CN115896168A - A method and application of rapidly obtaining and cultivating tobacco nicotine transformed strains - Google Patents

Abstract

The invention discloses a method for quickly obtaining a tobacco nicotine transformant for cultivation and application thereof. The method for quickly obtaining and cultivating the tobacco nicotine transformation strain is characterized in that genes related to tobacco nicotine transformation are knocked out by using a CRISPR/Cas9 system, the genes are NtNCP1 and NtNCP2 genes, the sequence of the NtNCP1 gene is shown in SEQ ID No.1, and the sequence of the NtNCP2 gene is shown in SEQ ID No. 3. The tobacco NtNCP1 and NtNCP2 genes are knocked out simultaneously by using a CRISPR/Cas9 mediated gene editing technology to obtain an editing material with reduced nicotine content and increased nornicotine content, so that a genetic material and a theoretical basis are provided for the function research of a tobacco nicotine transformation gene and the directional improvement of a new tobacco variety regulated by nicotine content.

Description

A method and application of rapidly obtaining and cultivating tobacco nicotine transformed strains

technical field

The invention relates to the technical field of plant genetic engineering, in particular to a method and application for rapidly obtaining cultivated tobacco nicotine transformed strains.

Background technique

Tobacco alkaloids mainly include nicotine, nornicotine, anatabine and pseudobasine. The composition and content of tobacco alkaloids are crucial to the quality and safety of tobacco leaves. The nicotine content of tobacco is generally above 94%, and the ratio of nornicotine does not exceed 2.5%. In the tobacco plant population of cultivated varieties, some plants will form nicotine demethylase due to gene mutation, and nicotine will be demethylated under the action of this enzyme to form nornicotine, resulting in a significant increase in nornicotine content. Nicotine was significantly reduced. Such plants are called transformants. Nornicotine is prone to oxidation, acylation and nitrosation reactions to produce mesmin, acyl nornicotine and nitroso nornicotine (NNN). The formation of these substances is important for the taste and safety of tobacco leaves. adverse effects.

At present, genes such as CYP82E5v2, CYP82E2, CYP82E10 and CYP82E have been isolated and identified to catalyze the conversion of nicotine to nornicotine. Under normal cultivation conditions, some tobacco plants will undergo gene mutations to obtain nicotine transformed strains, but natural mutations are difficult to determine the mutant genes, and the main agronomic traits do not change, making selection and breeding difficult.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method and application for rapidly obtaining cultivated tobacco nicotine transformed strains, which provide a theoretical basis for studying the function of tobacco nicotine transformed genes and directional improvement of cultivated tobacco varieties.

The technical problem to be solved by the present invention is achieved through the following technical solutions:

A method for rapidly obtaining and cultivating tobacco nicotine transformed strains, the method is to utilize the CRISPR/Cas9 system to simultaneously knock out genes related to tobacco nicotine transformation, the genes are NtNCP1 and NtNCP2 genes, and the NtNCP1 gene sequence is SEQ ID Shown in No.1, described NtNCP2 gene sequence is shown in SEQ ID No.3.

Preferably, after the sequence of the NtNCP1 gene is translated, its encoded protein sequence is shown in SEQ ID No.2.

Preferably, after the sequence of the NtNCP2 gene is translated, its encoded protein sequence is shown in SEQ ID No.4.

Preferably, the following steps are included:

(1) Creation of T0 generation editing materials;

(2) Homozygous editing materials were obtained from T0 generation plants through homozygous selfing;

(3) Planting and trait observation of homozygous edited materials;

(4) GC-MS was used to detect the alkaloid content of the leaves of the homozygous knockout material of NtNCP1 and NtNCP2 genes 14 days after topping, and calculate the conversion rate of nicotine.

Preferably, in step (1), the sgRNA sequence used by the CRISPR/Cas9 system is TCATAATGCATATAGGTTGGAGG, and the primer sequence used by the sgRNA sequence is:

Upstream primer sgRNA-F: GATTGTCATAATGCATATAGGTTGG;

Downstream primer sgRNA-R: AAACCCCAACCTATATGCATTATGA.

Preferably, in step (1),

The primer sequences used in NtNCP1 target editing detection are as follows:

Upstream primer NtNCP1-F: CCATCAGAAGCCACCACCAA;

Downstream primer NtNCP1-R: TGCTCTGGTTCTTCGGCTAAG;

The primer sequences used in NtNCP2 target editing detection are as follows:

Upstream primer NtNCP2-F: CCATCAGAAGCCACCACCAA;

Downstream primer NtNCP2-R: GCGTTTATTTGCTCCGGTTCTT.

Preferably, step (1) is specifically:

Design the sgRNA guide sequence, the upstream primer sgRNA-F and the downstream primer sgRNA-R anneal to form a double strand, and the restriction endonuclease BsaI-HF digests the CRISPR/Cas9 vector pORE-Cas9; The good vector backbone was ligated with T4 ligase; the ligated product was transformed into E. coli competent cells, positive clones were detected and recombinant plasmids were extracted to obtain CRISPR-Cas9 expression vectors;

Soak the Agrobacterium LBA4404 carrying the CRISPR/Cas9-sgRNA expression vector to infect tobacco leaf discs, and obtain T0 generation plants. After target editing and detection, the NtNCP1 and NtNCP2 genes are simultaneously edited plants, and the T0 generation is harvested. seed.

Step (2) is specifically as follows: the T0 generation seeds are self-crossed and homozygously multiplied and planted, and the NtNCP1 and NtNCP2 genes are homozygously edited plants are obtained through target editing and detection, and the T1 generation seeds are harvested, and the NtNCP1 and NtNCP2 genes are harvested. The primer sequences used in target editing detection are the same as in step (1).

An application of a gene related to tobacco nicotine conversion in rapidly preparing nicotine-transformed plants, wherein the genes related to tobacco nicotine conversion are NtNCP1 gene and NtNCP2 gene.

Application of a method for rapidly obtaining and cultivating tobacco nicotine transformed strains in preparing tobacco nicotine transformed strains.

Preferably, the total amount of various alkaloids in the leaves of the NtNCP1 and NtNCP2 gene knockout editing plants 14 days after topping is close to that of the control plants, but the nicotine content is extremely significantly lower than that of the control plants, and the nornicotine content is extremely significantly higher than that of the control plants plants, the conversion rate of nicotine is higher than 20%.

The technical scheme of the present invention has the following beneficial effects:

The present invention constructs a CRISPR/Cas9 editing vector for simultaneous knockout of tobacco NtNCP1 and NtNCP2 genes through CRISPR/Cas9-mediated gene editing technology, and obtains simultaneous knockout of tobacco NtNCP1 and NtNCP2 genes after creation of editing materials and molecular detection and identification Remove the safflower dajin yuan edited plants.

Compared with the control (unedited), the main agronomic traits of the tobacco plants with simultaneous knockout of the tobacco NtNCP1 and NtNCP2 genes provided by the present invention are not significantly different in traits such as plant height, waist leaf length, waist leaf width, and stem circumference.

The genes NtNCP1 and NtNCP2 related to the transformation of tobacco nicotine provided by the present invention were detected by gas chromatography-mass spectrometry and found that the total amount of various alkaloids in the leaves of the edited plants 14 days after topping was close to that of the control , but the nicotine content was extremely significantly lower than that of the control plants, the nornicotine content was extremely significantly higher than that of the control plants, and the conversion rate of nicotine was higher than 20%.

In summary, the use of CRISPR/Cas9-mediated gene editing technology to knock out and knock out tobacco NtNCP1 and NtNCP2 genes at the same time resulted in edited material with reduced nicotine content and increased nornicotine content, which provides a basis for the study of the function of tobacco nicotine conversion genes. To provide genetic materials and theoretical basis for directional improvement of new tobacco varieties regulated by nicotine content.

Description of drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention.

Figure 1 is a comparison of the main agronomic traits of the edited plants of the present invention and the control (unedited).

Figure 2 is a comparison of the nicotine conversion rate between the edited plants of the present invention and the control (unedited).

Detailed ways

Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangements of components and steps, numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless specifically stated otherwise.

All the experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained through commercial channels unless otherwise specified.

The acquisition of embodiment 1 NtNCP1 gene

Using the whole plant of the cultivar Tobacco Safflower Dajinyuan as the experimental material, the total RNA from the root of tobacco was extracted with an RNA extraction kit, and reverse-transcribed into cDNA for future use:

Tobacco total RNA was extracted according to the instructions of the plant RNA extraction kit.

1 μg of total RNA was extracted from leaves for reverse transcription, and the transcription system was as follows:

Total RNA 1 μg;

Oligo(dT)(10μM) 1.5μL;

ddH ₂ O up to 15 μL.

Mix the above system and place it in PCR, incubate at 70°C for 5 minutes, immediately place it on ice for 5 minutes after removal, and then add the following reagents to the system:

The above system was placed in a PCR instrument, kept at 42°C for 65 minutes, 65°C for 10 minutes, kept at 4°C, and then stored in a -20°C refrigerator for use.

Through the method of homology comparison, refer to the known partial gene sequence of tobacco, and design the amplification primer sequence as follows:

F: 5'-ATGGCGTCAAAGCTCTTTTTGG-3' (SEQ ID No.5);

R: 5'-TCAAAAATAATCAACTACAGGAG-3' (SEQ ID No. 6).

Using the cDNA prepared above as a template, PCR amplification was performed using the above primers:

Amplification system (50μL):

After mixing and centrifuging, perform PCR amplification. The PCR reaction conditions are: 95°C for 10 sec, 52°C for 30 sec, 72°C for 2 min, a total of 30 cycles; 72°C for 10 min; 25°C Hold.

The amplified product was purified and then sequenced to obtain the sequence of the gene NtNCP1 related to tobacco nicotine conversion, whose base sequence is shown in SEQ ID No.1, including a total of 690 bases. After the gene sequence is translated, the encoded protein sequence is shown in SEQ ID No. 2, including a total of 229 amino acid residues.

The acquisition of embodiment 2NtNCP2 gene

Using the whole plant of the cultivar Tobacco Safflower Dajinyuan as the experimental material, the total RNA from the root of tobacco was extracted with an RNA extraction kit, and reverse-transcribed into cDNA for future use:

Tobacco total RNA was extracted according to the instructions of the plant RNA extraction kit.

1 μg of total RNA was extracted from leaves for reverse transcription, and the transcription system was as follows:

Total RNA 1 μg;

Oligo(dT)(10μM) 1.5μL;

ddH ₂ O up to 15 μL.

Mix the above system and place it in PCR, incubate at 70°C for 5 minutes, immediately place it on ice for 5 minutes after removal, and then add the following reagents to the system:

The above system was placed in a PCR instrument, kept at 42°C for 65 minutes, 65°C for 10 minutes, kept at 4°C, and then stored in a -20°C refrigerator for use.

Through the method of homology comparison, refer to the known partial gene sequence of tobacco, and design the amplification primer sequence as follows:

F: 5'-ATGGCCCAATGTATGGATGCTG-3' (SEQ ID No. 7);

R: 5'-TCAAAAATAATCAATTACAGGAG-3' (SEQ ID No. 8).

Using the cDNA prepared above as a template, PCR amplification was performed using the above primers:

Amplification system (50μL):

After mixing and centrifuging, perform PCR amplification. The PCR reaction conditions are: 95°C for 10 sec, 52°C for 30 sec, 72°C for 2 min, a total of 30 cycles; 72°C for 10 min; 25°C Hold.

The amplified product was purified and then sequenced to obtain the sequence of NtNCP2, a gene related to tobacco nicotine conversion, whose base sequence is shown in SEQ ID No.3, including a total of 1083 bases. After the gene sequence is translated, the encoded protein sequence is shown in SEQ ID No.4, including a total of 360 amino acid residues.

The construction of embodiment 3 expression vector

Using the genes NtNCP1 and NtNCP2 related to nicotine conversion obtained in Example 1, the present invention further constructed a CRISPR/Cas9 vector.

(1) Design and synthesis of sgRNA sequences of NtNCP1 and NtNCP2 genes:

The sgRNA guide sequence was designed using the online software CRISPR-P 2.0 (http://cbi.hzau.edu.cn/crispr/), and the guide sequence with a higher score and located at the appropriate position of the NtNCP1 and NtNCP2 gene sequences was selected. The sgRNA sequence selected in this application is: TCATAATGCATATAGGTTGGAGG (SEQ ID No.9).

(2) Design the forward and reverse primers of the sgRNA sequence and submit them to the design company for synthesis:

Upstream primer sgRNA-F: GATTGTCATAATGCATATAGGTTGG (SEQ ID No.10);

Downstream primer sgRNA-R: AAACCCCAACCTATATGCATTATGA (SEQ ID No.11).

(3) Primer annealing: Dilute the synthesized target sequence primers (upstream primer and downstream primer) with sterilized ddH ₂ O to a concentration of 100 ng/μL, then take 5 μL of each of the upstream and downstream primers and mix them evenly in a PCR tube, place in Annealing is carried out on the PCR instrument, so that the upstream and downstream Oligo single strands are annealed to form double strands.

The annealing program of the PCR instrument is: 95°C for 2min, -0.1°C/8s, annealed to 25°C, and the annealed product was diluted to 10 ng/µL by adding 90 μL of sterile water.

(4) Digestion and connection

a. Digest the CRISPR/Cas9 vector pORE-Cas9 (provided by Southwest University) with restriction endonuclease BsaI-HF.

Enzyme digestion system (50μL):

Enzyme digestion at 37°C overnight, followed by 1.5% agarose gel electrophoresis to cut the target fragment band, and recover the backbone fragment with a gel extraction kit.

b. to connect

The double-stranded product formed by annealing is ligated to the backbone of the digested carrier.

Ligation System (10μL):

The connection conditions are: 16°C for 2 hours.

(5) transform Escherichia coli:

a. Take out Trans-T1 competent cells from -80°C, freeze and thaw on ice, and divide into 50 μL/portion;

b. After the competent cells are thawed, add 10 μL of the ligation product to the competent cells, mix gently, and bathe in ice for 10 minutes;

c. After ice-bathing, heat-shock in a 42°C water bath for 90 seconds, and quickly put the competent cells back on ice for 2 minutes.

d. Evenly spread 60 μL of the transformation product on LB solid medium containing 16 mg/L kanamycin, and incubate in a bacterial incubator at 37° C. for 12 hours.

(6) Positive clone screening:

a. After a single colony grows on the plate, pick a single colony of E. coli into LB liquid medium containing 50 mg/L kanamycin, and shake it overnight on a shaker at 37°C;

b. Take part of the bacterial liquid for bacterial liquid PCR, and check whether it is a positive clone by nucleic acid electrophoresis;

c. Escherichia coli plasmids were extracted from the remaining part of the bacterial solution initially detected as positive clones. Send the plasmid to Novogene for sequencing to confirm the correctness of the positive clone.

The acquisition and detection of the T0 generation plant of embodiment 4

(1) Transformation of Agrobacterium

Using the CRISPR/Cas9-NtNCP1-NtNCP2 editing vector plasmid constructed in the previous step, taking Honghua Dajinyuan as an example, carry out genetic transformation and tissue culture, and obtain plants with knockout and edited genes NtNCP1 and NtNCP2 related to tobacco nicotine transformation , and the related experimental procedures are briefly introduced as follows.

Sterilize the surface of the tobacco seeds and plant them on the MS medium. After they grow to 4 cotyledons (15-20 days), transfer them into a culture bottle containing MS solid medium, and place them at 25±1°C with a light intensity of 30-50 μmol/( m ² ·s), the light time is 16h/d, and the culture is continued for 35-40d, and it is set aside.

Transform the plasmid with the correct sequence into Agrobacterium, the specific steps are as follows:

① Take out the LBA4404 electroporation-competent Agrobacterium cells stored at -80°C, and freeze-thaw them on ice.

②When the competent cells are just thawed, add 2 μL of the CRISPR/Cas9-NtNCP1-NtNCP2 editing vector plasmid, mix well, and place on ice.

③Transfer the mixed competent state to a pre-cooled electric transfer cup, place the electric transfer cup in the electrotransfer instrument for transformation, add 1mL of YEB liquid medium to mix with the transformation solution after the transformation is completed, and then place it on a shaker at 28°C , 200rpm for 1.5-2h.

④Centrifuge the culture medium at 8,000rpm, discard the supernatant, then suspend the bacteria with 200μL of YEB liquid medium, and apply to YEB containing 50mg/L rifampicin, 50mg/L streptomycin and 50mg/L kanamycin Incubate in the dark at 28°C for 2-3 days on solid medium.

(2) Infect callus

①Make tobacco leaf discs with a side length of 1cm in the ultra-clean workbench, and use MS liquid to prepare the Agrobacterium colony suspension containing the CRISPR/Cas9-NtNCP1-NtNCP2 editing vector (OD ₆₀₀ ＝0.6-0.8) .

② Soak and infect tobacco leaf discs with suspended Agrobacterium liquid for 10 minutes.

③Put leaf discs on MS solid medium containing 2.0mg/L NAA+0.5mg/L 6-BA, co-cultivate for 3 days at 28°C in the dark.

④ For subculture, place on MS solid medium containing 2.0mg/L NAA+0.5mg/L 6-BA+250mg/LCb+50mg/L Kan.

The culture conditions are: light culture at 28°C for 16h/d, light intensity 30-50μmol/(m2 s), dark culture at 25°C for 8h/d, culture for 45-60d, until the formation of differentiated buds, replace the differentiation culture every 7-10d Replace the medium for 3-4 times; culture until the formation of differentiated shoots; excise the calli that have formed differentiated shoots, and place them on MS medium containing 500mg/L carbenicillin and 50mg/L kanamycin Carry out culture, until the differentiated shoots on the callus grow to 2-4cm high, the culture conditions are consistent with the differentiation culture conditions, and cultivate for 8-14d; the regenerated plants are rooted and cultured, the differentiated shoots are cut off, and inserted with 500mg/L carbenicillin Rooting culture was carried out on MS medium with 50mg/L kanamycin, the culture conditions were consistent with the differentiation culture conditions, cultured for 20-30 days, regenerated and transplanted to flowerpots for cultivation, and then transformed plant leaves were sampled and sent to Huada University For molecular detection of the gene, the NtNCP1 target detection primers are: upstream primer NtNCP1-F: CCATCAGAAGCCACCACCAA (SEQ ID No.12) and downstream primer NtNCP1-R: TGCTCTGGTTCTTCGGCTAAG (SEQ ID No.13); NtNCP2 target detection primer is: upstream Primer NtNCP2-F: CCATCAGAAGCCACCACCAA (SEQ ID No. 14) and downstream primer NtNCP2-R: GCGTTTATTTGCTCCGGTTCTT (SEQ ID No. 15) were determined to obtain T0 generation plants with simultaneous knockout of NtNCP1 and NtNCP2 genes, and harvested for later use.

Embodiment 5 Obtaining of Homozygous Editing Materials

The seeds of the T0 generation are self-crossed and homozygous at a rate of 96 times. When the plants grow to 5-6 leaves, the leaves of a single plant are sampled and sent to Huada Gene for molecular detection. The primers for NtNCP1 target detection are: upstream primer NtNCP1-F : CCATCAGAAGCCACCACCAA (SEQ ID No.12) and downstream primer NtNCP1-R: TGCTCTGGTTCTTCGGCTAAG (SEQ ID No.13); NtNCP2 target detection primers are: upstream primer NtNCP2-F: CCATCAGAAGCCACCACCAA (SEQ ID No.14) and downstream primer NtNCP2- R: GCGTTTATTTGCTCCGGTTCTT (SEQ ID No.15), determine the homozygous edited plants of NtNCP1 and NtNCP2 genes, and then harvest the T1 generation seeds of homozygous edited NtNCP1 and NtNCP2 genes.

Embodiment 6 material planting and character investigation

The seeds of the plants whose NtNCP1 and NtNCP2 gene homozygous knockouts were identified by molecular detection in Example 4 were propagated and planted in a potted manner, and 60 plants were planted, topped at the full flowering stage, and 10 days after topping, refer to "YCT 142-2010 Tobacco Agronomy" Traits Investigation and Measurement Method”, measuring and analyzing traits such as plant height, waist leaf length, waist leaf width, stem girth, and pitch.

Now for the tobacco plants 10 days after topping, collect index data such as plant height, waist leaf length, waist leaf width, stem circumference and other index data of 10 tobacco strains, and carry out statistical analysis.

The results showed that the plant height, stem girth, waist leaf length, waist leaf width and other indicators of the NtNCP1 and NtNCP2 gene knockout plants were close to those of the control without significant difference. The comparison of the main traits of the control and NtNCP1 and NtNCP2 gene homozygous edited tobacco plants is shown in Figure 1.

Embodiment 7GC-MS detects

Using the edited plants planted in Example 5, GC-MS was used to detect the alkaloid content of the leaves 14 days after topping of the NtNCP1 and NtNCP2 gene homozygous knockout materials.

Tobacco plants 14 days after topping were selected, and 5 control (unedited) tobacco plant samples were collected, and leaves at the same leaf position were collected; tobacco plants 10 days after topping were selected, and 5 plants homozygously edited for NtNCP1 and NtNCP2 genes were collected. Tobacco plant samples; the main tendons are removed from the leaves, wrapped in tin foil paper for storage and transportation, stored at ultra-low temperature (-70°C) in the laboratory, freeze-dried, ground and sieved.

Weigh 0.2g of sample into a 15mL centrifuge tube, accurate to 0.1mg, add 2.0mL of 5% sodium hydroxide solution, and then add 0.05mL of internal standard solution A (dimethylquinoline solution, prepared in methanol, dilute to 1.0 mg/mL) and internal standard solution B (2,2'-bipyridyl-d2 solution, prepared in methanol, diluted to 0.5mg/mL with dichloromethane), shake and mix well, let stand for 20min, and then add 10.0mL extraction solution (dichloromethane and methanol are mixed according to the volume ratio of 4:1), put the cover and seal it into the vortex shaker, shake and extract at a speed of 2000r/min for 40min, after standing for 1h, centrifuge for 8min, take the lower organic phase and transfer into a chromatographic vial for analysis by GC-MS.

The reference conditions of gas chromatography are: chromatographic column: DB-35MS or equivalent column efficiency capillary chromatographic column, specifications: 30mm (length) × 0.25mm (inner diameter) × 0.25m (film thickness); inlet temperature: 250°C; column Flow rate: 1.0mL/min; nicotine injection volume: 1.0L, split injection, split ratio 40:1; other alkaloid injection volume: 2.0L, split injection, split ratio 10:1; heating program : Initial temperature is 100°C, keep for 3min; increase to 260°C at a rate of 8°C/min, keep for 10min.

Mass spectrometry reference conditions: transfer line temperature: 280°C; ionization method: electron impact source (EI); ionization energy: 70eV; ion source temperature: 230°C; solvent delay: 8min; measurement method: selected ion monitoring (SIM) scanning.

Comparison of nicotine content in leaves of control (unedited) and NtNCP1 and NtNCP2 gene homozygous edited tobacco plants 14 days after topping (results are shown in Table 1). It was found that the total amount of 14 alkaloids in the NtNCP1 and NtNCP2 gene knockout edited plants was close to that of the control, but the nicotine content was significantly lower than that of the control plants, the nornicotine content was significantly higher than that of the control plants, and the nicotine conversion rate was 24.3% ( The control is 0.5%), exceeding the criterion of 2.5%, indicating that the nicotine transformed strain can be rapidly obtained by knocking out the NtNCP1 and NtNCP2 genes of the cultivated tobacco simultaneously by gene editing. This provides genetic material and theoretical basis for the research on the function of genes related to tobacco nicotine conversion and the cultivation of new tobacco varieties regulated by nicotine content.

Table 1 shows the alkaloid content (μg/g) of the edited plants of the present invention and the control (unedited) fresh tobacco leaves 14 days after topping

Although the present invention has been disclosed as above with the embodiments, it is not intended to limit the present invention, and any person skilled in the art can make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the present invention The scope of protection is defined by the claims and their equivalents.

Claims (10)

1. A method for rapidly obtaining a tobacco nicotine transformant for cultivation is characterized in that genes related to tobacco nicotine transformation are knocked out by a CRISPR/Cas9 system, the genes are NtNCP1 genes and NtNCP2 genes, the NtNCP1 gene sequence is shown in SEQ ID No.1, and the NtNCP2 gene sequence is shown in SEQ ID No. 3.

2. The method for rapidly obtaining the nicotiana tabacum nicotine transformant as claimed in claim 1, wherein the sequence of the encoded protein of the NtNCP1 gene is represented by SEQ ID No.2 after the NtNCP1 gene is translated.

3. The method for rapidly obtaining the nicotiana tabacum nicotine transformant as claimed in claim 1, wherein the sequence of the encoded protein of the NtNCP2 gene is represented by SEQ ID No.4 after the NtNCP2 gene is translated.

4. The method for rapidly obtaining a cultivated nicotine transformant according to claim 1, comprising the steps of:

(1) Creating a T0 generation editing material;

(2) The T0 generation plants are subjected to selfing homozygosis to obtain homozygous editing materials;

(3) Homozygous editing material planting and character observation;

(4) And detecting the alkaloid content of leaves 14 days after topping of NtNCP1 and NtNCP2 gene homozygous knockout materials by GC-MS, and calculating the nicotine conversion rate.

5. The method for rapidly obtaining the nicotine transformant of nicotiana tabacum as claimed in claim 4, wherein in step (1), the sgRNA sequence adopted by the CRISPR/Cas9 system is TCATAATGCATAGGTTGGAGG, and the primer sequence adopted by the sgRNA sequence is as follows:

an upstream primer sgRNA-F: gattgtcataatgcatatgg;

a downstream primer sgRNA-R: AAACCCAAACCTATATGCATTATGA.

6. The method for rapidly obtaining the nicotine transformant for cultivation of tobacco according to claim 5, wherein, in the step (1),

the primer sequences adopted by the editing detection of the NtNCP1 target point are as follows:

the upstream primer NtNCP1-F: CCATCAGAAGCAGCACACAAS;

the downstream primer NtNCP1-R: TGCTCTGGTTCTTCGGCTAAG;

the primer sequences adopted by the editing detection of the NtNCP2 target point are as follows:

the upstream primer NtNCP2-F: CCATCAGAAGCACCACAAA;

the downstream primer NtNCP2-R: GCGTTTATTTGCTCCGGTTCTT.

7. The method for rapidly obtaining a cultivated nicotine transformant of tobacco according to claim 6,

the step (1) is specifically as follows:

designing a sgRNA guide sequence, annealing an upstream primer sgRNA-F and a downstream primer sgRNA-R to form a double strand, and digesting a CRISPR/Cas9 vector pORE-Cas9 by using a restriction endonuclease BsaI-HF; connecting the double-chain product formed by annealing and the carrier skeleton which is well digested by enzyme by using T4 ligase; the ligation product is transformed into an escherichia coli competent cell, positive clone is obtained through detection, recombinant plasmid is extracted, and a CRISPR-Cas9 expression vector is obtained;

soaking and infecting a tobacco leaf disc with agrobacterium LBA4404 bacterial liquid carrying a CRISPR/Cas9-sgRNA expression vector to obtain a T0 generation plant, obtaining a plant with NtNCP1 and NtNCP2 genes simultaneously edited through target point editing and detection, and harvesting to obtain a T0 generation seed.

The step (2) is specifically as follows:

and (2) carrying out selfing homozygous expanded propagation planting on the T0 generation seeds, obtaining plants subjected to homozygous editing of NtNCP1 and NtNCP2 genes through target point editing detection, and harvesting seeds to obtain T1 generation seeds, wherein the primer sequences adopted by the NtNCP1 and NtNCP2 target point editing detection are the same as the step (1).

8. Use of a tobacco nicotine conversion-associated gene in the rapid production of nicotine converted plants, wherein the tobacco nicotine conversion-associated gene is the NtNCP1 gene and the NtNCP2 gene according to any one of claims 1 to 7.

9. Use of a method for rapidly obtaining a cultured nicotine tobacco transformant according to any one of claims 1 to 7 for the preparation of a nicotine tobacco transformant.

10. The use of claim 9, wherein the total amount of alkaloid in the leaves 14 days after the NtNCP1 and NtNCP2 genes are knocked out simultaneously is close to that of the control plant, but the nicotine content is significantly lower than that of the control plant, the nicotine content is significantly higher than that of the control plant, and the nicotine conversion rate is higher than 20%.

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