Apple gene editing system and application

Abstract

The invention relates to an apple gene editing system and application. According to the gene editing system, the promoter is adopted to drive sgRNA expression, and stable gene editing can be achieved in apple fruit calluses and tissue culture plants. According to the editing system, stable editing calluses and plants of apples can be obtained, albino phenotype calluses, albino plants and early blossoming apple materials are obtained, effective tools and technical supports are provided for apple molecular breeding, and the editing system can be applied to efficient cultivation of albino strains and early blossoming strains of the apples.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant gene improvement, and specifically relates to an apple gene editing system and application. Background Art

[0002] Apple (Malus×domestica Borkh.), a plant of the genus Malus in the Rosaceae family, is one of the important economic fruit trees in my country. It is loved by the public for its good flavor. my country is the world's largest apple producer and consumer. Apple breeding is crucial to the development of my country's apple industry. Current breeding methods mainly include conventional breeding techniques and assisted breeding techniques, and the long juvenile period of apples greatly limits the progress of apple breeding. In order to accelerate the development of varieties with better adaptability, new breeding technologies have become a promising alternative to meet the needs of the growing global population. Molecular-assisted breeding using transgenics and CRISPR / Cas genome editing has great potential in improving crop traits.

[0003] In woody plants, due to the difficulty of genetic transformation, the exploration of gene function mostly relies on transient transformation. In stable genetic transformation, siRNA interference is mainly used to silence gene expression, but siRNA molecules are easily degraded by nucleases and require a vector system to protect and effectively deliver them to the target cells. In addition, siRNA may cause non-specific gene silencing, so that the subsequent transformed plants are not stable. Through the CRISPR / Cas system, specific genes can be edited through specific PAM sequences and sgRNA, providing new ideas and directions for genetic improvement and molecular breeding of woody plants. At present, CRISPR / Cas9 technology has been used to edit target genes in many plants, but in most woody plants, due to low genetic transformation efficiency and extremely low gene editing efficiency, gene editing is difficult to be widely used. It is an urgent problem to optimize apple genetic transformation technology and modify CRISPR / Cas9 vectors to provide a fast and effective tool for apple germplasm improvement. Summary of the invention

[0004] In view of the defects in the prior art, the purpose of the present invention is to provide an apple gene editing system and application. The editing system uses a promoter to drive sgRNA expression, which can achieve stable gene editing in apple fruit callus and tissue culture plants. The editing system can be applied to the efficient cultivation of apple albino strains and early flowering strains.

[0005] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0006] An apple gene editing system, characterized by comprising a promoter and sgRNA.

[0007] On the basis of the above scheme,

[0008] The promoter is: the AtU6-1 promoter shown in SEQ ID NO.20 or the AtU6-26 promoter shown in SEQ ID NO.21.

[0009] On the basis of the above scheme,

[0010] The sgRNA is: the sgRNA shown in SEQ ID NO.1-7. Among them, SEQ ID NO.1-5 is PDSsgRNA; SEQ ID NO.6-7 is TFL1sgRNA.

[0011] The application of the above sgRNA is characterized in that: the sgRNA shown in SEQ ID NO.1-5 is used to edit apple callus to obtain the target gene sequence shown in SEQ ID NO.8-11, so as to obtain albino callus, specifically:

[0012] (1) The sequence shown in SEQ ID NO.8 is the nucleotide sequence of the target position where 5 bp (TCGGA) is deleted from the fourth base A upstream of the PAM sequence TGG in SEQ ID NO.1 ( Fig. 9 );

[0013] (2) The sequence shown in SEQ ID NO.9 is the nucleotide sequence of the target position after the deletion of 837 bp of bases between the fourth base G upstream of the PAM sequence AGG in the fragment shown in SEQ ID NO.3 and the fourth base T upstream of the PAM sequence GGG in the fragment shown in SEQ ID NO.4 (shown in SEQ ID NO.26, the 837 bp bases are located from the 808 bp to the 1644 bp in the full-length gene of MdPDS shown in SEQ ID NO.47) ( Fig.11 );

[0014] (3) The sequence shown in SEQ ID NO.10 is the nucleotide sequence of the target position for deletion of the fourth single base A upstream of the PAM sequence AGG in SEQ ID NO.5 ( Fig.12 );

[0015] (4) The sequence shown in SEQ ID NO.11 is the nucleotide sequence of the target position for deletion starting from the fourth base A upstream of the PAM sequence TGG in SEQ ID NO.1 ( Fig.13 );

[0016] The application of the above sgRNA is characterized in that: the sgRNA shown in SEQ ID NO.1-5 is used to edit apple tissue culture seedlings to obtain the target gene sequence shown in SEQ ID NO.12-15, so as to obtain albino apple plants, specifically:

[0017] (1) The sequence shown in SEQ ID NO. 12 is the nucleotide sequence of the target position after the 7 bp (TAGTGAT) from the first base T upstream of the PAM sequence GGG of SEQ ID NO. 4 is deleted ( Fig.14 );

[0018] (2) The sequence shown in SEQ ID NO. 13 is the nucleotide sequence of the target position after deleting 2 bp (TA) bases from the 4th base A upstream of the PAM sequence AGG in SEQ ID NO. 5 ( Fig.17 );

[0019] (3) The sequence shown in SEQ ID NO.14 is the nucleotide sequence of the target position after the 2 bp (AG) bases from the first base G upstream of the PAM sequence AGG in SEQ ID NO.5 are deleted and the third base C is mutated to G ( Fig.18 );

[0020] (4) The sequence shown in SEQ ID NO. 15 is the nucleotide sequence of the target position after deleting 3 bp (TAC) from the 3rd base C upstream of the PAM sequence AGG in SEQ ID NO. 5 ( Fig.18 );

[0021] The application of the above sgRNA is characterized in that: the sgRNA shown in SEQ ID NO.6-7 is used to edit apple tissue culture seedlings to obtain the target gene sequence shown in SEQ ID NO.16-19, so as to obtain early-flowering apple plants:

[0022] (1) The sequence shown in SEQ ID NO.16 is the nucleotide sequence of the target position after 1 bp (T) is inserted before the third base T upstream of the PAM sequence TGG in SEQ ID NO.6 ( Fig. 22 );

[0023] (2) The sequence shown in SEQ ID NO.17 is the nucleotide sequence of the target position after deleting 10 bp (CCTCTGGTTG) from the second base G upstream of the PAM sequence TGG in SEQ ID NO.6 ( Fig. 22 );

[0024] (3) The sequence shown in SEQ ID NO.18 is the nucleotide sequence of the target position after 3 bp (TTG) from the second base G of the PAM sequence TGG of SEQ ID NO.6 is deleted ( Fig.23 );

[0025] (4) The sequence shown in SEQ ID NO.19 is the nucleotide sequence of the target position after the fourth base T upstream of the PAM sequence TGG of SEQ ID NO.6 is mutated to G ( Fig.23 );

[0026] The application of the above-mentioned apple gene editing system is characterized in that the application is: in editing the apple genome, constructing apple mutants, and creating albino apple calli, albino apples or early-flowering apple plants.

[0027] The application of the above-mentioned sgRNA is characterized in that the application is: application in editing the apple genome, constructing apple mutants, and creating albino apple calli and albino apple plants.

[0028] The application of the above-mentioned sgRNA is characterized in that the application is: application in editing the apple genome, constructing apple mutants and creating early-flowering apple plants.

[0029] A gene editing method for apples, characterized by comprising the following steps:

[0030] Step 1, clone AtU6-1 and AtU6-26 promoters from Arabidopsis thaliana and construct them into CRISPR / Cas9 vector;

[0031] Step 2, designing multiple target sequences on different exons based on the nucleotide sequences of apple MdPDS and MdTFL1 genes;

[0032] Step 3, constructing apple MdPDS and MdTFL1 gene knockout vectors;

[0033] Step 4, transforming the constructed vector into EHA105 Agrobacterium;

[0034] Step 5, using the transformed Agrobacterium to perform stable genetic transformation on apple fruit callus or apple tissue culture seedlings.

[0035] The gene editing system and application of apples described in the present invention have the following beneficial effects:

[0036] 1. Provides a gene editing system for apple fruit callus and tissue culture seedlings, and provides a complete method for obtaining albino strains and early flowering apple materials. Positive callus can be obtained in 2 months, and positive apple plants can be obtained in 3 months. MdPDS gene-edited callus and apple seedlings appear white or light green; MdTFL1-edited apple seedlings can bloom after growing in tissue culture bottles for about 40 days.

[0037] 2. The gene editing vectors driven by AtU6-1 and AtU6-26 promoters to express sgRNA can be directly used for gene editing in apple callus and apple plants, providing a simple and efficient molecular tool for gene function verification and new germplasm creation, laying an important foundation for the exploration of key genes and the discovery of new genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The present invention has the following accompanying drawings:

[0039] Figure 1 Schematic diagram of the first nine exons of the MdPDS gene and the selected target sequence;

[0040] Figure 2 This is a simplified diagram of the gene editing vector structure;

[0041] Figure 3 This is the gel image of agarose gel electrophoresis detection of the recombinant vector;

[0042] Figure 4 Agarose gel electrophoresis image showing positive identification of Cas9-PDS-1-U6-26 transgenic callus;

[0043] Figure 5 Agarose gel electrophoresis image showing positive identification of Cas9-PDS-1-U6-1 transgenic callus;

[0044] Figure 6 Agarose gel electrophoresis image showing positive identification of Cas9-PDS-2-U6-26 transgenic callus;

[0045] Figure 7 Agarose gel electrophoresis image showing positive identification of Cas9-PDS-2-U6-1 transgenic callus;

[0046] Figure 8 Agarose gel electrophoresis image showing positive identification of Cas9-PDS-3-U6-26 transgenic callus;

[0047] Fig. 9 The phenotype and editing status of Cas9-PDS-1-U6-26 transgenic positive callus;

[0048] Fig.10Agarose gel electrophoresis detection gel image of target sequence fragment amplification of Cas9-PDS-2-U6-26 transgenic positive callus;

[0049] Fig.11 The phenotype and editing status of Cas9-PDS-2-AtU6-26 transgenic positive callus;

[0050] Fig.12 The phenotype and editing status of Cas9-PDS-3-AtU6-26 transgenic positive callus;

[0051] Fig.13 The phenotype and editing status of Cas9-PDS-1-AtU6-1 transgenic positive callus;

[0052] Fig.14 The phenotype and editing status of Cas9-PDS-2-AtU6-1 transgenic positive seedlings;

[0053] Fig.15 Agarose gel electrophoresis image showing positive identification of Cas9-PDS-3-AtU6-1 transgenic seedlings;

[0054] Fig.16 Agarose gel electrophoresis image showing positive identification of Cas9-PDS-3-AtU6-26 transgenic seedlings;

[0055] Fig.17 The phenotype and editing status of Cas9-PDS-3-AtU6-1 transgenic positive seedlings;

[0056] Fig.18 The phenotype and editing status of Cas9-PDS-3-AtU6-26 transgenic positive seedlings;

[0057] Fig.19 Schematic diagram of the four exons and target sequence selection of the MdTFL1 gene;

[0058] Fig. 20 This is the gel image of agarose gel electrophoresis detection for positive identification of Cas9-TFL1-U6-26 transgenic seedlings;

[0059] Fig.21 Agarose gel electrophoresis detection gel image for positive identification of Cas9-TFL1-U6-1 transgenic seedlings;

[0060] Fig. 22 Cas9-TFL1-U6-26 transgenic early flowering seedlings and editing status

[0061] Fig.23 Cas9-TFL1-U6-1 transgenic early flowering seedlings and editing status DETAILED DESCRIPTION

[0062] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0063] Apple phytoene dehydrogenase (MdPDS) is a key enzyme in the apple carotenoid biosynthesis pathway. It catalyzes the dehydrogenation of phytoene in plants to generate ζ-carotene, completing the transformation of plant carotenoids from colorless to colored. It plays a rate-limiting role in the biosynthesis of plant carotenoids. Inhibiting the expression of this enzyme will lead to a large accumulation of phytoene, which in turn affects the accumulation of carotenoids in plants and causes the plants to show a white phenotype. The apple Terminal Flower 1 (MdTFL1) gene is an apple flowering inhibition gene. Its silencing can promote apple flowering. Using CRISPR / Cas9 to knock it out can obtain early flowering materials, providing experimental materials for apple reproductive physiology research. The present invention establishes a CRISPR / Cas9 gene editing system for apple fruit callus and apple tissue culture plants, providing technical tools for apple germplasm improvement and molecular breeding.

[0064] Example 1 Apple MdPDS knockout

[0065] (1) Target gene sequence design

[0066] The nucleotide sequence of apple phytoene dehydrogenase MdPDS (MD04G1023800, shown in SEQ ID NO.47) was used as a reference, and its sequence was cloned in apple and connected to the PMD-19-T (Takara) intermediate vector. After sequencing and comparison, the first nine exons of the MdPDS gene (such as Figure 1 ).

[0067] Design target sequences on the website http: / / skl.scau.edu.cn / targetdesign and select targets with a GC content of 45-70% and an off-target estimate of less than 0.6 (e.g. Figure 1 ).

[0068] (2) Construction of gene editing vector

[0069] In this example, three vectors were constructed, including two dual-target vectors and one single-target vector: MdPDS sgRNA1 and MdPDS sgRNA2 are a group, and the vector name is Cas9-PDS-1 (dual-target vector); MdPDS sgRNA3 and MdPDSsgRNA4 are a group, and the vector name is Cas9-PDS-2 (dual-target vector); MdPDS sgRNA5 is a group, and the vector name is Cas9-PDS-3 (single-target vector). The primer sequences designed based on the above targets are shown in Table 1 below:

[0070] Table 1 Primer list

[0071]

[0072]

[0073] Among them, cagt is a protective base, GGTCTCa is a BsaI recognition sequence, TGCA is the sticky end of the vector tRNA, and AAAC is the reverse complementary sequence of the sticky end gttt of the vector gRNA scaffold. The forward primer end of the double-target vector primer is the first 20bp sequence of target 1 (spacer1) + the 20bp sequence of the gRNA scaffold head, and the reverse primer end is the 20bp sequence of the tRNA end + the reverse complementary sequence of the first 20bp of target 2 (spacer2).

[0074] Using gRNA scaffold-tRNA (SEQ ID NO.22) as a template, the target sequence was cloned to both ends of gRNA scaffold-tRNA by conventional PCR amplification using the above-mentioned synthesized primers to construct an sgRNA expression cassette with a fragment length of 233 bp. The single-target vector directly synthesized a double-stranded sgRNA expression cassette by primer annealing, with a fragment length of 60 bp (as shown in SEQ ID NO.23-25). pCAMBIA1300 / Cas9 was used as an empty vector. Figure 2 , the sgRNA expression cassette was recombined into the vector using the Golden gate method. The system is shown in Table 2 below, and the reaction procedure is shown in Table 3 below.

[0075] Table 2 Golden gate connection system

[0076]

[0077] Table 3 Golden gate reaction system

[0078]

[0079] Promoters AtU6-1 and AtU6-26 were cloned in Arabidopsis, and vector homology arms were added to the promoters by conventional PCR, and the homology arm sequences were shown in SEQ ID NO. 28-29. The constructed vector was cut with HindⅢ, and different promoters were recombined into the final vector by homologous recombination to construct a recombinant vector.

[0080] The recombinant vector was transformed into DH5α, and a single colony was picked. Conventional PCR was performed using vector-specific primers JC-F / JC-R (Table 1). After agarose gel electrophoresis, the vector was sent to the company for sequencing. The size of Cas9-PDS-U6-1 is 910 bp, and the size of Cas9-PDS-U6-26 is 1019 bp. Figure 3 The correctly sequenced gene editing vector was transformed into Agrobacterium tumefaciens EHA105 for later use.

[0081] (3) Stable genetic transformation of apple callus

[0082] Agrobacterium was cultured in YEP medium in a 28°C shaker and resuspended in resuspension solution (MS + 15 g / L sucrose + 5 g / L glucose) to an OD of 600 =0.5-0.8 for standby use. Take the fruit callus of 'Wanglin' ​​apple grown for 20-30 days on the subculture medium (MS+1.5mg / L 2,4D+0.4mg / L 6-BA+30g / L sucrose+8g / L agar powder; pH adjusted to 5.8) and put it in a 100mL conical flask, pour the resuspended bacterial solution into the inoculation for 15 minutes, use filter paper to absorb the bacterial solution on the surface of the callus, and then spread the callus on the co-culture medium (MS+1.5mg / L 2,4D+0.4mg / L 6-BA+30g / L sucrose+8g / L agar powder; pH adjusted to 5.8). After three days of dark culture, the calli were transferred to screening medium (MS+1.5 mg / L 2,4D+0.4 mg / L 6-BA+25 mg / L kan+250 mg / L Cef+30 g / L sucrose+8 g / L agar powder; pH adjusted to 5.8) and continued to be cultured in the dark for 30 days.

[0083] Newly grown calli were picked and transferred to a new screening medium for secondary screening. After the callus grew up, its DNA was extracted using the CTAB method, and JC-F / JC-R primers (Table 1) were used to identify whether the transgenic was successful. The identification results were as follows: Figure 4-8 , the ones with bands were identified as positive strains.

[0084] The Cas9-PDS-1 transgenic positive callus DNA was amplified by PCR using the target detection primers PDS-2eF / PDS-3eR (Table 1), the Cas9-PDS-2 identified as positive callus DNA was amplified by PCR using the target detection primers PDS-2eF / PDS-4eR (Table 1), and the Cas9-PDS-3 identified as positive callus DNA was amplified by PCR using the target detection primers PDS-7eF / PDS-7eR (Table 1). The PCR products were sequenced.

[0085] The comparison results showed that the Cas9 vector driven by the AtU6-26 promoter to express sgRNA could edit the target sequences SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 of the MdPDS gene in apple fruit callus and make the callus present an albino phenotype, among which the Cas9-PDS-2-U6-26 vector caused a deletion of up to 837 bp ( Figure 9-12 , SEQ ID NO 26).

[0086] The Cas9 vector driven by the AtU6-1 promoter to express sgRNA can edit the SEQ ID NO.1 of the MdPDS gene in apple fruit callus and make the callus present an albino phenotype ( Fig.13 ). The callus transgenic efficiency and gene editing efficiency are shown in Table 4.

[0087] Table 4 Statistics of apple callus transformation and editing numbers

[0088]

[0089] (4) Stable genetic transformation of apple tissue culture plants

[0090] Cultivate Agrobacterium in a shaking incubator at 28°C and resuspend it to an OD of 600 =0.5-0.8 for standby use. The leaves of 'Gala 3' apple were cut into squares, infected with the resuspended bacterial solution for 5 minutes, and the bacterial solution was dried and placed in co-culture medium (MS+2mg / L TDZ+0.5mg / L NAA+25g / L sucrose+5g / L acid hydrolyzed casein complex+0.5g / L+7g / L agar powder; pH adjusted to 5.8) for dark culture for 3 days, then transferred to delay medium (MS+2mg / L TDZ+0.5mg / L NAA+250mg / L Cef+30g / L sucrose+7g / L agar powder; pH adjusted to 5.8) for dark culture for 3 days, and finally transferred to screening medium (MS+2mg / L TDZ+0.5mg / L NAA+25mg / L kan+250mg / L Cef+30g / L sucrose+7g / L agar powder; pH adjusted to 5.8) for dark culture for 30 days, and then light cultured after the leaves differentiated and sprouted. The DNA of the regenerated resistant seedlings was extracted using the CTAB method, and the JC-F / JC-R primers were used to identify whether the transgenic was successful. The identification results were as follows: Figure 14-16 , the ones with bands were identified as positive strains.

[0091] The target detection primers PDS-2eF / PDS-4eR (Table 1) were used to perform PCR amplification on the DNA of the Cas9-PDS-2 transgenic positive plants, and the target detection primers PDS-7eF / PDS-7eR (Table 1) were used to perform PCR amplification on the DNA of the Cas9-PDS-3 transgenic positive plants. The products were connected to the PMD-19-T vector, transformed into DH5α, and single colonies were picked for sequencing.

[0092] The Cas9 vector driven by the AtU6-1 promoter to express sgRNA can edit the target sequences SEQ ID NO.3 and SEQ ID NO.5 of the MdPDS gene in apple tissue culture seedlings and make the regenerated buds show albino phenotype ( Fig.14 , 17 ). The transgenic efficiency and gene editing efficiency are shown in Table 5.

[0093] The Cas9 vector driven by the AtU6-26 promoter to express sgRNA can edit the target sequence SEQ ID NO.5 of the MdPDS gene in apple tissue culture seedlings and make the regenerated plants show an albino phenotype ( Fig.18 ).

[0094] Table 5 Statistics of transformation and editing numbers of apple tissue culture seedlings MdPDS

[0095]

[0096] Example 2 Apple MdTFL1 knockout

[0097] (1) Design of target gene sequence

[0098] The nucleotide sequence of apple MdTFL1 (MD12G1023900, shown in SEQ ID NO.48) was used as a reference, and its sequence was cloned in apple, and connected to the PMD-19-T (Takara) intermediate vector. After sequencing and comparison, the exons of the MdTFL1 gene were analyzed, and the MdTFL1 target sequence ( Fig.19 , SEQ ID NO.6, SEQ ID NO.7).

[0099] (2) Construction of gene editing vector

[0100] The vector constructed in this example is a dual-target vector, and MdTFL1 sgRNA1 and MdTFL1sgRNA2 are a set of designed target site primers as shown in Table 1. The method is the same as Example 1 (2) to construct an sgRNA expression cassette such as SEQ ID NO. 27. The vector name for driving sgRNA expression with the AtU6-1 promoter is Cas9-TFL1-U6, and the vector name for driving sgRNA expression with the AtU6-26 promoter is Cas9-TFL1-U6-26. The gene editing vector with the correct sequencing was transformed into Agrobacterium tumefaciens EHA105 for use.

[0101] (3) Stable genetic transformation of apple tissue culture plants

[0102] Referring to Example 1 (4), the apple seedlings were genetically transformed stably. The DNA of the regenerated resistant seedlings was extracted using the CTAB method, and the JC-F / JC-R primers were used to identify whether the transgenic success was successful. The positive strains were identified as having bands ( Fig. 20 , 21 ).

[0103] The target genes of the positive strains were subjected to conventional PCR using primers TFL-1eF / TFL-1eR (Table 1), and the PCR products were sequenced. The sequencing results showed that both Cas9-TFL1-U6 and Cas9-TFL1-U6-26 vectors were able to perform gene editing on the two target sites of the MdTFL1 gene in apple plants. The transgenic and editing conditions are shown in Table 6. Among the edited strains, Cas9-TFL1-U6-26-11#, Cas9-TFL1-U6-26-17#, and Cas9-TFL1-U6-1# all had an early flowering phenotype ( Fig. 22 , 23 ), U6-26 promoter has better editing efficiency, and apple early flowering material is successfully obtained by the method of the present invention.

[0104] Table 6 Statistics of transformation and editing numbers of apple tissue culture seedlings MdTFL1

[0105]

[0106] The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Any changes and variations made based on the research of the present invention are within the scope of protection of the present invention.

[0107] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.

Claims

1. An apple gene editing system, characterized in that: Including promoter and sgRNA.

2. An apple gene editing system according to claim 1, characterized in that: The promoter is: the AtU6-1 promoter shown in SEQ ID NO.20 or the AtU6-26 promoter shown in SEQ ID NO.

21.

3. The apple gene editing system according to claim 1, characterized in that: The sgRNA is: the sgRNA shown in SEQ ID NO.1-7.

4. The use of the sgRNA according to claim 2, characterized in that: The sgRNA shown in SEQ ID NO.1-5 was used for apple callus editing to obtain the target gene sequences shown in SEQ ID NO.8-11.

5. The use of the sgRNA according to claim 2, characterized in that: The sgRNA shown in SEQ ID NO.1-5 was used to edit apple tissue culture seedlings to obtain the target gene sequences shown in SEQ ID NO.12-15.

6. The use of the sgRNA according to claim 2, characterized in that: The sgRNA shown in SEQ ID NO.6-7 was used to edit apple tissue culture seedlings to obtain the target gene sequences shown in SEQ ID NO.16-19.

7. The use of an apple gene editing system as claimed in claim 2 or 3, characterized in that: The application is: use in editing the apple genome, constructing apple mutants, and creating albino apple callus, albino apple or early-flowering apple plants.

8. The use of the sgRNA according to claim 4 or 5, characterized in that The application is: application in editing apple genome, constructing apple mutants and creating albino apple callus and albino apple plants.

9. The use of sgRNA according to claim 6, characterized in that The application is: application in editing apple genome, constructing apple mutants and creating early-flowering apple plants.

10. A gene editing method for apple, characterized in that: The steps include: Step 1, clone AtU6-1 and AtU6-26 promoters from Arabidopsis thaliana and construct them into CRISPR / Cas9 vector; Step 2, designing multiple target sequences on different exons based on the nucleotide sequences of apple MdPDS and MdTFL1 genes; Step 3, constructing apple MdPDS and MdTFL1 gene knockout vectors; Step 4, transforming the constructed vector into EHA105 Agrobacterium; Step 5, using the transformed Agrobacterium to perform stable genetic transformation on apple fruit callus or apple tissue culture seedlings.

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