of leaf development. During leaf senescence, chlorophyll and biomacromolecular substances such as proteins, lipids, nucleic acids, etc. are degraded, the photosynthetic capacity of leaves is reduced, and nutrients in aging tissues are degraded and transformed and transported to young tissues and reproductive organs for further growth development or storage. Therefore, leaf senescence affects crop growth, nutrient accumulation and yield formation. Tobacco is a leaf economic crop, the leaf area size and senescence maturity directly affect the production yield and quality.

At present, the control of plant senescence mainly focuses on the regulation of plant hormones. Auxin, cytokinin, etc. can inhibit leaf senescence, and ethylene and abscisic acid can promote leaf senescence; Promote the synthesis or metabolism of hormones, on the other hand, it can also increase the synthesis or metabolism of aging-suppressing hormones. However, there are often defects in the effect of hormone regulation of exogenous effects. In view of this, the method of using genes to control senescence has begun to be studied. Currently, only a few tobacco senescence genes have been identified, and there are still a large number of plant senescence-related genes to be studied and utilized.

Contents of the invention

The purpose of the present invention is to provide the application of tobacco NtNAC096 gene to control tobacco aging, which can control the aging of tobacco through NtNAC096 gene, and delay the aging of tobacco through gene editing, thereby efficiently controlling the aging process of tobacco.

In order to achieve the above object, technical scheme of the present invention is:

The application of tobacco NtNAC096 gene to control tobacco aging, the genomic DNA sequence of tobacco NtNAC096 gene is 1203bp in full length, and its nucleotide sequence is as follows:

Among them, the atg marked with double underline in the sequence is the start codon, the tga marked with double underline is the stop codon, the part marked with single underline is the intron, and the tobacco NtNAC096 gene consists of 3 exons and 2 introns composition.

Further, the application of the tobacco NtNAC096 gene to control tobacco aging includes conducting gene function analysis on the tobacco NtNAC096 gene, knocking out the NtNAC096 gene, and obtaining tobacco plants that delay aging.

Further, the application of tobacco NtNAC096 gene to control tobacco aging includes the following steps: (1) tobacco NtNAC096 gene cloning and sequence analysis; (2) expression analysis of NtNAC096 gene;

(3) Carry out functional analysis of tobacco NtNAC096 gene by constructing tobacco NtNAC096 gene overexpression vector; (4) Use CRISPR/Cas9 gene editing technology to design the target site of tobacco NtNAC096 gene and construct NtNAC096 gene knockout vector; Genetic transformation to obtain tobacco plants with delayed senescence.

Further, the expression analysis of the NtNAC096 gene in step (2) is to use qRT-PCR to detect the expression patterns of the NtNAC096 gene in different tissues and different leaf growth stages of tobacco.

Further, the method for constructing the tobacco NtNAC096 gene overexpression vector in step (3) is to use the clontechinfusion homologous recombination method to construct NtNAC096 into the overexpression vector pRI101-AN, and construct the tobacco NtNAC096 gene overexpression vector pRI101-NtNAC096.

Further, after step (3) constructing the overexpression vector of the tobacco NtNAC096 gene, the Agrobacterium-mediated flower dipping method was used to infect Arabidopsis thaliana to obtain transgenic Arabidopsis, and the functional analysis of the tobacco NtNAC096 gene was performed.

Further, in step (4), two target sites are designed for the tobacco NtNAC096 gene, artificially synthesized and a linker sequence is added, and the target sites are connected to the vector pORE-Cas9 to obtain the pORE-Cas9/NtNAC096 vector.

Further, the specific operation of genetic transformation of tobacco species in step (4) is as follows: pORE-Cas9/NtNAC096 vector transforms Agrobacterium competent LBA4404, and uses Agrobacterium-mediated tobacco leaf disk transformation method to genetically transform tobacco to obtain delayed Aged tobacco plant.

Compared with the existing tobacco senescence control, the beneficial effects of the present invention are: 1. For the first time, the tobacco NtNAC096 gene is found to be used to control tobacco senescence; 2. The tobacco NtNAC096 gene can be used to accurately and quickly control the tobacco senescence process by using the gene editing technology, through mutation The senescence-delayed tobacco plant obtained by screening the nucleotide sequence of the gene has very important application in agricultural production, and has a very significant impact on the picking of tobacco leaves, quality control, selection of tobacco varieties and increase of yield.

Description of drawings

Fig. 1 is the electrophoresis graph of the DNA sequence amplification result of tobacco NtNAC096 gene of an embodiment of the present invention;

Fig. 2 is the electropherogram of the amplification result of the cDNA sequence of the tobacco NtNAC096 gene in one embodiment of the present invention;

Fig. 3 is the tissue expression analysis of tobacco NtNAC096 gene of an embodiment of the present invention;

Fig. 4 is the expression analysis of tobacco NtNAC096 gene tobacco leaf in different development stages of an embodiment of the present invention;

Fig. 5 is the electropherogram of the construction and verification of the plant overexpression vector pRI101-NtNAC096;

Figure 6 is the identification and phenotype diagram of pRI101-NtNAC096 transgenic Arabidopsis plants;

Figure 7 is the electrophoresis diagram of the target fragment containing the target site sequence in the NtNAC096 gene of tobacco CRISPR/Cas9 transgenic positive plants;

Figure 8 is a graph showing the results of target sequence analysis of the endogenous NtNAC096 gene of the transgenic tobacco strain;

Fig. 9 is a phenotype map of tobacco NtNAC096 knockout mutant.

detailed description

The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The embodiment of the present invention provides an application of the tobacco NtNAC096 gene to control tobacco aging. The nucleotide sequence of the tobacco NtNAC096 gene is shown in SEQ ID NO.1. Through analysis, the tobacco NtNAC096 gene consists of 3 exons and 2 endons. containing sub-composition. Tobacco plants with delayed senescence were obtained by knocking out the NtNAC096 gene after gene function analysis of the tobacco NtNAC096 gene.

The embodiment of the present invention also provides an application method for tobacco NtNAC096 gene to control tobacco aging, which specifically includes the following steps:

Cloning and sequence analysis of S1 tobacco NtNAC096 gene.

In this step, using tobacco genomic DNA and cDNA as templates, primers are designed to amplify the genomic DNA and cDNA of the tobacco NtNAC096 gene, and the intron and exon sequences of the genomic DNA are analyzed.

S2 The expression analysis of NtNAC096 gene.

In this step, the action site and action period of the gene can be obtained by analyzing the expression of the tobacco NtNAC096 gene.

S3 To analyze the function of tobacco NtNAC096 gene by constructing the overexpression vector of tobacco NtNAC096 gene.

In this step, through the construction of an overexpression vector, Agrobacterium-mediated preparation of transgenic Arabidopsis plants, functional analysis of the gene on Arabidopsis can verify the function of the tobacco NtNAC096 gene, and make a theory for its application Prepare.

S4 uses CRISPR/Cas9 gene editing technology to design the target site of the tobacco NtNAC096 gene, and constructs the NtNAC096 gene knockout vector; genetically transforms tobacco varieties to obtain tobacco that delays aging.

In this step, the gene editing technology can be used to randomly edit the tobacco NtNAC096 gene, and then through the transgenic technology, the aging of tobacco can be regulated, the aging process of tobacco can be changed, and late aging tobacco varieties can be cultivated to control the aging of tobacco as a whole.

As a preferred embodiment of the present invention, in step S2, the expression analysis of NtNAC096 gene can be performed, and the expression pattern of NtNAC096 gene in different tissues and different leaf growth stages of tobacco can be detected by qRT-PCR method. The qRT-PCR method is efficient and intuitive, but it can be understood that other methods for detecting gene expression in the field, such as RT-PCR, northern hybridization, etc., can also be used for the expression analysis of the above-mentioned NtNAC096 gene.

As a preferred embodiment of the present invention, the method for constructing the tobacco NtNAC096 gene overexpression vector is to construct NtNAC096 into the overexpression vector pRI101-AN by using the clontech infusion homologous recombination method, and construct the tobacco NtNAC096 gene overexpression vector pRI101-NtNAC096. It can be understood that the overexpression vector pRI101-AN is a suitable vector for the tobacco NtNAC096 gene after screening, and other gene overexpression vectors can also be used for the construction of the above-mentioned overexpression vector.

As a preferred embodiment of the present invention, Agrobacterium-mediated flower-dipping method is used to infect Arabidopsis thaliana to obtain T0 generation seeds, the medium is screened to obtain T1 generation positive seedlings, and the positive seedlings are selfed for one generation and continue to be screened to finally obtain transgenic Homozygous lines, the senescence phenotype analysis and the determination of aging indicators were carried out on the transgenic homozygous lines. It can be understood that other methods of introducing overexpression vectors into Arabidopsis to obtain transgenic Arabidopsis can also be used to verify the function of the above-mentioned tobacco NtNAC096 gene.

As a preferred embodiment of the present invention, CRISPR/Cas9 gene editing technology is used to design the target site of the tobacco NtNAC096 gene, and the method of constructing the NtNAC096 gene knockout vector is to design two target sites for the NtNAC096 gene, artificially synthesize and add a linker sequence, The target site was ligated to the vector to obtain the pORE-Cas9/NtNAC096 vector. It can be understood that the number of target sites designed for the NtNAC096 gene is not limited to two, but can be multiple, and the number of target sites can be determined as required.

As a preferred embodiment of the present invention, the pORE-Cas9/NtNAC096 vector vector is transformed into Agrobacterium competent LBA4404, and the tobacco leaf disk transformation method mediated by Agrobacterium is used to genetically transform tobacco to obtain positive transformation of the T0 generation resistant to kanamycin For the seedlings, the T0 generation transgenic positive seeds were collected, the T1 generation was screened on a kanamycin resistance plate, and the resistant seedlings were screened by PCR amplification and sequencing to obtain the homozygous mutants of the T1 generation, sequenced, and the aging-delayed tobacco was obtained. It can be understood that other methods of introducing vectors into tobacco to obtain transgenic tobacco can also be used to perform the above operations.

In order to introduce in detail the application of the tobacco NtNAC096 gene to control tobacco senescence provided by the embodiments of the present invention, the following will be described in conjunction with specific examples.

(1) Extraction of tobacco genomic DNA

(1) Put about 100 mg of fresh leaves into a centrifuge tube, quick-freeze in liquid nitrogen, grind in a grinder, add 650 μL of 2×CTAB extraction buffer (preheated to 65°C in advance), and mix quickly; (2 ) in a water bath at 65°C for 1 hour, during which time it was shaken gently every 15 minutes; (3) After the mixture was cooled to room temperature, an equal volume of phenol chloroform isoamyl alcohol (25:24:1) was added, mixed gently, and allowed to stand at room temperature for 15 minutes, at 4°C , centrifuge at 12000rpm for 15min; (4) transfer the supernatant to a new centrifuge tube; (5) add an equal volume of chloroform isoamyl alcohol (24:1), mix upside down, centrifuge at 12000rpm for 15min at 4°C, and carefully transfer the supernatant Transfer to a new centrifuge tube; (6) Take the supernatant, add an equal volume of isopropanol, mix well, place at room temperature for 10 minutes, centrifuge at 12,000 rpm for 15 minutes to precipitate DNA; (7) pour off the isopropanol, rinse twice with 75% ethanol, After the last rinsing, the remaining liquid was aspirated as much as possible, and after drying in a vacuum concentrator, an appropriate amount of sterile water was added to dissolve the DNA for later use.

2×CTAB extract formula: 100mM (PH8.0) Tris.Cl, 20mM (PH8.0) EDTA, 1.4M NaCl, 2% (w/v) CTAB, 40mM mercaptoethanol.

(2) Tobacco RNA extraction: RNAiso Reagent (TaKaRa) was used for routine extraction.

(3) Synthesis of cDNA by reverse transcription: PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, RR047A) was used.

(4) Tobacco NtNAC096 gene cloning

Design of primers for tobacco NtNAC096 gene

Primer NtNAC096-F:TATTTCTCCCTTCTATTTATTTTCCT (5'UTR area)

Primer NtNAC096-R: TCACTGGTATTGAAAGGCTGG (stop codon position)

PCR reaction system:

10×PCR Buffer for KOD-Plus-Neo 5μL

2mM dNTPs 5μL

25mM _MgSO4 3μL

Primer NtNAC096-F 1.25μL

Primer NtNAC096-R 1.25 μL

DNA (or cDNA) 1μL

KOD-Plus-Neo 1μL

ddH ₂ O to 50μL

PCR reaction program:

Step1 94℃2min

Step2 98℃10s

Step3 56℃30s

Step4 68℃1min20s Step2-4 35cycle

Step5 68℃8min

The genomic DNA and cDNA of Tobacco Safflower Dajinyuan were used as templates to amplify to obtain a 1203bp size fragment. See Figure 1 for the electrophoresis results. For the cDNA electrophoresis results, a 1079bp size fragment was obtained, see Figure 2. The DL5000marker molecular weight marker is on the right. Analysis was performed on the genome, which consists of 3 exons and 2 introns.

(5) The expression pattern of NtNAC096 gene in different tissues (roots, stems, young leaves, flowers, mature and senescent leaves, etc.) and different leaf growth stages (S1-S4) of tobacco was detected by fluorescent quantitative qRT-PCR method.

The FastStart Universal SYBR Green Master (ROX) Realtime kit (Roche) was used. The method of relative quantification was selected, the tobacco 18S gene was used as an internal reference gene, and the template was cDNA obtained from the above-mentioned different tissues or developmental stages. Three replicates were set up for each sample, and a negative control and a positive control were set at the same time.

Reaction system (20μL)

2×SYBR Green Master MIX 10μL

qRT-F primer (10μM)0.5μL

qRT-R primer (10μM)0.5μL

cDNA diluted template 1 μL

ddH ₂ O 8μL

After the reaction system was fully mixed, it was put into the ABI7500 fluorescent quantitative PCR system for PCR reaction. The three-step PCR reaction procedure was adopted, and the relative expression of the target gene was determined by the 2- ^ΔΔCt method.

qRT-PCR primer sequences:

NtNAC096-qRT-F:TTCACGACTATTTGATGACAATGCTAAC

NtNAC096-qRT-R: TATTGGTCATTGTGTTTTGGTTGTT

18S-qRT-F:GGTCCAGACATAGTAAGGATTGACAGA

18S-qRT-R:AGACAAATCGCTCCACCAACTAAG

Referring to Fig. 3 and Fig. 4, the results show that the expression level of NtNAC096 gene is highest in senescent leaves, followed by root tissue. And in the S2 phase, the NtNAC096 gene was significantly up-regulated by about 1300 times, and in the S3 phase by 37.7 times.

(6) Using clontech infusion homologous recombination method to construct NtNAC096 into the overexpression vector pRI101-AN, see Figure 5.

The plant overexpression vector pRI101-AN (Takara) was used. NdeI and EcoRI were used to double-digest pRI101-AN, and the NtNAC096 gene with a linker was amplified with high-fidelity enzymes. The large fragment of the vector and the target gene fragment were recovered separately, and the infusion HD was used to (clontech) seamless connection technology to connect the target fragments into pRI101-AN.

Enzyme cutting system:

pRI101-AN 2 μg

10×Cutsmart buffer 4μL

NdeI 2μL

EcoRI-HF 2μL

ddH ₂ O to 40μL

Enzyme digestion was carried out in a constant temperature water bath at 37°C for more than 3 hours.

The amplification system is the same as the above-mentioned PCR method, and the primer sequences are:

NtNAC096-35s-F:CACTGTTGATACATATGGTTGGGAAAAATAACTCCGAG

NtNAC096-35s-R: TGTTGATTCAGAATTCTCACTGGTATTGAAAGGCTGG

Restriction plasmids and target gene amplification were detected by agarose gel electrophoresis and recovered routinely.

Infusion connection system (5μL):

Linearized vector 3 μL

Target fragment 1 μL

In-fusion HD 1μL

50℃15min

Figure 5 shows that the left side of the Marker is the DNA molecular weight marker DL15000, the right side is DL2000, the NtNAC096 fragment is amplified with high fidelity enzyme, the left side of the left picture is the target fragment, pRI101-AN double enzyme digestion NdeI and EcoRI, the left picture right side enzyme digestion The linear pRI101-AN plasmid band, the right electrophoresis is the pRI101-NtNAC096 expression vector colony PCR verification NtNAC096 band.

Infect Arabidopsis thaliana by Agrobacterium-mediated flower dipping method to obtain T0 generation seeds, screen in 1/2MS medium with kanamycin resistance, obtain T1 generation positive seedlings, and self-fertilize the positive seedlings 1 generation, and continue to screen, and finally obtain two transgenic homozygous lines of OE1/OE2.

(7) Carry out aging phenotype analysis on OE1/OE2, and carry out relevant aging index determination:

Determination of chlorophyll content

30 Arabidopsis materials (transgenic OE and Col-0 wild-type plants) grown 45 days after seed germination were selected, and the sixth rosette leaf (counted from bottom to top) was cut from each plant to measure the chlorophyll content.

The experimental procedure is as follows: each group of 3 plants, put the leaves of each group into a 15mL centrifuge tube, and weigh the fresh weight of the leaves; add 10mL of absolute ethanol to each tube, so that the ethanol completely soaks the leaves; keep it in the dark overnight , make blade decolorize completely; Measure the absorbance value (with dehydrated alcohol as reference) of decolorization solution at 665nm and 649nm place respectively with spectrophotometer, be recorded as A665 and A649 respectively; Calculate the chlorophyll content of every group of leaves according to the following formula: Ca=13.95A665–6.88A649Cb=24.96A649–7.32A665

Total chlorophyll content (mg/g)=(Ca+Cb)×V/W. Among them, V is the volume of the extract, 10 mL; W is the fresh weight of the leaves.

Chlorophyll Fluorescence (Fv/Fm) Determination

Use the OS5p+ pulsed chlorophyll fluorometer of OPTI-SCIENCES Company to measure the Fv/Fm of the leaves. The operation steps are as follows: 5 Arabidopsis plant materials that have grown for 45 days after seed germination are selected, and placed in a dark environment for 20 minutes. ;Select the sixth rosette leaf for measurement; connect all parts of the instrument, turn on the instrument switch, find and enter the Fv/Fm measurement program on the main interface; clamp the leaf with the leaf clip, insert the measuring probe into the leaf clip, and adjust the parameter Ft , so that the value is within the range of 150-250; click the measurement mark on the interface or press the measurement button on the probe, the measurement value will be automatically recorded and displayed; after each set of data is measured, the data will be saved to the fuselage SD card; after all the measurements are completed, disassemble and organize the instrument components, and copy the data in the SD card for analysis.

See Figure 6 for the measurement results. In Figure 6A, WT is the wild type Col-0; OE1/OE2 are the two strains transformed with the pRI101-NtNAC096 gene. The phenotype picture is a picture of the phenotype of Arabidopsis thaliana during the growth period of 45 days. It can be seen that the OE strain exhibits a premature senescence phenotype. Fig. 6B is the analysis of chlorophyll and photosynthetic rate of transgenic Arabidopsis lines. Figure 6C is the analysis of NtNAC096 and senescence-related marker gene SAG12 mRNA levels in transgenic Arabidopsis lines. In the two lines of OE1/OE2, the expression level of NtNAC096 was significantly up-regulated, and it showed obvious premature aging phenotype 45 days after sowing. According to the measurement of physiological indicators, the chlorophyll content and photochemical rate of OE1/OE2 were significantly lower than that of VC, which is an aging-related marker gene. The expression of SAG12 was significantly higher than that of VC.

(8) Using CRISPR/Cas9 gene editing technology to design the target site of the tobacco NtNAC096 gene and construct the NtNAC096 gene knockout vector; genetically transform the tobacco variety to obtain tobacco plants that delay aging.

Design 2 target sites:

Target1: GCTATAAAGGAAAGCCCCCTA

Target2: TATAAAGGAAAGCCCCCTAA

Synthetic linker sequence

Target1-F: GATTGCTATAAAGGAAAGCCCCCTA

Target1-R: AAACTAGGGGGCTTTTCCTTATAGC

Target2-F: GATTGTATAAAGGAAAGCCCCCTAA

Target1-R: AAACTTAGGGGGCTTTCCTTTATAC

Knockout vector construction

①Annealing of single-stranded oligo DNA to form double-stranded DNA

The synthesized 2 single-stranded primers were diluted to 50 μM and then annealed.

Annealing reaction system:

Incubate the reaction system in a PCR instrument at 95°C for 3 min, and cool to room temperature naturally after incubation.

② Digestion of expression vector

Expression vector: pORE-Cas9/gRNA

Digest the expression vector with BsaI

Carrier 1μg

10×CutSmart Buffer 5μL

Enzyme 1 μL

Add _ddH2O to 50μL

Digest at 37°C for 1 hour, and recover the digested product.

③ Ligate the target site to the expression vector, ligation system: 5 μL linearized expression vector, 2 μL annealed double-stranded DNA, 2 μL T4 ligase buffer, 1 μL T4 ligase, Add ddH ₂ O to 20 μL, ligate at 25°C for 10 minutes.

④The ligation product was transformed into competent cells, and positive clones were obtained after PCR verification.

The constructed pORE-Cas9/NtNAC096 vector was transformed into Agrobacterium competent LBA4404, and the cultivated tobacco variety K326 was genetically transformed using the Agrobacterium-mediated tobacco leaf disc transformation method, and the T0 generation positive with kanamycin resistance was obtained transformed seedlings.

Extract the genomic DNA of positive tobacco seedling leaves, design primers on both sides of the two gRNA sequences, use the genomic DNA as a template, perform PCR amplification on the target fragment, purify and recover the PCR product, connect the cloning vector for sequencing, and detect the target Site fragment mutation form.

Harvest the T0 transgenic positive plant seeds whose target sites were edited, and screen the T1 generation on a kanamycin resistance plate. The resistant seedlings were then screened for homozygous mutants of the T1 generation by PCR amplification and sequencing. See Figure 7 for the electrophoresis. The genomic DNA of the leaves was extracted, the target fragment was amplified by PCR and the product was sequenced. See Figure 8 for the sequencing results. Figure 8 shows that the NtNAC096 gene in both tobacco plants ntnac096-1 and ntnac096-2 has been successfully edited, and a base "C" insertion was detected in the NtNAC096 target segment in the ntnac096-1 plant, and ntnac096- In 2 plants, a base "T" insertion was detected in the target region of NtNAC096, which caused changes in the protein coding frame, resulting in loss of protein function, and thus obtained plants that delayed tobacco senescence.

Using the above method for measuring chlorophyll content and chlorophyll fluorescence of Arabidopsis thaliana, the leaf position calculation starts from the top, and three points are randomly selected from the 1st to 6th fully developed leaves for measurement. The measurement results are shown in Fig. 9. As shown in Fig. 9A, the mutant exhibits an obvious aging-delaying phenotype starting from the group stage. Calculate the leaf position from the top, and measure the chlorophyll content and photosynthetic rate of the 1st to 6th fully developed leaves in turn. The chlorophyll content decreases significantly with the increase in the number of leaves, and the chlorophyll content of the mutant is significantly higher than that of the control K326. See Fig. 9B; Similarly, the photosynthetic rate was not significantly different in the 1-4 leaf position, and the photosynthetic rate of the mutant in the 5-6 leaf position was much higher than that of the control K326, see Fig. 9C. Through phenotypic analysis and determination of relevant physiological indicators, the senescence process of the tobacco NtNAC096 gene was significantly delayed after the knockout of the tobacco NtNAC096 gene, indicating that the method of the present invention utilizes the tobacco NtNAC096 gene to successfully obtain late-senescent tobacco plants.

<110> Institute of Tobacco, Chinese Academy of Agricultural Sciences

<120> Application of tobacco NtNAC096 gene to control tobacco senescence

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1203

<212> DNA

<213> Tobacco NtNAC096 gene

<400> 1

atggttggga aaaataactc cgagcagctt cctcctggat ttaggttcca tcctactgat 60

gaagaattaa tcatgtatta tcttcgaaat caagctacct cgaggccttg tcctgtttca 120

atcatccccg aagttgatgt ctataagttt gatccctggg aattgcctgg ttagtatact 180

cgttaaatcg cgtttaattc aaagaattaa tttgtagttg atttttaagt cttgaattaa 240

ttttttctta tgaaattctt gtagagaaag ctgaatttgg ggaaagggaa tggtactttt 300

tcacccctcg tgataggaag tacccaaatg gagttaggcc aaatagagca gctgtatcag 360

gttattggaa ggctacaggc acagataaag caatatatag tggatcaaaa tatgttggta 420

ttaagaaggc tcttgttttc tataaaggaa agccccctaa gggtattaag actgattgga 480

tcatgcatga atatcgatta agtgaatcca ggtctcaacc aatcaggcca aatggctcca 540

tgagggtaag acttgaattc ttggaattgt tttctactat agtttaactt gtatactatg 600

ttagcataga cgtttttact ctatcagatc attttaaaag gtaattacat gcaataatct 660

atattattga aaatatggca agtaactttc tagaatatgt taagtcgtac tcgtgtaaaa 720

atttcactat aatacatggc agtgttctac catatattat aagagttttt gtcatgacta 780

aaagtacaat tttttcgcca tttttttcag ttggatgatt gggtgctttg tagaatttat 840

aagaagaaga atttgggaaa agctatggag atgatgaaag ttgaagaaga gacacaacag 900

cctgaaatat tgagtactaa tcctgttgaa attattgcta ctactggacc acaaacattg 960

aaattgccaa ggacttgttc actgtctcat ctattggaaa tagattattt gggtcaatt 1020

tcacgactat ttgatgacaa tgctaacaac caaaacacaa tgaccaatat taatattgga 1080

aatatgcatc atcctgccct ggaaaaattt cagctagggg aattgtcaca ccagtacatg 1140

actagtacca acgttaatgg aaatacgcca atttttgtga atccagcctt tcaataccag 1200

tga 1203

Claims (8)

1. the old and feeble application of Nicotiana tabacum L. NtNAC096 Gene Handling Nicotiana tabacum L. is it is characterised in that the genome of Nicotiana tabacum L. NtNAC096 gene DNA sequence total length 1203bp, its nucleotide sequence is as follows：

Wherein, the atg that in sequence, double underline indicates is start codon, and the tga that double underline indicates is termination codon, single The part that underscore indicates is intron, and Nicotiana tabacum L. NtNAC096 gene is made up of 3 exons and 2 introns.

2. the old and feeble application of Nicotiana tabacum L. NtNAC096 Gene Handling Nicotiana tabacum L. according to claim 1 is it is characterised in that above-mentioned should With including Nicotiana tabacum L. NtNAC096 gene is carried out gene function analysis, knock out NtNAC096 gene, obtain the Nicotiana tabacum L. of slow down aging Plant.

3. the old and feeble application of Nicotiana tabacum L. NtNAC096 Gene Handling Nicotiana tabacum L. according to claim 2 is it is characterised in that above-mentioned should With comprising the steps：(1) Nicotiana tabacum L. NtNAC096 gene cloning, sequence analysis；(2) NtNAC096 gene is carried out with expression point Analysis；(3) pass through to build Nicotiana tabacum L. NtNAC096 gene overexpression carrier, carry out Nicotiana tabacum L. NtNAC096 gene function analysis；(4) utilize CRISPR/Cas9 gene editing technology is designed to Nicotiana tabacum L. NtNAC096 gene target site, builds NtNAC096 gene knockout Carrier；Tobacco bred is carried out with genetic transformation, obtains the tobacco plant of slow down aging.

4. the old and feeble application of Nicotiana tabacum L. NtNAC096 Gene Handling Nicotiana tabacum L. according to claim 3 is it is characterised in that step (2) NtNAC096 gene is carried out expression analysis be with qRT-PCR method detection NtNAC096 gene in Nicotiana tabacum L. different tissues and The expression pattern of different leaves growthdevelopmental stage.

5. the old and feeble application of Nicotiana tabacum L. NtNAC096 Gene Handling Nicotiana tabacum L. according to claim 3 is it is characterised in that step (3) method building Nicotiana tabacum L. NtNAC096 gene overexpression carrier is will using clontech infusion methods of homologous recombination NtNAC096 builds and enters over-express vector pRI101-AN, builds Nicotiana tabacum L. NtNAC096 gene overexpression carrier pRI101- NtNAC096.

6. the old and feeble application of the Nicotiana tabacum L. NtNAC096 Gene Handling Nicotiana tabacum L. according to claim 3 or 5 is it is characterised in that walk Suddenly, after (3) build Nicotiana tabacum L. NtNAC096 gene overexpression carrier, infect arabidopsiss using agriculture bacillus mediated flower-dipping method and turned Gene arabidopsiss, carry out Nicotiana tabacum L. NtNAC096 gene function analysis.

7. the old and feeble application of Nicotiana tabacum L. NtNAC096 Gene Handling Nicotiana tabacum L. according to claim 3 is it is characterised in that step (4) to two target sites of Nicotiana tabacum L. NtNAC096 gene design, synthetic adjunction header sequence, target site is connected to carrier pORE- On Cas9, obtain pORE-Cas9/NtNAC096 carrier.

8. the old and feeble application of Nicotiana tabacum L. NtNAC096 Gene Handling Nicotiana tabacum L. according to claim 7 is it is characterised in that step (4) concrete operations carrying out genetic transformation to tobacco bred are：PORE-Cas9/NtNAC096 carrier converts Agrobacterium competence LBA4404, carries out genetic transformation using agriculture bacillus mediated tobacco leaf disc conversion method to Nicotiana tabacum L., and the Nicotiana tabacum L. obtaining slow down aging is planted Strain.