CN112080517A - Screening system for improving probability of obtaining gene editing plants, construction method and application thereof - Google Patents

Abstract

The present invention relates to a method for increasing the probability of obtaining a gene-edited plant and its application. The invention links the expression cassette of a novel naked-eye visual reporter system under visible light and the expression cassette of the gene editing system to a vector to carry out genetic transformation of plants. Because the reporter system linked with the gene editing system can produce red betalain in plants, and the successful expression of the gene editing expression cassette in plant cells is a prerequisite for the plant to be edited, it can be directly indicated by the production of betalainin The gene editing expression cassette is expressed normally, so that the gene-edited plants can be selected directly by the color of the plant body under visible light with the naked eye. The invention effectively avoids the situation that some plants have no target site editing in the plants obtained through antibiotic screening, and successfully achieves the efficient acquisition of plants with edited target sites through the betalain reporting system.

Description

A screening system, construction method and method for improving the probability of obtaining gene-edited plants application

technical field

The invention belongs to the field of genetic engineering, and in particular relates to a new screening system for improving the probability of obtaining gene-edited plants and its application. The screening system of the present invention uses pigments produced in plants that can be directly observed with the naked eye as markers to screen to obtain gene-edited plants. The present invention also relates to the construction and application of the above-mentioned screening system.

Background technique

The core of gene editing technology is the use of sequence-specific nucleases (Sequence-specific nucleases, SSNs) to induce DNA double-strand breaks (DSBs), followed by targeted site-directed mutagenesis of the genome. Because after DSBs occur in the DNA of the organism, its self-repair mechanism will be activated to repair the broken DNA. At present, there are mainly two repair forms. First, in most cases, the repair mechanism will directly connect the chromosomes at the broken position, resulting in non-homologous end joining (NHEJ). Because NHEJ cannot guarantee very precise repair, the deletion or insertion of nucleotides at the break position will lead to gene mutation, so this feature can be used to create mutants of target site-directed mutagenesis. Second, in the presence of a homologous nucleic acid template, the organism can use the homologous nucleic acid as a template to perform homologous recombination repair (HDR) on the broken position when repairing DSBs. Because of the existence of the template, HDR produces precise repairs that, if engineered into the template, will introduce these mutations precisely into the organism's genome. However, the current research results show that even in the presence of homologous nucleic acid templates, the probability of NHEJ occurrence is much higher than that of HDR (Wyman C, Kanaar R. DNA double-strand break repair: all's well that ends well. Annu Rev Genet , 2006, 40: 363-383; Symington LS, Gautier J. Double-strand break end resection and repair pathway choice. Annu Rev Genet, 2011, 45: 247-271).

Currently, there are three main types of SSNs that are actually used in gene editing: Zinc finger nucleases (ZFNs) (Kim YG, Cha J, Chandrasegaran S. Hybrid restriction enzymes: zincfinger fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A, 1996, 93(3): 1156-1160), Transcription activator-like effector nucleases (TALEN) (Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF. Targeting DNA double-strand breaks with TAL effectornucleases. Genetics, 2010, 186(2):757-761; Miller JC, Tan S, Qiao G, Barlow KA, WangJ, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, Dulay GP, Hua KL, Ankoudinova I, Cost GJ, Urnov FD, Zhang HS, Holmes MC, Zhang L, Gregory PD, Rebar EJ.A TALEnuclease architecture for efficient genome editing.Nat Biotechnol,2011,29( 2): 143-148; Zhang F, Cong L, Lodato S, Kosuri S, Church GM, Arlotta P. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat Biotechnol, 2011, 29(2): 149-153) and clustered regularly interspaced short palindromic repeats and their related systems, namely the CRISPR/Cas system (Clustered regularly interspaced short palindromic repeats/CRISPR associated p roteins, CRISPR/Cas system) (Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012,337(6096):816-821 ; Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, JiangW, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cassystems. Science, 2013, 339(6121): 819-823). Among them, the CRISPR/Cas system is the easiest to operate, so it is widely used for the creation of targeted mutants.

CRISPR/Cas gene editing was originally found in archaea and bacteria, and its mechanism of action was elucidated by biochemical means (Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmabledual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012, 337(6096):816-821). Subsequently, this technology has also been proved to be able to target DNA editing in animals (Cong L, RanFA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, ZhangF. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013, 339(6121):819-823). Theoretically, the normal function of CRISPR/Cas gene editing only needs to meet three requirements: 1. The nuclear-localized CAS9 protein; 2. The gRNA with 20 target sequence-specific nucleotides; 3. The adjacent target sequence in the genome The 3' end is the protospacer-adjacent motif (PAM), PAM is NGG in the CAS9 system, and PAM is almost simplified to NG in the Cas9 variant Cas9-NG or xCas9 (Hu JH,, et al. al. Evolved Cas9variants with broad PAM compatibility and high DNA specificity. Nature, 2018, 556(7699): 57-63; Nishimasu H, et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science, 2018, 361(6408): 1259-1262). According to the mechanism of action of CRISPR/Cas and the successful application of the technology in animals, the technology has been rapidly applied in plants. At present, the CRISPR/Cas system has successfully achieved site-directed mutagenesis in a variety of plants (Mao Y, Botella JR, Liu Y, Zhu J-K. Geneediting in plants: progress and challenges. National Science Review, 2019, 6(3): 421-437; Xu J, Hua K, Lang Z. Genome editing for horticultural cropimprovement. Hortic Res, 2019, 6 : 113; Zhang Y, Malzahn AA, Sretenovic S, Qi Y. Theemerging and uncultivated potential of CRISPR technology in plant science. Nat Plants, 2019, 5(8):778-794).

Because the CRISPR/Cas gene editing system produces targeted mutations mainly rely on Cas protein and gRNA complex to cut the target site, an error occurs when the cell repairs the DSB, and if it can be repaired normally, the mutation cannot be generated. Therefore, the complex of Cas protein and gRNA needs to continuously cleave the target site until the target site is mutated and the gRNA cannot be fully complementary paired for recognition. Therefore, the efficient functioning of the CRISPR/Cas gene editing system in organisms depends on the presence of a large amount of Cas/gRNA nucleic acid-protein complexes in cells. Currently, two methods are mainly used to achieve this goal. First, the Cas/gRNA nucleic acid-protein complex or the mRNA and gRNA that can express Cas or the DNA that can transcribe Cas mRNA and gRNA is introduced into the target cell by transient transformation; second, the Cas protein can be expressed by the method of stable transformation. The DNA that transcribes and transcribes the gRNA is inserted into the genome of the target cell, and the Cas protein expression and gRNA transcription are continuously driven by the highly expressed promoter. Since the second method is convenient for screening transgenic cells, and Cas protein and gRNA can be stably expressed and transcribed in target cells, respectively, gene editing by stable transformation is more efficient in practice. In the CRISPR/Cas gene editing of plants, the DNA with the expression of Cas protein and transcribed gRNA is mainly inserted into the genome of plant cells by the method of stable transformation, and then positive plants are identified by antibiotic or herbicide screening and PCR amplification. to increase the probability of obtaining gene-edited plants.

However, in actual plant gene editing experiments, there are often some plants with resistance or positive PCR identification that do not undergo editing of the target site. The reasons are mainly as follows: 1. There is leakage in antibiotic screening, and some non-resistant tissues are protected by adjacent resistant tissues; 2. The screening pressure generated by antibiotics makes only the existence of resistance genes incomplete. Inserted DNA fragments were screened for accumulation, while DNA in which Cas protein was expressed and gRNA transcribed was lost. 3. The insertion position of exogenous DNA is inappropriate, and the DNA expression in it is low. Although the expression of antibiotic resistance genes is low enough to maintain tissue resistance and survive, the expression of Cas protein and transcribed gRNA is insufficient, which will lead to inability to induce The target site is mutated; 4. The exogenous DNA is inserted into the silent region of the chromosome. Although the PCR identification is positive, the gene on the exogenous DNA cannot be transcribed. Therefore, how to improve the probability of obtaining gene-edited plants through more effective screening still needs further research and optimization. It is a feasible method to link the reporter gene with the gene editing element gene to improve the probability of obtaining gene editing plants through obvious characteristics.

It has been reported that green fluorescent protein (GFP) and its derivatives (such as RFP, mCherry, and YFP) are used as reporter genes to assist screening to improve the probability of obtaining gene-edited plants (Gao X, Chen J, Dai X, Zhang D, Zhao Y. An Effective Strategy for Reliably Isolating Heritable and Cas9-Free Arabidopsis Mutants Generated by CRISPR/Cas9-Mediated Genome Editing. Plant Physiol, 2016, 171(3):1794-1800). Although fluorescent proteins are easy to use, they require specific wavelengths of light to excite them to produce visible fluorescent signals at specific wavelengths. Since there are a large number of substances that can autofluoresce in plants, the wavelengths of fluorescence generated by these substances interfere with the wavelengths emitted by different fluorescent proteins (Donaldson L. Autofluorescence in Plants. Molecules, 2020, 25(10)). Therefore, it is generally necessary to use a specific filter to observe a specific plant tissue, or to observe the tissue after special treatment, and the fluorescent protein will decay rapidly under excitation light, which is not conducive to continuous observation.

Betaine is a class of plant natural products derived from tyrosine (Strack D, Vogt T, Schliemann W. Recent advances in betalain research. Phytochemistry, 2003, 62(3): 247-269; XuJ-J, Fang X, Li C-Y, Yang L, Chen X-Y. General and specialized tyrosine metabolism paths in plants. aBIOTECH, 2020, 1(2):97-105). The bright red color seen in beets, dragon fruit, and other plants is the result of a buildup of betalains. At present, the biosynthetic pathway of betalain has been studied in detail, and it was found that only three enzymatic reactions are needed to convert tyrosine into betalain (Polturak G, Aharoni A. Advances and future directions in betalain metabolic engineering. New Phytol, 2019, 224(4):1472-1478). Tyrosine is first hydroxylated on the benzene ring to generate L-3,4-dihydroxyphenylalanine (L-DOPA). This reaction is catalyzed by the P450 oxidase CYP76AD1. L-DOPA can be further oxidized to cyclo-DOPA by CYP76AD1. At the same time, L-DOPA is also catalyzed by L-DOPA 4,5-dioxygenase (DODA) to generate beta-acid, which is then condensed with cyclo-DOPA to form beta-aglycone. The condensation reaction does not require an enzyme. Finally, the sugar group is added to the betalain by GT (glucosyltransferase), resulting in the brightly colored betalain. Therefore, we speculate that this pigment can also serve as an obvious marker to indicate the occurrence of transgenic plants.

SUMMARY OF THE INVENTION

In view of the limitations and deficiencies of the existing methods for improving the probability of obtaining gene-edited plants, it is necessary to develop a new reporting system that can be widely used in continuous, convenient and fast observation and low-cost to improve the probability of obtaining gene-edited plants. The invention uses a beet red pigment visible to the naked eye under natural light as a reporter molecule, and designs a new reporter system that improves the probability of obtaining gene editing plants.

In order to achieve the above object, the present invention adopts the following technical solutions:

The first object of the present invention is to provide a screening system for improving the probability of obtaining gene-edited plants, the screening system being a plant genetic transformation vector including a RUBY gene expression cassette and a CRISPR/Cas gene editing system, the RUBY gene expression cassette Including sequentially arranged promoter, betalain biosynthesis gene RUBY gene, terminator, the RUBY gene expression cassette is used as the reporter element of the screening system, and the CRISPR/Cas gene editing system is used as the gene of the screening system Edit components.

The betalain biosynthesis gene RUBY gene is used as a reporter gene for the screening system, and the protein expressed by the RUBY gene can synthesize a red pigment in plant cells, which can be directly observed with the naked eye under visible light, and through the constructed The RUBY gene expression cassette obtains a reporter system capable of producing pigments. Using the screening system of the present invention to perform gene editing on plants, it is possible to visually judge whether the gene editing is successful under visible light with the naked eye. When the plant tissue expresses the color of betalain, it means the CRISPR/Cas gene editing linked to the RUBY gene expression cassette. The expression cassette can be expressed normally in plant cells. The gene editing plant is guaranteed to have target site mutation, thereby increasing the probability of obtaining the gene editing plant.

Further, the RUBY gene includes the CYP76AD1 gene, the DODA gene and the GT (glucosyltransferase) gene; the CYP76AD1 gene, the DODA gene and the GT gene respectively represent the three genes on the intracellular beta red pigment synthesis pathway, and the synthesized CYP76AD1 Protein, DODA protein and GT protein respectively represent three enzymes on the intracellular beta red pigment synthesis pathway.

The nucleotide sequence of the CYP76AD1 gene comprises the nucleotide sequence encoding the amino acid sequence of CYP76AD1 shown in SEQ ID NO.1,

The nucleotide sequence of the DODA gene comprises the nucleotide sequence encoding the DODA amino acid sequence shown in SEQ ID NO.2,

The nucleotide sequence of the GT gene comprises the nucleotide sequence encoding the GT amino acid sequence shown in SEQ ID NO.3;

Preferably, the nucleotide sequence of the CYP76AD1 gene is shown in SEQ ID NO. 5, the nucleotide sequence of the DODA gene is shown in SEQ ID NO. 6, and the nucleotide sequence of the GT gene is shown in SEQ ID NO. ID NO.7.

Further, the CYP76AD1 gene, the DODA gene and the GT gene are connected in any order through a DNA linking unit;

According to the literature on the synthesis of betalain in the previous technical background (Polturak G, et al. New Phytol, 2019), it can be known that only these three proteins are needed for the synthesis of betalain. Therefore, as long as the three proteins can be expressed simultaneously, betalains can be produced in plants, and the connection sequence of the three proteins does not affect the final function of the three proteins.

In a specific embodiment, in the sequence of CYP76AD1 gene-DODA gene-GT gene, the DNA connecting units are connected in sequence, and the RUBY gene structure is: CYP76AD1 gene-DNA connecting unit-DODA gene-DNA connecting unit-GT gene.

Further, the DNA linking unit is a DNA sequence that can be transcribed and translated into a self-cleaving functional polypeptide;

Preferably, the nucleotide sequence of the DNA linking unit comprises a DNA sequence encoding the amino acid sequence of the 2A peptide shown in SEQ ID NO.4;

Further preferably, the DNA linking unit is 2A1 whose nucleotide sequence is shown in SEQ ID NO.8, or 2A2 whose nucleotide sequence is shown in SEQ ID NO.9. Both 2A1 and 2A2 can express the 2A peptide according to the nucleotide sequence 2A1 and 2A2 inversely deduced from the 2A peptide.

In a specific embodiment, the RUBY gene uses 2A peptide-encoding nucleotide sequences 2A1 and 2A2 as DNA linking units, and the CYP76AD1 gene shown in SEQ ID NO. 5-SEQ ID NO. 8 shows 2A1-SEQ ID The DODA gene shown in NO. 6 - the 2A2 gene shown in SEQ ID NO. 9 - the GT (glucosyltransferase) gene shown in SEQ ID NO. 7 - the sequence of the stop codon are connected in order.

Further, the promoter is a promoter that can function in plants, and the terminator is a terminator that can function in plants;

Preferably, the promoter is a promoter capable of functioning in dicotyledonous plants or monocotyledonous plants, and the terminator is a terminator capable of functioning in dicotyledonous plants or monocotyledonous plants;

Further preferably, the promoter is a promoter that can function in rice, and the terminator is a terminator that can function in rice;

More preferably, the promoter is the promoter of OsActin1 shown in SEQ ID NO.10, and the terminator is tHsp shown in SEQ ID NO.11.

Further, the CRISPR/Cas gene editing system includes a Cas protein expression cassette and a gRNA transcription cassette. The connection sequence of the Cas protein expression cassette and the gRNA transcription cassette is not limited.

Further, the Cas protein expression cassette comprises a Cas gene, a promoter that can function in plants and a terminator that can function in plants; the Cas gene is located in the promoter of the Cas protein expression cassette and between terminators.

In the screening system of the present invention, the RUBY gene expression cassette, the Cas protein expression cassette and the gRNA transcription cassette are independently constructed on a plant genetic transformation vector.

The second object of the present invention is to provide a method for constructing a screening system for improving the probability of obtaining a gene-edited plant, comprising the following steps:

1) construct RUBY gene, described RUBY gene is to connect between CYP76AD1 gene, DODA gene and GT gene by DNA linking unit in any order, connect RUBY gene and terminator, obtain RUBY-terminator DNA fragment;

2) connecting the Cas protein expression cassette into a plant genetic transformation vector to construct a vector pCas with the Cas protein expression cassette;

3) connecting the promoter into the vector pCas obtained in step 2), where the promoter is connected outside the Cas expression cassette, to construct a p-promoter-Cas vector;

4) The RUBY-terminator DNA fragment is constructed on the p-promoter-Cas carrier, and the RUBY gene-terminator DNA fragment is connected to the downstream of the promoter in step 3) to form a RUBY gene expression cassette with the promoter to obtain pRUBY - CRISPR vector;

5) Select the target site of the target gene, construct the gRNA transcription cassette capable of targeting the target site on the pRUBY-CRISPR vector, and obtain the screening system.

Further, the specific operations of the step 1) are: respectively obtaining CYP76AD1 gene, DODA gene, GT gene and terminator fragments; using the method of in vitro overlap extension PCR to pass the three DNA molecules of CYP76AD1 gene, DODA gene and GT gene through DNA. The connecting units are combined into a RUBY gene in any order, and a terminator is connected after the RUBY gene to obtain a RUBY gene-terminator DNA fragment;

In a specific embodiment, the RUBY gene is connected in sequence through the DNA linking unit in the sequence of CYP76AD1 gene-DODA gene-GT gene, and the RUBY gene structure is: CYP76AD1 gene-DNA linking unit-DODA gene-DNA linking unit-GT Gene. In this embodiment, the specific operation of the step 1) is:

1-1) Synthesize the DNA of the CYP76AD1 gene, the DNA of the DODA gene, and the DNA of the GT gene-terminator integral fragment respectively by means of whole gene synthesis in vitro;

1-2) use the method of in vitro overlap extension PCR to connect the obtained CYP76AD1 gene, DODA gene, GT gene-terminator DNA fragment by DNA connection unit in sequence, and obtain RUBY gene-terminator DNA fragment;

Further, the specific operations of the step 2) are as follows: obtaining the DNA of the Cas gene; connecting the DNA of the Cas gene into a plant genetic transformation vector to construct a vector pCas with a Cas protein expression cassette; the Cas gene is any one. A Cas gene that can function in the CRISPR/Cas gene editing system; the Cas gene is in a complete expression cassette of the plant genetic transformation vector; the promoter and the terminator are on the plant genetic transformation vector Existing promoters and terminators, or newly added promoters or terminators during construction.

In a specific embodiment, the Cas gene is the Cas9 gene as shown in SEQ ID NO.12;

In a specific embodiment, the plant genetic transformation vector already has a promoter that is functional in plants and a terminator that is functional in plants.

Further, the specific operation of the step 3) is: the promoter is connected to the vector pCas obtained in the step 2), the promoter is connected outside the Cas expression cassette, and the promoter is one that can play a role in plants. A functional promoter, a p-promoter-Cas vector was constructed.

Further, the specific operation of the step 4) is: the RUBY-terminator DNA fragment is constructed on the p promoter-Cas carrier, and the RUBY gene-terminator DNA fragment is connected downstream of the promoter described in step 3), The RUBY gene expression cassette was formed with the promoter to obtain the pRUBY-CRISPR vector.

Further, the specific operation of the step 5) is: according to the target gene target site, design a primer that can be complementary paired with a 3' end protruding, and by in vitro overlapping PCR amplification, the target gene can be targeted to the gRNA transcription cassette of the target gene. The DNA molecule is linked to the pRUBY-CRISPR vector obtained in step 4), and the gRNA transcription cassette is outside the Cas expression cassette and the RUBY expression cassette to obtain the screening system.

The third object of the present invention is to provide the application of the aforementioned screening system for increasing the probability of obtaining gene-edited plants or the construction method of the aforementioned screening system for increasing the probability of obtaining gene-edited plants in increasing the probability of obtaining gene-edited plants.

Further, when using the screening system to perform gene editing on a plant, when the plant tissue expresses the color of betalain, that is, red, it indicates that the gene-edited plant has undergone a target site mutation, and the gene-edited plant is obtained by screening.

Further, gene editing refers to the direct introduction of DNA molecules of the screening system into plant cells, or the introduction of RNA molecules of the transcription components of the screening system into plant cells, or the introduction of proteins of the expression components of the screening system into plant cells. within plant cells.

Further, the gene editing can occur in all kinds of plant whole or part of plant tissues or organs or plant cells,

Preferably, the plant is a dicotyledonous plant and/or a monocotyledonous plant,

Further preferably, the plant is rice.

It has been reported in the literature that the CRISPR/Cas gene editing system can perform gene editing functions as long as Cas protein and gRNA can be produced, and its gene editing function is universal in various organisms (including eukaryotes and prokaryotes). Komor AC, Badran AH, Liu DR. CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes. Cell, 2017, 168(1-2):20-36; Sander JD, Joung JK. CRISPR-Cassystems for editing, regulating and targeting genomes .Nat Biotechnol, 2014, 32(4): 347-355), and it has been confirmed that it can play a role in gene editing in all kinds of plants that have been studied (MaoY, Botella JR, Liu Y, Zhu J-K.Gene editing in plants: progress and challenges. National Science Review, 2019, 6(3): 421-437); the three enzymes combined in the RUBY expression cassette use tyrosine widely present in plants as the initial substrate to synthesize betalain , and studies have found that as long as these three proteins can be expressed at the same time, the production of betalain in dicotyledonous plants can be guaranteed (Polturak G, et al. NewPhytol, 2019). We confirmed the effect of the present invention through the experimental results of combining Cas protein expression cassette, gRNA transcription cassette and RUBY expression cassette in rice, so it can be speculated that the present invention also has the same effect in other plants.

The present invention efficiently co-expresses the enzymes required for the entire betalain synthesis pathway using a single promoter. We removed the stop codons of CYP76AD1, DODA and GT, connected the three sequentially through the DNA sequence encoding the 2A peptide, and finally connected the stop codon to form an artificial gene named RUBY. In a specific embodiment, the RUBY gene adopts the nucleotide sequence 2A encoding the 2A peptide as the DNA linking unit, and is sequentially linked in the sequence of CYP76AD1 gene-2A1-DODA gene-2A2-GT gene.

As we all know, tyrosine is one of the most commonly used amino acids in all kinds of organisms, and it is abundant in all kinds of organisms. In plants, tyrosine is first hydroxylated on the benzene ring to generate L-3,4-dihydroxyphenylpropane amino acid (L-DOPA). This reaction is catalyzed by the P450 oxidase CYP76AD1. L-DOPA can be further oxidized to cyclo-DOPA by CYP76AD1. At the same time, L-DOPA is also catalyzed by L-DOPA 4,5-dioxygenase (DODA) to generate beta-acid, which is then condensed with cyclo-DOPA to form beta-aglycone. The condensation reaction does not require an enzyme. Finally, the glycosyl group is added to the betain aglycone by GT (Glycosyl transferase, glucosyl transferase) to generate the brightly colored betalain.

The three betalain biosynthesis genes are fused into an open reading frame that can be expressed using a single promoter and terminator.

A sequence encoding the 2A peptide was inserted between these three genes. The 2A peptide is a short peptide consisting of only about 20 amino acids. During protein translation, when the ribosome encounters the newly translated 2A peptide, the ribosome will occur. "Jump", which in turn leads to self-cleavage of the peptide chain at the end of the 2A peptide (Sharma P, Yan F, Doronina VA, Escuin-Ordinas H, Ryan MD, Brown JD. 2A peptides provide distinct solutions to driving stop-carryon translational recoding .Nucleic Acids Res, 2012, 40(7): 3143-3151), so the 2A peptide can simultaneously express multiple proteins under the control of a single promoter (Liu Z, Chen O, Wall JBJ, Zheng M, ZhouY, Wang L, Vaseghi HR, Qian L, Liu J. Systematic comparison of 2A peptides forcloning multi-genes in a polycistronic vector. Sci Rep, 2017, 7(1):2193). After the RUBY gene is transcribed, three independent enzymes can be produced to catalyze the synthesis of betalain from tyrosine. A functional promoter in plants can be placed in front of the RUBY DNA to drive the expression of RUBY, and subsequent successful plant gene editing is presumed to occur through the colored betalain.

In a specific embodiment, the RUBY expression cassette is introduced into the rice CRISPR/Cas gene editing vector pOsActin1-UCAS9. The RUBY gene expression cassette uses the promoter of the constitutively expressed gene OsActin1 in rice (McElroy D, Zhang W, Cao J, Wu R. Isolation of an efficient actin promoter foruse in rice transformation. Plant Cell, 1990, 2(2):163 -171) drives the expression of RUBY, and the transcription terminator of the RUBY gene expression cassette is tHsp (Nagaya S, Kawamura K, Shinmyo A, Kato K. The HSPterminator of Arabidopsis thaliana increases gene expression in plantcells. Plant Cell Physiol, 2010, 51 ( 2): 328-332). The new gene editing vector is pRUBY-CRISPR. After transforming pRUBY-CRISPR into rice, the constitutive expression of RUBY makes rice appear bright red, which is easier to observe and screen than traditional reporter systems such as fluorescent proteins. The probability of editing the colored rice material is 100%, which effectively avoids the situation that some plants do not undergo target site editing in the plants obtained through antibiotic screening.

The beneficial effects of the present invention are embodied in :

The invention links the artificially combined betalain biosynthesis gene RUBY expression cassette and the CRISPR/Cas gene editing expression cassette to a vector. Because the RUBY expression cassette can produce brightly colored betalains in plants, it is easier to observe and screen than traditional reporter systems such as fluorescent proteins. The screening system is used to perform gene editing on plants, and the red color produced by the RUBY gene in the screening system is used as a marker to screen for gene-edited plants, and the probability of obtaining gene-edited plants can reach 100% by naked-eye screening under visible light. This effectively avoids the situation that some plants do not undergo target site editing in the plants obtained by antibiotic screening. Since the present invention does not require special equipment or expensive substrates, the present invention provides a gene editing screening reporting system with convenient observation and cost saving.

Description of drawings

Figure 1 is a schematic structural diagram of the screening system for improving the probability of obtaining gene-edited plants according to the present invention

Figure 2 is a schematic diagram of the construction of the RUBY gene:

Fig. 2A is the chemical reaction flow chart of converting tyrosine into red betalain;

Figure 2B is a strategy for expressing the complete betalain biosynthetic pathway in a single expression cassette.

Fig. 3 is the comparison diagram after utilizing the screening system of the present invention and the conventional vector without RUBY marker to transform rice:

Figure 3A shows that OsActin1:RUBY vector transformed rice into successfully transformed callus with obvious red color.

Figure 3B shows that the resistant callus obtained after the transformation of rice with a conventional vector without RUBY marker has no obvious color difference.

Figure 4 shows the gene editing of the rice material selected by using the red color produced by the RUBY expression cassette in the screening system of the present invention as a marker: Figure 4A and Figure 4B respectively show the gene editing of two independent transformation events.

Detailed ways

In order to facilitate the understanding of the technical solutions of the present invention, the present invention is further described below with reference to specific embodiments.

Example 1. Combination of RUBY genes to obtain RUBY-tHsp DNA

The RUBY gene in this example uses the nucleotide sequence 2A that can encode the 2A peptide as the DNA linking unit, and is connected in sequence in the order of CYP76AD1 gene-2A1-DODA gene-2A2-GT (glucosyltransferase) gene (Fig. 2B) , wherein the nucleotide sequence of 2A1 is shown in SEQ ID NO.8, the nucleotide sequence of 2A2 is shown in SEQ ID NO.9, and both 2A1 and 2A2 are nucleotide sequences encoding the 2A peptide shown in SEQ ID NO.4 , constitutes the RUBY gene, and uses tHsp as the terminator.

The build method is:

(1) Synthesize by in vitro whole gene synthesis:

(a) CYP76AD1 gene without stop codon (SEQ ID NO. 5),

(b) DODA gene without stop codon (SEQ ID NO. 6),

(c) GT gene without stop codon including the GT gene shown in SEQ ID NO.7, 3 consecutive stop codons TGA, TAG, TGA and the terminator tHsp shown in SEQ ID NO.11 + stop codon + stop sub-tHsp fragment (SEQ ID NO. 13);

(2) The DNA of the 2A peptide is used as the DNA linking unit, and the partial sequence of the corresponding DNA of the 2A peptide is respectively added to the 5' end of the primer used for PCR amplification as a linker sequence, and assembled by the method of in vitro overlap extension PCR (overlap PCR) into the DNA of the complete 2A peptide, and the CYP76AD1 gene (SEQ ID NO. 5)-2A1 (SEQ ID NO. 8)-DODA gene (SEQ ID NO. 6)-2A2 (SEQ ID NO. 9)-GT gene ( SEQ ID NO.7)+stop codon+terminator tHsp (SEQ ID NO.11) (the nucleotide sequence of GT gene+stop codon+terminator tHsp fragment is shown in SEQ ID NO.13) followed by end-to-end combination Together, the DNA of RUBY-tHsp was obtained.

Specific build steps:

(1) PCR1 amplification to obtain CYP76AD1+ front part 2A1 DNA, where the front part 2A1 sequence is: 5'-GGTAGCGGAGCTACCAATTTTAGCCTCCTTAAGCAGGCAGGTG-3' (SEQ ID NO.36), CYP76AD1 without stop codon shown in SEQ ID NO.5 The gene is template DNA, using primer pairs:

CYP76AD1+ front part 2A1-F:

5'-CACTGATAGTTTAAACTAGTATGGATCATGCGACCCTCGC-3' (SEQ ID NO. 18)

CYP76AD1+ front part 2A1-R:

5'-CACCTGCCTGCTTAAGGAGGCTAAAATTGGTAGCTCCGCTACCGTAGCGCGGAATCGGGA-3' (SEQ ID NO. 19) was subjected to PCR amplification.

PCR reaction system:

2×PCR Buffer 10μl 2.5mM dNTPs 2μl 10 μM CYP76AD1+ front part 2A1-F 0.6μl 10 μM CYP76AD1+ front part 2A1-R 0.6μl template DNA 0.5μl KOD-FX polymerase 0.2μl Supplement with double distilled water and add water to 20μl

PCR amplification procedure:

The PCR product is: CYP76AD1 + front part 2A1, the size is 1554bp, which includes the CYP76AD1 gene sequence without stop codon of SEQ ID NO.5 and the front part 2A1 of SEQ ID NO.36, here the front part 2A1 sequence provides PCR use Additional linker sequence for overlap PCR on reverse primer. However, since the primers used for PCR add additional linker sequences for the final Gibson ligation, the PCR product is slightly larger than the total sequence length of the CYP76AD1 gene sequence without stop codon of SEQ ID NO.

(2) PCR2 amplification to obtain rear part 2A1+DODA+ front part 2A2 DNA, where the rear part 2A1 sequence is: 5'-CCTTAAGCAGGCAGGTGATGTAGAAGAGAACCCCGGGCCT-3' (SEQ ID NO. 37), the front part 2A2 sequence is: 5'-GGATCCGGAGCAACCAACTTTAGCCTGCTCAAGCAAGCAGGAG -3' (SEQ ID NO.38), using the DODA gene without stop codon shown in SEQ ID NO.6 as template DNA, using primer pair:

Rear part 2A1+DODA+front part

2A2-F: 5'-CCTTAAGCAGGCAGGTGATGTAGAAGAGAACCCCGGGCCTATGAAGATGATGAACGGCGA-3' (SEQ ID NO. 20)

Rear part 2A1+DODA+front part

2A2-R: 5'-CTCCTGCTTGCTTGAGCAGGCTAAAGTTGGTTGCTCCGGATCCGGCGGAGGTGAACTTGT-3' (SEQ ID NO. 21) was used as a primer for PCR amplification.

PCR reaction system:

2×PCR Buffer 10μl 2.5mM dNTPs 2μl 10 μM rear fraction 2A1 + DODA + front fraction 2A2-F 0.6μl 10 μM rear fraction 2A1 + DODA + front fraction 2A2-R 0.6μl template DNA 0.5μl KOD-FX polymerase 0.2μl Add double distilled water to 20μl

PCR amplification procedure:

The PCR product is: the rear part 2A1+DODA+ the front part 2A2 is 908bp in size, including the DODA gene sequence of SEQ ID NO.6 without stop codon, the rear part 2A1 of SEQ ID NO.37 and the front part of SEQ ID NO.38 Section 2A2. Since the rear part 2A1 sequence and the front part 2A2 sequence here respectively provide the forward primer and reverse primer used for PCR additional adapter sequences for overlap PCR, the size of the PCR product is equal to the non-stop codon of SEQ ID NO. 6 The total sequence length of the DODA gene sequence of the son and the rear part 2A1 of SEQ ID NO. 37 and the front part 2A2 of SEQ ID NO. 38 here.

(3) PCR3 amplification to obtain the rear part 2A2+GT+tHsp DNA, where the sequence of the rear part 2A2 is: 5'-GCTCAAGCAAGCAGGAGATGTTGAGGAAATCCTGGCCCC-3' (SEQ ID NO.39), shown in SEQ ID NO.13: GT gene without stop codon (SEQ ID NO. 7) + stop codon (TGA, TAG, TGA) + terminator tHsp (SEQ ID NO. 11) DNA as template DNA, using primer pair:

Rear part 2A2+GT+tHsp

DNA-F: 5'-GCTCAAGCAAGCAGGAGATGTTGAGGAAAATCCTGGCCCCATGACCGCCATCAAGATGAA-3' (SEQ ID NO. 22)

Rear part 2A2+GT+tHsp

DNA-R: 5'-GCTAGCTTACTCAGTTAGGTCTTATCTTTAATCATATTCC-3' (SEQ ID NO. 23) was used as a primer for PCR amplification.

PCR reaction system:

PCR amplification procedure:

The PCR product is: rear part 2A2+GT+tHsp, the size is 1819bp, including the rear part 2A2 sequence shown in SEQ ID NO.39 and the sequence shown in SEQ ID NO.13: GT gene without stop codon (SEQ ID NO. 13) NO.7)+stop codon+terminator tHsp (SEQ ID NO.11) sequence, the stop codon is: three consecutive stop codons TGA, TAG, TGA are connected in series.

The rear part 2A2 sequence of SEQ ID NO. 39 here provides an additional linker sequence for the overlap PCR on the forward primer used for PCR, but because the reverse primer used for PCR plus an additional additional linker sequence for the final Gibson The linker sequence is connected, so the PCR product is slightly larger than the total length of the GT gene without stop codon shown in SEQ ID NO.13+stop codon+terminator tHsp and the rear part 2A2 shown in SEQ ID NO.39.

(4) PCR amplification to obtain RUBY-tHspDNA: The three PCR products of PCR1, PCR2, and PCR3, CYP76AD1 + front part 2A1, rear part 2A1 + DODA + front part 2A2, rear part 2A2 + GT + stop codon + tHsp, were mixed as a template, using Primer pair:

CYP76AD1+ front part

2A1-F: 5'-CACTGATAGTTTAAACTAGTATGGATCATGCGACCCTCGC-3' (SEQ ID NO. 18);

Rear part 2A2+GT+tHsp

DNA-R: 5'-GCTAGCTTACTCAGTTAGGTCTTATCTTTAATCATATTCC-3' (SEQ ID NO. 23) was used as primer for PCR amplification to obtain RUBY-tHsp DNA.

PCR reaction system:

PCR amplification procedure:

The PCR product size was 4247bp.

Example 2. Construction of vector pCUCas9 with Cas protein expression cassette

In this example, the Cas protein expression cassette, that is, the Cas9 gene capable of encoding the Cas protein, was linked into the pCXUN vector to obtain the vector pCUCas9. Specific build steps:

(1) The Cas9 gene DNA was obtained by PCR amplification. It is derived from the codon-optimized Cas9 gene (sequence shown in SEQ ID NO.12, see literature: Miao J, Guo D, Zhang J, Huang Q, Qin G, Zhang X, Wan J, GuH, Qu LJ. Targeted mutagenesis in rice using CRISPR-Cas system. Cell Res, 2013, 23(10): 1233-1236; Cas9 gene DNA can be obtained from the author through the mailbox in the literature) as template DNA. Take primer pairs:

UCas9-F: 5'-CCCGGGGGATCCCCAATACTATGGCCCCAAAGAAGAAGCGCAAGG-3' (SEQ ID NO. 24)

UCas9-R: 5'-GAAATTCGGATCCCCAATACTTCAATCGCCGCCGAGTTGTGAGAGG-3' (SEQ ID NO. 25) was PCR amplified.

PCR reaction system:

PCR amplification procedure:

The PCR product is: Cas9 gene DNA, the size is 4172bp, which includes the Cas9 gene sequence of SEQ ID NO.12, because the primer used in PCR has added an extra linker sequence for Gibson connection, so the PCR product is slightly larger than SEQ ID NO. Sequence length of the Cas9 gene shown in 12.

(2) Construct the pCUCas9 vector: use Xcm I to digest the pCXUN vector plasmid (see the literature for pCXUN vector information: Chen S, Songkumarn P, Liu J, Wang GL. A versatile zero background T-vector system for gene cloning and functional genomics. Plant Physiol, 2009, 150(3): 1111-1121; the pCXUN vector plasmid can be obtained from the author through the mailbox in the literature), and the PCR product of the Cas9 gene DNA obtained in step (1) of this example is connected by Gibson (Gibson). The ligation is a currently popular and well-known method for rapid ligation of DNA. For the principle, detailed operation steps and reagent information, please refer to the literature: Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, 3rd, Smith HO. Enzymatic assembly of DNAmolecules up toseveral hundred kilobases. Nat Methods, 2009, 6(5): 343-345) was ligated into the Xcm I of the pCXUN vector to obtain the pCUCas9 vector.

Example 3. Construction of pOsActin1-UCas9 vector

(1) The OsActin1 promoter DNA was obtained by PCR amplification. With the OsActin1 promoter (sequence shown in SEQ ID NO.10, see literature: He Y, Zhang T, Yang N, Xu M, Yan L, Wang L, Wang R, Zhao Y. Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR/Cas9 mediated genome editing. J Genet Genomics, 2017, 44(9): 469-472; DNA containing the OsActin1 promoter can be obtained from the authors through the mailbox in the literature) as template DNA. Take primer pairs:

OsActin1P-F:

5'-TACGAATTCGAGCTCGGTACGCATACTCGAGGTCATTCATATGCTTGAG-3' (SEQ ID NO. 26)

OsActin1P-R: 5’-ATCCCCCTTTCGCCAGGGGTACCGAGATCGTCGTCCGGCAGC-3’

(SEQ ID NO. 27) PCR amplification was performed.

PCR reaction system:

2×PCR Buffer 10μl 2.5mM dNTPs 2μl 10 μM OsActin1P-F 0.6μl 10 μM OsActin1P-R 0.6μl template DNA 0.5μl KOD-FX polymerase 0.2μl Supplement with double distilled water and add water to 20μl

PCR amplification procedure:

The PCR product is: OsActin1 promoter DNA, with a size of 950bp, including the OsActin1 promoter sequence of SEQ ID NO. 10. Since the primers used in PCR add an additional linker sequence for Gibson ligation, the PCR product is slightly larger than the SEQ ID NO. 10. The sequence length of the OsActin1 promoter shown in ID NO. 10.

(2) Construct pOsActin1-UCas9 vector: use Kpn I to digest the pCUCas9 vector plasmid in Example 2, and connect the PCR product of the OsActin1 promoter DNA obtained in step (1) of this example into pCUCas9 by Gibson ligation At Kpn I of the vector, the pOsActin1-UCas9 vector was obtained.

Example 4. Construction of pRUBY-CRISPR vector

(1) RUBY-tHsp DNA was obtained by PCR amplification.

The RUBY-tHsp PCR product DNA in Example 1 was used as a template, and primer pairs were used:

OsActin1P-RUBY-F:

5'-TGCCGGACGACGATCTCGGTACCATGGATCATGCGACCCTCG-3' (SEQ ID NO. 28)

OsActin1P-RUBY-R:

5'-CACATCCCCCTTTCGCCAGGGTTAACCTTATCTTTAATCATATTCCATAGTCCATACCA-3' (SEQ ID NO. 29) was used as a primer for PCR amplification.

PCR reaction system:

2×PCR Buffer 10μl 2.5mM dNTPs 2μl 10 μM OsActin1P-RUBY-F 0.6μl 10 μM OsActin1P-RUBY-R 0.6μl template DNA 0.5μl KOD-FX polymerase 0.2μl Add double distilled water to 20μl

PCR amplification procedure:

The PCR product is: RUBY-tHsp DNA with a size of 4256bp.

(2) The pRUBY-CRISPR vector was constructed: the pOsActin1-UCas9 vector plasmid in Example 3 was digested with Kpn I, and the PCR product of the RUBY-tHsp DNA obtained in step (1) of this example was connected by Gibson connection. into the Kpn I of the pOsActin1-UCas9 vector to obtain the pRUBY-CRISPR vector.

Example 5. Construction of a target DNA-targeting gRNA transcription cassette into the pRUBY-CRISPR vector.

A specific DNA capable of transcribing gRNA was designed with SE5 gene in rice as the target gene, and the target sequence was 5'-GACGAGCTTGCTGTCGACG-3' (SEQ ID NO. 14). The pRUBY-CRISPR plasmid was digested with Pme I into linear DNA, and the DNA of the gRNA was introduced by the method of overlapping PCR (as a conventional method) amplification. The present embodiment adopts the OsU6 promoter as the promoter of the gRNA transcription unit, and the OsU3 terminator as the terminator of the gRNA transcription unit, and the specific steps are as follows:

OsU6P DNA (sequence as shown in SEQ ID NO.16), gRNA backbone DNA (sequence as shown in SEQ ID NO.15) and OsU3T DNA (sequence as shown in SEQ ID NO.17) have been constructed in our laboratory. The vector (for the vector containing OsU6PDNA, gRNA backbone DNA and OsU3T DNA, see literature: He Y, Zhang T, Yang N, Xu M, Yan L, Wang L, Wang R, Zhao Y. Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR/Cas9 mediated genome editing. JGenet Genomics, 2017, 44(9): 469-472; vectors containing OsU6P DNA, gRNA backbone DNA and OsU3T DNA can be obtained from the authors through the mailbox in the literature) as Template DNA, respectively OsU6PF:

5'-GTCGTTTCCCGCCTTCAGTTTATGTACAGCATTACGTAGG-3' (SEQ ID NO. 30) and Se5-U6-SalI-R: 5'-CGTCGACAGCAAGCTCGTCCAACCTGAGCCTCAGCGCAGCAGC-3'

(SEQ ID NO. 31) primer pair, and

OsU3TR: 5’-CTGTCAAACACTGATAGTTTAAACGCTGTGCCGTACGACGGTACG-3’

(SEQ ID NO.32) and Se5-U6-SalI-F: 5'-GGACGAGCTTGCTGTCGACGGTTTTAGAGCTAGAAATAGCAAGTTAAAAT-3' (SEQ ID NO.33) primer pair amplified two kinds of DNA, the above two kinds of DNA were cut into gel and recovered and mixed as Template, using OsU6PF and OsU6TR as primers to amplify the complete gRNA transcription unit DNA.

The above PCR reaction systems:

2×PCR Buffer 10μl 2.5mM dNTPs 2μl 10 μM F primer 0.6μl 10 μM R primer 0.6μl template DNA 0.5μl KOD-FX polymerase 0.2μl Add double distilled water to 20μl

PCR amplification procedure:

Then, the DNA of the PCR product was recovered by gel cutting, and the recovered DNA was ligated into the pRUBY-CRISPR plasmid cut by Pme I using the Gibson ligation method. The pRUBY-CRISPR-SE5-Sal I vector was obtained.

Example 6. Transformation of pRUBY-CRISPR-SE5-Sal I vector into rice callus

The sequenced positive plasmids pRUBY-CRISPR-SE5-Sal I and pOsActin1-UCAS9 were electro-transformed into Agrobacterium (EHA105), respectively, and then infected rice callus. The transformed variety is rice "Zhonghua 11" (also known as ZH11, from the Institute of Crop Science, Chinese Academy of Agricultural Sciences). For the basic formula, see literature: Hiei Y, Ohta S, Komari T, Kumashiro T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J, 1994, 6(2) :271-282). In the callus screening stage, the pRUBY-CRISPR-SE5-SalI transgenic rice with the RUBY expression cassette can be seen to have obvious red color, and some callus without normal expression of RUBY (no color) can also be seen in the antibiotics. Growth on selection medium (Figure 3A). However, after the conventional vector pOsActin1-UCAS9 without the RUBY expression cassette was transformed into rice, it was impossible to directly see whether the transgene was successfully expressed (Fig. 3B).

Example 7. Detection of editing status by pRUBY-CRISPR-SE5-Sal I transformed rice material

The editing of resistant calli after pRUBY-CRISPR-SE5-Sal I transformed rice was tested. The callus is divided into R with color and N without red according to color. In transformation event A, only red (R) callus was taken from callus block 1 for detection; callus blocks 2, 3, 4, 5, and 6 were all taken with red (R) and no red (N), respectively. Callus was tested; callus block 7 only had no red (N) callus for testing (Fig. 4A). In transformation event B, callus blocks 1, 2, and 9 with red (R) and no red (N) were selected for detection; callus blocks 3, 4, 5, and 6 were only picked with red ( R) Callus pieces were tested; callus pieces 7 and 8 only took non-red (N) callus for testing (Fig. 4B).

Using conventional CTAB method (He Y, Yan L, Ge C, Yao XF, Han X, Wang R, Xiong L, Jiang L, Liu CM, Zhao Y. PINOID Is Required for Formation of the Stigma and Style in Rice. Plant Physiol, 2019, 180(2):926-936) DNA was extracted from each callus respectively. use

SE5-SalF: 5'-CACGGAACGGAGACCCCTA-3' (SEQ ID NO. 34);

SE5-SalR: 5'-AGCGGTCCAAAGTAGAGCAC-3' (SEQ ID NO. 35) was used as a primer to amplify the DNA extracted from each callus, respectively.

PCR reaction system:

10×PCR Buffer 2μl 2.5mM dNTPs 2μl 10μM SE5-SalF 0.3μl 10μM SE5-SalR 0.3μl Rice genomic DNA 2μl rTaq polymerase 0.1μl Supplement with double distilled water and add water to 20μl

PCR amplification procedure:

The PCR product size was 550bp. Because the target site mutation position generated by the CRISPR/Cas9 gene editing system occurs at the position of 3 bp before the PAM (protospacer-adjacent motif) of the target site, and 2-7 bp before the PAM ( The position on the target sequence) is the recognition site (GTCGAC) of the restriction endonuclease Sal I, so the method of digesting the PCR product with Sal I can be used to identify whether the target site is mutated. If the DNA is mutated and the recognition site of Sal I is destroyed, the DNA of the PCR product cannot be digested by Sal I.

PCR product digestion system:

PCR-DNA 5μl 10×buffer 0.5μl Enzyme 0.1μl (1U) H₂O 5μl

Electrophoresis was performed after 3-4 hours of reaction at 37°C. Because there is a Sal I restriction site 39 bp downstream of the target site, the PCR product DNA of the mutant (Mut) and the wild type (WT) has a small difference in size after restriction. Therefore, Use 2% agarose gel electrophoresis for 30min to distinguish DNA bands of different sizes, and use the PCR product DNA of wild-type rice DNA as a control, which is different from the electrophoresis band type of the control number (WT). The numbered material is the edited target site.

The results showed that the probability of target site mutation (Mut) of the selected red callus (R) was 100%; while the callus without red color (N) had no target site mutation (WT) in more than half. )(Figure 4). It shows that although some callus can grow normally on dishes with antibiotics, some of them do not have target site mutation. The invention can directly select the material for gene editing by the color of the naked-eye visible plant body under visible light, effectively avoids the situation that some plants do not have the target site editing in the plants obtained by antibiotic screening, and successfully realizes the use of betalains. The reporter system efficiently obtains plants whose target sites are edited.

sequence listing

<110> Nanjing Agricultural University

<120> A screening system, construction method and application for improving the probability of obtaining gene-edited plants

<160> 39

<170> SIPOSequenceListing 1.0

<210> 1

<211> 497

<212> PRT

<213> Artificial Sequence

<400> 1

Met Asp His Ala Thr Leu Ala Met Ile Leu Ala Ile Trp Phe Ile Ser

1 5 10 15

Phe His Phe Ile Lys Leu Leu Phe Ser Gln Gln Thr Thr Lys Leu Leu

20 25 30

Pro Pro Gly Pro Lys Pro Leu Pro Ile Ile Gly Asn Ile Leu Glu Val

35 40 45

Gly Lys Lys Pro His Arg Ser Phe Ala Asn Leu Ala Lys Ile His Gly

50 55 60

Pro Leu Ile Ser Leu Arg Leu Gly Ser Val Thr Thr Ile Val Val Ser

65 70 75 80

Ser Ala Asp Val Ala Lys Glu Met Phe Leu Lys Lys Asp His Pro Leu

85 90 95

Ser Asn Arg Thr Ile Pro Asn Ser Val Thr Ala Gly Asp His His Lys

100 105 110

Leu Thr Met Ser Trp Leu Pro Val Ser Pro Lys Trp Arg Asn Phe Arg

115 120 125

Lys Ile Thr Ala Val His Leu Leu Ser Pro Gln Arg Leu Asp Ala Cys

130 135 140

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

145 150 155 160

Glu Cys Ala Gln Lys Gly Gln Ala Val Asp Ile Gly Lys Ala Ala Phe

165 170 175

Thr Thr Ser Leu Asn Leu Leu Ser Lys Leu Phe Phe Ser Val Glu Leu

180 185 190

Ala His His Lys Ser His Thr Ser Gln Glu Phe Lys Glu Leu Ile Trp

195 200 205

Asn Ile Met Glu Asp Ile Gly Lys Pro Asn Tyr Ala Asp Tyr Phe Pro

210 215 220

Ile Leu Gly Cys Val Asp Pro Ser Gly Ile Arg Arg Arg Leu Ala Cys

225 230 235 240

Ser Phe Asp Lys Leu Ile Ala Val Phe Gln Gly Ile Ile Cys Glu Arg

245 250 255

Leu Ala Pro Asp Ser Ser Thr Thr Thr Thr Thr Thr Asp Asp Val

260 265 270

Leu Asp Val Leu Leu Gln Leu Phe Lys Gln Asn Glu Leu Thr Met Gly

275 280 285

Glu Ile Asn His Leu Leu Val Asp Ile Phe Asp Ala Gly Thr Asp Thr

290 295 300

Thr Ser Ser Thr Phe Glu Trp Val Met Thr Glu Leu Ile Arg Asn Pro

305 310 315 320

Glu Met Met Glu Lys Ala Gln Glu Glu Ile Lys Gln Val Leu Gly Lys

325 330 335

Asp Lys Gln Ile Gln Glu Ser Asp Ile Ile Asn Leu Pro Tyr Leu Gln

340 345 350

Ala Ile Ile Lys Glu Thr Leu Arg Leu His Pro Pro Thr Val Phe Leu

355 360 365

Leu Pro Arg Lys Ala Asp Thr Asp Val Glu Leu Tyr Gly Tyr Ile Val

370 375 380

Pro Lys Asp Ala Gln Ile Leu Val Asn Leu Trp Ala Ile Gly Arg Asp

385 390 395 400

Pro Asn Ala Trp Gln Asn Ala Asp Ile Phe Ser Pro Glu Arg Phe Ile

405 410 415

Gly Cys Glu Ile Asp Val Lys Gly Arg Asp Phe Gly Leu Leu Pro Phe

420 425 430

Gly Ala Gly Arg Arg Ile Cys Pro Gly Met Asn Leu Ala Ile Arg Met

435 440 445

Leu Thr Leu Met Leu Ala Thr Leu Leu Gln Phe Phe Asn Trp Lys Leu

450 455 460

Glu Gly Asp Ile Ser Pro Lys Asp Leu Asp Met Asp Glu Lys Phe Gly

465 470 475 480

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

485 490 495

Tyr

<210> 2

<211> 275

<212> PRT

<213> Artificial Sequence

<400> 2

Met Lys Met Met Asn Gly Glu Asp Ala Asn Asp Gln Met Ile Lys Glu

1 5 10 15

Ser Phe Phe Ile Thr His Gly Asn Pro Ile Leu Thr Val Glu Asp Thr

20 25 30

His Pro Leu Arg Pro Phe Phe Glu Thr Trp Arg Glu Lys Ile Phe Ser

35 40 45

Lys Lys Pro Lys Ala Ile Leu Ile Ile Ser Gly His Trp Glu Thr Val

50 55 60

Lys Pro Thr Val Asn Ala Val His Ile Asn Asp Thr Ile His Asp Phe

65 70 75 80

Asp Asp Tyr Pro Ala Ala Met Tyr Gln Phe Lys Tyr Pro Ala Pro Gly

85 90 95

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

100 105 110

Phe Glu Thr Ala Glu Thr Asp Gln Lys Arg Gly Leu Asp His Gly Ala

115 120 125

Trp Val Pro Leu Met Leu Met Tyr Pro Glu Ala Asp Ile Pro Val Cys

130 135 140

Gln Leu Ser Val Gln Pro His Leu Asp Gly Thr Tyr His Tyr Asn Leu

145 150 155 160

Gly Arg Ala Leu Ala Pro Leu Lys Asn Asp Gly Val Leu Ile Ile Gly

165 170 175

Ser Gly Ser Ala Thr His Pro Leu Asp Glu Thr Pro His Tyr Phe Asp

180 185 190

Gly Val Ala Pro Trp Ala Ala Ala Phe Asp Ser Trp Leu Arg Lys Ala

195 200 205

Leu Ile Asn Gly Arg Phe Glu Glu Val Asn Ile Tyr Glu Ser Lys Ala

210 215 220

Pro Asn Trp Lys Leu Ala His Pro Phe Pro Glu His Phe Tyr Pro Leu

225 230 235 240

His Val Val Leu Gly Ala Ala Gly Glu Lys Trp Lys Ala Glu Leu Ile

245 250 255

His Ser Ser Trp Asp His Gly Thr Leu Cys His Gly Ser Tyr Lys Phe

260 265 270

Thr Ser Ala

275

<210> 3

<211> 500

<212> PRT

<213> Artificial Sequence

<400> 3

Met Thr Ala Ile Lys Met Asn Thr Asn Gly Glu Gly Glu Thr Gln His

1 5 10 15

Ile Leu Met Ile Pro Phe Met Ala Gln Gly His Leu Arg Pro Phe Leu

20 25 30

Glu Leu Ala Met Phe Leu Tyr Lys Arg Ser His Val Ile Ile Thr Leu

35 40 45

Leu Thr Thr Pro Leu Asn Ala Gly Phe Leu Arg His Leu Leu His His

50 55 60

His Ser Tyr Ser Ser Ser Gly Ile Arg Ile Val Glu Leu Pro Phe Asn

65 70 75 80

Ser Thr Asn His Gly Leu Pro Pro Gly Ile Glu Asn Thr Asp Lys Leu

85 90 95

Thr Leu Pro Leu Val Val Ser Leu Phe His Ser Thr Ile Ser Leu Asp

100 105 110

Pro His Leu Arg Asp Tyr Ile Ser Arg His Phe Ser Pro Ala Arg Pro

115 120 125

Pro Leu Cys Val Ile His Asp Val Phe Leu Gly Trp Val Asp Gln Val

130 135 140

Ala Lys Asp Val Gly Ser Thr Gly Val Val Phe Thr Thr Gly Gly Ala

145 150 155 160

Tyr Gly Thr Ser Ala Tyr Val Ser Ile Trp Asn Asp Leu Pro His Gln

165 170 175

Asn Tyr Ser Asp Asp Gln Glu Phe Pro Leu Pro Gly Phe Pro Glu Asn

180 185 190

His Lys Phe Arg Arg Ser Gln Leu His Arg Phe Leu Arg Tyr Ala Asp

195 200 205

Gly Ser Asp Asp Trp Ser Lys Tyr Phe Gln Pro Gln Leu Arg Gln Ser

210 215 220

Met Lys Ser Phe Gly Trp Leu Cys Asn Ser Val Glu Glu Ile Glu Thr

225 230 235 240

Leu Gly Phe Ser Ile Leu Arg Asn Tyr Thr Lys Leu Pro Ile Trp Gly

245 250 255

Ile Gly Pro Leu Ile Ala Ser Pro Val Gln His Ser Ser Ser Asp Asn

260 265 270

Asn Ser Thr Gly Ala Glu Phe Val Gln Trp Leu Ser Leu Lys Glu Pro

275 280 285

Asp Ser Val Leu Tyr Ile Ser Phe Gly Ser Gln Asn Thr Ile Ser Pro

290 295 300

Thr Gln Met Met Glu Leu Ala Ala Gly Leu Glu Ser Ser Glu Lys Pro

305 310 315 320

Phe Leu Trp Val Ile Arg Ala Pro Phe Gly Phe Asp Ile Asn Glu Glu

325 330 335

Met Arg Pro Glu Trp Leu Pro Glu Gly Phe Glu Glu Arg Met Lys Val

340 345 350

Lys Lys Gln Gly Lys Leu Val Tyr Lys Leu Gly Pro Gln Leu Glu Ile

355 360 365

Leu Asn His Glu Ser Ile Gly Gly Phe Leu Thr His Cys Gly Trp Asn

370 375 380

Ser Ile Leu Glu Ser Leu Arg Glu Gly Val Pro Met Leu Gly Trp Pro

385 390 395 400

Leu Ala Ala Glu Gln Ala Tyr Asn Leu Lys Tyr Leu Glu Asp Glu Met

405 410 415

Gly Val Ala Val Glu Leu Ala Arg Gly Leu Glu Gly Glu Ile Ser Lys

420 425 430

Glu Lys Val Lys Arg Ile Val Glu Met Ile Leu Glu Arg Asn Glu Gly

435 440 445

Ser Lys Gly Trp Glu Met Lys Asn Arg Ala Val Glu Met Gly Lys Lys

450 455 460

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

465 470 475 480

Lys Ala Ile Asp Asp Phe Leu Asp Ala Val Met Gln Ala Lys Leu Glu

485 490 495

Pro Ser Leu Gln

500

<210> 4

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 4

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 5

<211> 1491

<212> DNA

<213> Artificial Sequence

<400> 5

atggatcatg cgaccctcgc catgatcctc gcgatctggt tcatcagctt ccacttcatc 60

aagctgctgt tctcccagca gaccaccaag ctgcttccgc caggaccaaa gccgcttccg 120

atcatcggca acatccttga ggtgggcaag aagccgcatc ggtccttcgc caacctcgcc 180

aagattcacg gcccactcat ttccctcaga ctcggctctg tgaccaccat cgttgtgtcc 240

tctgccgacg tggccaaaga gatgttcctc aagaaggatc acccgctctc caaccgcacg 300

atcccgaata gtgttacagc cggcgaccac cacaagctca ccatgtcttg gctcccggtg 360

tctccgaagt ggcgcaactt ccgcaagatt accgccgtgc atctgctctc cccacagaga 420

ctcgatgcct gccagacatt caggcacgcc aaggtgcagc agctctacga gtacgttcaa 480

gagtgcgccc agaaaggcca ggccgtggat attggcaagg ccgcctttac gaccagcctc 540

aacctcctca gcaagctgtt cttcagcgtc gagctggcgc accacaagtc ccataccagc 600

caagagttca aagagctgat ctggaacatc atggaagata taggcaagcc gaactacgcc 660

gactacttcc cgattctcgg ctgcgttgac ccatctggca ttagaagaag gctcgcctgc 720

tccttcgaca agctgatcgc cgtgttccag ggcatcatct gcgagagact cgccccagat 780

tcctccacca caactaccac caccaccgac gacgtgctcg atgtgctcct ccagctgttc 840

aagcagaacg agctgacgat gggcgagatc aaccacctcc tcgtggacat cttcgacgcc 900

ggcaccgata ccacatcctc cacattcgag tgggtgatga ccgagctgat ccgcaatcca 960

gagatgatgg aaaaggccca agaggaaatc aagcaggtcc tcggcaagga caagcagatc 1020

caagagtccg acatcatcaa cctgccgtac ctccaggcga tcatcaaaga gacactccgc 1080

ctccatccgc cgaccgtgtt cttgctccca agaaaggccg acaccgatgt cgagctgtac 1140

ggctacatcg tgccgaagga tgcccagatc ctcgtgaacc tctgggccat tggcagggac 1200

ccaaacgcct ggcagaacgc cgatattttc agcccagagc gcttcatcgg ctgcgagatc 1260

gatgttaagg gccgcgattt cggcctcctt ccatttggcg ctggccgcag aatttgccca 1320

ggcatgaatc tcgccatcag gatgctcacc ctcatgctcg ccacactcct ccagttcttc 1380

aactggaagc tcgaaggcga catctccccg aaggacctcg acatggacga gaagttcggc 1440

attgcgctcc aaaagaccaa gccgctcaag ctcatcccga ttccgcgcta c 1491

<210> 6

<211> 822

<212> DNA

<213> Artificial Sequence

<400> 6

aagatgatga acggcgagga cgccaacgac cagatgatca aagagtcctt cttcatcacc 60

cacggcaacc cgatcctcac cgtcgaggat acacatccgc tcaggccgtt cttcgagaca 120

tggcgcgaga agattttctc caagaagccg aaggccatcc tcatcatctc cggccactgg 180

gagacagtga agccaaccgt gaacgccgtg cacatcaacg acaccatcca cgacttcgac 240

gactacccag ccgccatgta ccagttcaag tacccagctc caggcgagcc agagcttgcg 300

agaaaggtgg aagagatcct caagaagtcc gggttcgaga cagccgagac agaccaaaag 360

aggggccttg atcacggcgc ctgggttcca ctcatgctca tgtatccaga ggcggacatc 420

ccggtgtgcc agctctcagt tcagccacat ctcgacggca cctaccacta caatctcggc 480

agagccctcg cgccgctcaa gaatgatggc gtgctcatta ttggctccgg cagcgccaca 540

catccactcg atgagacacc gcactacttc gatggtgttg ccccttgggc cgctgccttc 600

gattcttggc ttaggaaggc cctcatcaac ggccgcttcg aggaagtgaa catctacgag 660

agcaaggccc cgaactggaa gctcgcccat ccatttccag agcacttcta cccgctccac 720

gttgtgctcg gcgctgctgg tgaaaagtgg aaggccgagc tgatccactc ctcctgggat 780

catggcacac tttgccacgg ctcctacaag ttcacctccg cc 822

<210> 7

<211> 1497

<212> DNA

<213> Artificial Sequence

<400> 7

accgccatca agatgaacac caacggcgag ggcgagacac agcacatcct catgatcccg 60

ttcatggcgc agggccacct caggccattt ctcgaactcg ccatgttcct ctacaagcgc 120

tcccacgtga tcatcaccct gctcacaact ccgctcaacg ccggcttcct caggcacctc 180

cttcaccacc attcctactc ctccagcggc atcaggatcg tcgagctgcc attcaactcc 240

accaaccacg gactcccacc gggcatcgag aacaccgata agctcacact cccgctcgtg 300

gtgtccctct tccattccac catcagcctc gatccgcacc tccgcgatta catctccagg 360

catttcagcc cagccaggcc accactctgc gtgatccatg atgtgttcct cggctgggtt 420

gaccaggtgg ccaaggatgt gggctctaca ggcgtggtgt tcacaacagg cggcgcttat 480

ggcacatccg cctacgtgtc catctggaac gatctcccgc accagaacta ctccgacgac 540

caagagttcc cgctgccagg cttcccagag aaccataagt tccgcaggtc ccagctccat 600

cggttcctca gatatgccga cggctccgac gattggtcca agtatttcca gccgcagctc 660

cgccagtcca tgaagtcttt tggctggctc tgcaactccg tggaagagat cgagacactc 720

ggcttctcca tcctccgcaa ctacaccaag ctgccgatct ggggcatcgg cccacttatt 780

gcttccccag tgcagcactc ctcctccgac aacaattcaa caggcgccga gttcgtgcag 840

tggctcagcc tcaaagagcc ggactccgtc ctctacatct ccttcggctc ccagaacacg 900

atcagcccga cgcagatgat ggaactcgct gctggccttg agtcctccga gaagccattc 960

ctctgggtga tcagagcccc gttcggcttc gacatcaacg aagagatgcg cccagagtgg 1020

ctgccagagg gctttgagga acgcatgaag gtgaagaaac agggcaagct cgtgtacaag 1080

ctcggcccgc agcttgagat cctcaaccat gaatccatcg gcggctttct cacccactgc 1140

ggatggaaca gcatccttga gtctcttcgc gagggcgttc cgatgcttgg atggccactt 1200

gctgccgagc aggcctacaa cctcaagtac ctcgaagatg agatgggcgt cgcggttgag 1260

cttgctagag gcctcgaagg cgagatctcc aaagagaagg tcaagcgcat cgtcgagatg 1320

atccttgagc gcaacgaggg ctccaaaggc tgggagatga agaatcgcgc cgtggaaatg 1380

ggcaaaaagc tcaaggacgc cgtgaacgag gaaaaagagc tgaagggctc ctccgtgaag 1440

gcgatcgacg atttcctcga cgccgtcatg caggccaaac ttgagccaag cctccag 1497

<210> 8

<211> 66

<212> DNA

<213> Artificial Sequence

<400> 8

ggtagcggag ctaccaattt tagcctcctt aagcaggcag gtgatgtaga agagaacccc 60

gggcct 66

<210> 9

<211> 66

<212> DNA

<213> Artificial Sequence

<400> 9

ggatccggag caaccaactt tagcctgctc aagcaagcag gagatgttga ggaaaatcct 60

ggcccc 66

<210> 10

<211> 907

<212> DNA

<213> Artificial Sequence

<400> 10

gcatactcga ggtcattcat atgcttgaga agagagtcgg gatagtccaa aataaaacaa 60

aggtaagatt acctggtcaa aagtgaaaac atcagttaaa aggtggtata aagtaaaata 120

tcggtaataa aaggtggccc aaagtgaaat ttactctttt ctactattat aaaaattgag 180

gatgtttttg tcggtacttt gatacgtcat ttttgtatga attggtttttt aagtttattc 240

gcttttggaa atgcatatct gtatttgagt cgggttttaa gttcgtttgc ttttgtaaat 300

acagagggat ttgtataaga aatatcttta aaaaaaccca tatgctaatt tgacataatt 360

tttgagaaaa atatatattc aggcgaattc tcacaatgaa caataataag attaaaatag 420

ctttcccccg ttgcagcgca tgggtatttt ttctagtaaa aataaaagat aaacttagac 480

tcaaaacatt tacaaaaaca acccctaaag ttcctaaagc ccaaagtgct atccacgatc 540

catagcaagc ccagcccaac ccaacccaac ccaacccacc ccagtccagc caactggaca 600

atagtctcca caccccccca ctatcaccgt gagttgtccg cacgcaccgc acgtctcgca 660

gccaaaaaaa aaaaaagaaa gaaaaaaaag aaaaagaaaa aacagcaggt gggtccgggt 720

cgtgggggcc ggaaacgcga ggaggatcgc gagccagcga cgaggccggc cctccctccg 780

cttccaaaga aacgcccccc atcgccacta tatacatacc cccccctctc ctcccatccc 840

cccaacccta ccaccaccac caccaccacc tccacctcct cccccctcgc tgccggacga 900

cgatctc 907

<210> 11

<211> 250

<212> DNA

<213> Artificial Sequence

<400> 11

atatgaagat gaagatgaaa tatttggtgt gtcaaataaa aaggttgtgt gcttaagttt 60

gtgttttttt cttggcttgt tgtgttatga atttgtggct ttttctaata ttaaatgaat 120

gtaacatctc attataatga ataaacaaat gtttctataa tccattgtga atgttttgtt 180

ggatctcttc tccagcatat aactactgta tgtgctatgg tatggactat ggaatatgat 240

taaagataag 250

<210> 12

<211> 4131

<212> DNA

<213> Artificial Sequence

<400> 12

atggccccaa agaagaagcg caaggtcgac aagaagtact ccatcggcct cgacatcggc 60

accaattctg ttggctgggc cgtgatcacc gacgagtaca aggtgccgtc caagaagttc 120

aaggtcctcg gcaacaccga ccgccactcc atcaagaaga atctcatcgg cgccctgctg 180

ttcgactctg gcgagacagc cgaggctaca aggctcaaga ggaccgctag acgcaggtac 240

accaggcgca agaaccgcat ctgctacctc caagagatct tctccaacga gatggccaag 300

gtggacgaca gcttcttcca caggctcgag gagagcttcc tcgtcgagga ggacaagaag 360

cacgagcgcc atccgatctt cggcaacatc gtggatgagg tggcctacca cgagaagtac 420

ccgaccatct accacctccg caagaagctc gtcgactcca ccgataaggc cgacctcagg 480

ctcatctacc tcgccctcgc ccacatgatc aagttcaggg gccacttcct catcgagggc 540

gacctcaacc cggacaactc cgatgtggac aagctgttca tccagctcgt gcagacctac 600

aaccagctgt tcgaggagaa cccgatcaac gcctctggcg ttgacgccaa ggctattctc 660

tctgccaggc tctctaagtc ccgcaggctc gagaatctga tcgcccaact tccgggcgag 720

aagaagaatg gcctcttcgg caacctgatc gccctctctc ttggcctcac cccgaacttc 780

aagtccaact tcgacctcgc cgaggacgcc aagctccagc tttccaagga cacctacgac 840

gacgacctcg acaatctcct cgcccagatt ggcgatcagt acgccgatct gttcctcgcc 900

gccaagaatc tctccgacgc catcctcctc agcgacatcc tcagggtgaa caccgagatc 960

accaaggccc cactctccgc ctccatgatc aagaggtacg acgagcacca ccaggacctc 1020

acactcctca aggccctcgt gagacagcag ctcccagaga agtacaagga gatcttcttc 1080

gaccagtcca agaacggcta cgccggctac atcgatggcg gcgcttctca agaggagttc 1140

tacaagttca tcaagccgat cctcgagaag atggacggca ccgaggagct gctcgtgaag 1200

ctcaatagag aggacctcct ccgcaagcag cgcaccttcg ataatggctc catcccgcac 1260

cagatccacc tcggcgagct tcatgctatc ctccgcaggc aagaggactt ctacccgttc 1320

ctcaaggaca accgcgagaa gattgagaag atcctcacct tccgcatccc gtactacgtg 1380

ggcccgctcg ccaggggcaa ctccaggttc gcctggatga ccagaaagtc cgaggagaca 1440

atcaccccct ggaacttcga ggaggtggtg gataagggcg cctctgccca gtctttcatc 1500

gagcgcatga ccaacttcga caagaacctc ccgaacgaga aggtgctccc gaagcactca 1560

ctcctctacg agtacttcac cgtgtacaac gagctgacca aggtgaagta cgtgaccgag 1620

gggatgagga agccagcttt ccttagcggc gagcaaaaga aggccatcgt cgacctgctg 1680

ttcaagacca accgcaaggt gaccgtgaag cagctcaagg aggactactt caagaaaatc 1740

gagtgcttcg actccgtcga gatctccggc gtcgaggata ggttcaatgc ctccctcggg 1800

acctaccacg acctcctcaa gattatcaag gacaaggact tcctcgacaa cgaggagaac 1860

gaggacatcc tcgaggacat cgtgctcacc ctcaccctct tcgaggaccg cgagatgatc 1920

gaggagcgcc tcaagacata cgcccacctc ttcgacgaca aggtgatgaa gcagctgaag 1980

cgcaggcgct ataccggctg gggcaggctc tctaggaagc tcatcaacgg catccgcgac 2040

aagcagtccg gcaagacgat cctcgacttc ctcaagtccg acggcttcgc caaccgcaac 2100

ttcatgcagc tcatccacga cgactccctc accttcaagg aggacatcca aaaggcccag 2160

gtgtccggcc aaggcgattc cctccatgag catatcgcca atctcgccgg ctccccggct 2220

atcaagaagg gcattctcca gaccgtgaag gtggtggacg agctggtgaa ggtgatgggc 2280

aggcacaagc cagagaacat cgtgatcgag atggcccgcg agaaccagac cacacagaag 2340

ggccaaaaga actcccgcga gcgcatgaag aggatcgagg agggcattaa ggagctgggc 2400

tcccagatcc tcaaggagca cccagtcgag aacacccagc tccagaacga gaagctctac 2460

ctctactacc tccagaacgg ccgcgacatg tacgtggacc aagagctgga catcaaccgc 2520

ctctccgact acgacgtgga ccatattgtg ccgcagtcct tcctgaagga cgactccatc 2580

gacaacaagg tgctcacccg ctccgacaag aacaggggca agtccgataa cgtgccgtcc 2640

gaagaggtcg tcaagaagat gaagaactac tggcgccagc tcctcaacgc caagctcatc 2700

acccagagga agttcgacaa cctcaccaag gccgagagag gcggcctttc cgagcttgat 2760

aaggccggct tcatcaagcg ccagctcgtc gagacacgcc agatcacaaa gcacgtggcc 2820

cagatcctcg actcccgcat gaacaccaag tacgacgaga acgacaagct catccgcgag 2880

gtgaaggtca tcaccctcaa gtccaagctc gtgtccgact tccgcaagga cttccagttc 2940

tacaaggtgc gcgagatcaa caactaccac cacgcccacg acgcctacct caatgccgtg 3000

gtgggcacag ccctcatcaa gaagtaccca aagctcgagt ccgagttcgt gtacggcgac 3060

tacaaggtgt acgacgtgcg caagatgatc gccaagtccg agcaagagat cggcaaggcg 3120

accgccaagt acttcttcta ctccaacatc atgaatttct tcaagaccga gatcacgctc 3180

gccaacggcg agattaggaa gaggccgctc atcgagacaa acggcgagac aggcgagatc 3240

gtgtgggaca agggcaggga tttcgccaca gtgcgcaagg tgctctccat gccgcaagtg 3300

aacatcgtga agaagaccga ggttcagacc ggcggcttct ccaaggagtc catcctccca 3360

aagcgcaact ccgacaagct gatcgcccgc aagaaggact gggacccgaa gaagtatggc 3420

ggcttcgatt ctccgaccgt ggcctactct gtgctcgtgg ttgccaaggt cgagaagggc 3480

aagagcaaga agctcaagtc cgtcaaggag ctgctgggca tcacgatcat ggagcgcagc 3540

agcttcgaga agaacccaat cgacttcctc gaggccaagg gctacaagga ggtgaagaag 3600

gacctcatca tcaagctccc gaagtacagc ctcttcgagc ttgagaacgg ccgcaagaga 3660

atgctcgcct ctgctggcga gcttcagaag ggcaacgagc ttgctctccc gtccaagtac 3720

gtgaacttcc tctacctcgc ctcccactac gagaagctca agggctcccc agaggacaac 3780

gagcaaaagc agctgttcgt cgagcagcac aagcactacc tcgacgagat catcgagcag 3840

atctccgagt tctccaagcg cgtgatcctc gccgatgcca acctcgataa ggtgctcagc 3900

gcctacaaca agcaccgcga taagccaatt cgcgagcagg ccgagaacat catccacctc 3960

ttcaccctca ccaacctcgg cgctccagcc gccttcaagt acttcgacac caccatcgac 4020

cgcaagcgct acacctctac caaggaggtt ctcgacgcca ccctcatcca ccagtctatc 4080

acaggcctct acgagacacg catcgacctc tcacaactcg gcggcgattg a 4131

<210> 13

<211> 1756

<212> DNA

<213> Artificial Sequence

<400> 13

accgccatca agatgaacac caacggcgag ggcgagacac agcacatcct catgatcccg 60

ttcatggcgc agggccacct caggccattt ctcgaactcg ccatgttcct ctacaagcgc 120

tcccacgtga tcatcaccct gctcacaact ccgctcaacg ccggcttcct caggcacctc 180

cttcaccacc attcctactc ctccagcggc atcaggatcg tcgagctgcc attcaactcc 240

accaaccacg gactcccacc gggcatcgag aacaccgata agctcacact cccgctcgtg 300

gtgtccctct tccattccac catcagcctc gatccgcacc tccgcgatta catctccagg 360

catttcagcc cagccaggcc accactctgc gtgatccatg atgtgttcct cggctgggtt 420

gaccaggtgg ccaaggatgt gggctctaca ggcgtggtgt tcacaacagg cggcgcttat 480

ggcacatccg cctacgtgtc catctggaac gatctcccgc accagaacta ctccgacgac 540

caagagttcc cgctgccagg cttcccagag aaccataagt tccgcaggtc ccagctccat 600

cggttcctca gatatgccga cggctccgac gattggtcca agtatttcca gccgcagctc 660

cgccagtcca tgaagtcttt tggctggctc tgcaactccg tggaagagat cgagacactc 720

ggcttctcca tcctccgcaa ctacaccaag ctgccgatct ggggcatcgg cccacttatt 780

gcttccccag tgcagcactc ctcctccgac aacaattcaa caggcgccga gttcgtgcag 840

tggctcagcc tcaaagagcc ggactccgtc ctctacatct ccttcggctc ccagaacacg 900

atcagcccga cgcagatgat ggaactcgct gctggccttg agtcctccga gaagccattc 960

ctctgggtga tcagagcccc gttcggcttc gacatcaacg aagagatgcg cccagagtgg 1020

ctgccagagg gctttgagga acgcatgaag gtgaagaaac agggcaagct cgtgtacaag 1080

ctcggcccgc agcttgagat cctcaaccat gaatccatcg gcggctttct cacccactgc 1140

ggatggaaca gcatccttga gtctcttcgc gagggcgttc cgatgcttgg atggccactt 1200

gctgccgagc aggcctacaa cctcaagtac ctcgaagatg agatgggcgt cgcggttgag 1260

cttgctagag gcctcgaagg cgagatctcc aaagagaagg tcaagcgcat cgtcgagatg 1320

atccttgagc gcaacgaggg ctccaaaggc tgggagatga agaatcgcgc cgtggaaatg 1380

ggcaaaaagc tcaaggacgc cgtgaacgag gaaaaagagc tgaagggctc ctccgtgaag 1440

gcgatcgacg atttcctcga cgccgtcatg caggccaaac ttgagccaag cctccagtga 1500

tagtgaatat gaagatgaag atgaaatatt tggtgtgtca aataaaaagg ttgtgtgctt 1560

aagtttgtgt ttttttcttg gcttgttgtg ttatgaattt gtggcttttt ctaatattaa 1620

atgaatgtaa catctcatta taatgaataa acaaatgttt ctataatcca ttgtgaatgt 1680

tttgttggat ctcttctcca gcatataact actgtatgtg ctatggtatg gactatggaa 1740

tatgattaaa gataag 1756

<210> 14

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 14

gacgagcttg ctgtcgacg 19

<210> 15

<211> 76

<212> DNA

<213> Artificial Sequence

<400> 15

gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60

ggcaccgagt cggtgc 76

<210> 16

<211> 457

<212> DNA

<213> Artificial Sequence

<400> 16

tatgtacagc attacgtagg tacgttttct ttttcttccc ggagagatga tacgataatc 60

atgtaaaccc agaatttaaa aaatattctt tactataaaa attttaatta gggaacgtat 120

tattttttac atgacacctt ttgagaaaga gggacttgta atatgggaca aatgaacaat 180

ttctaagaaa tgggcatatg actctcagta caatggacca aattccctcc agtcggccca 240

gcaatacaaa gggaaagaaa tgagggggcc cacaggccac ggcccacttt tctccgtggt 300

ggggagatcc agctagaggt ccggcccaca agtggccctt gccccgtggg acggtgggat 360

tgcagagcgc gtgggcggaa acaacagttt agtaccacct cgctcacgca acgacgcgac 420

cacttgctta taagctgctg cgctgaggct caggttg 457

<210> 17

<211> 252

<212> DNA

<213> Artificial Sequence

<400> 17

tttttttttt cgttttgcat tgagttttct ccgtcgcatg tttgcagttt tattttccgt 60

tttgcattga aatttctccg tctcatgttt gcagcgtgtt caaaaagtac gcagctgtat 120

ttcacttatt tacggcgcca cattttcatg ccgtttgtgc caactatccc gagctagtga 180

atacagcttg gcttcacaca acactggtga cccgctgacc tgctcgtacc tcgtaccgtc 240

gtacggcaca gc 252

<210> 18

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 18

cactgatagt ttaaactagt atggatcatg cgaccctcgc 40

<210> 19

<211> 60

<212> DNA

<213> Artificial Sequence

<400> 19

cacctgcctg cttaaggagg ctaaaattgg tagctccgct accgtagcgc ggaatcggga 60

<210> 20

<211> 60

<212> DNA

<213> Artificial Sequence

<400> 20

ccttaagcag gcaggtgatg tagaagagaa ccccgggcct atgaagatga tgaacggcga 60

<210> 21

<211> 60

<212> DNA

<213> Artificial Sequence

<400> 21

ctcctgcttg cttgagcagg ctaaagttgg ttgctccgga tccggcggag gtgaacttgt 60

<210> 22

<211> 60

<212> DNA

<213> Artificial Sequence

<400> 22

gctcaagcaa gcaggagatg ttgaggaaaa tcctggcccc atgaccgcca tcaagatgaa 60

<210> 23

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 23

gctagcttac tcagttaggt cttatcttta atcatattcc 40

<210> 24

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 24

cccgggggat ccccaatact atggccccaa agaagaagcg caagg 45

<210> 25

<211> 46

<212> DNA

<213> Artificial Sequence

<400> 25

gaaattcgga tccccaatac ttcaatcgcc gccgagttgt gagagg 46

<210> 26

<211> 49

<212> DNA

<213> Artificial Sequence

<400> 26

tacgaattcg agctcggtac gcatactcga ggtcattcat atgcttgag 49

<210> 27

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 27

atcccccttt cgccaggggt accgagatcg tcgtccggca gc 42

<210> 28

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 28

tgccggacga cgatctcggt accatggatc atgcgaccct cg 42

<210> 29

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 29

cacatcccccc tttcgccagg gttaacctta tctttaatca tattccatag tccatacca 59

<210> 30

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 30

gtcgtttccc gccttcagtt tatgtacagc attacgtagg 40

<210> 31

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 31

cgtcgacagc aagctcgtcc aacctgagcc tcagcgcagc agc 43

<210> 32

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 32

ctgtcaaaca ctgatagttt aaacgctgtg ccgtacgacg gtacg 45

<210> 33

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 33

ggacgagctt gctgtcgacg gttttagagc tagaaatagc aagttaaaat 50

<210> 34

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 34

cacggaacgg agaccccta 19

<210> 35

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 35

agcggtccaa agtagagcac 20

<210> 36

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 36

ggtagcggag ctaccaattt tagcctcctt aagcaggcag gtg 43

<210> 37

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 37

ccttaagcag gcaggtgatg tagaagagaa ccccgggcct 40

<210> 38

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 38

ggatccggag caaccaactt tagcctgctc aagcaagcag gag 43

<210> 39

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 39

gctcaagcaa gcaggagatg ttgaggaaaa tcctggcccc 40

Claims (12)

1. a screening system that improves the probability of obtaining a gene-edited plant, is characterized in that, the screening system is a plant genetic transformation vector comprising a RUBY gene expression cassette and a CRISPR/Cas gene editing system; the RUBY gene expression cassette comprises sequentially Set promoter, RUBY gene, terminator. 2. The screening system for improving the probability of obtaining a gene-edited plant according to claim 1, wherein the RUBY gene comprises CYP76AD1 gene, DODA gene and GT gene; The nucleotide sequence of the CYP76AD1 gene comprises the nucleotide sequence encoding the amino acid sequence of CYP76AD1 shown in SEQ ID NO.1, The nucleotide sequence of the DODA gene comprises the nucleotide sequence encoding the DODA amino acid sequence shown in SEQ ID NO.2, The nucleotide sequence of the GT gene comprises the nucleotide sequence encoding the GT amino acid sequence shown in SEQ ID NO.3; preferably, the nucleotide sequence of the CYP76AD1 gene is shown in SEQ ID NO.5, and the The nucleotide sequence of the DODA gene is shown in SEQ ID NO.6, and the nucleotide sequence of the GT gene is shown in SEQ ID NO.7. 3 . The screening system for increasing the probability of obtaining gene-edited plants according to claim 2 , wherein the CYP76AD1 gene, the DODA gene and the GT gene are linked in any order by a DNA linking unit. 4 . 4. The screening system for improving the probability of obtaining a gene-edited plant according to claim 3, wherein the DNA linking unit is a DNA sequence capable of being transcribed and translated into a self-cleaving functional polypeptide; Preferably, the nucleotide sequence of the DNA linking unit comprises the nucleotide sequence encoding the amino acid sequence of the 2A peptide shown in SEQ ID NO.4; Further preferably, the DNA linking unit is 2A1 whose nucleotide sequence is shown in SEQ ID NO.8, or 2A2 whose nucleotide sequence is shown in SEQ ID NO.9. 5. The screening system for improving the probability of obtaining gene-edited plants according to claim 1, wherein the promoter is a promoter capable of functioning in plants, and the terminator is a promoter capable of functioning in plants terminator; Preferably, the promoter is a promoter capable of functioning in dicotyledonous plants or monocotyledonous plants, and the terminator is a terminator capable of functioning in dicotyledonous plants or monocotyledonous plants; Further preferably, the promoter is a promoter that can function in rice, and the terminator is a terminator that can function in rice; More preferably, the promoter is the promoter of OsActin1, and the terminator is tHsp. 6. The screening system for improving the probability of obtaining gene-edited plants according to claim 1, wherein the CRISPR/Cas gene-editing system comprises a Cas protein expression cassette and a gRNA transcription cassette. 7. A method for constructing a screening system for improving the probability of obtaining a gene-edited plant, comprising the following steps: 1) construct RUBY gene, connect RUBY gene and terminator to obtain RUBY-terminator DNA fragment; 2) connecting the Cas protein expression cassette into a plant genetic transformation vector to construct a vector pCas with the Cas protein expression cassette; 3) connecting the promoter into the vector pCas obtained in step 2), where the promoter is connected outside the Cas expression cassette, to construct a p-promoter-Cas vector; 4) The RUBY-terminator DNA fragment obtained in step 1) is constructed on the p-promoter-Cas carrier, and the RUBY gene-terminator DNA fragment is connected to the downstream of the promoter in step 3), forming the RUBY gene with the promoter. Expression cassette to obtain pRUBY-CRISPR vector; 5) Select the target site of the target gene, construct the gRNA transcription cassette capable of targeting the target site on the pRUBY-CRISPR vector, and obtain the screening system. 8. The method for constructing a screening system for improving the probability of obtaining gene-edited plants according to claim 7, wherein the specific operation of the step 1) is: respectively obtaining CYP76AD1 gene, DODA gene, GT gene, termination Sub-fragment; using the method of in vitro overlap extension PCR, three DNA molecules of CYP76AD1 gene, DODA gene and GT gene are combined into the RUBY gene in any order through the DNA linking unit, and the terminator is connected after the RUBY gene to obtain the RUBY gene-terminator DNA fragment; Preferably, the specific operation of the step 1) is as follows: respectively obtaining CYP76AD1 gene, DODA gene and GT gene-terminator DNA fragments; using the method of in vitro overlap extension PCR to obtain CYP76AD1 gene, DODA gene, GT gene-terminator The DNA fragments are sequentially connected through a DNA linking unit to obtain RUBY gene-terminator DNA fragments. 9. The construction method of a screening system for improving the probability of obtaining a gene-edited plant according to claim 7, wherein the specific operation of the step 5) is: according to the target gene target site, design a 3'-end overhang The primers that can be complementary paired are amplified by overlapping PCR in vitro, and the gRNA transcription cassette that can target the target gene is connected to the pRUBY-CRISPR vector obtained in step 4). The gRNA transcription cassette is in the Cas expression cassette and the RUBY expression cassette. In addition, the screening system is obtained. 10. The application of the screening system for increasing the probability of obtaining gene-edited plants according to claim 1 or the construction method of the screening system for increasing the probability of obtaining gene-edited plants according to claim 7 in increasing the probability of obtaining gene-edited plants. 11 . The application according to claim 10 , wherein the screening system is used to perform gene editing on plants, and when the plant tissue expresses the color of betalain, it indicates that the gene-edited plant has undergone a target site mutation. 12 . 12. The application according to claim 10, wherein the gene editing can occur in all kinds of plant whole or plant parts or plant cells, Preferably, the plant is a dicotyledonous plant and/or a monocotyledonous plant, Further preferably, the plant is rice.

Images