CN111560389A - Tobacco mitogen-activated protein kinase gene NtMAPK8 and its application - Google Patents

Abstract

The invention discloses a tobacco mitogen activated protein kinase geneNtMAPK8And application thereof, namely the tobacco mitogen-activated protein kinaseNtMAPK8The gene nucleotide sequence is shown as SEQ ID NO. 1 or SEQ ID NO. 3, and the coded amino acid sequence is shown as SEQ ID NO. 2 or SEQ ID NO. 4; the invention clones the gene of the tobacco mitogen activated protein kinase for the first timeNtMAPK8Constructing a cloning vector and constructing a transgenic mutant strain with NtMAPK8 gene deletion by using a gene editing vector, so that the color of the tobacco leaves is changed from normal green to white; therefore, a gene editing vector can be usedThe NtMAPK8 gene is not expressed, and plant variety breeding is carried out to obtain ornamental plant varieties of plant mosaic.

Description

Tobacco mitogen-activated protein kinase gene NtMAPK8 and its application

technical field

The invention relates to the technical field of molecular biology, in particular to a tobacco mitogen-activated protein kinase gene NtMAPK8 and its application.

Background technique

Among plants, tobacco is an important economic crop. The color of tobacco leaves directly affects the color of tobacco after curing, which is an important indicator to measure the quality of tobacco leaves.

The MAPKKK-MAPKK-MAPK cascade pathway is very conserved in plants, and it not only plays an important role in the defense system of plants, but also plays a very important role in the regulation of plant growth and development. However, there is no report about MAPK gene in leaf color.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the present invention provides a tobacco mitogen-activated protein kinase gene NtMAPK8 , which is shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the amino acid sequence encoded by the gene is shown in SEQ ID NO: 3 or SEQ ID NO:4.

Another object of the present invention is to provide a recombinant vector containing the above-mentioned tobacco mitogen-activated protein kinase gene NtMAPK8 .

The recombinant vector of the present invention includes a cloning vector and an expression vector; the preparation method of the cloning vector includes using the cDNA of tobacco as a template and using primers as shown in SEQ ID NO: 5 and SEQ ID NO: 6 to carry out PCR Amplify; connect the obtained PCR product to T vector, and transform E. coli to obtain a cloning vector.

Another object of the present invention is to provide a gene knockout vector, which is obtained by using CRISPR/Cas9 gene editing technology to design a target site for the tobacco NtMAPK8 gene, and the nucleotide sequence of the target site knockout sequence is as shown in SEQID NO: 7; the nucleotide sequences of the primer pairs obtained by adding artificially synthesized linker sequences on both sides of the target site are shown in SEQ ID NO: 8 and SEQ ID NO: 9; the primers After the pair was annealed, it was connected to the vector pORE-Cas9 digested with BsaI to obtain the gene knockout vector pORE-Cas9/NtMAPK8.

Another object of the present invention is to provide a transgenic plant, the preparation method of which includes: transforming the gene knockout vector pORE-Cas9/NtMAPK8 into Agrobacterium competent cells, and then transforming the infected plant.

The transgenic plant is transgenic tobacco; the pORE-Cas9/NtMAPK8 vector is used to transform Agrobacterium-competent when transforming and infecting the transgenic tobacco, and the tobacco leaf disc transformation method mediated by Agrobacterium is used to genetically transform the tobacco to obtain tobacco plants of different colors. . Specifically, the color part of its leaves changed from green to light green.

Another object of the present invention is to apply the above-mentioned tobacco mitogen-activated protein kinase gene NtMAPK8 in the preparation of plant varieties with different leaf colors.

Compared with the prior art, the present invention has the following advantages and effects:

The present invention clones and obtains the tobacco mitogen-activated protein kinase gene NtMAPK8 for the first time, and constructs a cloning vector and a knockout vector; the NtMAPK8 protein participates in MAPK signal transmission, and a transgenic mutant strain with the deletion of the NtMAPK8 gene can be constructed through the gene editing vector, so that the tobacco The leaf color changed from normal green to whitish, and the exact reason is still unclear.

The NtMAPK8 gene knockout mutant line obtained by the present invention has the color part of its leaves turned white, so the gene editing vector can be used to make the NtMAPK8 gene not express, and plant variety breeding can be performed to obtain ornamental plant varieties of plant flowers and leaves.

Description of drawings

Fig. 1 is a graph showing the expression level of NtMAPK8 gene in different tissues in the experiment of the present invention, picture A is the result of transcript MAPK8a; picture B is the result of transcript MAPK8b;

Figure 2 is a schematic diagram of the target site design of NtMAPK8 gene knockout in the experiment of the present invention;

Fig. 3 is a phenotypic diagram of the NtMAPK8 mutant of the T0 generation transgenic line in the experiment of the present invention.

Detailed ways

The present invention is described in further detail below in conjunction with the accompanying drawings and examples, but the protection scope of the present invention is not limited to the content, and the reagents and methods used in the examples, unless otherwise specified, all adopt conventional reagents and use conventional methods.

The biological materials, experimental reagents and consumables involved in this embodiment are as follows:

Tobacco variety: Safflower Dajinyuan, the seeds of which were provided by the State Key Laboratory of Silkworm Genome Biology, Southwest University;

Vector: The CRISPR/Cas9 basic knockout vector was provided by the State Key Laboratory of Silkworm Genome Biology, Southwest University; the pEASY®-Blunt Zero Cloning Kit vector was purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

Strain: Trans1-T1 chemically competent cells, LBA4404 Agrobacterium chemically competent cells, all purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

Primers and sequencing: primer synthesis and DNA sequencing were completed by Beijing BGI;

Experimental reagents: Plant DNA extraction kit EasyPure Plant Genomic DNA Kit (containing RNase A) and DNA purification kit EasyPure® PCR Purification Kit PCR were purchased from Beijing Quanshi Gold Company; Trizol® reagent (Invitrogen, USA) for RNA extraction; Reverse transcription kit, TransScript® One-Step gDNARemoval and cDNA Synthesis SuperMix, purchased from Beijing Quanshijin Company; T4 ligase, restriction endonuclease BsaI, DNA amplification enzyme, purchased from NEB Company; PrimeSTAR Max DNA polymerase Purchased from Takara Corporation.

Experimental equipment: PCR amplification instrument Veriti (Gene Co., Ltd.); gel imaging system UV gel imaging instrument (BIO-RAD); quantitative PCR instrument, ABI 7500 fast real-time PCR system (Thermo Fisher).

Example 1: Cloning and sequence analysis of tobacco mitogen-activated protein kinase gene NtMAPK8

The present invention obtains the tobacco mitogen-activated protein kinase gene NtMAPK8 based on the sequencing of the tobacco whole genome, performs a large number of bioinformatics analysis and screening, and then uses the tobacco whole genome cDNA as a template to clone the tobacco mitogen-activated protein kinase gene NtMAPK8 , specifically:

1. cDNA template preparation

Total RNA was extracted from fresh tissues of tobacco plants using reagents (Invitrogen, USA) following the manufacturer's instructions. Total RNA was reverse transcribed into cDNA using TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (Quanshijin, China).

2. PCR product amplification and recovery

Primers were designed with Primer5, and the primer sequences were as follows:

CDS-F: 5'-ATGGGAAGTTTCAGGAGATGCATGGAAATT-3' (represented by SEQ ID NO: 5)

CDS-R: 5'-CTAGTTGTCAACAACCTCAATCAAGCTTG-3' (shown in SEQ ID NO: 6)

The cDNA prepared in step 1 was used as the template, and the designed primers were used for PCR amplification. The conditions were: 98°C for 2 min, 28 cycles (98°C for 5s, 55°C for 15s, 72°C for 15s), and 72°C for 5min.

3. Connection

The PCR product was purified and recovered with the EasyPure® PCR Purification Kit PCR kit, and then ligated to the pEASY®-Blunt Zero vector. The system (5 μL): 1 μL of the T vector and 4 μL of the fragment; reaction conditions: Mix gently and react at 25°C for 10 min.

4. Conversion

Transform into Trans1-T1 competent cells (ice bath for 30 min → 42 °C heat shock for 30 s → ice bath for 2 min), add 250 μL of anti-LB liquid medium, culture at 220 rpm, 37 ° C for 1 h, centrifuge at 1500 rpm for 1 min, discard part of the supernatant, Take 100-150 μL of the coated plate.

5. Spot

Take a 1.5mL centrifuge tube, add 10μL ddH ₂ O, pick spots and hit the white pipette tip into the centrifuge tube, then shake the plate back and forth, discard the white pipette tip; aspirate 4μL ddH ₂ O from 10μL ddH ₂ O for colony PCR .

6. Sequencing verification

After the colony PCR-positive clones were shaken and mixed, they were sent to BGI for verification by sequencing.

After being sequenced by the company, two transcripts of the gene NtMAPK8 were obtained by analysis: The full-length NtMAPK8a gene sequence is 2976 bp, and its nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded tobacco NtMAPK8a protein is shown in SEQ ID NO. : shown in 2; NtMAPK8b gene sequence full-length 3947 bp, and its nucleotide sequence is shown in SEQ ID NO: 3. The amino acid sequence of the encoded tobacco NtMAPK8b protein is shown in SEQ ID NO:4.

Example 2 Characteristic analysis of the expression pattern of tobacco NtMAPK8 gene in different tissues

The expression patterns of NtMAPK8 gene in different tobacco tissues (roots, stems, leaves, flowers, etc.) were detected by fluorescence quantitative qRT-PCR. FastStart Universal SYBR Green Master (ROX) Realtime kit (Roche) was used. The relative quantitative method was used, the tobacco EF1α gene was used as the internal reference gene, and the template was the cDNA obtained from the above-mentioned different tissues. Three replicates were set for each sample, and a negative control and a positive control were set at the same time. The reaction system is shown in Table 1.

Table 1

The two transcripts of the NtMAPK8 gene are MAPK8a and MAPK8b, respectively; qRT-PCR primer sequences:

NtMAPK8a-qRT-F: CGACCCAAAGGACCGACCCA is shown in SEQ ID NO: 10;

NtMAPK8a-qRT-R: CCTCAGGTCCTCCTTCGTCAC is shown in SEQ ID NO: 11;

NtMAPK8b-qRT-F: TTGCTAAGTTCTCACTGGGA is shown in SEQ ID NO: 12;

NtMAPK8b-qRT-R: CAGTGGATCAGCATTTGGAAA is shown in SEQ ID NO: 13;

NtEF1α-qRT-F: GCATTGCTTGCTTTCACCCTT is shown in SEQ ID NO: 14;

NtEF1α-qRT-R: AACCTCCTTCACGATTTCATCATACC is shown in SEQ ID NO: 15;

The reaction system was fully mixed and put into ABI7500 fluorescence quantitative PCR system for PCR reaction. The three-step PCR reaction program was used, and the 2- ^ΔΔCt method was used to determine the relative expression level of the target gene.

The results are shown in Figure 1. The results showed that both transcripts of the NtMAPK8 gene had the highest expression levels in the anthers.

Example 3: Construction of gene editing vector

Using CRISPR/Cas9 gene editing technology to design the target site of tobacco NtMAPK8 gene to construct NtMAPK8 gene knockout vector; genetically transform tobacco varieties to obtain transgenic tobacco plants;

1. Design target sites:

Target: GAGGCTAATGATGACTTGACA (as shown in SEQ ID NO: 7);

Synthetic plus linker sequence

Target-F: GATTGAGGCTAATGATGACTTGACA (as shown in SEQ ID NO: 8);

Target-R: AAACTGTCAAGTCATCATTAGCCTC (as shown in SEQ ID NO:9);

2. Knockout vector construction

① Single-stranded oligo DNA annealing to form double-stranded DNA

The synthesized two single-stranded primers (Target-F and Target-R) were diluted to 50 μmol/L, and then annealed. The annealing reaction system is shown in Table 2:

Table 2

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

②Enzyme cleavage of expression vector

Expression vector: pORE-Cas9/gRNA, digest the expression vector with BsaI, the digestion system is shown in Table 3, digest at 37°C for 1 h, and recover the digested product;

table 3

③ The target site is connected to the expression vector, the connection system is shown in Table 4, and the connection is performed at 25°C for 10 minutes;

Table 4

④ The ligation products were transformed into competent cells, and positive clones were obtained by PCR verification; sequencing and comparison showed that the clones were successful.

Experimental Example 4 Obtainment of tobacco NtMAPK8 knockout plants

1. The constructed pORE-Cas9/NtMAPK8 vector was transformed into Agrobacterium-competent LBA4404, and the cultivated tobacco variety K326 was genetically transformed by Agrobacterium-mediated transformation of tobacco leaf discs to obtain kanamycin-resistant T0 Generation of positive transformed seedlings, the specific transformation steps are:

(1) Agrobacterium transformation

① Add the constructed knockout vector to 100 μL of Agrobacterium competent cells LBA-4404;

② Ice bath for 30 minutes, liquid nitrogen quick-freeze for 1 minute, and then water bath at 37°C for 5 minutes;

③ Add 1ml of YEB liquid antibacterial medium, treat at 28°C, 200rpm for 3h and take out;

④ After centrifugation at 3000 rpm for 3 min, part of the supernatant was removed, and 150 μL was spread on a plate containing rifampicin, streptomycin and kanamycin;

⑤ 28°C, invert in the dark for 2-3 days;

(2) Tobacco genetic transformation

Suspend Agrobacterium in MS liquid medium to an OD value of 0.6-0.8, infect tobacco leaf discs for 10 min, co-cultivate in the dark for 3 days, select and cultivate until callus grows and cut shoots.

2. Molecular detection

Use specific primers to amplify whether the genome sequence near the sgRNA in the regenerated tobacco genome is edited, specifically: extract the genomic DNA of positive tobacco seedling leaves, use primers as shown in SEQ ID NO: 8 and SEQ ID NO: 7, and use the primers shown in SEQ ID NO: 8 and SEQ ID NO: 7. DNA is used as a template, the target fragment is amplified by PCR, the PCR product is purified and recovered, and the cloned vector is connected for sequencing to detect the mutant form of the target site fragment.

The T0 generation transgenic positive plant seeds whose target sites were edited were collected. In this experiment, the gene editing situation of the T0 generation transgenic lines knocking out the target site is shown in Table 5;

table 5

It can be seen from the above that the NtMAPK8 gene was successfully edited in the mutant line of tobacco plants.

3. Phenotypic observation of transgenic lines

Figure 3 is a phenotype diagram of the T0 generation transgenic line in the experiment of the present invention. It can be seen from Figure 3 that the color of the tobacco leaves of the NtMAPK8 gene knockout mutant line changed from normal green to white.

To sum up, in this example, based on the gene editing vector CRISPR/Cas9, the gene knockout vector was transformed into tobacco leaf discs by using the leaf disc genetic transformation method, and a knockout line of the NtMAPK8 gene was obtained, and the color of the leaves was partially changed. Therefore, the gene editing vector can be used to make the NtMAPK8 gene not express, and plant variety breeding can be carried out to obtain ornamental plant varieties of plant flowers and leaves.

The description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection of the present invention. within the range.

sequence listing

<110> Yunnan China Tobacco Industry Co., Ltd.

<120> Tobacco mitogen-activated protein kinase gene NtMAPK8 and its application

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actagaatat tgtctaggaa taagatcaaa tcttcccttt tacatgccat ttggcataaa 2820

gcgttttgaa tggattgttg gtgctttcaa gaaaatcatt tgacctttca gcttggttgc 2880

cccctatgcg cacttatgtt gtgattctgt gtttatgccc atctttctgt atattttgtt 2940

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<211> 610

<212> PRT

<213> Tobacco (Nicotiana tabacum L.)

<400> 3

Met Asp Phe Phe Ser Glu Tyr Gly Asp Ala Asn Arg Tyr Lys Ile Gln

1 5 10 15

Glu Val Ile Gly Lys Gly Ser Tyr Gly Val Val Cys Ser Ala Ile Asp

20 25 30

Thr His Thr Gly Glu Lys Val Ala Ile Lys Lys Ile His Asp Ile Phe

35 40 45

Glu His Ile Ser Asp Ala Ala Arg Ile Leu Arg Glu Ile Lys Leu Leu

50 55 60

Arg Leu Leu Arg His Pro Asp Ile Val Glu Ile Lys His Ile Met Leu

65 70 75 80

Pro Pro Ser Arg Arg Asp Phe Lys Asp Ile Tyr Val Val Phe Glu Leu

85 90 95

Met Glu Ser Asp Leu His Gln Val Ile Lys Ala Asn Asp Asp Leu Thr

100 105 110

Arg Glu His Tyr Gln Phe Phe Leu Tyr Gln Leu Leu Arg Ala Leu Lys

115 120 125

Tyr Ile His Thr Ala Asn Val Tyr His Arg Asp Leu Lys Pro Lys Asn

130 135 140

Ile Leu Ala Asn Ala Asn Cys Lys Leu Lys Ile Cys Asp Phe Gly Leu

145 150 155 160

Ala Arg Val Ala Phe Asn Asp Thr Pro Thr Thr Ile Phe Trp Thr Asp

165 170 175

Tyr Val Ala Thr Arg Trp Tyr Arg Ala Pro Glu Leu Cys Gly Ser Phe

180 185 190

Tyr Ser Lys Tyr Thr Pro Ala Ile Asp Ile Trp Ser Ile Gly Cys Ile

195 200 205

Phe Ala Glu Val Leu Thr Gly Lys Pro Leu Phe Pro Gly Lys Asn Val

210 215 220

Val His Gln Leu Asp Leu Met Thr Asp Leu Leu Gly Thr Pro Ser Met

225 230 235 240

Asp Thr Ile Ser Arg Val Arg Asn Asp Lys Ala Arg Arg Tyr Leu Thr

245 250 255

Ser Met Arg Lys Lys Gln Pro Val Ser Phe Ala Gln Lys Phe Pro Asn

260 265 270

Ala Asp Pro Leu Ala Leu Lys Leu Leu Glu Arg Leu Leu Ala Phe Asp

275 280 285

Pro Lys Asp Arg Pro Thr Ala Glu Glu Ala Leu Ala Asp Pro Tyr Phe

290 295 300

Lys Gly Leu Ala Lys Ser Glu Arg Glu Pro Ser Cys Lys Pro Ile Ser

305 310 315 320

Lys Met Glu Phe Glu Phe Glu Arg Arg Arg Val Thr Lys Glu Asp Leu

325 330 335

Arg Glu Leu Ile Phe Arg Glu Ile Leu Glu Tyr His Pro Gln Leu Arg

340 345 350

Lys Asp Tyr Met Asn Gly Val Glu Arg Thr Asn Phe Leu Tyr Pro Ser

355 360 365

Ala Val Asp Gln Phe Arg Lys Gln Phe Ala His Leu Glu Glu Asn Gly

370 375 380

Gly Asn Gly Gly Pro Val Val Pro Met Asp Arg Lys His Val Ser Leu

385 390 395 400

Pro Arg Ser Thr Val Leu His Ser Asn Thr Ile Pro Ser Lys Glu Gln

405 410 415

Pro Ile Ala Ala Ala Asn Met Arg Asp Arg Gln Asn Gly Glu Glu Ser

420 425 430

Cys Ser Arg Asn Cys Arg Asp Ser Glu Gly Leu Ala Asn Ser Leu Thr

435 440 445

Arg Thr Met Gln Ala Gln Pro Arg Ile Ala Leu Ala Lys Thr Gly Lys

450 455 460

Val Val Gly Pro Val Leu Ala Tyr Asp Ser Gly Asn Lys Glu Lys Tyr

465 470 475 480

Asp Pro Arg Ser Gln Val Arg Asn Thr Val Phe Pro Ser Gln Ile Met

485 490 495

Pro Ser Ala Tyr Cys Tyr Asp Arg Ser Gly Met Val Lys Gln Glu Arg

500 505 510

Pro Val Val Glu Thr Glu Arg Asp Val Ser Ser His Ala Lys Pro Met

515 520 525

Pro Pro Cys Gly Met Ala Ala Lys Leu Ala Pro Asp Ile Ala Ile Asn

530 535 540

Ile Asp Ser Asn Pro Phe Tyr Met Met Arg Ala Gly Val Thr Lys Pro

545 550 555 560

Asp Arg Val Glu Asp Arg Ile Thr Ile Asp Thr Asn Leu Leu Gln Ala

565 570 575

Lys Ser Gln Tyr Gly Gly Ile Gly Val Ala Ala Ala Ala Ala Thr Thr

580 585 590

Ala Ala Ala His Arg Lys Val Gly Thr Val Gln Tyr Gly Ile Ser Arg

595 600 605

Met Tyr

610

<210> 4

<211> 610

<212> PRT

<213> Tobacco (Nicotiana tabacum L.)

<400> 4

Met Asp Phe Phe Ser Glu Tyr Gly Asp Ala Asn Arg Tyr Lys Ile Gln

1 5 10 15

Glu Val Ile Gly Lys Gly Ser Tyr Gly Val Val Cys Ser Ala Ile Asp

20 25 30

Thr His Thr Gly Glu Lys Val Ala Ile Lys Lys Ile His Asp Ile Phe

35 40 45

Glu His Ile Ser Asp Ala Ala Arg Ile Leu Arg Glu Ile Lys Leu Leu

50 55 60

Arg Leu Leu Arg His Pro Asp Ile Val Glu Ile Lys His Ile Met Leu

65 70 75 80

Pro Pro Ser Arg Arg Asp Phe Lys Asp Ile Tyr Val Val Phe Glu Leu

85 90 95

Met Glu Ser Asp Leu His Gln Val Ile Lys Ala Asn Asp Asp Leu Thr

100 105 110

Arg Glu His Tyr Gln Phe Phe Leu Tyr Gln Leu Leu Arg Ala Leu Lys

115 120 125

Tyr Ile His Thr Ala Asn Val Tyr His Arg Asp Leu Lys Pro Lys Asn

130 135 140

Ile Leu Ala Asn Ala Asn Cys Lys Leu Lys Ile Cys Asp Phe Gly Leu

145 150 155 160

Ala Arg Val Ala Phe Asn Asp Thr Pro Thr Thr Ile Phe Trp Thr Asp

165 170 175

Tyr Val Ala Thr Arg Trp Tyr Arg Ala Pro Glu Leu Cys Gly Ser Phe

180 185 190

Tyr Ser Lys Tyr Thr Pro Ala Ile Asp Ile Trp Ser Ile Gly Cys Ile

195 200 205

Phe Ala Glu Val Leu Thr Gly Lys Pro Leu Phe Pro Gly Lys Asn Val

210 215 220

Val His Gln Leu Asp Leu Met Thr Asp Leu Leu Gly Thr Pro Ser Met

225 230 235 240

Asp Thr Ile Ser Arg Val Arg Asn Asp Lys Ala Arg Arg Tyr Leu Thr

245 250 255

Ser Met Arg Lys Lys Gln Pro Val Ser Phe Ala Gln Lys Phe Pro Asn

260 265 270

Ala Asp Pro Leu Ala Leu Lys Leu Leu Glu Arg Leu Leu Ala Phe Asp

275 280 285

Pro Lys Asp Arg Pro Thr Ala Glu Glu Ala Leu Ala Asp Pro Tyr Phe

290 295 300

Lys Gly Leu Ala Lys Ser Glu Arg Glu Pro Ser Cys Lys Pro Ile Ser

305 310 315 320

Lys Met Glu Phe Glu Phe Glu Arg Arg Arg Val Thr Lys Glu Asp Leu

325 330 335

Arg Glu Leu Ile Phe Arg Glu Ile Leu Glu Tyr His Pro Gln Leu Arg

340 345 350

Lys Asp Tyr Met Asn Gly Val Glu Arg Thr Asn Phe Leu Tyr Pro Ser

355 360 365

Ala Val Asp Gln Phe Arg Lys Gln Phe Ala His Leu Glu Glu Asn Gly

370 375 380

Gly Asn Gly Gly Pro Val Val Pro Met Asp Arg Lys His Val Ser Leu

385 390 395 400

Pro Arg Ser Thr Val Val His Ser Asn Thr Ile Pro Ser Lys Glu Gln

405 410 415

Pro Ile Ala Ala Ala Asn Met Arg Asp Arg Gln Asn Gly Glu Glu Ser

420 425 430

Cys Ser Arg Asn Cys Arg Asp Ser Glu Gly Phe Ala Asn Ser Leu Thr

435 440 445

Arg Thr Met Gln Ala Gln Pro Arg Ile Ala Leu Ala Lys Pro Gly Lys

450 455 460

Val Val Gly Pro Val Leu Ala Tyr Asp Ser Gly Asn Lys Glu Lys Tyr

465 470 475 480

Asp Pro Arg Ser Gln Val Arg Asn Thr Val Pro Pro Ser Gln Ile Met

485 490 495

Pro Ser Ala Tyr Cys Tyr Asp Lys Ser Gly Met Val Lys Gln Glu Arg

500 505 510

Ala Val Val Glu Thr Glu Arg Asp Leu Ser Ser His Ala Lys Pro Met

515 520 525

Pro Pro Cys Gly Met Ala Ala Lys Leu Gly Pro Asp Ile Ala Ile Asn

530 535 540

Ile Asp Ser Asn Pro Phe Tyr Met Met Arg Ala Gly Val Thr Lys Pro

545 550 555 560

Asp Arg Val Asp Asp Arg Ile Thr Ile Asp Thr Asn Leu Leu Gln Ala

565 570 575

Lys Ser Gln Tyr Gly Gly Ile Gly Val Ala Ala Ala Ala Ala Thr Thr

580 585 590

Ala Ala Ser His Arg Lys Val Gly Thr Val Gln Tyr Gly Met Ser Arg

595 600 605

Met Tyr

610

<210> 5

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 5

atgggaagtt caggagatgc atggaaatt 29

<210> 6

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 6

ctagttgtca acaacctcaa tcaagcttg 29

<210> 7

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 7

gaggctaatg atgacttgac a 21

<210> 8

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 8

gattgaggct aatgatgact tgaca 25

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 9

aaactgtcaa gtcatcatta gcctc 25

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 10

cgacccaaag gaccgaccca 20

<210> 11

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 11

cctcaggtcc tccttcgtca c 21

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 12

ttgctgaagt tctcactggg a 21

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 13

cagtggatca gcatttggaa a 21

<210> 14

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 14

gcattgcttg ctttcaccct t 21

<210> 15

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 15

aacctccttc acgatttcat catacc 26

Claims (5)

1. Tobacco mitogen activated protein kinase geneNtMAPK8The method is characterized in that: the nucleotide sequence is shown in SEQ ID NO. 1 or SEQ ID NO. 2.

2. The tobacco mitogen-activated protein kinase gene according to claim 1NtMAPK8The method is characterized in that: the amino acid sequence of the gene code is shown in SEQ ID NO. 3 or SEQ ID NO. 4.

3. A knock-out vector, comprising: the gene is obtained by designing a target site knockout sequence for the tobacco NtMAPK8 gene by using a CRISPR/Cas9 gene editing technology and then carrying out gene editing.

4. The knock-out vector according to claim 3, wherein: the nucleotide sequence of the knockout sequence of the target site is shown as SEQ ID NO. 7, after artificially synthesized linker sequences are added on two sides of the target site, primer pairs with the nucleotide sequences shown as SEQ ID NO. 8 and SEQ ID NO. 9 are obtained, and after the primer pairs are annealed, the primer pairs are connected to a Bsa I enzyme-digested vector pORE-Cas9, and the gene knockout vector pORE-Cas9/NtMAPK8 is obtained.

5. The tobacco mitogen-activated protein kinase gene of claim 1NtMAPK8Or the use of the gene knockout vector of claim 3 or 4 in the preparation of different color leaf plant varieties.

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