CN113667689A - Vector capable of carrying out efficient gene editing in tobacco and application thereof - Google Patents

Abstract

The present disclosure relates to a vector capable of performing efficient gene editing in tobacco, in which a 35Spro promoter, a gRNA insertion region, an eu terminator, a calreticulin promoter element, a spCas9 protein expression element, a ubp terminator and a hygromycin selection marker are sequentially inserted. The vector is a pDC45 vector system, can obviously improve the gene editing efficiency of tobacco, can greatly improve the editing efficiency of multiple target spots, homozygosis/biallelism, and lays a foundation for creating plant whole genome editing strains in high flux.

Description

Vector capable of carrying out efficient gene editing in tobacco and application thereof

Technical Field

The disclosure relates to the field of genetic engineering, in particular to a vector capable of performing efficient gene editing in tobacco and application thereof.

Background

The genome editing technology is a modern biotechnology for directionally changing species genomes, and has the characteristics of high modification efficiency, strong specificity, no species limitation and the like, so that the genome editing technology is widely concerned and applied. The gene editing technology developed and utilized at the earliest is Zinc Finger Nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), which can realize genome modification of different species, but have the disadvantages of complex design, large workload, low efficiency, high cost and the like. The CRISPR-Cas technology is the latest 3-generation gene editing technology, is composed of clustered and regularly spaced palindromic repeats and a CRISPR nuclease protein Cas, and is an acquired autoimmune defense system formed by archaea in evolution (Ishino Y, 1987). The CRISPR-Cas system is widely applied to gene function analysis, new germplasm creation and genetic improvement of animals, plants and microorganisms due to the advantages of simple and convenient design and operation, high editing efficiency, accuracy, no species specificity and the like.

In recent years, genome-directed editing has been achieved in plants such as rice, maize, arabidopsis thaliana, tomato and the like, using the type II CRISPR-Cas9 system. Although scientists have also achieved gene editing in polyploid plants such as wheat and tobacco, since polyploid plants must knock out multiple copies of 1 gene at the same time to achieve the effect of the gene in a specific development process, for example, knock out the MLO genes of wheat A, B and D genome at the same time to obtain powdery mildew resistance of wheat (Wang el al, 2014, Nature biotech). Tobacco is an important model plant for polyploid research, and has important functions in the aspects of metabolism, antibody production, foreign protein expression and the like. In 2015, Gao et al first achieved gene editing in tobacco cultivars, 77.8%, 81.8%, and 87.5% mutation efficiencies were achieved in NtCCD8, NtPDS, and NtPDR6 genes, with biallelic mutation efficiencies of 16.7%, 36.4%, and 6.25%, respectively (Gao, et al, 2015,2018). In the aspect of tobacco polygene editing, Jansing et al (Jansing et al, 2018) target 4 nicotiana benthamiana a-1, 3-fuse genes to obtain editing efficiency of 10% -14%, and target beta-1, 2-xylose to obtain editing efficiency of 62% -67%. Xixiaoeng et al (2019) use 5u 6-26 promoters to serially knock out 5 genes simultaneously, the single gene cutting efficiency is 76.9-92.3%, and the simultaneous editing efficiency is only 53.8%, but the homozygous/biallelic editing efficiency is not detected.

The traditional gene editing vector realizes about 10-85% of haploid gene editing efficiency, but homozygous or biallelic editing is only 6.3-36.4%. Meanwhile, due to the redundancy of gene functions, for polyploid plants, it is often necessary to edit a homologous gene and a gene family simultaneously to obtain a desired developmental phenotype, so that there is a defect of low editing efficiency of polyploid plants.

Disclosure of Invention

It is an object of the present disclosure to improve polyploid plant editing efficiency.

The inventors of the present invention have unexpectedly found that: in the same vector, the 35Spro promoter is used for driving gRNA, and the calreticulin promoter element is used for driving spCas9 protein, so that the editing efficiency of polyploid plants can be greatly improved, and the invention is obtained.

Therefore, in order to achieve the above objects, the present disclosure provides in a first aspect a vector capable of performing highly efficient gene editing in tobacco, the vector having inserted therein, in order, a 35Spro promoter, a gRNA insertion region, an eu terminator, a calreticulin promoter element, a spCas9 protein expression element, a ubp terminator, and a hygromycin selection marker, the calreticulin promoter element having a nucleotide sequence of SEQ ID No. 1.

Preferably, the nucleotide sequence of the 35Spro promoter is SEQ ID NO. 10; the nucleotide sequence of the gRNA insertion region is SEQ ID NO. 11; the nucleotide sequence of the eu terminator is SEQ ID NO. 12; the nucleotide sequence of the spCas9 protein expression element is SEQ ID NO. 13; the nucleotide sequence of the ubp terminator is SEQ ID NO. 14; the nucleotide sequence of the hygromycin screening marker is SEQ ID NO. 15.

Preferably, at least 2 tRNA cleavage sequences are inserted into the gRNA insertion region; the nucleotide sequence of the tRNA cutting sequence is SEQ ID NO. 8; the spCas9 protein expression element has an NLS element and a PA50 element.

Preferably, the nucleotide sequence of the vector is SEQ ID NO. 9.

In a second aspect, the present disclosure provides a method for efficient gene editing in tobacco, the method comprising the steps of:

(1) inserting gRNA of a target gene to be edited into a gRNA insertion region of the vector to obtain a gene editing vector;

(2) transforming agrobacterium with the gene editing vector to obtain a transformant;

(3) infecting the pre-cultured tobacco leaves with the transformant to obtain infected tobacco leaves;

(4) and culturing the infected tobacco leaves in a culture medium containing hygromycin.

Preferably, the nucleotide sequence of the gRNA targeting the gene to be edited is SEQ ID No.4, 5, 6 or 7.

Preferably, the nucleotide sequence of the gene editing vector is obtained by replacing positions 9754-9769 of SEQ ID NO.9 with SEQ ID NO.2, or is obtained by replacing positions 9754-9769 of SEQ ID NO.9 with SEQ ID NO. 3.

In a third aspect of the present disclosure, there is provided a calreticulin promoter element having the nucleotide sequence of SEQ ID No. 1.

The fourth aspect of the present disclosure provides a use of a calreticulin promoter element in constructing a gene editing vector, wherein a nucleotide sequence of the calreticulin promoter element is SEQ ID No. 1.

A fifth aspect of the present disclosure provides a gRNA targeting a gene to be edited, the nucleotide sequence of the gRNA targeting the gene to be edited being SEQ ID No.4, 5, 6, or 7.

By the technical scheme, the gene editing efficiency can be remarkably improved, the overall editing efficiency of the MAX4 gene can reach 100%, the homozygous/biallelic ratio reaches 85.7%, and the efficiency is improved by 22.2% and 69% respectively compared with the efficiency reported in the literature.

Additional features and advantages of the disclosure will be set forth in the detailed description which follows.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure without limiting the disclosure. In the drawings:

FIG. 1 shows the tobacco PDS gene editing vector and transformation regeneration.

A is a vector skeleton schematic diagram of pDC45 and pDC45_ PDSsg; b is a schematic diagram of the locus of the NtPDS gene; c is albinism of 18-day and 30-day regenerated plantlets.

FIG. 2 is a tobacco multi-site editing and transformation regeneration phenotype analysis.

A and B are schematic diagrams of NtBARC 1 and NtMAX4 genes and gRNA sites; c, comparing the plant types of the edited seedling stage and the non-edited seedling stage; d, removing the regenerated seedlings of the leaves.

Sequence Listing information

SEQ ID NO. 1: nucleotide sequence of calreticulin promoter PCE8pro968 element.

SEQ ID NO.2 or 3: the nucleotide sequence of the gene editing vector is obtained by replacing 9754 th to 9769 th positions of SEQ ID NO.9 with SEQ ID NO.2, or is obtained by replacing 9754 th to 9769 th positions of SEQ ID NO.9 with SEQ ID NO. 3.

SEQ ID No.4, 5, 6 or 7: the nucleotide sequence of the gRNA that targets the gene to be edited.

SEQ ID NO. 8: the tRNA cleaves the nucleotide sequence of the sequence.

SEQ ID NO. 9: nucleotide sequence of the vector without inserted gRNA coding sequence.

SEQ ID NO. 10: 35Spro promoter.

SEQ ID NO. 11: nucleotide sequence of gRNA insertion region.

SEQ ID NO. 12: nucleotide sequence of eu terminator.

SEQ ID NO. 13: nucleotide sequence of spCas9 protein expression element.

SEQ ID No. 14: ubp nucleotide sequence of terminator.

SEQ ID NO. 15: hygromycin selection marker nucleotide sequence.

SEQ ID No. 16: NLS element nucleotide sequence.

SEQ ID NO. 17: PA50 element nucleotide sequence.

Detailed Description

The following detailed description of specific embodiments of the present disclosure is provided in connection with the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.

The first aspect of the disclosure provides a vector capable of performing efficient gene editing in tobacco, wherein a 35Spro promoter, a gRNA insertion region, an eu terminator, a calreticulin promoter element, a spCas9 protein expression element, a ubp terminator and a hygromycin screening marker are sequentially inserted into the vector, and the nucleotide sequence of the calreticulin promoter element is SEQ ID No. 1.

In the invention, the calreticulin promoter element shown in SEQ ID No.1 is from the genome of Nicotiana tabacum, and the specific source is an Nsyl _ KD957634.1 segment.

Wherein the 35Spro promoter functions to initiate transcription of the gRNA coding sequence inserted downstream thereof, preferably having the nucleotide sequence of SEQ ID No. 10. Wherein, the gRNA insertion region is used for inserting a gRNA coding sequence which contains an enzyme cutting site sequence, and preferably, the nucleotide sequence of the gRNA insertion region is SEQ ID NO. 11. The eu terminator is used to terminate transcription of the gRNA coding sequence, preferably, its nucleotide sequence is SEQ ID No. 12. The spCas9 protein expression element is used to encode a spCas9 protein, preferably, the nucleotide sequence of which is SEQ ID No. 13. The ubp terminator is used to terminate transcription of the coding sequence of spCas9 protein, preferably, its nucleotide sequence is SEQ ID No. 14. The hygromycin screening marker is used for screening transformants and invaders, and preferably, the nucleotide sequence of the hygromycin screening marker is SEQ ID NO. 15.

According to the present disclosure, preferably, at least 2 tRNA cleavage sequences are inserted into the gRNA insertion region, for example, 1 tRNA cleavage sequence at each end of the gRNA insertion region, so that the transcribed RNA molecule containing the gRNA can be cleaved from the gRNA by a nuclease in a cell; the nucleotide sequence of the tRNA cutting sequence is SEQ ID NO. 8; the spCas9 protein expression element is provided with an NLS element and a PA50 element, the NLS element has the function of interacting with an introduced vector and has the sequence of SEQ ID NO.16, and the PA50 element has the function of enhancing the stability of Cas9 in plants and has the sequence of SEQ ID NO. 17.

According to the present disclosure, it is particularly preferred that the nucleotide sequence of the vector is SEQ ID No. 9. The vector shown in SEQ ID NO.9, named pDC45 vector, after being cut by SpeI-HF and SbfI-HF enzymes, can be ligated with gRNA coding sequence cut by SpeI-HF and SbfI-HF enzymes to form a vector capable of efficient gene editing in tobacco, i.e., binary vector pDC-2x35S-PTG-EU-NtPCE8pro: Cas 9-A50.

In a second aspect, the present disclosure provides a method for efficient gene editing in tobacco, the method comprising the steps of: (1) inserting a gRNA coding sequence of a targeted gene to be edited into a gRNA insertion region of the vector to obtain a gene editing vector; (2) transforming agrobacterium with the gene editing vector to obtain a transformant; (3) infecting the pre-cultured tobacco leaves with the transformant to obtain infected tobacco leaves; (4) and culturing the infected tobacco leaves in a culture medium containing hygromycin.

According to the disclosure, the nucleotide sequence of the gRNA targeting the gene to be edited is SEQ ID No.4, 5, 6 or 7. The gRNA shown in SEQ ID No.4 is the gRNA of the PDS gene of the targeted forest tobacco. The gRNA shown in SEQ ID No.5 is a gRNA of a PDS gene of targeted villous tobacco. The gRNA shown in SEQ ID No.6 is a gRNA of the NtMAX4 gene of the targeted forest tobacco. The gRNA shown in SEQ ID No.7 is a gRNA targeting the NtBARC 1 gene of villous tobacco.

According to the disclosure, the nucleotide sequence of the gene editing vector is obtained by replacing positions 9754-9769 of SEQ ID NO.9 with SEQ ID NO.2, or is obtained by replacing positions 9754-9769 of SEQ ID NO.9 with SEQ ID NO. 3.

In a third aspect of the present disclosure, there is provided a calreticulin promoter element having the nucleotide sequence of SEQ ID No. 1.

The fourth aspect of the present disclosure provides a use of a calreticulin promoter element in constructing a gene editing vector, wherein a nucleotide sequence of the calreticulin promoter element is SEQ ID No. 1.

A fifth aspect of the present disclosure provides a gRNA targeting a gene to be edited, the nucleotide sequence of the gRNA targeting the gene to be edited being SEQ ID No.4, 5, 6, or 7.

Example 1

This example illustrates the construction of the Gene editing vector backbone pDC01

The pDC01 binary expression vector was constructed with pCAMBIA1300 with BsaI and NheI sites removed as a backbone vector, and contained U6-sgRNA, Arabidopsis UBQ promoter, hspCas9, UBQ terminator (Zhang et. al,2016) and PolyA tail (50 bp).

The original pCAMBIA1300 vector (purchased from vast Ling plasmid platform, cat # P0439) was subjected to site-directed mutagenesis to remove BsaI site (GGCTCC changed to GGCTCg) and NheI site (GCTAGC-GCaAGC) from the vector backbone, wherein the obtained plasmid P1300 was double-digested with the restriction enzymes HindIII-HF and EcoRI-HF of NEB, the product after double-digestion and U6-sNA insert were recovered according to the standard procedure of gel recovery Kit (all-type gold), the recovered fragments were ligated with homologous recombination Kit (ClonEx II One Step Cloning Kit, Norzaar, C112-01), and the correctly sequenced plasmid, designated P1300-U6_ sg, was stored at-20 ℃ for subsequent experiments.

In order to increase the stability of Cas9 in plants, 18T-U6-sg-Cas9 vector in the article "A multiplex CRISPR/Cas9 platform for fast and effective identification of multiple genes in Arabidopsis" published in zhang et al,2016 is used as a template to amplify a Cas9-PA50 fragment, a forward primer Cas9-Nco-F sequence and a reverse primer Cas9-PA50-R sequence.

The 2ug 18T-U6-sg-Cas9 vector was digested simultaneously with restriction enzymes NcoI-HF and BamHI-HF, the product after digestion and the Cas9-PA50 fragment were recovered according to the standard procedures of gel recovery kit (all-purpose gold), the product after recovery was ligated with NEB T4 ligase (M0202S), and the ligation product was transformed into DH5 a. Positive clones were sequenced using M13R and the plasmid containing the intact polyA was selected and named 18T-U6-sg-Cas9-PA50 and stored at-20 ℃ for further experiments.

The method comprises the following steps of carrying out double enzyme digestion on 2ug p1300-U6-sg and 2ug 18T-U6-sg-Cas9-PA50 by XmaI and EcoRI-HF respectively, digging gel to recover an upper band, carrying out connection by using T4 ligase, carrying out transformation, and carrying out clone identification, wherein the specific operation is as above. The final sequencing identified plasmid was the pDC01 vector as a subsequent backbone vector.

Example 2

This example demonstrates the construction of a NtPCE8 promoter-driven Gene editing vector

And carrying out XmaI and NcoI-HF double enzyme digestion and gel digging recovery on the pDC01 vector to obtain a linear framework.

Designing NtPCE8 promoter derived from Nsyl _ KD957634.1 segment of forest tobacco genome and amplification primer (the primer direction is 5 ' -3 ') of CDS near 5 ' end sequence, using NtPCE8 promoter forward primer as NtPCE8pro-F and NtPCE8pro-R, making amplicon undergo the process of agarose electrophoresis detection, connecting PCR purified product and connecting them

A Blunt Zero Cloning Kit vector (gold full, CB501), the ligation product is transformed into Escherichia coli DH5a, and Cloning is picked and plasmid sequencing is carried out to obtain an NtPCE8 sequence with the length of 1046bp, wherein the promoter is a promoter sequence with the length of 968bp (PCE8pro968, SEQ ID NO. 1).

A promoter PCE8pro968(SEQ ID NO.1) is connected with a linearized pDC01 vector by using a homologous recombination Kit (Cloneexpress II One Step Cloning Kit, Novozan), and the pDC-NtPCE8pro:: Cas9-A50 is finally obtained through sequencing and identification.

Example 3

This example demonstrates the use of calreticulin promoter-driven gene editing vectors in plants

In order to test the editing activity of the editing vector constructed above in plants, heterotetraploid common tobacco was selected as an editing receptor, and phytoene dehydrogenase gene (NtPDS) was selected as a target gene.

1. Target gene gRNA design

Design 20bp gRNA sequence PDS-sg 5: GAGGCAAGAGATGTCCTAGG (SEQ ID NO.5), can simultaneously target 2 copies of PDS from both Lin tobacco and Villus tobacco.

2. Editing vector construction of target PDS gene

In order to obtain the gRNA sequence of the double-stranded PDS, 5ul 10uM PDS-sg5 forward primer PDS-sg 5-sense and reverse primer PDS-sg5-R-anti were added with TE buffer, mixed well and placed in a PCR instrument, heated at 98 ℃ for 4min, and then the PCR tube was directly taken out and cooled to room temperature to obtain the double-stranded DNA of the target site of the sticky end.

pDC-NtPCE8pro was linearized with BsaI Cas9-A50 vector, the linearized vector was T4 ligated to a double-stranded PDS-gRNA (PDS-sg5), the ligation product was transformed into DH5a and screened on LB plates supplemented with kanamycin. Performing colony PCR identification by using a primer 1300-gRNA-F2 and pds-sg5-R-anti, extracting plasmid DNA by positive cloning, transforming agrobacterium tumefaciens EH105 by a binary vector with correct sequencing, and storing at-80 ℃ for later use; placing the pre-cultured leaf disc into an agrobacterium infection solution for infection, wherein the infection time is 5min, and intermittently and gently shaking; culturing the infected tobacco leaves in a hygromycin-containing culture medium for 3 generations to obtain robust seedlings; removing the bottom expanded part and the lower yellow leaves of the robust plantlets, inoculating the robust plantlets into an MS culture medium containing 250mg/L of thienamomycin, and culturing for 1-2 weeks by light to promote the plantlets to root as soon as possible; when the plantlets are rooted for 5-10 and grow for 2-3cm, the cover of the culture bottle is opened, and the plantlets are hardened for 3 days. Transplanting the seedlings into a nutrition pot filled with sterile soil. After being moisturized for 2 days by a plastic film, the plastic film is placed in a greenhouse, and after about one week, the preservative film can be removed according to the growth condition of the seedlings, so that the seedlings can grow under the natural condition.

3. Editing detection analysis of plants

The leaves developed from rooted shoots were taken, genomic DNA of the edited plants was extracted with a genome extraction kit (all-plant gold, EE111-12), and the editing of both PDS gene copies was examined using the editing detection primers PDS-g5-seq-F and PDS-g 5-seq-R. PCR product purification and connection after different T0 generation regeneration plants are amplified

Blunt Zero Cloning Kit Blunt-ended vector, after transformation of E.coli, 12 clones were picked each for sanger sequencing. The sequencing result is aligned to the reference sequence. The specific results are as follows:

TABLE 1 NtPCE8pro Cas9-A50 vector T0 generation transgenic tobacco mutation efficiency analysis

The result shows that the editing efficiency of the NtPCE8 promoter is 88.9%, and the mutation frequency of the NtPCE8 promoter reaches 37.5% in the aspect of producing homozygous or double-allelic plants. Thus, the NtPCE8pro:: Cas9-A50 vector was used as a binary vector for further experimental analysis.

Example 4

This example demonstrates the use of the construction of an efficient multigene editing vector

1. gRNA framework driven by II type promoter and binary vector construction

In order to construct the gRNA sequence driven by pol II type promoter, 2X35S promoter fragment was amplified by using pCHF3 plasmid as a template, 35Spro-SpeI-F and 35Spro-SalI-R as a template, and EU-F and EU-R as primers to obtain EU terminator (Rosenthal et al, 2018Plant Molecular Biology), and meanwhile, tRNA-BsaI-sg _ scanfold-tRNA sequence was synthesized by Huada and amplified 2X35Spro and EU sequence were used to construct pMV-2X35S-PTG-EU intermediate vector. The intermediate vector is used as a template, a forward primer 35S-CE-F and a reverse primer EU-CE-R are used for amplifying a 2x35S-PTG-EU sequence as an insert fragment, and a SpeI-HF and an SbfI-HF double enzyme digestion is carried out on a 2ug pDC-NtPCE8pro vector to obtain a linearized vector; 60ng of linearized vector and 50ng of 2X35S-PTG-EU insert were subjected to homologous recombination ligation and transformation. And finally sequencing the correct binary vector pDC-2x35S-PTG-EU-NtPCE8pro, wherein Cas9-A50 is named as pDC45, and the sequence is SEQ ID NO. 9.

2. Construction of Multi-target binary vector

To test the editing efficiency of the pDC45 vector at multiple sites, 2 grnas were tested targeting 2 genes (table 2, pDC45_ PDSsg) and 2 grnas targeting 4 genes (table 2, pDC45_ MAX4_ BRC1sg), and the corresponding binary vectors were constructed to transform nicotiana tabacum. The target information and corresponding primer information are as follows:

TABLE 2 Multi-target binary vector targets and primer information

The 2x35S-PTG-EU fragment is used as a template, and forward and reverse primers of a multi-target sequence are used for amplification to obtain PDS _ g4g5 and MAX4_ BRC1_ sg fragments.

After purification of the PCR product, a gold gate ligation was performed to obtain a ligation product. The ligation product was transformed into E.coli. Colony PCR was performed using 35S-PCR-F and EU-50R. Selecting bacterial colony with band about 570bp for sanger sequencing, converting agrobacterium EH105 after the result is compared correctly, and then introducing the vector into common tobacco Honghua Dajinyuan through agrobacterium-mediated genetic transformation to obtain stable transgenic plant.

In order to visually display the editing efficiency of a vector system, a gRNA capable of simultaneously targeting two copies of PDS is designed to construct a pDC45_ PDSsg editing vector, and albino seedlings generated by infecting explants with the pDC45_ PDSsg vector at 18 days and 30 days are recorded. At 18 days 2-6 fully albino shoots were obtained per explant, after 30 days 1-4 albino regenerated shoots were produced per explant. Finally, 32 completely albino plants, 3 spotted albino (mosaic) plants and 2 wild type (green) plants are obtained. Sequencing of these regenerated plants revealed an NtPDS gene editing efficiency of 94.6% (35/37), with a homozygous/biallelic editing efficiency of 86.5% (32/37).

To further test the editing efficiency of this system, 2 targets were designed, targeting 4 genes of tobacco simultaneously (table 3). Randomly selecting 14T 0 generation regenerated seedlings on a rooting culture medium, extracting genome DNA, and utilizing high-throughput sequencing primers MAX4-hitom-F6 and MAX 4-hitom-R6; primers BRC1-hitom-F4 and BRC1-hitom-R4 analyze gene editing conditions of NtMAX4 and NtBARC 1 in regenerated seedlings of different strains through a hi-tom method. The results show that: the efficiency of simultaneous editing of four genes in the tested strain was 100%, wherein the percentage of NtMAX4-S and NtMAX4-T homozygous or biallelic was 92.9% and 85.7%, and the percentage of NtBARC 1-S and NtBARC 1-T homozygous or biallelic was 78.6% and 42.9%. The percentage of simultaneous editing of NtMAX4-S and-T reaches 85.7%. The number of plants producing both homozygous and biallelic editing for the four genes was 3, with a percentage of 21.4%.

TABLE 3 high throughput sequencing analysis of the editing efficiency of NtMAX4 and NtBARC 1 sites

Analysis of the NtBARC 1-T chimeric line revealed that all chimeric plants also exhibited edits, 3-6 edit types appeared in the chimeric line, and no unedited wild type was found. Phenotypic analysis of knockout lines showed that homozygous editing produced a multi-branched phenotype compared to non-edited regenerated shoots.

The results show that the pDC45 vector system can significantly improve the gene editing efficiency of tobacco, greatly improve the editing efficiency of multi-target, homozygous/biallelic genes, and lay a foundation for creating plant whole genome editing strains in high throughput.

The preferred embodiments of the present disclosure are described in detail with reference to the accompanying drawings, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

Sequence listing

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cttcaaagca agtggattga tgtgataaca tggtggagca cgacactctc gtctactcca 600

agaatatcaa agatacagtc tcagaagacc aaagggctat tgagactttt caacaaaggg 660

taatatcggg aaacctcctc ggattccatt gcccagctat ctgtcacttc atcaaaagga 720

cagtagaaaa ggaaggtggc acctacaaat gccatcattg cgataaagga aaggctatcg 780

ttcaagatgc ctctgccgac agtggtccca aagatggacc cccacccacg aggagcatcg 840

tggaaaaaga agacgttcca accacgtctt caaagcaagt ggattgatgt gatatctcca 900

ctgacgtaag ggatgacgca caatcccact atccttcgca agaccttcct ctatataagg 960

aagttcattt catttggaga ggacacgctg aaatcaccag tctctctcta caaatctatc 1020

tctctcgagc tttcgcagat cccggagggc aatgagatat gaaaaagcct gaactcaccg 1080

cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc 1140

tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc 1200

tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg 1260

catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggagtttagc gagagcctga 1320

cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac 1380

tgcccgctgt tctacaaccg gtcgcggagg ctatggatgc gatcgctgcg gccgatctta 1440

gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc 1500

gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg 1560

acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact 1620

gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca 1680

atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg 1740

aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct 1800

acttcgagcg gaggcatccg gagcttgcag gatcgccacg actccgggcg tatatgctcc 1860

gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt 1920

gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac 1980

aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata 2040

gtggaaaccg acgccccagc actcgtccga gggcaaagaa atagagtaga tgccgaccgg 2100

atctgtcgat cgacaagctc gagtttctcc ataataatgt gtgagtagtt cccagataag 2160

ggaattaggg ttcctatagg gtttcgctca tgtgttgagc atataagaaa cccttagtat 2220

gtatttgtat ttgtaaaata cttctatcaa taaaatttct aattcctaaa accaaaatcc 2280

agtactaaaa tccagatccc ccgaattaat tcggcgttaa ttcagtacat taaaaacgtc 2340

cgcaatgtgt tattaagttg tctaagcgtc aatttgttta caccacaata tatcctgcca 2400

ccagccagcc aacagctccc cgaccggcag ctcggcacaa aatcaccact cgatacaggc 2460

agcccatcag tccgggacgg cgtcagcggg agagccgttg taaggcggca gactttgctc 2520

atgttaccga tgctattcgg aagaacggca actaagctgc cgggtttgaa acacggatga 2580

tctcgcggag ggtagcatgt tgattgtaac gatgacagag cgttgctgcc tgtgatcacc 2640

gcggtttcaa aatcggctcc gtcgatacta tgttatacgc caactttgaa aacaactttg 2700

aaaaagctgt tttctggtat ttaaggtttt agaatgcaag gaacagtgaa ttggagttcg 2760

tcttgttata attagcttct tggggtatct ttaaatactg tagaaaagag gaaggaaata 2820

ataaatggct aaaatgagaa tatcaccgga attgaaaaaa ctgatcgaaa aataccgctg 2880

cgtaaaagat acggaaggaa tgtctcctgc taaggtatat aagctggtgg gagaaaatga 2940

aaacctatat ttaaaaatga cggacagccg gtataaaggg accacctatg atgtggaacg 3000

ggaaaaggac atgatgctat ggctggaagg aaagctgcct gttccaaagg tcctgcactt 3060

tgaacggcat gatggctgga gcaatctgct catgagtgag gccgatggcg tcctttgctc 3120

ggaagagtat gaagatgaac aaagccctga aaagattatc gagctgtatg cggagtgcat 3180

caggctcttt cactccatcg acatatcgga ttgtccctat acgaatagct tagacagccg 3240

cttagccgaa ttggattact tactgaataa cgatctggcc gatgtggatt gcgaaaactg 3300

ggaagaagac actccattta aagatccgcg cgagctgtat gattttttaa agacggaaaa 3360

gcccgaagag gaacttgtct tttcccacgg cgacctggga gacagcaaca tctttgtgaa 3420

agatggcaaa gtaagtggct ttattgatct tgggagaagc ggcagggcgg acaagtggta 3480

tgacattgcc ttctgcgtcc ggtcgatcag ggaggatatc ggggaagaac agtatgtcga 3540

gctatttttt gacttactgg ggatcaagcc tgattgggag aaaataaaat attatatttt 3600

actggatgaa ttgttttagt acctagaatg catgaccaaa atcccttaac gtgagttttc 3660

gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 3720

tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 3780

gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 3840

accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 3900

accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 3960

gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 4020

ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 4080

atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 4140

gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 4200

cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 4260

gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 4320

gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 4380

tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 4440

cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct 4500

tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga 4560

tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg 4620

ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc 4680

gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca 4740

tcaccgaaac gcgcgaggca gggtgccttg atgtgggcgc cggcggtcga gtggcgacgg 4800

cgcggcttgt ccgcgccctg gtagattgcc tggccgtagg ccagccattt ttgagcggcc 4860

agcggccgcg ataggccgac gcgaagcggc ggggcgtagg gagcgcagcg accgaagggt 4920

aggcgctttt tgcagctctt cggctgtgcg ctggccagac agttatgcac aggccaggcg 4980

ggttttaaga gttttaataa gttttaaaga gttttaggcg gaaaaatcgc cttttttctc 5040

ttttatatca gtcacttaca tgtgtgaccg gttcccaatg tacggctttg ggttcccaat 5100

gtacgggttc cggttcccaa tgtacggctt tgggttccca atgtacgtgc tatccacagg 5160

aaacagacct tttcgacctt tttcccctgc tagggcaatt tgccctagca tctgctccgt 5220

acattaggaa ccggcggatg cttcgccctc gatcaggttg cggtagcgca tgactaggat 5280

cgggccagcc tgccccgcct cctccttcaa atcgtactcc ggcaggtcat ttgacccgat 5340

cagcttgcgc acggtgaaac agaacttctt gaactctccg gcgctgccac tgcgttcgta 5400

gatcgtcttg aacaaccatc tggcttctgc cttgcctgcg gcgcggcgtg ccaggcggta 5460

gagaaaacgg ccgatgccgg gatcgatcaa aaagtaatcg gggtgaaccg tcagcacgtc 5520

cgggttcttg ccttctgtga tctcgcggta catccaatca gcaagctcga tctcgatgta 5580

ctccggccgc ccggtttcgc tctttacgat cttgtagcgg ctaatcaagg cttcaccctc 5640

ggataccgtc accaggcggc cgttcttggc cttcttcgta cgctgcatgg caacgtgcgt 5700

ggtgtttaac cgaatgcagg tttctaccag gtcgtctttc tgctttccgc catcggctcg 5760

ccggcagaac ttgagtacgt ccgcaacgtg tggacggaac acgcggccgg gcttgtctcc 5820

cttcccttcc cggtatcggt tcatggattc ggttagatgg gaaaccgcca tcagtaccag 5880

gtcgtaatcc cacacactgg ccatgccggc cggccctgcg gaaacctcta cgtgcccgtc 5940

tggaagctcg tagcggatca cctcgccagc tcgtcggtca cgcttcgaca gacggaaaac 6000

ggccacgtcc atgatgctgc gactatcgcg ggtgcccacg tcatagagca tcggaacgaa 6060

aaaatctggt tgctcgtcgc ccttgggcgg cttcctaatc gacggcgcac cggctgccgg 6120

cggttgccgg gattctttgc ggattcgatc agcggccgct tgccacgatt caccggggcg 6180

tgcttctgcc tcgatgcgtt gccgctgggc ggcctgcgcg gccttcaact tctccaccag 6240

gtcatcaccc agcgccgcgc cgatttgtac cgggccggat ggtttgcgac cgctcacgcc 6300

gattcctcgg gcttgggggt tccagtgcca ttgcagggcc ggcagacaac ccagccgctt 6360

acgcctggcc aaccgcccgt tcctccacac atggggcatt ccacggcgtc ggtgcctggt 6420

tgttcttgat tttccatgcc gcctccttta gccgctaaaa ttcatctact catttattca 6480

tttgctcatt tactctggta gctgcgcgat gtattcagat agcagctcgg taatggtctt 6540

gccttggcgt accgcgtaca tcttcagctt ggtgtgatcc tccgccggca actgaaagtt 6600

gacccgcttc atggctggcg tgtctgccag gctggccaac gttgcagcct tgctgctgcg 6660

tgcgctcgga cggccggcac ttagcgtgtt tgtgcttttg ctcattttct ctttacctca 6720

ttaactcaaa tgagttttga tttaatttca gcggccagcg cctggacctc gcgggcagcg 6780

tcgccctcgg gttctgattc aagaacggtt gtgccggcgg cggcagtgcc tgggtagctc 6840

acgcgctgcg tgatacggga ctcaagaatg ggcagctcgt acccggccag cgcctcggca 6900

acctcaccgc cgatgcgcgt gcctttgatc gcccgcgaca cgacaaaggc cgcttgtagc 6960

cttccatccg tgacctcaat gcgctgctta accagctcca ccaggtcggc ggtggcccat 7020

atgtcgtaag ggcttggctg caccggaatc agcacgaagt cggctgcctt gatcgcggac 7080

acagccaagt ccgccgcctg gggcgctccg tcgatcacta cgaagtcgcg ccggccgatg 7140

gccttcacgt cgcggtcaat cgtcgggcgg tcgatgccga caacggttag cggttgatct 7200

tcccgcacgg ccgcccaatc gcgggcactg ccctggggat cggaatcgac taacagaaca 7260

tcggccccgg cgagttgcag ggcgcgggct agatgggttg cgatggtcgt cttgcctgac 7320

ccgcctttct ggttaagtac agcgataacc ttcatgcgtt ccccttgcgt atttgtttat 7380

ttactcatcg catcatatac gcagcgaccg catgacgcaa gctgttttac tcaaatacac 7440

atcacctttt tagacggcgg cgctcggttt cttcagcggc caagctggcc ggccaggccg 7500

ccagcttggc atcagacaaa ccggccagga tttcatgcag ccgcacggtt gagacgtgcg 7560

cgggcggctc gaacacgtac ccggccgcga tcatctccgc ctcgatctct tcggtaatga 7620

aaaacggttc gtcctggccg tcctggtgcg gtttcatgct tgttcctctt ggcgttcatt 7680

ctcggcggcc gccagggcgt cggcctcggt caatgcgtcc tcacggaagg caccgcgccg 7740

cctggcctcg gtgggcgtca cttcctcgct gcgctcaagt gcgcggtaca gggtcgagcg 7800

atgcacgcca agcagtgcag ccgcctcttt cacggtgcgg ccttcctggt cgatcagctc 7860

gcgggcgtgc gcgatctgtg ccggggtgag ggtagggcgg gggccaaact tcacgcctcg 7920

ggccttggcg gcctcgcgcc cgctccgggt gcggtcgatg attagggaac gctcgaactc 7980

ggcaatgccg gcgaacacgg tcaacaccat gcggccggcc ggcgtggtgg tgtcggccca 8040

cggctctgcc aggctacgca ggcccgcgcc ggcctcctgg atgcgctcgg caatgtccag 8100

taggtcgcgg gtgctgcggg ccaggcggtc tagcctggtc actgtcacaa cgtcgccagg 8160

gcgtaggtgg tcaagcatcc tggccagctc cgggcggtcg cgcctggtgc cggtgatctt 8220

ctcggaaaac agcttggtgc agccggccgc gtgcagttcg gcccgttggt tggtcaagtc 8280

ctggtcgtcg gtgctgacgc gggcatagcc cagcaggcca gcggcggcgc tcttgttcat 8340

ggcgtaatgt ctccggttct agtcgcaagt attctacttt atgcgactaa aacacgcgac 8400

aagaaaacgc caggaaaagg gcagggcggc agcctgtcgc gtaacttagg acttgtgcga 8460

catgtcgttt tcagaagacg gctgcactga acgtcagaag ccgactgcac tatagcagcg 8520

gaggggttgg atcaaagtac tttgatcccg aggggaaccc tgtggttggc atgcacatac 8580

aaatggacga acggataaac cttttcacgc ccttttaaat atccgttatt ctaataaacg 8640

ctcttttctc ttaggtttac ccgccaatat atcctgtcaa acactgatag tttaaactga 8700

aggcgggaaa cgacaatctg atccaagctc aagctgctct agcattcgcc attcaggctg 8760

cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa 8820

gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt 8880

tgtaaaacga cggccagtgc caagcttact agttacggag gtcaaacatg gtggagcacg 8940

acacacttgt ctactccaaa aatatcaaag atacagtctc agaagaccaa agggcaattg 9000

agacttttca acaaagggta atatccggaa acctcctcgg attccattgc ccagctatct 9060

gtcactttat tgtgaagata gtggaaaagg aaggtggctc ctacaaatgc catcattgcg 9120

ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag tggtcccaaa gatggacccc 9180

cacccacgag gagcatcgtg gaaaaagaag acgttccaac cacgtcttca aagcaagtgg 9240

attgatgtga taacatggtg gagcacgaca cacttgtcta ctccaaaaat atcaaagata 9300

cagtctcaga agaccaaagg gcaattgaga cttttcaaca aagggtaata tccggaaacc 9360

tcctcggatt ccattgccca gctatctgtc actttattgt gaagatagtg gaaaaggaag 9420

gtggctccta caaatgccat cattgcgata aaggaaaggc catcgttgaa gatgcctctg 9480

ccgacagtgg tcccaaagat ggacccccac ccacgaggag catcgtggaa aaagaagacg 9540

ttccaaccac gtcttcaaag caagtggatt gatgtgatat ctccactgac gtaagggatg 9600

acgcacaatc ccactatcct tcgcaagacc cttcctctat ataaggaagt tcatttcatt 9660

tggagaggac gtcgacaaca aagcaccagt ggtctagtgg tagaatagta ccctgccacg 9720

gtacagaccc gggttcgatt cccggctggt gcaagagacc gaggtctcgg ttttagagct 9780

agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc 9840

ggtgcaacaa agcaccagtg gtctagtggt agaatagtac cctgccacgg tacagacccg 9900

ggttcgattc ccggctggtg cagtcgacaa agcagaatgc tgagctaaaa gaaaggcttt 9960

ttccattttc gagagacaat gagaaaagaa gaagaagaag aagaagaaga agaagaagaa 10020

aagagtaaat aataaagccc cacaggaggc gaagttcttg tagctccatg ttatctaagt 10080

tattgatatt gtttgcccta tattttattt ctgtcattgt gtatgttttg ttcagtttcg 10140

atctccttgc aaaatgcaga gattatgaga tgaataaact aagttatatt attatacgtg 10200

ttaatattct cctcctctct ctagctagcc ttttgttttc tctttttctt atttgatttt 10260

ctttaaatca atccatttta ggagagggcc agggagtgat ccagcaaaac atgaagatta 10320

gaagaaactt ccctcttttt tttcctgaaa acaatttaac gtcgagattt atctcttttt 10380

gtaatggaat catttctaca gttatgaccc tgcaggcccg ggaatagatt cattgtttac 10440

ttttattatg tatacactat ataattatta tacatgtact atacttattt tatacctcca 10500

aaagttattt ttaattgaag aggttgggtt gacaactatt taggttaatt gactttttag 10560

gaattcaccc ttttttagct tcaatctcaa aaggtaaata gaaaaacaga aagaataaaa 10620

tcattttgat tgtacaagtg ctaattattc ttttagtaaa caataaattc taagattaat 10680

agaatcgtta tatgcacgcc attgtatgcg aaattcatgt gaaagaaaaa agagagcaaa 10740

aaaccaatgg ctaaattcgt tattttgaaa aaaccatcgc acaagtgcca acttaattgt 10800

ggtcactttc aactcgtgga gatacccaac aagcttcagc cacgtcaagt caatgacact 10860

ctaattacaa atgcctcttt ttaattcgtc cacgtcatcg aacgtagaca cgtcggacta 10920

agaccaaacg ctgccctacg tatctaccaa aatcctctcg ccagtataaa agagcattca 10980

tcagagatct tttagagtca gtatttcgat ctcacaacag tggccatgga ctataaggac 11040

cacgacggag actacaagga tcatgatatt gattacaaag acgatgacga taagatggcc 11100

ccaaagaaga agcggaaggt cggtatccac ggagtcccag cagccgacaa gaagtacagc 11160

atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag 11220

gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac 11280

ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga 11340

accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc 11400

agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg 11460

gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg 11520

gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc 11580

gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc 11640

cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc 11700

cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg 11760

gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc 11820

gcccagctgc ccggcgagaa gaagaatggc ctgttcggca acctgattgc cctgagcctg 11880

ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg 11940

agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac 12000

gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg 12060

agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac 12120

gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag 12180

tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga 12240

gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc 12300

gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac 12360

aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag 12420

gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc 12480

cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc 12540

agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct 12600

tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag 12660

gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa 12720

gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag 12780

gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag 12840

gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg 12900

ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc 12960

ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt 13020

gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa 13080

gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg 13140

atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac 13200

ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag 13260

gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat 13320

ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag 13380

ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag 13440

aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag 13500

ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg 13560

cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag 13620

gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt 13680

ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag 13740

agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg 13800

ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc 13860

ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag 13920

atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat 13980

gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc 14040

cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac 14100

gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc 14160

gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag 14220

caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc 14280

aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac 14340

ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg 14400

ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc 14460

aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg 14520

gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg 14580

gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc 14640

accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc 14700

tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg 14760

gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg 14820

gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag 14880

ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg 14940

gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat 15000

ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc 15060

gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac 15120

tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc 15180

ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga 15240

ggcgacaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagtaagga 15300

tcaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaggatccag 15360

agactcttat caagaatccc atctcttgct tgcttttttt tgttgcttcc ctttgatagg 15420

gtttgttttt cttgtttcag tgactttcta tgttaaaaga taatgtcagt aaaaggattt 15480

ggttttctat tattctgaat cgattacgga agattcttgc ttaattccaa tctatacaag 15540

tatcgtgaaa taatgaccgt ttatgtggta ccgagctcga attc 15584

<210> 10

<211> 757

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tacggaggtc aaacatggtg gagcacgaca cacttgtcta ctccaaaaat atcaaagata 60

cagtctcaga agaccaaagg gcaattgaga cttttcaaca aagggtaata tccggaaacc 120

tcctcggatt ccattgccca gctatctgtc actttattgt gaagatagtg gaaaaggaag 180

gtggctccta caaatgccat cattgcgata aaggaaaggc catcgttgaa gatgcctctg 240

ccgacagtgg tcccaaagat ggacccccac ccacgaggag catcgtggaa aaagaagacg 300

ttccaaccac gtcttcaaag caagtggatt gatgtgataa catggtggag cacgacacac 360

ttgtctactc caaaaatatc aaagatacag tctcagaaga ccaaagggca attgagactt 420

ttcaacaaag ggtaatatcc ggaaacctcc tcggattcca ttgcccagct atctgtcact 480

ttattgtgaa gatagtggaa aaggaaggtg gctcctacaa atgccatcat tgcgataaag 540

gaaaggccat cgttgaagat gcctctgccg acagtggtcc caaagatgga cccccaccca 600

cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat 660

gtgatatctc cactgacgta agggatgacg cacaatccca ctatccttcg caagaccctt 720

cctctatata aggaagttca tttcatttgg agaggac 757

<210> 11

<211> 98

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ggtgcaagag accgaggtct cggttttaga gctagaaata gcaagttaaa ataaggctag 60

tccgttatca acttgaaaaa gtggcaccga gtcggtgc 98

<210> 12

<211> 479

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

aagcagaatg ctgagctaaa agaaaggctt tttccatttt cgagagacaa tgagaaaaga 60

agaagaagaa gaagaagaag aagaagaaga aaagagtaaa taataaagcc ccacaggagg 120

cgaagttctt gtagctccat gttatctaag ttattgatat tgtttgccct atattttatt 180

tctgtcattg tgtatgtttt gttcagtttc gatctccttg caaaatgcag agattatgag 240

atgaataaac taagttatat tattatacgt gttaatattc tcctcctctc tctagctagc 300

cttttgtttt ctctttttct tatttgattt tctttaaatc aatccatttt aggagagggc 360

cagggagtga tccagcaaaa catgaagatt agaagaaact tccctctttt ttttcctgaa 420

aacaatttaa cgtcgagatt tatctctttt tgtaatggaa tcatttctac agttatgac 479

<210> 13

<211> 4101

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc 60

accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 120

agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 180

acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 240

ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 300

gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 360

atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 420

ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 480

atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 540

gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 600

aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 660

ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggcaacctg 720

attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 780

gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 840

atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 900

ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 960

atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1020

cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1080

tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1140

aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1200

cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1260

attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 1320

aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1380

ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1440

gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1500

ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1560

aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1620

ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1680

aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1740

ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1800

aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1860

accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 1920

ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 1980

ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2040

ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 2100

ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2160

gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2220

aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2280

gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2340

aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2400

gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2460

atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc 2520

gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 2580

aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2640

tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2700

aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2760

gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2820

aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 2880

ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 2940

caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3000

cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3060

atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3120

atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct 3180

ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 3240

accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3300

acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 3360

agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 3420

tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3480

gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 3540

ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3600

tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 3660

aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 3720

tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3780

cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 3840

ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 3900

atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 3960

gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 4020

gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 4080

ctgtctcagc tgggaggcga c 4101

<210> 14

<211> 208

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

agagactctt atcaagaatc ccatctcttg cttgcttttt tttgttgctt ccctttgata 60

gggtttgttt ttcttgtttc agtgactttc tatgttaaaa gataatgtca gtaaaaggat 120

ttggttttct attattctga atcgattacg gaagattctt gcttaattcc aatctataca 180

agtatcgtga aataatgacc gtttatgt 208

<210> 15

<211> 1026

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac 60

agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat 120

gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat 180

cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt 240

ggggagttta gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg 300

caagacctgc ctgaaaccga actgcccgct gttctacaac cggtcgcgga ggctatggat 360

gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga 420

atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat 480

cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag 540

ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc 600

tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg 660

atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct 720

tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgcca 780

cgactccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac 840

ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga 900

gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc 960

tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag 1020

aaatag 1026

<210> 16

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atggactata aggaccacga cggagactac aaggatcatg atattgatta caaagacgat 60

gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc 120

<210> 17

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ggatcaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 55

Claims (10)

1. A vector capable of performing efficient gene editing in tobacco is characterized in that a 35Spro promoter, a gRNA insertion region, an eu terminator, a calreticulin promoter element, a spCas9 protein expression element, a ubp terminator and a hygromycin screening marker are sequentially inserted into the vector, and the nucleotide sequence of the calreticulin promoter element is SEQ ID No. 1.

2. The vector of claim 1, wherein the nucleotide sequence of the 35Spro promoter is SEQ ID No. 10; the nucleotide sequence of the gRNA insertion region is SEQ ID NO. 11; the nucleotide sequence of the eu terminator is SEQ ID NO. 12; the nucleotide sequence of the spCas9 protein expression element is SEQ ID NO. 13; the nucleotide sequence of the ubp terminator is SEQ ID NO. 14; the nucleotide sequence of the hygromycin screening marker is SEQ ID NO. 15.

3. The vector of claim 1, wherein the gRNA insertion region has at least 2 tRNA cleavage sequences inserted therein; the nucleotide sequence of the tRNA cutting sequence is SEQ ID NO. 8; the spCas9 protein expression element has an NLS element and a PA50 element.

4. The vector of claim 1, wherein the nucleotide sequence of the vector is SEQ ID No. 9.

5. A method for efficient gene editing in tobacco, comprising the steps of:

(1) inserting a gRNA coding sequence targeting a gene to be edited into a gRNA insertion region of the vector of any one of claims 1 to 4 to obtain a gene editing vector;

(2) transforming agrobacterium with the gene editing vector to obtain a transformant;

(3) infecting the pre-cultured tobacco leaves with the transformant to obtain infected tobacco leaves;

(4) and culturing the infected tobacco leaves in a culture medium containing hygromycin.

6. The method according to claim 5, wherein the nucleotide sequence of the gRNA targeting the gene to be edited is SEQ ID No.4, 5, 6 or 7.

7. The method according to claim 5, wherein the nucleotide sequence of the gene editing vector is a sequence obtained by replacing positions 9754 to 9769 of SEQ ID NO.9 with SEQ ID NO.2, or a sequence obtained by replacing positions 9754 to 9769 of SEQ ID NO.9 with SEQ ID NO. 3.

8. The calreticulin promoter element, wherein the nucleotide sequence of the calreticulin promoter element is SEQ ID No. 1.

9. The application of the calreticulin promoter element in constructing a gene editing vector, wherein the nucleotide sequence of the calreticulin promoter element is SEQ ID No. 1.

10. The gRNA of the targeted gene to be edited has a nucleotide sequence of SEQ ID No.4, 5, 6 or 7.

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